Gut microbiota-driven imbalance of the gut-brain-immune homeostatic network in constipation: Pathogenic mechanisms and evolving therapeutic prospects

Summary

Chronic constipation (CC) is increasingly recognized as a systems-level disorder that cannot be explained by impaired intestinal motility alone. Here, we integrate evidence from clinical cohorts, animal models, and mechanistic studies to examine how gut microbial dysbiosis and altered microbial metabolites perturb epithelial barrier integrity, immune homeostasis, enteric neural activity, and gut-brain signaling. We propose a “gut-brain-immune homeostatic network” in which short-chain fatty acids (SCFAs), tryptophan metabolites, bile acids, and other microbial signals may sustain self-reinforcing interactions linking dysmotility, inflammation, and neurobehavioral disturbances. We further evaluate microbiota-directed interventions, including prebiotics, probiotics, postbiotics, fecal microbiota transplantation, engineered bacteria, and multi-omics-guided personalized strategies. This framework reframes CC as a multisystem homeostatic disorder and provides a mechanistic basis for patient stratification, therapeutic target discovery, and precision interventions for constipation and related gut-brain-immune disorders.

Graphical abstract

Disease; Omics

Introduction

Chronic constipation (CC) is a highly prevalent yet frequently underestimated gastrointestinal disorder,^1,2 characterized by varied clinical manifestations and multifactorial etiologies.^3,4 Conventional views typically attribute CC to linear mechanisms, such as impaired motility, defecatory outlet obstruction, or dysfunctional neuro-reflex pathways.^5,6,7 However, emerging interdisciplinary research suggests that CC pathogenesis extends beyond isolated disturbances in intestinal motility. Instead, it arises from a coordinated breakdown of gut microbial ecology, neural regulation, and immune homeostasis.⁸ This paradigm shift represents a transition from symptom-oriented frameworks to mechanistic reconstruction, providing an opportunity to re-conceptualize CC within the broader context of systems medicine.

The gut microbiota constitutes the most extensive and intricate symbiotic ecological system within the human body, critically involved in regulating mucosal immunity, metabolic pathways, and neural functions.^9,10 In individuals with good health, microbiota equilibrium is a biologically sophisticated and dynamically modulated process, reflecting cooperation between trillions of microorganisms and their host. This equilibrium, termed “gut homeostasis”, primarily involves maintaining the diversity of microbial populations, their functional robustness, and immune tolerance.¹¹ Under normal physiological circumstances, the gut microbial landscape is predominantly characterized by Firmicutes and Bacteroidetes, whose stable proportions are essential for optimal intestinal transit, thereby preventing gastrointestinal disturbances such as constipation or diarrhea.^12,13 Beyond its roles in vitamin production (e.g., vitamin K and B-complex vitamins) and nutrient metabolism, this microbial community actively participates in immune modulation, preserves the mucosal barrier, and prevents colonization by pathogens, thereby protecting host health.¹¹ Particularly noteworthy are microbiota-derived metabolites, including short-chain fatty acids (SCFAs), indole-based compounds, γ-aminobutyric acid (GABA), and 5-hydroxytryptamine (5-HT),^14,15,16,17 which mediate chemical signaling between microbes and the host. These metabolites play significant roles in modulating epithelial barrier integrity, neuronal excitability within the enteric system, and the rhythmic patterns of gut motility, as well as exerting distal physiological effects on mood regulation and behavior.¹⁸

The enteric nervous system (ENS), as the largest and most structurally complex component of the peripheral nervous system, is often referred to as the “second brain”. It consists of hundreds of millions of neurons and a large number of glial cells embedded within the walls of the gastrointestinal tract, forming two major networks: the myenteric plexus and the submucosal plexus. These networks are capable of autonomously regulating basic functions such as gastrointestinal motility, secretion, absorption, and local blood flow, independently of the central nervous system (CNS).¹⁹

Recent studies suggest that constipation onset and persistence may result from an imbalance in the gut-brain-immune homeostatic loop.^20,21,22 The core components of this circuit consist of three parts: the gut microbiota acts as the “sensing end” and a source of metabolic signals, the immune system serves as the “effector end” and a hub for regulating inflammation, and the central nervous system establishes a high-speed “upstream-downstream” pathway with the ENS via the vagus nerve, forming the circuit’s “neuro-regulatory axis”. These components interact through multilayered and multifaceted signaling pathways, resulting in mutual modulation and complementary regulation. As an essential theoretical framework in recent life science research, it highlights the highly integrated physiological interactions among gut microbiota, immunity, and the CNS. Specifically, the three systems are interconnected and work in concert through the following mechanisms of interaction. The gut, as the largest immune organ and a critical neuroendocrine interface,^23,24 conveys microbial ecosystem changes to the CNS through microbial metabolites (e.g., SCFAs, tryptophan derivatives), gut hormones, and vagal afferent pathways.^{25,26,27,28,29,30} This communication process affects neurotransmitter synthesis, neural plasticity, and behavioral states.^31,32 Simultaneously, the brain influences gut barrier integrity, motility, and mucosal immune responses via the autonomic nervous system and hypothalamic-pituitary-adrenal (HPA) axis, thus reshaping the gut environment.^33,34,35,36 The immune system, serving as a third regulatory hub, connects neural activity with gut homeostasis through inflammatory mediators, cytokine networks, and pattern-recognition receptors (PRRs).^37,38 Immune responses originating in the gut can alter inflammatory sensitivity and neuroimmune balance within the CNS, while neuroendocrine signals from the brain modulate peripheral immune cell activation and effector functions.^21,39,40,41 Continuous interaction among these systems creates a complex, finely regulated feedback mechanism enabling homeostasis under environmental stress, microbial perturbations, or neuropsychological challenges.

At the level of physiological plasticity, this three-dimensional interactive network maintains dynamic homeostasis through multilevel coordination. The ENS, as the central integrative hub, senses the physicochemical properties of intestinal contents, microbial metabolism, and immune signals, and regulates gastrointestinal motility. SCFAs, such as butyrate, enhance excitatory transmission by activating G protein-coupled receptors (GPR41/43) on ENS neurons, thereby promoting propulsive colonic peristalsis. Concurrently, the ENS releases inhibitory neurotransmitters such as vasoactive intestinal peptide (VIP) to modulate peristalsis according to varying physiological demands, thereby achieving a dynamic balance between propulsion and absorption.⁴² The brain-gut axis, mediated by the vagus nerve, provides higher-level central regulation. During stress, the HPA axis is activated, while inhibition of the amygdala by the prefrontal cortex limits its excessive response, preventing elevated glucocorticoids from causing intestinal barrier disruption and changes in the composition and function of the microbiome.⁴³ Furthermore, the intestinal immune system also maintains homeostasis. Myenteric macrophages participate in ENS synaptic pruning and secrete osteopontin during adulthood to sustain neuronal survival, while simultaneously regulating local inflammation to prevent runaway neurogenic inflammation.^44,45 Disruption of this feedback loop represents a common pathological basis for various metabolic, neuropsychiatric, and inflammatory conditions. Therefore, the gut-brain-immune homeostatic loop illustrates not only the integrative nature of physiological regulation but also provides an essential theoretical framework for understanding and intervening in multisystemic disorders.

Within this paradigm, CC is no longer regarded as a localized single-organ disorder, but rather as a systemic homeostatic imbalance resulting from multisystem disruptions. Constipated patients frequently demonstrate decreased microbial diversity, altered metabolite profiles, and disrupted neurotransmitter metabolism.^{46,47,48,49,50} These systemic changes not only compromise gastrointestinal motility but also reshape signaling within the gut-brain-immune loop, consequently affecting central neural activities and immune responses. Therefore, therapeutic strategies targeting gut microbiota have become critical approaches for managing CC. Methods such as probiotics, prebiotics, fecal microbiota transplantation (FMT), and traditional Chinese medicine formulations can help restore microbial equilibrium, normalize metabolic activities, and regulate neuroimmune interactions, promoting systemic homeostasis rather than merely alleviating symptoms.^51,52 Alongside these methods, next-generation precision microbiome therapies, including multi-omics-based patient stratification and AI-driven personalized microbiome interventions, are attracting increased attention.

Collectively, this review utilizes the concept of a disrupted gut-brain-immune homeostatic loop as a central theoretical model, integrating previously fragmented mechanistic insights (Figure 1). Our aim is to elucidate the systemic pathogenic basis of CC, establish stratification frameworks based on metabolic phenotypes, and foster microbiota-targeted therapeutic strategies. This integrated perspective could facilitate a transition from structural microbiome modulation to functional restoration, laying a foundation for future therapeutic interventions in CC and associated neuro-metabolic disorders.

Figure 1.

Role of the gut-brain-immune homeostatic triad in constipation onset and treatment

In individuals with constipation, dysbiosis, characterized by reduced beneficial taxa and overgrowth of pathogenic species, leads to increased microbial production of harmful metabolites such as NH₃, H₂S, and LPS, accompanied by diminished levels of 5-HT, indoles, and GABA. These alterations disrupt the intestinal epithelial barrier and activate mucosal immune cells, initiating inflammatory cascades that further impair epithelial integrity. Peripheral inflammation subsequently influences central neural circuits. Decreased beneficial microbial metabolites impact the brain through the blood-brain barrier and neural pathways, including vagal-associated nuclei such as the NTS, PBN, and PFC. The brain, in turn, modulates gut motility through neuroendocrine signaling involving the hypothalamic-pituitary-adrenal axis (CRH, ACTH, cortisol) and neurotransmitters such as norepinephrine and acetylcholine. Collectively, these processes establish a self-reinforcing cycle reflecting the disrupted gut-brain-immune homeostatic triad in constipation. The figure highlights therapeutic strategies including dietary modulation, traditional Chinese medicine, probiotics, postbiotics, prebiotics, fecal microbiota transplantation, emerging interventions, and AI-integrated multi-omics approaches aimed at restoring homeostatic balance. The figure was created using Figdraw.

Gut microbiota and its metabolites in the pathogenesis of constipation

The gut microbiota has a critical role in the initiation and progression of various functional gastrointestinal disorders.⁵³ Studies have demonstrated that the microbiota influences gut motility, epithelial barrier function, and neurotransmitter regulation through diverse pathways, including metabolic products, neural signals, and immune modulation, which are summarized in Figure 2.⁵⁴

Figure 2.

Mechanistic roles of gut microbiota and metabolites in constipation

(A) In patients with constipation, beneficial taxa (e.g., Lactobacillus, Bifidobacterium, Acidaminococcus) markedly decrease, whereas pathogenic or motility-inhibiting bacteria (e.g., Turicibacter and Gardnerella) become enriched.

(B) Depletion of Bifidobacterium and Lactobacillus reduces 5-HT synthesis, impairing neurotransmitter release from enterochromaffin cells and weakening vagal signaling, ultimately slowing intestinal transit. Reduced microbial indole production decreases AhR activation in EGCs, altering NF-κB-associated pathways that regulate mucosal inflammation and barrier integrity. Microbial or host-derived GABA modulates gut motility by binding receptors in the myenteric plexus. The figure was created using Figdraw.

(C) Under physiological conditions, SCFAs modulate gut motility directly or via G protein-coupled receptors (GPR41, GPR43). Secondary bile acids regulate motility through activation of FXR and GPCR TGR5. During dysbiosis, reduced SCFA production suppresses intestinal motility by upregulating aquaporins and promoting ATP depletion in cells. Altered bile acid metabolism further disrupts receptor-mediated signaling, impairing intestinal motility.

Reduced microbial diversity and homeostatic disruption of gut microbiota

In the intestinal ecosystem, microbiota diversity and stability significantly influence the host’s gut metabolism and immune function. Patients with CC often display dysbiosis,⁵⁵ typically characterized by decreased commensal microbiota populations and increased pathogenic species, causing substantial alterations in microbial composition. For instance, in a rat model of constipation, changes in the abundance of Lactobacillus, Bifidobacterium, and Bacteroides correlate closely with constipation development.⁵⁶ Table 1 summarizes the mechanisms and roles of representative gut microbiota in normal defecation and constipation. Given that changes in the abundance of these specific microbial communities are directly associated with intestinal function and pathological changes, they are considered potential biomarkers for assessing intestinal homeostasis and can be precisely quantified through fecal 16S rRNA sequencing.⁶³

Table 1.

Mechanisms and roles of representative gut microbiota in normal defecation and constipation

Bacterial taxon	Physiological mechanisms	Abundance in constipation	Reference
Lactobacillus	enhances multiple intestinal enzymes, inhibits harmful enzymes, increases fecal water content and luminal Lactobacillus levels, improves intestinal environment, and maintains microbial balance.	decreased	Zhang et al.57, Lee et al.58
Bifidobacterium	regulates water and electrolyte absorption, improves stool consistency, promotes intestinal peristalsis, increases fecal water content, and reshapes gut microbiota structure.	decreased	Zhang et al.57, Qiao et al.59, Wang et al.60, Ríos-Covián et al.61, Lin et al.62
Ruminococcus spp.	modulates key metabolic pathways linked to diarrhea and constipation, significantly contributes to dietary fiber degradation and SCFAs production.	increased	Ren et al.63
Acidaminococcus spp.	maintains microbial balance and activates immune cells, promoting probiotic dominance in the intestinal ecosystem.	decreased	Qiao et al.59
Shigella (peristaltic-inhibiting bacterium)	suppresses gut motility through the production of docosapentaenoic acid (DPA), a lipid mediator that reduces colonic contractions.	increased	Chen et al.64
Turicibacter	lowers fecal water content by upregulating aquaporin 8 (AQP8), a water channel protein, thus further reducing stool hydration.	increased	Chai et al.65
Gordonibacter	disrupts intestinal homeostasis by decreasing acetate production and thereby lowers SCFA levels, ultimately impairing gut balance.	increased	Zhang et al.57

The intestinal epithelial barrier represents an essential defensive interface that maintains homeostasis between the gut lumen and the internal bodily environment. It consists of epithelial cells, mucus layers, immune components, and gut microbiota.⁶⁶ The central structural components of this barrier are tight junctions between epithelial cells, including key proteins such as zonula occludens and claudins.⁶⁷ Homeostatic disruptions in the gut microbiota can compromise epithelial barrier integrity, trigger immune activation, and lead to chronic inflammation.⁶⁸ Pathogenic bacteria can degrade the mucus layer or secrete specific enzymes and toxins, damaging the epithelial barrier.^69,70 The resulting inflammatory responses further attract pathogenic bacteria, exacerbating intestinal barrier damage and forming a vicious cycle.⁷¹ Over time, pathogenic bacteria may establish biofilms resistant to immune clearance, propagate antibiotic resistance genes, induce epithelial cell transformation, and inhibit commensal microbiota growth.⁷² This further limits the commensal microbiota’s ability to produce bioactive substances, shape mucosal immunity, and strengthen the gut barrier.^69,70 Studies have shown that Lactobacillus and Bifidobacterium play crucial roles in intestinal barrier maintenance by promoting tight junction protein expression. Given that both directly improve the intestinal environment by regulating enzyme activity and increasing fecal water content,^57,58 changes in their abundance levels can serve as sensitive diagnostic indicators of intestinal function and the severity of constipation.^59,60 Conversely, certain pathogenic bacteria exacerbate constipation by suppressing intestinal motility.

Overall, the studies have established the specific manifestations of dysbiosis in patients with slow-transit constipation (STC), the considerable heterogeneity in gut microbial composition between individuals remains a major challenge. Firstly, specific bacterial genera (such as Lactobacillus and Turicibacter) may play a dual role in regulating the intestinal barrier and inhibiting intestinal motility.^57,59,65 Furthermore, mechanistic studies have largely relied on rodent models,^57,60 which struggle to fully replicate the complex neuropathological changes observed in human STC, hindering successful translation from the laboratory to the clinical setting. There is an urgent need to integrate advanced technologies, such as single-cell sequencing, to elucidate the co-evolutionary mechanisms between the host and the microbiota in gastrointestinal motility disorders, thereby providing new insights for precision interventions.

Dysregulated metabolites of gut microbiota

In addition to the ecological imbalance of gut microbiota, microbial-derived metabolites play an essential role in the initiation and progression of constipation. Recent studies have extensively explored these metabolites, confirming their significance in gut motility, epithelial function, and neurotransmitter regulation.

Impaired regulation of gut motility

Gut microbiota-derived metabolites serve as critical signaling molecules and significantly influence gut motility regulation. Among these metabolites, SCFAs and secondary bile acids have gained considerable attention as central regulators of intestinal motility mechanisms. These two types of metabolites are not only key regulatory molecules but, owing to their significant pathological changes and the fact that they can be detected, have already been used as potential diagnostic biomarkers for patients with constipation in a number of studies.⁵³

SCFAs (e.g., acetate, propionate, and butyrate), produced by commensal SCFA-producing bacteria through dietary fiber fermentation, have established roles as gut motility regulators, exerting dual effects.⁷³ In an in vitro model of the intermuscular plexus of the mouse ileum, SCFAs indirectly regulate colonic motility by activating G-protein-coupled receptors (such as GPR41/43) on enteric neurons and smooth muscle cells.⁷⁴ In IBS mouse models, SCFAs also directly enhance smooth muscle contractions, thereby precisely modulating gut motility.⁷⁵ In patients with constipation, SCFA concentrations are significantly reduced compared to healthy individuals, with a notable decrease in butyrate.⁷⁶ Based on experimental observations in mouse models of constipation induced by loperamide, reduced butyrate levels not only upregulate genes encoding specialized water channels, thus reducing fecal water content, but may also cause cellular ATP depletion, further impairing smooth muscle contractions.⁷⁶ Furthermore, patients with constipation also exhibit significantly reduced levels of propionic acid in their feces, which show a synergistic relationship with the decrease in primary and secondary bile acid levels. This specific metabolic change can be directly quantified through fecal metabolomics, offering the potential for non-invasive diagnosis.⁵⁵

Additionally, through the cultivation of cell lines and the development of molecular biology models, researchers have found that secondary bile acids (including lithocholic acid and deoxycholic acid) activate Farnesoid X receptor (FXR) and G-protein-coupled bile acid receptor (GPBAR1, also known as TGR5), triggering the FXR-TGR5 signaling pathway.⁷⁷ Activation of this pathway stimulates 5-HT secretion from enterochromaffin cells, further enhancing smooth muscle contraction and coordinating gut motility. Studies have shown that the expression of both TGR5 and FXR is significantly downregulated in the colon tissue of patients with constipation.⁵⁵ Recent studies indicate that secondary bile acids, as signaling molecules, in a primary culture model of interstitial cells of Cajal (ICCs) in the small intestine of mice and a model of the distal colon mucosa in rats, influence the electrical frequency of ICCs and synaptic interactions between neurons and smooth muscle cells,^78,79,80 thus reshaping patterns of gut motility.⁸¹ Moreover, research has revealed that patients with CC exhibit significantly reduced total fecal bile acids and primary bile acids compared to healthy individuals.⁸¹ Altered bile acid metabolism, leading to decreased secondary bile acids (e.g., deoxycholic acid), further disrupts receptor-mediated signaling and impairs intestinal motility.⁸² Furthermore, a study developed a predictive model for patients with slow-transit constipation based on nine differentially expressed metabolites, including taurocholic acid and deoxycholic acid. Validated using a random forest algorithm (with an AUC value of 0.81), this study confirmed the feasibility of secondary bile acids as potential high-precision diagnostic biomarkers.⁵⁵

Neurotransmitter dysregulation

Gut microbiota metabolites also influence defecation through modulation of neurotransmitter systems. A systematic review in 2025 suggested that constipation’s core neuropathology arises from a combination of microbial dysbiosis, metabolic imbalance, and neurotransmitter dysregulation, a concept increasingly supported by current research.⁸³ The metabolic fingerprint corresponding to the specific metabolic pathways and molecular changes involved in this tripartite imbalance has the potential to serve as a biomarker for the classification and diagnosis of functional constipation and for the assessment of treatment efficacy, and can be accurately detected using high-throughput metabolomics techniques. Specifically, microbial metabolites, including 5-HT, indole derivatives, and GABA, directly or indirectly affect enterochromaffin cells, the ENS, and vagal nerve pathways, influencing intestinal motility, sensory transmission, and gut-brain communication.

The monoamine neurotransmitter 5-HT is primarily synthesized through gut microbiota-mediated alterations in tryptophan metabolism.^84,85 Accumulating evidence indicates that the microbiota influences gastrointestinal motility by regulating the tryptophan-5-HT metabolic pathway, playing a significant role in constipation pathogenesis. Clinical evidence suggests that fluctuations in 5-HT levels in the serum and feces of patients with constipation are closely related to the function of vagal nerve signaling, this suggests that the ratio of 5-HT to its precursor (tryptophan) may serve as an important indicator for quantifying gut neurodynamic dysfunction.⁸⁶ In patients with IBS undergoing transcutaneous auricular vagus nerve stimulation (taVNS), reduced abundance of microbiota promoting 5-HT synthesis, such as Bifidobacterium and Lactobacillus, results in decreased neurotransmitter release from enterochromaffin cells, weakened vagal signaling, and delayed intestinal transit.⁸⁶

In addition to 5-HT, microbiota-derived indole metabolites (e.g., indole-3-acetate and indole-3-propionate) function as endogenous ligands for the aryl hydrocarbon receptor (AhR). These compounds activate the AhR signaling pathway, regulating enteric glial cells (EGCs) functions. As the metabolic levels of indole compounds directly reflect the activation status of the AhR pathway and intestinal barrier function, changes in their concentrations have been shown to serve as effective diagnostic markers for distinguishing between different subtypes of constipation and assessing the status of intestinal inflammation.⁸⁷ In patients with IBS, deficiencies in indole compounds may reduce AhR pathway activation, thus impacting gut inflammation and barrier integrity through pathways such as NF-κB, ultimately contributing to constipation. Wang et al. preliminarily validated this phenomenon.⁸⁷

Furthermore, GABA and tyrosine-derived metabolites (e.g., dopamine and tyramine) modulate enteric neuron excitability through distinct pathways, affecting gut motility and potentially contributing to constipation. New research indicates that the gut microbiota can influence gastrointestinal motility by regulating the GABA signaling pathway, and it has been demonstrated that the expression levels of GABA and its receptors can serve as potential biomarkers reflecting an imbalance in local neural excitability in the gut.⁸⁸ Studies have demonstrated that GABA, a primary inhibitory neurotransmitter, in the in vitro electrophysiological models of the intermuscular plexus of the mouse ileum, reduces myenteric neuron excitability in the mouse ileum by mediating calcium signaling through GABA receptors.⁸⁹ Dopamine inhibits neurotransmitter release from cholinergic motor neurons, reducing postganglionic circuit excitability and modulating colonic contraction patterns.⁹⁰ Additionally, microbial-derived tyramine promotes 5-HT secretion from enterochromaffin cells, indirectly enhancing overall enteric neuronal circuit excitability.⁹¹

Although previous studies have clearly elucidated, at the molecular level, the regulatory networks of short-chain fatty acids, secondary bile acids, and various neurotransmitters on intestinal function, a significant disconnect still exists between microscopic metabolic signals and macroscopic physiological phenotypes. Existing studies on intestinal motility, such as the mouse ileal myenteric plexus model and the primary ICC culture system,^78,79,80 have confirmed that metabolites can regulate smooth muscle contraction and pacemaker cell discharge. However, no direct quantitative correlation has yet been established with overall propulsive functions such as colonic peristaltic complex movements. At the same time, metabolomics largely relies on static fecal analysis, making it difficult to reflect the spatial heterogeneity and dynamic flux of metabolites along the colonic axis.^55,75,81,82 Research on neurotransmitters is also limited by insufficient source identification. It is currently difficult to distinguish between microbial and host sources of 5-HT, GABA, and dopamine,^84,89 which hinders the elucidation of the mechanisms underlying the “microbiota-metabolite-neurotransmitter” axis. Future efforts should combine in vivo imaging with isotope tracing to enable simultaneous analysis of metabolic spatial distribution and intestinal motility, and to clarify the microbial contribution to neurotransmitters, thereby bridging the critical gap between mechanistic understanding and translational applications.

Mechanistic role of the “gut-brain-immune homeostatic tri-axis” in CC

Different subtypes of constipation exhibit significant differences in terms of imbalances in the composition and function of the gut microbiota, abnormal neural regulation, immune and inflammatory activation, and their interactions. To facilitate a systematic understanding of their multidimensional pathological characteristics and corresponding intervention approaches, Table 2 summarizes common types of constipation along with their core mechanisms and treatment strategies.

Table 2.

Characteristics of microbiome changes, immune and neural mechanisms, and treatment strategies for common subtypes of constipation

Subtypes of constipation		Changes in the gut microbiome	Immune mechanisms	Neural mechanisms	Neuroimmune pathway	Treatment strategies	Reference
Functional constipation	normal transit constipation (NTC)	an increase in the abundance of the genus Ruminococcus promotes the synthesis of SCFAs and is negatively correlated with intestinal transit time	increased intestinal permeability, elevated levels of CD3+, CD4+, and CD25+ T cells, enhanced spontaneous lymphocyte proliferation, and elevated levels of antibacterial antibodies	normal ENS structure or sensory input impairment, mild inflammation	a slight imbalance in the gut microbiota mildly activates the TLR pathway, leading to mast cell activation and the release of trace amounts of IL-6 and TNF-α, which disrupt the enteric nervous system, resulting in visceral hypersensitivity and abnormal bowel movements.	osmotic laxatives as the primary treatment, combined with cognitive/psychological interventions	Wan et al.20, Ren et al.63, Ihara et al.92, Burgell et al.93, Khalif et al.94, Bilgic et al.95, Kabouridis and Pachnis96
	slow-transit constipation (STC)	a decrease in the Firmicutes phylum, an increase in the Bacteroidetes phylum, and a decrease in the F/B ratio; a reduction in Bifidobacterium and Lactobacillus, and an increase in Bacteroides and Prevotella	myenteric macrophages and iNOS, IL-6, and TNF-α alter the microenvironment; macrophage-derived BMP2 influences enteroneurons and motility via the BMP2/Smad pathway.	loss of ENS neurons, disruption of the ICC network	overexpression of the TLR5/IL-17A pathway leads to intestinal neurofunctional disorders and slowed colonic motility.	volumic/osmotic laxatives as the primary treatment, with prokinetic/secretagogue agents added as needed	Zhuang et al.54, Fan et al.55, Ren et al.63, Wang and Yao97, Tang et al.98, Anorectal Branch of Chinese Medical Doctor Association99, Liang et al.100, Ren et al.101
	defecation disorders (DD)	decreased butyrate-producing bacteria (overlapping with functional constipation)	/	abnormal central regulation/pelvic floor neuromuscular coordination disorder	abnormal TLR4 signaling and dysregulation of mast cells and macrophages lead to visceral hypersensitivity, impaired nitrogen-mediated neurotransmission, and dysfunction of the pelvic floor muscles.	with biofeedback as the core approach, combined with lubricating/osmotic laxatives and pelvic floor exercises; for those who do not respond, sacral nerve stimulation or surgery may be considered.	Zhuang et al.54, Horowitz et al.67, Wang and Yao97, Anorectal Branch of Chinese Medical Doctor Association99, Li et al.102, Habib et al.103
irritable bowel syndrome with constipation (IBS-C)		a decrease in Bifidobacterium and Lactobacillus, an increase in Veillonella, Escherichia coli, and Shigella, and impaired metabolism of indole derivatives	abnormal levels of pro-inflammatory factors such as IL-6, IL-8, and TNF-α were observed; following lactic acid bacteria intervention, these pro-inflammatory factors decreased and constipation improved.	high visceral sensitivity, abnormal neuro-immune interactions	TLR signaling is activated, mast cells are activated, and sustained action of IL-6 and TNF-α leads to sensitization of sensory neurons and disruption of intestinal motility.	osmotic laxatives are used as first-line treatment; if symptoms are not adequately controlled, treatment should be escalated to secretagogues (such as linaclotide, prucalopride, or tinapanol) or 5-HT4 receptor agonists (such as tegaserod).	Wang et al.87, Ihara et al.92, Mokhtar et al.104, Yuan et al.105, Wang et al.106, Vasant et al.107
Secondary constipation	opioid-induced constipation (OIC)	in the loperamide model, there is a reduction in Bifidobacterium and Lactobacillus, a decrease in butyrate, and disruption of the neurotransmitter profile (e.g., 5-HT)	morphine induces GFAP/Ca2+-related activation via μ-opioid receptors on enteric glial cells, promoting the release of pro-inflammatory factors.	μ-opioid receptor-mediated inhibition of the ENS	the drug activates μ-opioid receptors, suppresses immune cell activity, alters the gut microbiota, damages the intestinal barrier, and disrupts the ENS, leading to slowed motility and reduced secretion.	start with osmotic laxatives; if ineffective, escalate to peripheral μ-receptor antagonists (PAMORAs)	Makizaki et al.56, Zhang et al.57, Qiao et al.59, Wang et al.60, Chai et al.65, Wobbe et al.108, Serra et al.109, Lin et al.110, Gao et al.111, Portincasa et al.112
	neurogenic constipation (Parkinson’s disease, spinal cord injury, etc.)	low gut microbiota diversity, a reduction in butyrate-producing bacteria (such as the genus Faecalibacterium), and disruption of the microbiota-gut-brain axis (MGBA) signaling affect the function of the vagus nerve and enteric nervous system.	following injury, changes occur in the gut microbiota and metabolic signaling, leading to atrophy of the ENS and reduced intestinal motility. Dietary fiber or SCFA interventions protect the ENS via IL-10 signaling, thereby limiting neurodegeneration and motility disorders.	degeneration of the dorsal nucleus of the vagus nerve, ENS α-synaptophysin deposits (Parkinson’s disease); disruption of the brain-sacral spinal cord pathway for defecation (spinal cord injury)	central nervous system lesions (α-synuclein deposits, disruption of neural pathways) lead to secondary ENS dysfunction and intestinal immune dysregulation, resulting in motility disorders.	centered on an individualized bowel movement plan, combining oral and rectal medications with manual evacuation techniques	O’Riordan et al.83, Shi et al.86, Johns et al.113, Sakakibara et al.114, Han et al.115
	metabolic/endocrine constipation (diabetes, hypercalcemia, hypokalemia, etc.)	an imbalance in the ratio of Firmicutes to Bacteroidetes leads to a decrease in butyrate-producing bacteria, and their metabolic products inhibit the excitability of cholinergic neurons.	systemic low-grade inflammation and cytokine abnormalities in diabetes increase enteric neurotoxicity; IL-8 is associated with delayed gastric emptying, and IL-10 is associated with prolonged colonic transit.	diabetic autonomic neuropathy (diabetes)	metabolic abnormalities such as hyperglycemia and electrolyte imbalances trigger intestinal autonomic neuropathy and systemic inflammation, disrupting neuro-immune homeostasis and leading to reduced motility.	first, correct electrolyte imbalances and control blood glucose levels; then select a laxative based on symptoms.	Koussoulas et al.89, Dehkhoda et al.90, Meling et al.116, Andreyev et al.117, Okdahl et al.118
	structural/mechanical constipation (colorectal tumors, intestinal adhesions, strictures, etc.)	a decline in microbial diversity and the overgrowth of potentially pathogenic bacteria (such as Clostridium difficile and Pseudomonas aeruginosa) exacerbate local inflammation and barrier damage.	damage to mesothelial cells following injury to the peritoneum or intestinal wall triggers an imbalance in coagulation, inflammation, and fibrinolysis; infiltration by neutrophils, macrophages, T cells, and mast cells promotes the release of IL-6, TNF-α, and TGF-β, as well as collagen deposition and fibrin exudation, leading to the formation of adhesions or strictures.	/	physical intestinal obstruction causes intestinal distension, which activates immune cells and the TLR pathway; bacterial translocation occurs, and local inflammation damages the ENS, leading to secondary motility disorders.	with the goal of relieving the obstruction, surgical or interventional treatment as needed	Ochieng et al.70, Grishin et al.71, Andreyev et al.117, Faggin et al.119, Hu et al.120

Gut-brain communication imbalance: A self-amplifying pathological loop in CC

Considerable epidemiological evidence indicates that individuals with anxiety or depressive disorders are significantly more susceptible to developing CC than the general population.¹²¹ Traditional views typically describe CC as a localized colonic motility disorder. However, this narrow definition fails to explain its strong comorbidity with psychological conditions. With improved understanding of bidirectional gut-brain interactions, CC is increasingly recognized as a psychosomatic disorder, characterized by imbalanced neural, endocrine, and metabolic signaling within the gut-brain axis (Figure 3). Gut-derived chemical signals may fail to be correctly interpreted by the CNS, whereas neural commands originating in the brain become disrupted. Together, these disturbances intensify impaired motility and visceral sensory abnormalities.¹²²

Figure 3.

Gut-brain communication and CC: integrated bottom-up microbial signaling and top-down neuroendocrine regulation

(A) SCFAs activate GPR43 receptors on enterochromaffin cells, triggering phospholipase C-β, PIP₂ hydrolysis, IP₃ formation, and intracellular Ca²⁺ release, promoting 5-HT secretion. Concurrently, GABA-B receptor activation promotes K⁺ efflux and suppresses Ca²⁺ influx. Released 5-HT signals reach the NTS, PBN, prefrontal cortex, and amygdala, modulating mood, stress responses, and gut motility.

(B) Emotional stress activates the HPA axis via the CRH-ACTH-glucocorticoid cascade, disturbing microbial homeostasis and altering gut motility. Sympathetically mediated release of norepinephrine inhibits intestinal transit, whereas acetylcholine release from DMV promotes smooth muscle contraction and gut motility. Figure created using Figdraw.

In the early stages of the disease, CC is primarily characterized by functional signaling dysregulation and remains within a reversible compensatory range. However, as it progresses to the long-term, refractory stage, CC evolves into an organic neuropathy, manifested by significant neuronal loss in the enteric nervous system (ENS), disruption of the network of ICCs, and a shift in the excitatory-inhibitory balance toward an inhibitory dominance.^123,124 This progression from functional compensation to structural damage may well be the pathological basis for the “self-reinforcing” nature of CC and its tendency to become intractable. At the same time, emotional stress can exacerbate constipation symptoms, while persistent difficulties in defecation further aggravate mood disturbances.^125,126 These interactions form a self-reinforcing pathological cycle.

Bottom-up signaling: Gut microbiota-driven disruption of CNS function

Gut microbiota and their metabolites serve as key long-distance modulators of CNS activity. They regulate neuroactive molecule synthesis, reshape ENS functions, and influence vagal afferent pathways that transmit chemical and mechanical signals from the gut lumen to the brain. These microbiota-derived signals alter activity in brainstem, limbic, and hypothalamic regions, which are crucial for mood control and autonomic regulation. Alterations within these neural circuits strongly correlate with anxiety and depression. Mood and autonomic disturbances can heighten visceral sensitivity, causing patients to perceive normal colonic distension as discomfort, thus leading to defecation avoidance.¹²⁷ Emotional disorders are also commonly associated with decreased physical activity, lower dietary fiber intake, and altered bowel habits, further exacerbating CC.^128,129

The vagus nerve represents the primary conduit for ascending gut-to-brain signaling. SCFAs, critical microbial metabolites, activate G-protein-coupled receptor GPR43, highly expressed on enterochromaffin cells.¹³⁰ GPR43 couples to both Gq and Gi/o signaling pathways.¹³¹ Gq-mediated PLC-β/IP₃ signaling elevates intracellular Ca²⁺ levels, promoting 5-HT release. The Gi/o βγ subunits further enhance calcium transients, facilitating 5-HT secretion.^132,133

By contrast, the causal relationship between microbiota-derived GABA and constipation is not yet firmly established. In vitro studies have shown that, microbial-derived GABA activates GABA-B receptors (Gi/o) on enterochromaffin cells, triggering potassium efflux and membrane hyperpolarization. This process inhibits Ca²⁺ influx, reducing neurotransmitter release.^134,135,136 As a central mediator in gut-brain communication, 5-HT stimulates vagal afferent terminals, projecting signals through the nucleus tractus solitarius (NTS) to the parabrachial nucleus (PBN), amygdala, and prefrontal cortex, thus modulating mood, stress response, and bowel function.^137,138 However, the aforementioned mechanism lacks in vivo, real-time, cell-type-specific causal validation and requires further investigation. On this basis, various components of the GABA signaling pathway (such as the expression levels of GABA-B receptors in intestinal chromaffin cells and the 5-HT/GABA ratio) may be worthy of preliminary clinical evaluation as exploratory biomarkers. However, their clinical utility cannot be established until causal relationships have been clarified.

In addition, the microbiota generates small metabolites that can enter the systemic circulation, some of which may cross the blood-brain barrier (BBB) and influence CNS activity. Microbial GABA and 5-HT modulate local ENS activity and may affect rhythmic neuronal firing within the dorsal motor nucleus of the vagus (DMV) and limbic circuits, influencing gut reflexes and emotional stress responses.^139,140,141 However, the human evidence currently supporting this view is derived mainly from cross-sectional or case-control studies, and remains essentially correlational. Taking SCFAs as an example, numerous epidemiological studies have demonstrated reduced SCFA levels in patients with CC.⁸ However, such designs cannot distinguish whether the reduction in SCFAs is the cause or the consequence of constipation, or whether both are driven by third factors (such as intestinal transit time or dietary composition).

Furthermore, as epigenetic regulators, SCFAs inhibit histone deacetylases (HDACs), stimulating brain-derived neurotrophic factor (BDNF) expression,¹⁴² which enhances emotional resilience and stability. Reduced SCFA availability weakens this protective effect, reinforcing the reciprocal cycle between constipation and mood disorders. This further supports the theoretical possibility that SCFAs may serve as potential biomarkers for gut-brain axis dysregulation associated with constipation. At present, quantitative analysis of fecal SCFAs using gas chromatography-mass spectrometry (GC-MS) has been implemented in some research hospitals to assess patients’ gut microbiota metabolic function and to guide the development of personalized prebiotic or probiotic intervention programs.¹⁴³ For example, a clinical trial involving Bifidobacterium HN019 confirmed that its efficacy in alleviating constipation was positively correlated with increased levels of fecal SCFAs.¹⁴⁴ However, the aforementioned epigenetic mechanisms currently lack causal validation in humans: whether the upregulation of BDNF is a direct effect of SCFAs or an indirect compensatory response remains to be further investigated.

Dysbiosis also redirects tryptophan metabolism away from 5-HT synthesis toward the kynurenine pathway.¹⁴⁵ This metabolic shift may elevate neurotoxic metabolites such as quinolinic acid, accumulating in the brain and impairing neuronal function.¹⁴⁶ Quinolinic acid-induced excitotoxicity via N-methyl-D-aspartate (NMDA) receptors is a central pathological mechanism observed in several neurodegenerative disorders, including multiple sclerosis.¹⁴⁷ Lower availability of substrates for 5-HT synthesis weakens serotonergic signaling, exacerbating anxiety and depression, and further impairing gut function through emotional, behavioral, and autonomic pathways, ultimately exacerbating constipation. The above discussion is limited to the unilateral damage caused by 5-HT to the nervous system, further experimental evidence is required to establish a complete causal link between 5-HT, the central nervous system and constipation.

Top-down regulation: Stress and cortical control in shaping intestinal homeostasis

As a complex neural network embedded within the intestinal wall, the ENS acts both as the final executor of central commands and as a hub for the integration of local intestinal signals. Psychological and social stress signals originating from higher cortical areas, including the amygdala and prefrontal cortex, regulate intestinal homeostasis by activating the HPA axis and autonomic nervous system. Maintaining a balance between sympathetic and parasympathetic outputs is critical and closely influenced by gut microbiota function and the role of ENS in integration.

Animal studies provide direct evidence of a causal relationship between stress and CC. Prolonged exposure to light can induce a constipation phenotype in zebrafish larvae, characterized by elevated cortisol levels, reduced intestinal neuronal activity and decreased fecal output.¹⁴⁸ Chronic Unpredictable Mild Stress (CUMS) can also induce constipation-like phenotypes in rodents, such as reduced defecation and slowed colonic transit.¹⁴⁹ Notably, disruptions in the gut microbiota composition are consistently observed in rodent models, and these disruptions often precede damage to the intestinal barrier.¹⁵⁰ To further validate the causal relationship between the two, researchers conducted intervention studies by manipulating the microbiota and found that supplementation with probiotics such as Bifidobacterium longum or microbial metabolites such as butyrate could effectively reverse the aforementioned stress-induced constipation phenotypes.^148,151 These results collectively demonstrate that the gut microbiota plays a crucial mediating role in the causal chain of stress-induced CC.

During emotional stress, electrophysiological and microdialysis studies in rodent models, as well as functional magnetic resonance imaging (fMRI) and cerebrospinal fluid analyses in healthy volunteers, have all shown that activation of the locus coeruleus-noradrenergic system enhances central sympathetic tone, significantly increasing norepinephrine (NE) release.¹⁵² Research based on an in vitro co-culture system has found that NE modulates microbial gene expression, growth dynamics, and virulence factors,¹⁵³ shifting microbial communities toward pathogenic species (e.g., Escherichia coli) and suppressing Escherichia coli, thus disrupting ecological stability.¹⁵⁴ For example, research has shown that noradrenaline significantly promotes the proliferation of F4⁺ enterotoxigenic Escherichia coli (ETEC) isolates and enhances the susceptibility of F4⁺ ETEC to antibiotics, thereby disrupting the ecological stability of the microbiota.¹⁵⁵ NE also acts on adrenergic receptors expressed by enteric neurons, inhibiting intestinal motility.^156,157 This inhibition likely results from suppressed enteric neuronal excitability and reduced firing frequency, diminishing high-amplitude propagating contractions and slowing intestinal transit.¹⁵⁸

Parasympathetic withdrawal, particularly diminished vagal tone, plays an equally important role during stress. Descending vagal fibers from the DMV regulate intestinal motility through neurotransmitters activating muscarinic acetylcholine receptors within the myenteric plexus.¹⁵⁹ Constipation is highly prevalent among patients with Alzheimer’s disease (AD).¹⁶⁰ Postmortem studies revealed β-amyloid accumulation and neuronal loss within the colonic ENS, especially in the myenteric plexus, disrupting integrity of the motility reflex arc.¹⁶¹ In Tg2576 mouse models of AD, reduced colonic M1 and M3 muscarinic receptor expression occurs alongside endoplasmic reticulum stress activation, suggesting vagal inhibition and impaired cholinergic signaling underlie constipation associated with AD.¹⁶²

In recent years, two independent experiments have provided strong support for the aforementioned “top-down” regulatory pathway. Using chemogenetics and optogenetics, Ma et al. identified a neural circuit in mice that originates from glutamatergic neurons in the paraventricular nucleus (PVN) of the hypothalamus and transmits signals to the small intestine via cholinergic neurons in the dorsal motor nucleus of the DMV.¹⁶³ Artificial inhibition of this circuit induces a constipation-like phenotype, whereas activation of the circuit alleviates morphine-induced constipation. Dai et al. further found in the mice of depression that electroconvulsive therapy (ECT) improves colonic motility, while subphrenic vagotomy completely blocks this effect.¹⁶⁴ These two studies corroborate each other and collectively suggest a regulatory pathway extending from the emotional centers to the gut via the vagus nerve. However, the causal relationship of this pathway in CC remains to be further verified.

Functional connectivity deficits within cortical and hypothalamic areas further compromise bowel regulation. Studies of patients with Parkinson’s disease (PD) showed decreased PVN connectivity correlated with constipation severity.¹⁶⁵ Similar impairments observed in patients with AD suggest that neuroendocrine pathways contribute to intestinal dysfunction.

Persistent HPA axis activation is another hallmark of stress physiology. Elevated glucocorticoid levels during chronic psychological stress or mood disorders inhibit epithelial proliferation, reduce tight junction protein synthesis, and increase gut permeability. These changes alter the luminal environment and microbial composition. Glucocorticoids also directly suppress motility rhythms and propulsive activity by acting on receptors expressed by enteric neurons and smooth muscle cells, ultimately exacerbating constipation.^166,167,168

Collectively, these findings indicate emotional stress profoundly influences gut homeostasis through hierarchical regulation involving cortical, hypothalamic, and autonomic pathways. CC thus emerges as a self-sustaining pathological loop. Understanding these mechanisms may provide opportunities to overcome existing therapeutic obstacles and enhance insight into the integrated dynamics of the gut-brain-immune homeostatic tri-axis.

Interactions between gut and immune system in CC

CC does not result from a single motor abnormality. Rather, it represents a complex pathological condition arising from persistent disturbances among the intestinal immune barrier, mucosal immune homeostasis, and gut motility. Recent evidence suggests that epithelial barrier disruption enhances luminal microbial and metabolite translocation, triggering mucosal inflammation and disturbing neural-muscular regulation in the intestine. Continuous immune activation further damages barrier integrity and impairs responsiveness of enteric ganglia and smooth muscles. This cyclical effect is summarized in Figure 4. This cycle progressively weakens propulsive motility, leading to refractory CC. Therefore, clarifying the regulatory interactions among gut barrier, immune function, and motility is critical for understanding the chronic nature of constipation and developing novel therapeutic approaches.

Figure 4.

Mechanisms linking epithelial barrier disruption, immune dysregulation, and impaired smooth muscle contraction in CC

(A) Following disruption of the intestinal epithelial barrier, bacterial LPS binds more readily to TLR4 on the surface of epithelial cells or immune-related cells, activating the TLR4/NF-κB-mediated inflammatory pathway and promoting the expression of pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α, thereby amplifying the local mucosal inflammatory response.

(B) The disruption of tight junctions and increased epithelial permeability can further promote the release of inflammatory factors, among which IL-1β and TNF-α can exacerbate immune dysregulation and lead to abnormalities in neuromuscular regulation by affecting signals related to enteric neurons, glial cells, and smooth muscle. MLCK activation may also contribute to tight junction disruption and impaired barrier function, creating a positive feedback loop between inflammation and barrier damage.

(C) In intestinal smooth muscle cells, IL-1β-related signaling can influence the RGS4, Gαq-PLC-β, Ca²⁺/CaM-MLCK, and CPI-17/MLC phosphatase pathways. Upregulation of RGS4 may attenuate Gαq-PLC-β-mediated calcium signaling and MLCK activity, reducing MLC20 phosphorylation levels and leading to weakened initial contraction. Concurrently, decreased CPI-17 levels may reduce inhibition of MLC phosphatase, enhancing MLC20 dephosphorylation and further weakening sustained contraction. These changes may ultimately reduce propulsive intestinal motility and contribute to the development and maintenance of CC. This figure was created using Figdraw.

Intestinal immune barrier disruption amplifies inflammation and drives constipation

The intestinal barrier, as the primary structure sustaining luminal homeostasis, relies on coordinated functions of microbiota, mucosal immune cells, and ENS to maintain epithelial integrity, nutrient absorption, and immune tolerance.¹⁶⁹ When compromised, bacteria, toxins, and microbial metabolites cross the epithelium more readily, activating mucosal immunity and intensifying inflammation. Reduced expression of tight junction proteins, disrupted local microenvironment, and persistent inflammatory exposure collectively destabilize barrier architecture, elevating permeability. Barrier damage and immune activation reinforce each other and disrupt gut neural-muscular circuits. This suppresses motility and reduces defecation efficacy, establishing a critical pathological mechanism underlying CC.

Tight junction proteins, including occludin, claudin-1, and ZO-1, maintain epithelial integrity. Their decreased expression or mislocalization significantly increases epithelial permeability.¹⁷⁰ In patients with CC, impaired motility results in prolonged stool retention,¹⁷¹ altering luminal pH, increasing oxidative stress, and accumulating toxic metabolites.^172,173,174 These factors directly damage epithelial cells and disrupt tight junctions,⁴⁹ further exacerbating constipation. Inflammatory mediators are central to this process: TNF-α suppresses occludin and claudin-1 expression via NF-κB signaling,¹⁷⁵ whereas IL-1β disrupts tight junction structure through myosin light-chain kinase (MLCK) activation.¹⁷⁶ These pathways synergistically worsen barrier damage. Genetic alterations also compromise barrier stability. For example, mice harboring mutations in PTPN2 lose T cell protein tyrosine phosphatase activity, significantly reducing tricellulin expression, weakening tricellular junctions, and increasing permeability, thereby underscoring the gene’s critical role in epithelial homeostasis.¹⁷⁷ Research on the Otud3 global knockout mouse models indicates that loss of OTUD3 disrupts ubiquitin regulation in epithelial cells, increasing stress sensitivity and susceptibility to inflammation, thereby impairing barrier repair. Although increased mucosal permeability and enhanced spontaneous pro-inflammatory signaling were observed in OTUD3-mutant duodenal and colonic organoids in vitro, intestinal inflammation in vivo was confined to the colon, suggesting that additional interacting factors, such as the microbiome and immune compartments, are required for disease pathogenesis.¹⁷⁸ Once barrier integrity is compromised, pathogen-associated molecular patterns (e.g., flagellin, bile acids) penetrate submucosal tissues and activate innate immunity, initiating chronic low-grade inflammation that further impairs permeability and gut function.^179,180,181 The gut microbiota can influence the composition and metabolism of bile acids, which, in turn, via receptors such as FXR and TGR5, may be involved in the regulation of the intestinal barrier, mucosal immunity, and intestinal motility. Therefore, in CC, abnormalities in bile acids may represent one of the metabolic pathways through which alterations in the microbiota affect the intestinal microenvironment.¹⁸² Furthermore, relevant studies have shown that FXR signaling is associated with NF-κB-mediated inflammatory responses, whilst TGR5 may also be involved in the regulation of the NOD-like receptor protein 3 (NLRP3) inflammasome and macrophage responses, suggesting that bile acid receptor signaling may play a role in the link between local inflammation and alterations in motility.¹⁸³ This cycle of barrier injury and inflammation disrupts neuroimmune signaling within the gut wall, diminishing smooth muscle excitability and contractile function, thus promoting constipation persistence.¹⁸⁴

Barrier disruption and microbial dysbiosis form a reciprocal amplification cycle that perpetuates constipation progression. Microbiota dysbiosis should not be viewed merely as a concomitant change following delayed intestinal transit. It may also exert a reciprocal influence on intestinal motility. Ge et al. performed fecal microbiota transplantation from patients with STC into pseudo-germ-free mice and observed that the recipient mice exhibited decreased defecation frequency, reduced fecal water content, delayed gastrointestinal and colonic transit, and weakened colonic smooth muscle contractility, suggesting that STC-associated microbiota can confer a constipation-like motility disorder phenotype under experimental conditions.¹⁷¹ Furthermore, under normal conditions, commensal bacteria metabolize tryptophan into indoles and related derivatives, which preserve barrier integrity and modulate mucosal immunity.¹⁵ Indoles act as AhR ligands, regulating epithelial structure and immune responses via the AhR-IL-22 axis. They promote barrier repair, defend against pathogens, and sustain intestinal stem cell renewal.¹⁸⁵ Bifidobacterium species also generate indole-3-lactic acid, interacting with AhR to suppress IL-8 transcription, thus exerting anti-inflammatory effects.¹⁸⁶ Upon barrier disruption, microbial penetration into deeper tissues activates local immune responses, promoting pathogenic species growth and reducing beneficial commensals, causing dysbiosis.¹⁸⁷ Dysbiosis further impairs barrier healing. For instance, decreased abundance of butyrate-producing bacteria reduces SCFA availability, diminishing epithelial energy sources and protective functions, exacerbating barrier damage. Although there is currently limited evidence to suggest that SCFA-based therapies lead to clinical improvement in patients with CD, the supplementation of specific probiotics to promote butyrate production may nevertheless offer a new avenue for controlling disease activity.¹⁸⁸

Persistent inflammatory exposure accelerates intestinal epithelial apoptosis, further weakening the barrier. In patients with Behçet’s syndrome, monocyte-derived TNF-α activates the IFI16-STING-TBK1 signaling pathway and subsequently Polo-like kinase 1, driving epithelial apoptosis.¹⁸⁹

Collectively, barrier disruption, microbiota-associated perturbations, and enhanced inflammation form a fundamental pathological basis for CC development. This underscores the interdependence of epithelial barrier function, microbiota composition, and immune responses. These insights suggest therapeutic possibilities: strategies aimed at strengthening barrier repair, restoring microbial balance, or modulating inflammation represent promising directions for managing CC. In animal studies, Tang et al. used loperamide-induced constipation mouse models and found that intervention with Bifidobacterium lactis TY-S01 improved intestinal propulsion, fecal water content, barrier damage, inflammatory factors, and gastrointestinal motility-related indicators such as 5-HT, MTL, and SP, accompanied by changes in the gut microbiota and metabolites.¹⁷⁴ These results suggest that microbiota modulation may influence intestinal motility and the barrier-immune status in constipation-like animal models. However, this evidence does not yet directly prove that microbiota dysbiosis is the primary cause of CC in humans.¹⁷⁴

Immunological dysregulation drives gut inflammation and constipation

In pathological models of CC, disturbances in immune homeostasis often represent critical initiating events in disease progression. Abnormal activation of the immune system triggers mucosal inflammation, characterized by elevated inflammatory mediators (e.g., IL-1β, IL-6, TNF-α), and also remodels the ENS. Such remodeling disrupts mucosal barrier integrity and impairs smooth muscle contractility, establishing an immune-driven pathological foundation for CC onset. Consequently, the immune signaling pathways described in this section should not be viewed simply as entirely independent, parallel pathways, but rather as a network in which multiple signaling pathways interact with one another.¹⁹⁰ Damage to the barrier, changes in the microbiota and its metabolites, and TLR/NF-kB-mediated inflammatory responses may each contribute to local immune activation and may further influence neuro-smooth muscle function via epithelial cells, immune cells and enteric glial cells.^191,192,193

In the normal intestinal wall of mice and in a DSS-induced colitis model, research has demonstrated the presence of an intermuscular neural plexus barrier in the intestinal wall of mice, functionally analogous to the blood-brain barrier in the CNS. This barrier effectively limits peripheral immune molecules from entering enteric ganglia.¹⁹⁴ Under immune dysregulation, macrophages secrete matrix metalloproteinases and related enzymes that degrade this barrier, increasing its permeability.¹⁹⁴ Consequently, TNF-α, IFN-γ, and IL-1β released by local immune cells in the gut directly interact with the enteric ganglia.¹⁹⁵ These cytokines can bind to receptors on the surface of EGCs and reprogram their metabolic and signaling pathways, inducing them to adopt a reactive enteric glial state rather than a fixed M1-like phenotype.¹⁹⁶ Using models of postoperative ileus-associated motility disorders and EGC-restricted IL-1R1-deficient mice, Schneider et al. found that selective blockade of IL-1R1 signaling in EGCs reduced enteric gliosis, decreased immune cell infiltration, and improved motility disorders. These findings suggest that the IL-1β-EGC signaling axis is not only associated with inflammatory responses but may also be directly involved in immune-neural pathway-mediated motility abnormalities.¹⁹⁶ It is worth noting that this reactive state does not conform to simple M1/M2 binary models, but rather represents a pattern co-regulated by the specific microenvironment and transcriptional programs.¹⁹⁷ The state change in EGCs is not merely a shift in inflammatory phenotype. It may also further transmit local immune homeostasis disruption to the functional level of the ENS circuitry by modulating neuron-glial communication and chemokine release. Studies have shown that persistent inflammation affects both the duration and amplitude of slow excitatory postsynaptic potentials, reducing neuronal excitability and synaptic transmission efficiency.¹⁹⁸ Additionally, EGCs regulate chemokines (e.g., CXCL5) that recruit neutrophils, exacerbating local neuroinflammation.¹⁹⁹ Ultimately, immune-mediated barrier disruption and reactive glial transformation severely impair the ENS’s integrative capacity and information transmission, reducing propulsive motility and elevating the threshold for defecation, thus creating a neuroregulatory basis for constipation. It is worth noting that gut microbiota metabolites can also influence communication between immune and neural cells via intestinal barrier inflammation, the vagus nerve, and neurotransmitter pathways. Signals such as SCFAs, tryptophan derivatives, and bile acids may be involved in T cell differentiation, macrophage responses, and the regulation of glial cells.²⁰⁰

Immune dysregulation is also critical in STC. Yan et al. observed downregulation of T cell receptor signaling, NF-κB pathways, and immune synapse-related genes in STC colon tissues, while inflammation, cell migration, and matrix remodeling pathways were upregulated. This paradoxical state indicates impaired immune sensing accompanied by inflammatory activation, suggesting STC involves systemic immune imbalance, not merely defective propulsion.²⁰¹ Wang et al. found miR-129-3p expression decreased in STC tissues from both human and mouse models, blocking the AKT/mTOR signaling pathway and reducing ICCs' proliferation and repair capacity, thus increasing their susceptibility to apoptosis and pacemaker dysfunction under inflammation.²⁰² Persistent ICC dysfunction reduces rhythmic colonic contractions, impairing coordinated smooth muscle motility and ultimately causing STC.

An imbalance in gut microbiota may also initiate immune activation, exacerbating local chronic inflammation, impairing intestinal motility, and promoting constipation development. At the molecular level, proliferation of conditionally pathogenic bacteria significantly elevates lipopolysaccharide (LPS) concentrations in the gut lumen.²⁰³ LPS binds Toll-like receptor 4 (TLR4) on intestinal epithelial and immune cells, activating MyD88-dependent pathways and subsequent NF-κB nuclear translocation.²⁰⁴ This cascade induces pro-inflammatory cytokine production (e.g., IL-1β, IL-6, TNF-α).^203,205 In colonic smooth muscle tissues, these cytokines upregulate regulator of G protein signaling 4 (RGS4) and downregulate CPI-17, a myosin light-chain phosphatase (MLCP) inhibitor. Loss of MLCP inhibition reduces phosphorylation of myosin light chain (MLC20), decreasing smooth muscle calcium sensitivity and impairing contractility. Thus, a vicious cycle of microbial imbalance, aberrant immune activation, and gut dysmotility forms a key pathological basis for CC.²⁰⁶ However, the current evidence is insufficient to conclude that LPS-related immune activation necessarily occurs prior to motility dysfunction. In CC, delayed intestinal transit itself may also contribute to the retention of microbiota and metabolites. Therefore, it is more likely that there is a reciprocal relationship between microbiota dysbiosis, LPS accumulation, immune activation, and reduced motility.

It should be noted that there are currently few studies directly comparing immunological differences between early-stage constipation and long-term, refractory constipation. Therefore, it is not appropriate to state outright that early-stage or mild constipation is characterized by stable mucosal immune activation. Some studies have indicated that certain patients with FC do not exhibit significant elevations in CD3⁺ lymphocytes, D-lactate, or zonulin, suggesting that their immunological changes may not be pronounced.²⁰⁷ Other articles have also noted that in FC, the gut microbiota and intestinal transport may exhibit a bidirectional relationship, influencing colonic motility, secretion, and absorption through metabolic activities such as bile acids, SCFAs, 5-HT, and methane.²⁰⁸ In contrast, chronic or refractory constipation, particularly STC, is more likely to present with abnormalities in the immune microenvironment, which, through interactions between immune cells, fibroblasts, the ENS, ICCs and smooth muscle, contributes to the maintenance of motility disorders.²⁰⁹ Based on these findings, it is conceivable to develop a combination of biomarkers to clinically stratify patients according to different types of homeostatic disruption. Signals related to fecal SCFAs, bile acids and exhaled methane have the potential to provide supplementary information for identifying patients with microbial metabolic disturbances and alterations in mucosal signaling.^53,55,210 For STC, inflammatory factors and immune cell-related transcriptomic features may help characterize the constipation phenotype.²¹¹ However, these markers are currently still at the research stage rather than established clinical biomarkers. They require further validation and standardization of detection thresholds before they can be incorporated into clinical stratification.

Gut-brain-immune tri-dimensional interactions driven by gut microbial metabolites in CC

Recent studies increasingly recognize gut microbial metabolites not merely as passive metabolic by-products, but as central signaling molecules regulating host neural, immune, and metabolic activities. These metabolites traverse or modify the intestinal mucosal barrier, establishing bidirectional interactions with the ENS and CNS, while modulating mucosal immune responses and inflammation (Figure 5). Particularly in motility-related disorders such as constipation, these microbiota-driven signaling pathways intricately link changes in gut motility regulation, barrier homeostasis, and immune responses. Thus, a multidimensional communication network centered on microbial metabolites emerges as a pivotal mechanism for understanding the gut-brain-immune homeostatic loop and its role in constipation pathogenesis.

Figure 5.

Mechanistic overview of microbiota-derived metabolites governing gut-brain-immune interactions in constipation

(A) Peripheral inflammatory signals propagate via vagal afferents to the NTS, subsequently activating higher integrative centers, particularly the hypothalamus (PVN). This activation enhances SNS output, impairing peripheral organ function and further exacerbating tissue injury and motility disturbances.

(B) Accumulated luminal toxins (NH3, LPS, H2S) activate inflammatory pathways, stimulating macrophages and dendritic cells to release pro-inflammatory cytokines (IL-1β, IL-6, TNF-α) and inducing chronic intestinal inflammation.

(C) Gut-derived tryptophan metabolism elevates 5-HT levels, accompanied by reduced intestinal expression of SERT, decreasing 5-HT clearance and causing its local accumulation. This disrupts gut motility and compromises barrier integrity. Microbial metabolites (GABA, glutamate) modulate epithelial membrane potentials and ion channel permeability, adjusting postsynaptic enteric neuronal excitability and overall enteric neural activity. The interaction between these three factors forms a pathological cycle leading to the progressive worsening of CC. The figure was created using FigDraw.

Interaction network of 5-HT and GABA in constipation

Gut microbial metabolites, as key signaling molecules, dynamically interact with neuroprecursor cells in the CNS and ENS, directly or indirectly regulating their proliferation, differentiation, migration, and maturation.²⁷ This process significantly influences the host’s neurochemical environment. Additionally, these metabolites critically modulate cytokine production and inflammatory responses, contributing significantly to intestinal immune homeostasis. Thus, three-dimensional signaling among microbial metabolites, neural, and immune systems constitutes a critical regulatory mechanism within the gut-brain-immune tri-dimensional homeostatic loop.

Current evidence suggests this interaction depends on two parallel, closely coupled core mechanisms. The first involves microbiota-driven reprogramming of tryptophan metabolism, controlling 5-HT biosynthesis in intestinal mucosa, particularly in the colon. Tryptophan metabolism represents a central hub in host-microbe interactions, branching into the indole-AhR axis, the 5-HT axis, and the kynurenine-NMDA axis. A dynamic balance among these pathways precisely regulates neural, immune, and metabolic processes.²¹² Specifically, 5-HT, a monoamine neurotransmitter primarily synthesized in the gut (∼90% of total body 5-HT), depends heavily on microbial-regulated tryptophan metabolism.^84,85,213 Under physiological conditions, 5-HT binds receptor subtypes on enteric neurons, smooth muscle cells, and enterochromaffin cells (such as 5-HT3/4 receptors), facilitating gut reflexes that enhance motility and secretion.^214,215 However, abnormally high 5-HT levels can excessively activate downstream signaling or alter gut sensitivity, disrupting normal motility patterns.²¹⁶ The serotonin transporter (SERT), expressed on enterocytes, regulates this pathway by removing excess 5-HT. SERT dysfunction reduces myenteric excitability, disrupts mucosal barriers, and contributes to constipation.^217,218 Research indicates inflammatory diseases can disturb this signaling axis, inducing constipation. For example, in Crohn’s disease with stenosing colitis, chronic inflammation promotes enterochromaffin cell hyperplasia and excessive 5-HT production, while significantly reducing gastrointestinal SERT expression. This reduces 5-HT clearance and causes local accumulation, impairing motility and mucosal integrity.^219,220,221 Tryptophan metabolism can shift between the 5-HT, kynurenine, and indole pathways, and inflammatory factors can also enhance signaling in the kynurenine pathway via IDO-related mechanisms. This suggests that mucosal immune abnormalities in CC may simultaneously affect both 5-HT-related motor regulation and neuroimmune signaling.²²²

The second mechanism involves a neuro-immune-metabolic network mediated by microbial metabolites, particularly glutamate and its derivative, GABA. This network activates specific ionotropic receptors (e.g., NMDA receptors) on postsynaptic enteric neurons, dynamically modulating their excitability by altering membrane potentials and ion permeability. Additionally, these metabolites influence immune responses by activating specific receptors on immune cells, suppressing NLRP3 inflammasome activation, decreasing pro-inflammatory cytokine release, and enhancing anti-inflammatory factor expression. Consequently, this reshapes the local neuroinflammatory microenvironment.^223,224 Moreover, GABA and associated cytokines can influence brain regions (e.g., prefrontal cortex) characterized by higher blood-brain barrier permeability, thus regulating neural plasticity and emotional stability.^225,226,227 Nonetheless, these remote regulatory pathways, mediated through circulatory routes, require further direct experimental validation.

This abnormality in neuro-immune regulation, centered on microbial metabolites, is particularly pronounced in the pathological progression of irritable bowel syndrome (especially the constipation-predominant type, IBS-C) and exhibits bidirectional feedback characteristics. On the one hand, studies have found that mucosal “low-grade inflammation” mediated by gut microbial dysbiosis directly influences the neurotransmitter system via immune factors. It has been observed that in the intestinal mucosa of the majority of patients with IBS-C, the area occupied by mast cells is significantly increased. Upon abnormal activation, these cells release trypsin-like enzymes, histamine, and pro-inflammatory cytokines (such as TNF-α, IL-6).²²⁸ These mediators not only stimulate intestinal chromaffin cells to release excessive 5-HT but also directly sensitize afferent nerve endings via protease-activated receptor PAR2, leading to motility disorders and visceral hypersensitivity.^229,230 On the other hand, abnormal neurotransmitter expression further exacerbates immune dysregulation. 5-HT secreted by intestinal chromaffin cells binds to 5-HT receptors on the surface of mast cells, potentially leading to desensitization and internalization of 5-HT₃ and 5-HT₄ receptors on enteric neurons, thereby further exacerbating constipation.²³¹ Conversely, excessive activation of 5-HT₇ receptors may promote the activation of the NF-κB pathway, further aggravating local inflammation and creating a positive feedback cascade.^231,232 At the same time, an imbalance in GABA also plays an indispensable role in immune dysregulation. A significant reduction in bacterial diversity in the intestines of patients with IBS-C leads to a decrease in microbiota-derived GABA.^233,234 As a key inhibitory immunomodulator, GABA exerts a negative regulatory effect on the production of pro-inflammatory cytokines and the proliferation of immune cells.²³⁵ A decline in its concentration or a weakening of local GABAergic signaling, as described above, will directly lift the inhibitory constraints on the activation of macrophages and NLRP3 inflammasomes, leading to the persistent worsening of inflammation. Consequently, IBS-C is not merely a motility disorder, but rather a concrete manifestation of a vicious cycle between the nervous and immune systems arising from an imbalance in the 5-HT and GABA regulatory networks.

Interestingly, recent research indicates significant interactions between the tryptophan-5-HT and glutamate-GABA axes.²³⁶ Tryptophan metabolites, particularly those via the 5-HT pathway, influence 5-HT synthesis in enterochromaffin cells and indirectly set the overall excitability threshold within the gut neural network by modulating GABAergic neuron activity.²³⁷ Such cross-axis regulation demonstrates complex interactions between microbial metabolism and neuronal signaling. It not only clarifies the pathological link between gut immune dysregulation and neuropsychiatric disorders but also identifies potential mechanisms contributing to constipation through combined modulation of enteric neuronal excitability and local inflammation. These findings offer critical intervention points for future therapeutic strategies focusing on multi-pathway, gut microbiota-neuro-immune interactions.

Accumulation of gut toxins forms a vicious cycle, aggravating constipation

Numerous studies have demonstrated that microbial-derived toxins from the gut act through neuro-reflex and immune signaling pathways, establishing a persistent positive-feedback loop and exacerbating constipation.²³⁸ Ammonia, a major microbial metabolite from protein and urea degradation,²³⁹ accumulates significantly due to prolonged fecal retention during constipation, enhancing intestinal protein fermentation.²⁴⁰ Elevated ammonia concentrations strengthen GABAergic neurotransmission by interfering with GABA receptor function or altering GABA synthesis and metabolism, and inhibit smooth muscle contractions, reducing colonic motility.²⁴¹ Additionally, ammonia directly damages enterocytes, disrupts tight junction integrity, and impairs barrier function.²⁴² Studies have shown ammonia activates inflammatory signaling (NF-κB, MAPK pathways), prompting macrophages and dendritic cells to release pro-inflammatory cytokines (IL-1β, IL-6, TNF-α), inducing chronic low-grade inflammation and further exacerbating tissue injury and motility dysfunction.²⁴³

In addition to ammonia, abnormal accumulation of hydrogen sulfide (H2S) significantly affects inflammation and neuronal regulation. Under physiological conditions, both intestinal cells and microbiota produce H2S through enzymes including cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), and 3-mercaptopyruvate sulfurtransferase (3MST).^244,245 Physiological H2S levels suppress NF-κB activation, decreasing pro-inflammatory cytokine (TNF-α, IL-6, IL-1β) production and exerting anti-inflammatory effects.²⁴⁶ However, excessive H2S under pathological conditions promotes inflammation and neurotoxicity. For example, in hemorrhagic shock reperfusion mouse models, elevated H2S activates NMDA receptors in the colon, exacerbating gastrointestinal inflammation and inducing anxiety-like behaviors.²⁴⁷ Furthermore, high concentrations of H₂S activate TRPV1 channels in central cholinergic nuclei (e.g., medullary raphe), inhibiting gut motility.²⁴⁸ Thus, supra-physiological H₂S levels may regulate gastrointestinal motility via central-peripheral cholinergic networks, forming a persistent negative-feedback loop.

Another key gut toxin, LPS, is a critical component of Gram-negative bacterial cell walls. Dysbiosis conditions result in abnormal LPS release. Serum LPS levels in functional constipation patients are significantly higher than in healthy individuals, highlighting its key pathological role.²⁴⁹ LPS activates TLR4 signaling, causing local and systemic low-grade intestinal inflammation, damaging the mucosal barrier, and reducing tight junction protein expression. This enhances intestinal permeability (“leaky gut”), allowing more endotoxins into systemic circulation, triggering widespread inflammatory responses.²⁵⁰ Importantly, these inflammatory mediators affect peripheral tissues and also cross peripheral-central interfaces, influencing brain function. On the one hand, inflammatory mediators directly increase blood-brain barrier permeability and induce endothelial cell adhesion molecules. On the other hand, peripheral inflammatory signals reach the brainstem via vagal afferents, then ascend to higher integrative centers such as the hypothalamus.^251,252 The hypothalamic PVN activates sympathetic nervous system (SNS) signaling upon receiving inflammatory input, regulating peripheral organ function.²⁵³ Enhanced sympathetic activity itself suppresses smooth muscle contractions and slows intestinal transit.^253,254,255 This “inflammation-autonomic imbalance-motility impairment” pathway thus represents a crucial mechanism underlying constipation.

Targeting the gut-brain-immune tri-dimensional homeostatic loop for constipation treatment: Strategies and future directions

Traditional treatments for CC include pharmacological therapy, biofeedback, dietary adjustments, and psychological interventions. However, these methods exhibit significant limitations regarding long-term efficacy, dose dependence, and consistency across patients. Advances in microbiome research have increasingly recognized the critical role of gut microbiota in constipation pathogenesis. Studies have demonstrated that patients with CC often exhibit notable dysbiosis, characterized by overgrowth of specific microbial populations (e.g., Helicobacter pylori, spirochetes, and Desulfovibrio species). Such microbial alterations are closely associated with impaired gut motility, mucosal immune dysfunction, and disrupted gut-brain communication.²⁵⁶ Consequently, restoring gut microbiota balance has emerged as a pivotal therapeutic strategy for constipation.^192,257 This approach seeks to improve the gut-brain-immune homeostatic loop by regulating microbial composition, restoring microbiota-metabolism pathways, and enhancing gut neural signaling and immune responses. These interventions aim to improve intestinal motility, providing a mechanistically supported treatment avenue for refractory constipation (Table 3).⁸⁶ Currently, interventions such as probiotics, prebiotics, synbiotics, postbiotics, fecal microbiota transplantation (FMT), personalized nutrition, and traditional Chinese medicine have demonstrated varying degrees of microbiota modulation. Furthermore, the integration of emerging techniques, including neuromodulation, multi-omics analyses, artificial intelligence (AI), and engineered bacteria, advances the field toward more precise and systematic treatment models (Figure 6). Table 4 summarizes the classification, principles, clinical applications, future prospects, and current limitations of each treatment method.

Table 3.

FDA-approved clinical trials for constipation treatment via gut microbiota modulation

NCT number	Study title	Status	Disease indication	Intervention(s)	Phase	Experimental models
NCT00788658	effects of cognitive behavioral therapy in patients with IBS	completed	IBS, constipation, abdominal pain, depression	dietary modification; cognitive behavioral therapy	not applicable	randomized, parallel-group controlled RCT
NCT04457791	individualized dietary advice to increase fiber intake and improve constipation	completed	chronic constipation	personalized dietary intervention	not applicable	single-group before-and-after design (quasi-experimental)
NCT04282551	oligosaccharide-based dietary intervention for functional constipation	status unknown	functional constipation	oligosaccharide supplementation vs. placebo	not applicable	randomized, double-blind, three-arm, parallel-group, placebo-controlled RCT
NCT01333787	mixed dietary fiber intervention in children with constipation	completed	pediatric constipation	mixed dietary fiber supplementation	phase 4	randomized, double-blind, two-arm, parallel-group, placebo-controlled RCT (true experimental)
NCT05923723	dietary intervention in elderly patients with chronic constipation	not yet recruiting	idiopathic chronic constipation	dietary intervention vs. placebo	not applicable	single-group before-and-after quasi-experimental design
NCT03100851	effects of probiotics on constipation	completed	constipation	lactobacillus-based probiotic supplementation	phase 3	randomized, double-blind, parallel-group, placebo-controlled RCT
NCT04667884	effects of prebiotic or probiotic supplementation on symptoms of functional constipation	completed	functional constipation	prebiotic or probiotic supplementation	not applicable	randomized, parallel-group, placebo-controlled RCT

Figure 6.

Therapeutic strategies targeting the gut-brain-immune homeostasis loop in constipation

(A) Prebiotics and wheat peptides derived from fermentation engineering remodel the functional coupling of the gut-brain-immune axis by regulating the synthesis and release of endogenous neurotransmitters, thereby restoring its dynamic homeostasis and enhancing systemic regulatory capacity.

(B) Electroacupuncture promotes the conversion of 5-HTP to 5-HT and increases the bioavailability of 5-HT in the enteric nervous system, thereby enhancing intestinal motility and improving the constipation phenotype.

(C) Prebiotics and engineered bacteria optimize the intestinal metabolic microenvironment by enhancing the biosynthesis of short-chain fatty acids such as butyrate, thereby further promoting the structural and functional integrity of the intestinal epithelial barrier.

(D) Traditional Chinese medicines (such as ginseng), dietary fiber, and fecal microbiota transplantation can increase the abundance of Bacteroides and Phylum Firmicutes, and reshape the gut microbiome structure.

(E) Acupuncture and postbiotics participate in the regulation of gastrointestinal motility-related signaling pathways by modulating host microRNA expression and providing microbiota-derived bioactive metabolites, respectively, thereby alleviating constipation symptoms.

(F) Active components derived from moringa leaves and ginseng can modulate immune cell activity and suppress abnormal inflammatory responses, thereby improving the inflammatory microenvironment of the gut.

(G) Fecal microbiota transplantation improves gastrointestinal motility by reducing levels of pro-inflammatory cytokines and suppressing local inflammatory responses in the gut. The figure was created using Figdraw.

Table 4.

Classification, principles, clinical applications, future prospects, and current limitations of common treatment methods

Name of treatment method	Categories of treatment methods	Specific principles and mechanisms	Clinical applications	Future outlook	Knowledge gaps	Reference
Probiotics	drug therapy	SCFA-mediated regulation of motility and secretion; intestinal barrier function and anti-inflammatory effects; neurotransmitters and remodeling of the enteric nervous system; aquaporin regulation; novel enteric signaling molecules	functional constipation in children; chronic constipation in adults	precision matching and the “Synthetic Biology 2.0” Approach; Exploration of New Substrates Such as Microbial Extracellular Polysaccharides (EPS)	optimal strain/substrate combinations and standardization; human validation of mechanisms of action; assessment of long-term safety and dependence; biomarkers for predicting responders; pathways for integration with conventional therapies	Araújo and Botelho258, Zhen et al.259, Wang et al.260, Shan et al.261, Razzak et al.262, Abot et al.263
Prebiotics	daily health care	SCFA-mediated regulation of intestinal motility and secretion, activation of 5-HT-related pathways, anti-inflammatory and immunomodulatory effects, enhancement of the mucus layer and barrier function, and novel enteric signaling molecules	chronic constipation in adults; functional constipation; functional constipation in children; constipation associated with IBS-C			Araújo and Botelho258, Abot et al.263
Biostime	drug therapy treatment with probiotics	SCFA-mediated regulation of intestinal motility and secretion, activation of 5-HT-related pathways, anti-inflammatory and immunomodulatory effects, enhancement of the mucus layer and barrier function, and novel enteric signaling molecules	functional constipation in children, chronic constipation in adults			Abot et al.263
Post-Gen	drug therapy treatment with probiotics	activates the succinic acid-SCFA axis, regulates the tryptophan metabolic pathway, improves the intestinal barrier and mucus secretion, modulates bile acid and water-salt metabolism, exerts anti-inflammatory and immunomodulatory effects, targets the enrichment of beneficial microbiota, and acts directly on intestinal smooth muscle cells	functional constipation in children, chronic constipation in adults	precise matching and clarification of ingredients, synergy with dietary patterns, and expansion to include patients with refractory constipation and comorbidities	personalized prediction and stratified treatment; long-term safety and dependency assessment; dose-response relationships and optimal dosing regimens; precise identification of active ingredients and targets; standardized production and quality control systems	Chen et al.264, Ma et al.265, Cai et al.266, Zhao et al.267, Mosca et al.268
Fecal Microbiota Transplantation	probiotic therapy	microbiome restructuring and colonization, neurotransmitter- and transporter-mediated weight programming, restoration of immune-barrier homeostasis, activation of protein digestion and absorption pathways, and metabolite-mediated regulation of motility and secretion (SCFA axis and bile acid axis)	slow-transit constipation in adults, mixed-type constipation in adults, constipation in children	precise donor selection and the concept of “Super Donors”, long-term/sequential treatment strategies, and optimization of oral capsules and delivery technologies	predictability and standardization of donor efficacy; long-term safety and long-term outcomes; optimal administration regimens and reproducibility; validation of mechanisms; and	Xie et al.269, Liu et al.270, Pan et al.271, Settanni et al.272, Kamath et al.273, Wen et al.274
Dietary Fiber	basic nutritional therapy, dietary supplementation therapy	fecal bulking and softening, mechanical stimulation and propulsion, physiological effects driven by fermentation products, gel formation and osmotic effects, neuroendocrine and ion channel regulation	chronic constipation in adults, constipation in children, and occasional constipation	targeted dietary fiber interventions, the use of modified dietary fiber materials, and the integration and optimization of lifestyle changes	determining the optimal fiber types and ratios; long-term adherence and real-world efficacy; developing personalized predictive models; mechanisms of adverse reactions and strategies for prevention and control; and gathering additional evidence for specific populations	Cho et al.275, McKeown et al.276, Wang et al.277, Zhang et al.278, Procházková et al.279, van der Schoot et al.280, Dimidi et al.281
Chewing Behavior	behavioral intervention therapy	activation of the vagal reflex, promotion of gut hormone and neurotransmitter-related signaling, improvement of dietary fiber intake and digestive efficiency, maintenance of gut microbiota and SCFA homeostasis, and reduction of large food particle residue and bacterial overgrowth	association between recovery from postoperative intestinal paralysis/intestinal obstruction, masticatory ability, and risk of constipation	as a low-cost adjunct to Enhanced Recovery After Surgery (ERAS), incorporating comprehensive management for older adults and individuals with swallowing disorders, and exploring “chewing training” as part of a lifestyle intervention	evidence of efficacy in individuals with functional constipation; optimal intervention parameters have not yet been determined; long-term safety and definition of the target population; quantification of the benefits for constipation following improved chewing ability; and in-depth validation at the mechanistic level	Yaoita et al.282, Malek Hosseini et al.283, Wang et al.284, Kumar et al.285
Fermented Foods	dietary intervention, nutritional therapy	regulation of gut microbiota composition; promotion of SCFA production and utilization; enhancement of intestinal motility and neuroendocrine regulation; improvement of the intestinal barrier and anti-inflammatory effects; regulation of water channels and transporters	functional constipation in children, chronic constipation in adults	from “black-box” fermented foods to “precision” interventions; the standardized development of postbiotics and inactivated bacterial products; and the combined use of fermented foods with other therapies	the optimal strains/combinations and dosages have not yet been established; the relationship between fermented foods and “real-world” dietary patterns remains unclear; there is insufficient data on long-term safety and tolerability; there is a lack of biomarkers for individualized prediction; and there is insufficient causal validation of the mechanistic pathways.	Ma et al.265, Huang et al.286, Zhang et al.287
Traditional Chinese Medicine	traditional Chinese medicine, acupuncture, tuina, dietary therapy	regulate the functions of the internal organs and the balance of qi, blood, and body fluids; restore the gut microbiome and barrier function; and improve intestinal motility and secretion	functional constipation in children, chronic constipation in adults	moving from empirical pattern differentiation to integrated precision treatment based on “syndrome-target-pathway”, developing an integrated treatment model combining “Traditional Chinese Medicine and modern medicine”, and expanding the applications of non-pharmacological therapies	there remains a lack of high-quality clinical evidence; the mechanism of action has not yet been fully elucidated; standards for personalized treatment need to be refined; and research on long-term safety and drug interactions is insufficient.	Wu et al.288, Wang et al.289
Neuromodulation	psychological intervention therapy, electroacupuncture therapy	① a combination of psychological intervention and biofeedback therapy improves pelvic floor muscle function, regulates the brain-gut axis, and enhances intestinal motility. ② combined treatment with herbal decoctions and electroacupuncture increases the levels of 5-HTP, a 5-HT precursor, in the colon and promotes 5-HT production.	functional constipation; functional constipation accompanied by mental health conditions such as anxiety and depression	by expanding the treatment of constipation from regulating intestinal function to the brain-gut neural network, and by identifying specific neural biomarkers for constipation, we aim to achieve a breakthrough in moving from adjunctive interventions to preventive treatment methods.	there is insufficient evidence of long-term efficacy, and research has not yet addressed the specific mechanisms of action and signaling pathways.	Hongyu et al.290, Chen et al.291, Marks et al.292
Multi-omics Integration	precision diagnosis and treatment technologies	to accurately identify patients with constipation by analyzing changes in their gut microbiome, metabolites, and related protein expression, and to investigate the pathophysiological mechanisms of constipation	constipation associated with Parkinson’s disease, IBS-C-related constipation, chronic transit constipation, and constipation-related defecation syndrome, etc.	moving from single-omics analyses to systematic studies that integrate molecular network technologies and network pharmacology; further optimizing the analysis of various omics data types; establishing standards for analytical workflows; and reducing the cost of omics research	in metabolomics, metabolites cannot be quantified; single-omics analyses alone cannot fully elucidate the specific pathophysiological mechanisms of constipation; and multi-omics integration faces challenges such as high research costs and the need for large sample sizes.	Shin and Kashyap293, Hasin et al.294, Zhang et al.295, Wang et al.296, Huang et al.297
Artificial Intelligence	adjuvant therapies	automatically analyze image features and colon-related data to classify constipation subtypes, optimize lesion detection, and provide personalized dietary interventions based on patients’ gut microbiomes.	functional constipation in adults and children, refractory constipation in children, chronic constipation	from auxiliary analyses of the pathology and gut microbiome characterization in patients with constipation to precision treatment approaches open to human intervention	research on artificial intelligence models has small sample sizes, low accuracy in identifying patients with various types of constipation, and limited generalizability.	Su et al.298, Huang et al.299, Ianiro et al.300, Zhang et al.301
Engineered bacteria	novel microbial-targeted therapies	genetically modified, protein-coated “programmable strains” enhance butyrate production in the gut, improve intestinal mucosal inflammation and barrier function, and reduce systemic side effects.	functional constipation in the elderly, medication-induced constipation	combined with AI-powered personalized analysis models, we develop innovative, patient-specific strains; by integrating omics research, we fine-tune these strains to maximize their efficacy.	issues such as disruptions to the microbiome balance, the spread of antibiotic resistance, and opportunistic infections caused by engineered bacteria have not yet been fully resolved.	He et al.48, Hashemi302, Deng et al.303, Xue et al.304, Duan et al.305

Probiotics, prebiotics, synbiotics, and postbiotics in therapeutic approaches

Probiotics

Probiotics are live microorganisms that, when administered in sufficient quantities, confer health benefits to the host. They modulate the gut microenvironment through multiple mechanisms, including microbial regulation, enhancement of the mucosal barrier, and modulation of hormone secretion and gene expression. Their therapeutic efficacy depends largely on successful gut colonization. Studies showed that Lactobacillus plantarum and Lactobacillus rhamnosus exhibit greater colonization potential than Lactobacillus acidophilus.³⁰⁶ In constipation management, Bifidobacterium species can reduce the abundance of potential pathogens such as Alistipes and Clostridium. For instance, Bifidobacterium longum TY-S01 exerts dual anti-inflammatory and metabolic regulatory functions by decreasing Proteobacteria while enriching Spirochaetaceae, Bacteroidaceae, and butyrate-producing taxa.^60,174 Combination probiotic therapies may also produce synergistic benefits. Co-administration of Streptococcus thermophilus MG510 and Lactobacillus plantarum LRCC5193 enhances the abundance of Lactobacillus and Bifidobacterium. Bacillus subtilis TO-A improves substrate utilization via amylase secretion, indirectly promoting probiotic growth.^307,308

Prebiotics

Prebiotics are dietary substrates selectively utilized by host microorganisms to modulate gut microbiota composition. Oligosaccharides are among the most extensively studied prebiotics. Galactooligosaccharides (GOS) promote Bifidobacterium growth, enhance SCFA production, and cooperate with anaerobic bacteria to generate butyrate and hydrogen, thereby optimizing metabolic conditions in the gut.³⁰⁹ Xylooligosaccharides (XOS) stimulate Enterococcus and Ruminococcus proliferation, increase butyrate synthesis, activate the AKT-NF-κB pathway, and improve gut motility and immune regulation, providing multifaceted relief from constipation.^310,311 Notably, konjac mannan oligosaccharides may modulate neurotransmitter secretion, offering a novel strategy for managing neurogenic constipation via the gut-brain-immune homeostatic loop.³¹²

Marine and plant-derived resources also provide valuable prebiotic candidates.^313,314 For example, brown algal extract (SPE) significantly increases cecal Bifidobacterium abundance. Its metabolic products, such as butyrate, acetate, and propionate, reduce intestinal pH and activate FFA2/FFA3 receptors on enteric chromaffin cells, thereby stimulating 5-HT release and promoting gut motility.³¹⁵

Synbiotics

Synbiotics combine probiotics and prebiotics to maximize synergistic effects.³¹⁶ In in vitro fermentation models, Bifidobacterium longum BL-99 together with fructooligosaccharides (FOS) enriches beneficial taxa such as Faecalibacterium and Blautia, boosts SCFA production, and inhibits pathogenic bacteria including Shigella.³¹⁷ Clinical studies further confirmed that the synbiotic formulation MPRO3 selectively increases Bifidobacterium and Lactobacillus populations, leading to systematic improvements in gut motility and host physiological status. These findings mark a transition from simple microbial modulation toward functional optimization in microbiota-targeted therapy.^318,319

Postbiotics

Postbiotics, preparations containing non-viable microorganisms and their components, have recently attracted growing attention in microbiome research.³²⁰ Evidence shows that postbiotics regulate gut motility, maintain mucosal immune balance, and preserve epithelial barrier function, making them promising candidates for constipation therapy. Postbiotics derived from Crataegus (hawthorn) probiotics alleviate constipation by inhibiting epithelial apoptosis, modulating local immune responses, and influencing the expression of key genes.²⁶⁴ Additionally, Ma et al. identified bioactive components in the novel postbiotic formulation Probio-Eco, which significantly improved constipation by stimulating mucin 2 (MUC2) secretion, regulating intestinal transport hormones, and enhancing anti-inflammatory pathways.²⁶⁵ These findings provide strong support for the clinical potential of postbiotic-based therapeutics.

FMT

FMT involves introducing functional microbiota from a healthy donor to restore microbiome homeostasis in the recipient. Its application has expanded beyond Clostridium difficile infection to include CC treatment.³²¹ Multi-omics analyses demonstrated that FMT significantly enriched Bacteroides species in the gut of patients with constipation, decreased the ratio of Proteobacteria and Actinobacteria, and reactivated metabolic pathways related to amino acids and energy metabolism.²⁶⁹ FMT also increases microbiota α-diversity, promotes beneficial clostridial populations, and reduces pro-inflammatory cytokines (e.g., IL-8), thereby enhancing gut motility.³²² Clinical evidence suggests successful colonization by “keystone species” such as Fusicatenibacter and Paraprevotella is essential for sustained therapeutic outcomes.³²² Periodic supplementation with frozen or lyophilized FMT capsules can prolong beneficial microbiota retention, delaying efficacy loss.³²³ Combining FMT with biofeedback therapy results in sustained bowel function improvement,³²⁴ advancing toward a multimodal therapeutic model. Importantly, FMT effects are not limited to symptom relief. Recent findings indicate that FMT reshapes microbiota ecological structure and metabolic activities, restoring intestinal ecological function. Transplanted microbiota metabolites may modulate gut-brain communication via the gut-brain-immune homeostatic loop,³²⁵ providing a biological foundation for understanding systemic constipation pathology and FMT mechanisms.

Regarding the selection of optimal fecal donors, the following three practical challenges currently exist. First, while the concept of “super donors” has been proposed in FMT studies for conditions such as IBS, the overall evidence is inconsistent, and no specific donor selection criteria for constipation have yet been established.³²⁶ Second, existing donor screening primarily focuses on safety assessments, and no donor selection strategy based on microbiome composition has yet been developed.³²⁷ Third, the use of frozen preparations and capsule formulations from fecal microbiota banks is becoming increasingly widespread.³²⁸ Among these, frozen preparations are easier to control for quality, and their efficacy is comparable to that of fresh preparations, while capsule formulations offer the advantages of being minimally invasive and convenient for repeated administration.³²⁹

FMT carries a certain risk of adverse events in clinical practice. According to statistics, the overall incidence of adverse events (AEs) is approximately 39.3%, with serious adverse events (SAEs) accounting for 5.3%. Among acute adverse events, abdominal pain is the most common, followed by diarrhea; the most serious AE is Clostridioides difficile infection. Studies have further found that FMT administration via the upper gastrointestinal tract is associated with an increased incidence of total AEs, but does not significantly increase the risk of SAEs.³³⁰ Based on the current literature, clinicians must remain highly vigilant for various related adverse events when using FMT to treat diseases.³³¹ Additionally, antibiotic pretreatment could theoretically enhance the efficacy of FMT by suppressing the patient’s own gut microbiota and promoting the colonization of donor microbiota. However, this strategy also carries potential risks of inducing adverse reactions and enriching drug-resistant genes.^332,333 Currently, there are no uniform standards regarding the use of antibiotic pretreatment, specific dosing regimens, or the timing of discontinuation. In the treatment of constipation, most studies have combined antibiotics with bowel-cleansing agents; coupled with certain flaws in trial design, there remains insufficient evidence to support the independent efficacy of FMT, and its clinical application remains highly controversial.³³⁴

Regulation of dietary structure

Dietary fiber supplementation

Dietary fiber regulates gut function through prebiotic actions and fermentation metabolites such as SCFAs. A chronic low-fiber diet promotes opportunistic pathogen proliferation (e.g., Enterococcus), whereas appropriate fiber supplementation significantly improves spontaneous bowel movement frequency in patients with CC.³³⁵ However, excessive fiber intake may induce bloating, and fiber types vary in their effects on stool properties.³³⁶ Recent studies introduced nanocrystalline cellulose (NCC), a novel nano dietary fiber that enhances microbiota diversity and mucosal barrier integrity at low doses, demonstrating high tolerance and offering a promising therapeutic direction for refractory constipation.³³⁷ Clinically, fiber supplementation should be tailored according to constipation subtype, underlying conditions, and individual microbiota profiles, aiming for precision medicine.³³⁸

Improvement of mastication behavior

Food physical properties influence gut function through the oral-gut axis. Animal studies demonstrated that impaired mastication reduces colonic motility, disrupts mucosal immunity, and alters expression of AQP4 and nNOS, causing a constipation phenotype.²⁸² Restoration of normal chewing normalizes these molecular markers and enhances metabolic activities of butyrate-producing bacteria, suggesting mastication benefits gut function through a multi-pathway mechanism involving mechanical stimulation, neurohormonal regulation, and microbial metabolism.^282,339

Supplementation with fermented foods

Fermented foods containing lactic acid bacteria, Bifidobacterium, prebiotics, and bioactive peptides are natural microbiota modulators with high patient compliance. For example, the traditional Japanese fermented rice beverage “Amazake” selectively enriches Lactobacillus and Bifidobacterium, inhibits potential pathogens (Proteobacteria, Enterobacteriaceae, Shigella), and optimizes intestinal pH, demonstrating bidirectional microbiota regulation. Additionally, wheat peptides from fermentation products promote intestinal epithelial repair, modulate neurotransmitter release, and restore the gut-brain-immune homeostatic loop. This effectively improves multiple symptoms, alleviates inflammation, and restores motility.^340,341 The effectiveness of fermented foods still depends on baseline microbiota composition and dosage. Future research should clarify the mechanisms underlying their specific efficacy, supporting personalized nutrition strategies.

Traditional Chinese medicine (TCM) approaches

TCM provides unique advantages in managing CC due to its multi-target systemic regulation and relatively low adverse effects. For instance, Atractylodes enhances gastrointestinal motility by promoting the secretion of SP, VIP, and 5-HT, thus improving enteric nerve conduction and smooth muscle contraction. Moringa leaf extract regulates the ENS-ICCs-SMCs functional unit, repairs the intestinal mucosal barrier, and optimizes the SCFA microenvironment.^342,343 Compared with single-compound treatments, TCM formulas achieve more stable therapeutic effects due to the synergistic interactions of their components. Furthermore, innovations in administration methods, such as transdermal delivery, bypass the first-pass effect, allowing faster onset and higher compliance, making these methods promising for long-term CC management. For example, the ZBVO formula, composed of dried tangerine peel, enoki mushrooms, and immature bitter orange, delivered transdermally, can regulate gastrointestinal hormones, suppress inflammation, and achieve systemic improvement through the brain-gut-immune homeostatic loop.³⁴⁴ Recent studies also suggest that active ingredients of ginseng can modulate the abundance of Bacteroides and Firmicutes, improving colonic inflammation and motility dysfunction, thereby alleviating constipation symptoms.³⁴⁵

Acupuncture, as a non-pharmacological therapy, is increasingly utilized in patients with constipation.³⁴⁶ Research demonstrates that acupuncture directly enhances gastrointestinal motility and promotes microbiota restoration.³⁴⁷ Epigenetic studies further indicate that acupuncture regulates molecules such as miR-let-7f, influencing the density and excitability of NaV1.5 channels in colonic smooth muscle cells, thus precisely modulating colonic contraction rhythms.^348,349

Emerging therapies: From systematic understanding to precision intervention

Neurological regulation

Neuroregulation, employing methods such as psychological interventions and electrical stimulation, has emerged as a novel therapeutic approach for alleviating constipation. Studies indicate that neuroregulatory interventions not only improve constipation symptoms but also offer new perspectives and strategies for personalized treatment. For instance, Zhou et al. reported that patients with constipation receiving biofeedback therapy and psychological interventions experienced significantly greater symptom relief compared to those treated with conventional gastrointestinal motility drugs or anxiolytic and antidepressant medications.²⁹⁰ This research confirmed the critical role of the nervous system in functional gastrointestinal disorders and introduced a promising translational therapeutic approach for CC management, particularly for patients poorly responsive to standard medications. It should be noted that the long-term efficacy of biofeedback and psychological interventions for patients with constipation still needs to be determined through long-term, large-scale studies. Furthermore, Yi et al. explored how the CNS regulates gastrointestinal motility via electroacupuncture. They discovered that electroacupuncture modulates the TPH2/5-HT signaling pathway, effectively alleviating depressive behaviors and constipation symptoms in mice.²⁹¹ This study provided molecular evidence of the brain’s role in gastrointestinal motility disorders, highlighting the close connection between the nervous system and gut function. However, there are currently no definitive studies detailing the specific pathways by which 5-hydroxytryptophan (5-HTP) is synthesized into 5-HT across the blood-brain barrier. Future research could focus on 5-HT precursors to address this gap. These findings suggest that brain regulation could enhance symptom relief and offer novel biomarkers and therapeutic targets for personalized treatment. However, before it can be put into actual clinical use, the limitations of a large number of preclinical studies still need to be addressed in order to achieve more precise targeted therapy. Future clinical research could leverage CNS regulation to develop precise treatments, especially for patients with complex or refractory constipation, expanding targeted therapeutic options.

Multi-omics integration

Multi-omics technologies are reshaping the theoretical framework of constipation research. By integrating metagenomic, metabolomic, and proteomic data, researchers can clarify causal networks between gut microbiota and the host, providing the basis for precise, actionable therapeutic strategies.³⁵⁰

Metagenomics enables analysis of microbial structural changes at both species and functional levels, providing key insights into constipation mechanisms. Zhang et al. reconstructed microbial genomes from metagenomic sequences and identified gut microbiota features strongly linked to PD, a condition frequently associated with constipation. This suggests that modulating gut microbiota may simultaneously alleviate neurological and gastrointestinal symptoms. Another study on gut stress syndrome indicated that microbial dysbiosis and reduced SCFA production could directly trigger constipation, further highlighting the potential of metagenomics in mechanistic exploration and therapy development.³⁵¹

Metabolomics reflects pathological changes through shifts in metabolites and can link abnormalities in gut, brain, and metabolic functions. Research indicated that blood metabolites could serve as biomarkers for depression-related constipation. Additionally, analysis of fecal amino acids and energy metabolites showed disruptions in the TCA cycle associated with energy metabolism disorders in patients with functional constipation.^296,352 Despite the potential value of metabolomics as an adjunct in the diagnosis of functional gastrointestinal disorders such as constipation, its clinical translation still faces significant challenges. One of the most prominent limitations lies in the complexity of metabolite profiles and the issues of reproducibility and standardization in their quantitative analysis.²⁹³ Metabolites exhibit highly dynamic changes under the influence of different physiological states, gut microbiota composition, and exogenous dietary factors, resulting in a lack of uniform quantitative standards across platforms and cohorts. This, to some extent, undermines their potential for clinical application as stable biomarkers.

Proteomics, through the analysis of disease-related proteins, helps identify critical signaling pathways and therapeutic targets. For example, studies on STC demonstrated that certain polysaccharides regulate protein expression in colonic tissues, thereby improving motility dysfunction.³⁵³ This highlights the value of proteomics in drug mechanism studies and new target identification.

Given the limitations of metabolomics in elucidating the mechanisms of complex diseases from a single dimension, multi-omics integration has been regarded as a key strategy to bridge this information gap. This strategy systematically elucidates the molecular basis of the onset and progression of constipation by integrating multi-level biological information, including genomic, transcriptomic, proteomic, and metabolomic data. Although this approach demonstrates significant advantages in mechanism elucidation, there remain notable gaps in its translation to clinical practice. First, multi-omics studies rely heavily on large-scale sample cohorts to enhance statistical robustness and reproducibility. However, the resulting high costs and resource consumption significantly limit their accessibility in routine clinical research.²⁹⁴ Second, while large-sample studies enhance statistical power, they inevitably introduce complex individual heterogeneity and confounding factors, including differences in gender, age, dietary patterns, and gut microbiota. These factors may cause inconsistent perturbations across different omics levels, thereby affecting the stability of cross-omics integration inferences.

Furthermore, the inherent differences among heterogeneous omics data mean that cross-omics fusion analysis still lacks unified methodological standards.³⁵⁴ Inappropriate integration strategies can compromise the reliability of final conclusions. Therefore, future research urgently needs to achieve breakthroughs in standardized data processing workflows, low-cost multi-omics data collection strategies, and stratified analysis frameworks tailored to individual differences, in order to drive the true transformation of multi-omics integration from a “tool for explaining mechanisms” to a “precision clinical decision support system”.

AI-driven personalized intervention

Artificial intelligence (AI) is becoming crucial for developing precision medicine strategies for constipation. With its ability to integrate multimodal data, AI has shown significant advantages in screening, subclassification, treatment selection, and prognosis management. In diagnosis, deep learning combined with imaging and clinical data has significantly reduced misdiagnosis rates for gastrointestinal diseases and enabled rapid differentiation of constipation subtypes, such as pediatric refractory constipation and functional constipation, supporting early clinical decision-making.^298,299 It should be noted that the current study sample is drawn exclusively from a cohort of children with functional abdominal pain, and the sample structure is relatively homogeneous. Consequently, the models’ generalization ability has not yet been validated in healthy populations or across a broader range of clinical phenotypes, which limits its potential for application in universal early screening settings. AI also demonstrates value in microbiome-based treatments. Research by Ianiro et al. revealed that algorithmic matching of donor and recipient microbiota profiles could predict “super donors” in FMT, significantly improving colonization efficiency and therapeutic outcomes.³⁰⁰ Moreover, artificial intelligence has begun to demonstrate potential value in personalized interventions for functional constipation. For example, AI can dynamically generate time-segmented, personalized dietary plans and eating recommendations based on a patient’s lifestyle history, thereby optimizing the underlying nutritional environment of the gut microbiome. Such dietary intervention strategies can, to a certain extent, increase gut microbial diversity and promote the restoration of microbiome homeostasis, thereby improving patients’ overall symptom burden and quality of life.³⁵⁵ Furthermore, AI can identify potential pathogenic strains closely associated with the onset of constipation. Building on this, AI holds promise for driving a shift from “empirical dietary adjustments” toward “targeted microbial interventions”, thereby paving the way for the implementation of personalized treatment strategies based on microbiota regulation, such as targeted probiotic supplementation, postbiotic interventions and dietary restructuring.³⁵⁶

However, there remain several common bottlenecks in the current application of AI to constipation-related research and clinical translation. Firstly, due to limitations in sample size and the lack of sufficient data diversity, the application of existing AI models in the field of constipation is still in the early exploratory stages, and their clinical accessibility and real-world applicability remain relatively limited. Existing research indicates that, in tasks such as identifying anxiety levels in patients with functional constipation, machine learning models typically demonstrate high classification accuracy within the training set. However, their performance often declines significantly when validated in independent clinical cohorts or across centers, thereby limiting their generalization to complex clinical phenotypes.³⁰¹ Secondly, current AI models struggle to fully integrate and analyze individual variations in the gut microbiome driven by multidimensional confounding factors such as age, gender, dietary patterns, and geographical environment. This high-dimensional heterogeneity significantly increases uncertainty during the model training process and may undermine the robustness and reproducibility of their predictive outcomes. Furthermore, most current models still rely primarily on comparative learning using large sample datasets, whilst lacking the capacity to analyze the causal mechanisms underlying constipation. This results in research findings that lack scientific rigor.³⁵⁷ Consequently, the future development of AI in constipation research should not be confined to pattern recognition and phenotypic classification, but should extend further toward interpretable modeling, causal inference frameworks, and mechanism analysis driven by multi-omics integration, thereby facilitating its transformation from a “data-driven predictive tool” to an “intervention-ready precision medicine decision-making system”.

Engineered bacteria: Programmable living medicines

Engineered bacteria, innovative products of synthetic biology, offer novel therapeutic models for constipation treatment.³⁵⁸ By introducing SCFA biosynthesis pathways into safe probiotic strains, programmable bacteria can specifically release metabolites in the colon.³⁰² Compared to traditional oral supplements, this approach prevents metabolite degradation in the upper gastrointestinal tract, significantly enhancing local effective concentrations. In preclinical studies, engineered butyrate-producing Clostridium strains encapsulated in hyaluronic acid nanoparticles exhibited improved mucosal adhesion and butyrate production, effectively alleviating intestinal mucosal inflammation and enhancing barrier function. This represents a typical therapeutic paradigm for microbial metabolic remodeling.³⁰³ Additionally, programmable probiotics can release targeted proteins, peptides, and immunomodulatory molecules, enabling precise local treatment with minimal systemic side effects. Such strategies provide more accurate therapeutic possibilities for constipation and associated gastrointestinal dysfunctions.³⁰⁴ Despite these promising prospects, the clinical translation of engineered bacteria faces potential risks, including disruption of microbiota homeostasis, antibiotic resistance transmission, and opportunistic infections. These issues necessitate further advancements in safety evaluation and ecological stability monitoring.³⁰⁵

Summary and future perspectives

Research on CC is shifting from a focus on “local motility disturbances” to addressing “systemic homeostatic imbalances”. This review integrates rapidly accumulating multidisciplinary evidence, confirming that gut microbiota, neuroregulation, and the mucosal-immune system do not function independently. Instead, they collectively maintain physiological gut rhythms through highly interconnected mechanisms, facilitating normal bowel movements. When microbial metabolic functions are disrupted, such as insufficient SCFAs, altered tryptophan metabolism, or toxic metabolite accumulation, the effects extend beyond the intestinal environment. These disruptions amplify through the CNS, ENS, and immune signaling networks, ultimately causing a persistent structural misalignment of the gut-brain-immune homeostatic loop. This multi-level imbalance can form a self-reinforcing pathological cycle, where reduced motility, dysbiosis, mucosal inflammation, and mood disorders mutually exacerbate, rendering the disease chronic, recurrent, and highly heterogeneous.

The concept of the “three-dimensional gut-brain-immune homeostasis loop” marks a significant theoretical expansion and mechanistic deepening of the traditional “gut-brain axis” binary model. Although the classical gut-brain axis has elucidated the basic framework of bidirectional communication between the gut and the central nervous system via neural, endocrine, and immune pathways, its limitation lies in treating the immune system as a passive responder or auxiliary pathway, failing to fully reveal the integrative regulatory logic underlying the dynamic interactions among the three systems. By contrast, the “Gut-Brain-Immune Three-Dimensional Homeostatic Loop” not only elevates the immune system to a core dimension on a par with the nervous system and the microbiome, but also emphasizes the pivotal role of the ENS as a central processing unit in real-time integration of signals from all three components and in maintaining the dynamic homeostasis of the internal environment, thereby surpassing the traditional model in terms of structural organization, functional mechanisms, and pathological explanatory power. In this context, CC has gradually moved beyond the classical definition of organ-specific dysfunction toward being recognized as a systemic homeostatic disorder. This shift explains why patients with similar clinical symptoms may have entirely different underlying pathologies and clarifies why single-target treatments often fail to produce lasting results. Traditional treatments usually target specific neural or metabolic pathways, offering only temporary interventions to isolated segments of the pathological network, without correcting the systemic homeostatic imbalance.

Future research should explore the structural basis and dynamic interactions within the gut-brain-immune homeostatic loop, clarifying causal relationships and identifying critical intervention points. The integration of multi-omics tools, including metagenomics, metabolomics, single-cell immune profiling, and spatial transcriptomics, provides unprecedented insights into the heterogeneity of constipation.³⁵⁹ These technologies will facilitate the transition from broad classifications based on clinical symptoms toward mechanism-driven precision subtyping, enabling diagnosis, prognosis, and treatment based on quantifiable biological evidence.³⁶⁰ Additionally, treatment strategies must shift from merely altering microbiota composition to restoring the coordinated functions of the microbiota metabolism-CNS-immune loop. Postbiotic therapies focused on metabolic functions, engineered delivery systems for targeted metabolites, and programmable microbes designed to precisely modulate specific metabolic, inflammatory, or neural circuits all show promising translational potential.^265,361 Additionally, previous studies often underestimated the impact of long-term psychological stress, sleep architecture, dietary rhythms, lifestyle, and genetic background on gut homeostasis.³⁶² These factors influence disease onset, persistence, treatment efficacy, and relapse risk, thus necessitating their inclusion in systemic intervention models. With advancements in artificial intelligence, multimodal bioinformatics, wearable monitoring devices, and synthetic biology, a new therapeutic framework centered around “systemic homeostasis restoration” is emerging. This framework could overcome the limitations of traditional constipation treatments, fundamentally improving disease control and providing a scalable theoretical and therapeutic model for additional gut-brain-immune coupling diseases, including functional gastrointestinal disorders, mood disorders, and chronic inflammatory conditions.

Acknowledgments

This work was supported by the Major Programs of Natural Science Foundation of higher education in Jiangsu Province (19KJA480003), the Natural Science Foundation of Jiangsu Province (BK20171290), the Administration of Traditional Chinese Medicine Project of Jiangsu Province (MS2021081), and the Industry-University-Research Collaboration of Jiangsu Province (BY20240547).

Author contributions

All authors contributed to the conception and design of the study. J.Z. and B.Z. contributed to literature investigation, conceptualization, and writing – original draft. W.Z. contributed to literature investigation and writing – original draft. Y.L., X.L., Y.Z., Z.X., Q.X., W.W., J.L., and X.S. contributed to literature collection, manuscript revision, and visualization. T.Z. contributed to writing-review and editing. F.L. and Y.Q. contributed to conceptualization, supervision, project administration, and writing-review and editing. All authors have read and approved the final manuscript.

Declaration of interests

The authors declare no competing interests.

Declaration of generative AI and AI-assisted technologies in the manuscript preparation process

The authors declare that no artificial intelligence tools were utilized in the preparation of this manuscript.