NOD-like receptors mediate homeostatic intestinal epithelial barrier function: promising therapeutic targets for inflammatory bowel disease

Abstract

Inflammatory bowel disease (IBD) is a chronic gastrointestinal inflammatory disease that involves host genetics, the microbiome, and inflammatory responses. The current consensus is that the disruption of the intestinal mucosal barrier is the core pathogenesis of IBD, including intestinal microbial factors, abnormal immune responses, and impaired intestinal mucosal barrier. Cumulative data show that nucleotide-binding and oligomerization domain (NOD)-like receptors (NLRs) are dominant mediators in maintaining the homeostasis of the intestinal mucosal barrier, which play critical roles in sensing the commensal microbiota, maintaining homeostasis, and regulating intestinal inflammation. Blocking NLRs inflammasome activation by botanicals may be a promising way to prevent IBD progression. In this review, we systematically introduce the multiple roles of NLRs in regulating intestinal mucosal barrier homeostasis and focus on summarizing the activities and potential mechanisms of natural products against IBD. Aiming to propose new directions on the pathogenesis and precise treatment of IBD

Introduction

Inflammatory bowel disease (IBD) is an illness with an unknown etiology and pathogenesis characterized by chronic inflammation of the intestinal mucosa with recurrent and persistently delayed episodes, encompassing ulcerative colitis (UC) and Crohn’s disease (CD). IBD patients are mainly dispersed in developed countries (>0.3%), but the incidence of IBD in newly industrialized countries is rapidly growing (the annual growth rate percentage has reached 14.9%). ¹ IBD has thus evolved into a global disease, and the number of IBD patients is expected to reach 30 million by the year 2025. ² The number of IBD patients is increasing year by year, and there is an urgent need to explore the pathogenesis of IBD and find potential targets and drugs for the treatment of IBD.

IBD is known to be caused by interactions between genetics, host immunity, the mucosal barrier, and the gut microbiome, but its exact etiology remains unclear. ³ Impaired mucosal barrier function is considered to be the primary pathogenic factor in the development of IBD.^4,5 As the primary defense barrier of the body, the intestinal mucosal barrier has made outstanding contributions to regulating and maintaining the homeostasis of the internal environment. ⁶ Once the intestinal mucosal barrier function is disrupted by various factors, the immune balance is disrupted, triggering an inflammatory response that is ultimately involved in the development of IBD. ⁷ It is believed that NOD-like receptors (NLRs) are the main mediators in maintaining intestinal mucosal barrier homeostasis,^8,9 including regulation of intestinal microbiota, inflammatory response, cellular senescence, and cell death. ¹⁰ In particular, the role of NLRs in modulating the intestinal mucosal barrier and immune mechanisms has become a hot topic of research and is considered a valuable therapeutic target for the treatment of IBD.

It is thought that aberrant activation of NLRs is the main cause of intestinal mucosal barrier dysfunction. Blocking NLR-inflammasome activation by botanicals or synthetic small molecules may be a valuable way to prevent IBD progression. Natural bioactive compounds are an important resource for drug discovery and modification. Several studies suggest that natural products (NPs), including dietary, and herbal formulations or their extracts, have unique efficacy in the health management of chronic inflammatory diseases.^11,12 In this review, we elucidate the composition and function of the intestinal mucosal barrier and discuss the potential role of NLRs in regulating the intestinal mucosal barrier and inflammatory mechanisms. We also summarize the original extracts and traditional medicines targeting NLRs against IBD with the potential to develop NPs.

Literature search strategy

Studies were identified by searching electronic medical databases: Web of Science, PubMed, SciFinder, OvidSP, Scopus, MEDLINE, ScienceDirect, IMBIOMED, and Cochrane database, from January 1983 to February 2023. All human and animal studies are included to evaluate the role of NOD-like receptors in the pathogenesis of IBD, ensuring attention to the latest and comprehensive literature. MeSH terms and/or text words included the broad terms NOD-like Receptors, inflammatory bowel disease, intestinal mucus barrier, and Botanicals. We also searched specific pathogens including NLRP1, NOD2, NLRP3, NLRP6, NLRC4, NOD1, NLRP12, biological barrier, gut microbiota, chemical barrier, mechanical barrier, immunological barrier, botanicals, natural products, flavonoids, terpenoids, plant extracts, alkaloids, phenylpropanoids, treatment, therapeutic, pathogenesis, epidemiology, combined with ‘AND inflammatory bowel disease OR UC OR Crohn disease OR colitis’. We also reviewed the references of relevant IBD disease review articles and studies that met the inclusion criteria of other relevant articles.

The composition and function of the intestinal mucosal barrier

Biological barrier

The gut microbiome is mainly composed of bacteria, archaea, fungi, viruses, and bacteriophages. ¹³ The gut microbiota is dominated by bacteria, especially members of the phylum Bacteroidetes, Firmicutes, Actinobacteria, and Proteobacteria, the sum of which exceeds 90%. ¹⁴ The gut microbiota plays an important role in several aspects of host homeostasis. The gut microbiota plays an important role in several aspects of host homeostasis: (1) Aid the host digest and metabolize indigestible nutrients such as xylan, cellulose, and resistant starch. (2) Provide substrates, enzymes, and energy for host metabolic processes. (3) Prevent the invasion of harmful bacteria by secreting bacterial toxins, producing short-chain fatty acids, promoting intestinal peristalsis, occupying the adhesion sites of pathogenic bacteria, and competing for nutrients. (4) Suppresses the gut immune system’s hyperreactive immune response to antigens to maintain immune tolerance and homeostasis.

Chemical barrier

The intestinal chemical barrier consists of mucus secreted by intestinal epithelial cells, digestive juice fluids (gastric acid, bile), and antimicrobial peptides (AMPs), which cooperate to form a complex protective mechanism. ¹⁵ The role of mucus is to insulate intestinal bacteria from contact with the intestinal epithelium, thereby avoiding tissue damage and microbiota translocation. ¹⁶ Stomach acid and bile acid could inhibit the growth and reproduction of bacteria, thus protecting the intestinal tract from pathogenic bacteria infection. ¹⁷ AMPs can have multiple effects on intestinal mucosal homeostasis; in addition to killing ingested pathogens, controlling the composition of the gut microbiota can also coordinate adaptive immune responses. ¹⁸ The intestinal chemical barrier is a good support for the intestinal microbial barrier and plays an important role in maintaining the intestinal microecological balance.

Mechanical barrier

The intestinal mucosal mechanical barrier is composed of intestinal mucosal epithelial cells, the apical junction complex (AJC), and the lamina propria. The AJC is located at the apical junction between adjacent intestinal epithelial cells and is composed of adherens junctions (AJs) and tight junctions (TJs). ¹⁹ AJs and TJs include two cell-cell adhesion modes with different functions. The adhesion junction contains intracellular components such as α-catenin, β-catenin, and p120-catenin, as well as the transmembrane protein E-cadherin, which triggers and regulates contact between cells in addition to regulating maturation. ²⁰ TJs are composed of claudin, the transmembrane proteins occludin, and the cytoplasmic scaffolding proteins ZO-1, -2, and -3, which, together, regulate the paracellular pathways of ion and solute movement between cells. ²¹ Mechanical barriers have several critical physiological functions: (1) Preventing harmful entities in the lumen from invading the body, including foreign antigens, microorganisms, and toxins. (2) Selectively allowing essential dietary nutrients, electrolytes, and water to enter the circulation through the intestinal cavity. (3) Secretion of mucus, antimicrobial proteins (AMPs), and immunoglobulin A (SIgA), which provides the basis for the intestinal chemical barrier, immune barrier, and other barrier functions.

Immunological barrier

The intestinal mucosal immune barrier is mainly composed of gut-associated lymphocyte tissue (GALT) and diffuse immune cells [innate immune cells (neutrophils, monocyte/macrophages, dendritic cells, mast cells, and innate lymphoid cells) and adaptive immune cells (B and T cells)]. ²² Moreover, epithelial cells are increasingly viewed as an integral part of the immune barrier. ²³ The GALT secretes immunoglobulin A (IgA) antibodies, and IgA forms IgA – antigen complexes with antigenic substances. The IgA antigen complexes bind to the receptors on M cells. The antigens are then transferred to the lamina propria and subsequently presented to dendritic cells. ²⁴ Antigen-presenting cells in the lamina propria receive immune-stimulating antigens from the cavity, process those antigens, and present them to T cells. The IL-12 secreted by antigen-presenting cells can promote the production of Th1 by T cells, but the simultaneous production of IL-10 limits Th1 immune responses. ²⁵ Additionally, IgA promotes the renewal of intestinal epithelial cells, and repairs damage over time, stabilizing the intestinal mucosal barrier. ²⁶ In summary, the main function of the intestinal mucosal immune barrier is to recognize antigens in the body, generate humoral and cellular immunity, and effectively remove antigens.

In conclusion, the intestinal mucosal barrier is an important protective mechanism and defense system for maintaining body health. Moreover, it is worth mentioning that the interaction between each barrier forms a complex regulatory network, which fully plays the role of preventing the invasion of pathogenic microorganisms, regulating antigen import, and inhibiting inflammation (Figure 1).

Figure 1.

Components of the intestinal mucosal barrier.

NLRs family and signaling mechanisms in the intestinal epithelial barrier

NLRs are composed of multiple domains, including a C-terminal domain, a series of leucine-rich repeats (LRRs), a central nucleotide-binding NACHT domain, and a variable N-terminal effector domain. Specifically, NLRs are divided into five subfamilies based on their unique N-terminal effecter domains: NLRAs with unique acidic activation domains, NLRBs feature a baculovirus inhibitor of apoptosis repeat (BIR)-like domain, NLRCs that have a caspase activation and Death domain or a recruitment domain (CARD), and the NOD and leucine-rich-repeat-containing protein (NLRP) subfamily NLRAs embody a PYRIN domain. ²⁷ The NOD and leucine-rich-repeat-containing protein X (NLRX) subfamily contains only one member, which has an undefined N-terminal domain and has no significant homology with the N-terminal domain of other NLR subfamily members. The LRR domains are responsible for recognizing molecular patterns, including the corresponding components of PAMPs/DAMPs. The characteristic NACHT domain has dNTPase activity and mainly regulates ATP-dependent oligomerization. The N-terminal effector domain is the most prominent component of the NLRs and binds to various linker molecules and downstream effectors to mediate signal transduction.

NLRs are widely expressed in the gut, and their role in the pathogenesis of IBD was previously demonstrated (Table 1).^28,29 NOD1 and NOD2 are involved in the recognition of infectious bacteria, induction of immune responses, and protection of the intestinal mucosal barrier from bacterial erosion through the activation of nuclear factor kappa B (NF-κB).^30,31 The NLRP3 and NLRC4 assemblages respond to some microorganisms or parasites by activating caspase-1, producing IL-1β and IL-18, and initiating inflammatory processes. NLRP6 recognizes Listeria monocytogenes and recruits caspases, and Gasdermin D (GSDMD) cleavage further enhances NLR’s role as an important molecule in cell death. ³² Similarly, NLRP12 was demonstrated to be crucial in the regulation of interferon and cytokine production, as well as the maintenance of immune homeostasis. ³³ Moreover, cell death mediated by NLR-inflammasome activation protects host cells from attack by pathogens, by impeding the nutrient supply to the pathogens, ³⁴ and the activated bystander cells can also secrete antimicrobial agents that arrest pathogen expansion and disease aggravation. ³⁵ However, the specific molecular basis of the causal relationship between NLRs and IBD remains enigmatic. Nonetheless, due to the central roles of NLRs in immune regulatory signaling, they are considered as potential therapeutic targets for IBD.

Table 1.

NLRs in IBD.

Subfamily	NLR		Cells	Effect	References
NLRC	NOD1		Intestinal epithelial cells and dendritic cells	Protection	Chen et al. 36 Le Bourhis et al. 37
	NOD2		Intraepithelial lymphocyte cells (IELs), enterocytes, Paneth cells, and goblet cells	Protection	Al Nabhani et al. 38 Jiang et al. 39 Richardson et al. 40 Shanahan et al. 41
	NOD2		/	Aggravation	Corridoni et al. 42
	NLRC4		Intestinal epithelial cells and phagocytes	Protection	Sellin et al. 43 Franchi et al. 44
	NLRC4		/	Aggravation	Steiner et al. 45
NLRP	NLRP1		Intestinal epithelial cells	Protection	Williams et al. 46
	NLRP1		Macrophages	Aggravation	Wang et al. 47
	NLRP3		Macrophages and neutrophils	Aggravation	Ranson et al. 48 Chen et al. 49
	NLRP3		Macrophages and neutrophils	Protection	Yao et al. 50
	NLRP6		Intestinal epithelial cells	Protection	Kempster et al. 51 Yin et al. 52
	NLRP12		Macrophages and dendritic cells	Protection	Chen et al. 53
LRR PYD NACHT FIIND CARD

IBD, inflammatory bowel disease.

NOD-like receptors regulate homeostatic intestinal epithelial barrier function

Role of NLRs in modulating intestinal biologic barriers

The gut microbiota is equivalent to a ‘hidden organ’ that helps maintain a healthy mucosal barrier. The composition and diversity of the intestinal microbiota are influenced by host genes.^54,55 To assess the contribution of NLRs to the intestinal microbiota in IBD, Elinav et al. analyzed the fecal microbiota based on 16S rRNA. The results showed that the frequency of Prevotella in NLRP6−/− mice were higher. Compared to wild-type mice, NLRP6-/- mice exhibit a higher susceptibility to colitis. ⁵⁶ Similarly, the study of Seregin et al. demonstrated that NLRP6 could alter Akkermansia muciniphila colonization, preventing IBD, in IL10−/− mice. ⁵⁷ Interestingly, NLRP6−/− mice are resistant to systemic Escherichia coli, Salmonella typhimurium, and Listeria monocytogenes infections. ⁵⁸ Gut mucosal microbiome dysbiosis was also reported in mice deficient in NLRP3. Hirota et al. reported that NLRP3−/− mice exhibited enhanced susceptibility to colitis and lower levels of B-defensins compared to wild-type mice, most likely due to altered gut microbiota compositions. Notably, the levels of potentially pathogenic bacteria increased; for example, Enterobacteriaceae, Genus mycobacterium, Collinsella, Subdoligranulum, Clostridium, and Ralstonia increased significantly. ⁵⁹ However, recent studies contradicted these observations by showing that NLRP3−/− mice were resistant to colitis. ⁶⁰ Further research showed that mice lacking the NLR components NLRP3, NLRC4, and caspase-1 were highly sensitive to Citrobacter. rodentium-induced gastrointestinal inflammation and could not control C. rodentium due to defects in the generation of IL-18 and IL-1β. ⁶¹ Moreover, NLRP3-mutated mice showed reshaping of the gut microbiota and the appearance of regional regulatory T cells (Tregs) to maintain homeostasis and compensate for otherwise-harmful intestinal inflammation. ⁴⁴ The NLRP3 inflammasome plays a significant role in the pathogenesis of colitis, although NLRP3 was found to have both protective and disadvantageous functions in studies on mucosal immunity. Different results are associated, for example, with the mouse/human genetic backgrounds, ⁶² according to the composition of the gut microbiota, ⁶³ with different colitis models, ⁶⁴ or according to the method of inducing colitis (the concentration of DSS, the continuance of DSS delivery, and the number of cycles).

In addition, the regulation of the microbiota of the ileum and the bactericidal activity of ileal crypts are largely determined by NOD2, mainly by altering the interaction between mucosal immunity and ileal microbiota. Biswas et al. reported that NOD2−/− mice presented increased loads of commensal bacteria and pathogens at the terminal ileum. Further analysis showed that NOD2−/− mice experienced a remarkable increase in the amount of Bacteroides, Firmicutes, and Bacillus in the terminal ileum compared to wild-type littermate controls. ⁶⁵ Notably, increased intestinal inflammation in NOD2-deficient mice and patients with CD was attributed to the impaired bactericidal activity of Paneth cells. ⁶⁶ Furthermore, reciprocal microbiota transplantation was found to reduce the risk of colitis or colorectal cancer in Nod2−/− mice. ⁶⁷ Lastly, NLRP12, a negative regulator of inflammation, was identified in an experimental colitis model and patients with IBD. ⁶⁸ NLRP12 suppresses the excessive production of inflammatory cytokines in the intestines and decreases the susceptibility to colitis by maintaining commensal variety and protective microbiota. The sequencing of bacterial 16S rRNA confirmed that NLRP12−/− may result in the loss of protective gut commensal strains (Lachnospiraceae) and an increase in Escherichia coli strains (Erysipelas), which can be reversed via the administration of beneficial commensal Lachnospira isolates or antibodies targeting inflammatory cytokines. Additionally, NLRP12-deficient mice could be treated with microbiome transplants or antibodies targeting inflammatory cytokines. ⁵³ The above studies on the microbiome in NLRP12-deficient mice resemble those of human IBD patients.^{69 -71}

To date, accumulating evidence has demonstrated the impact of NLRs on the gut microbiota and IBD and illustrated the potential of manipulating the gut microbiota in the treatment of IBD. Therefore, there is great interest in the possible benefits of microbiome-modulating interventions, such as probiotics, prebiotics, antibiotics, fecal microbiota transplantation, and the role of genetic manipulation in the treatment of IBD. However, the underlying mechanisms of how genetic alterations affecting NLRs affect gut microbiota biodiversity are still not fully understood. Additionally, the relationship between gut dysbiosis and IBD is complex and dynamic, rather than a simple causal relationship.

Role of NLRs in modulating the intestinal mechanical barrier

Gut mechanical barrier dysfunction is a consistent feature of IBD. ⁷² This barrier is composed of intact intestinal epithelial cells, the AJC, and the lamina propria. ⁷³ AJC maintains intestinal homeostasis mainly by controlling the permeability of intestinal epithelial cells to limit the transport of relatively large molecules.^74,75 Accumulating data support a correlation between NLRs and the integrity of the intestinal mechanical barrier. protects mice against increased intestinal mucosal permeability caused by AJC dysfunction by regulating myosin light chain kinase (MLCK) signaling^76,77 In Caco-2, a small intestinal eNOD2pithelial cell similar in structure and function to differentiation, NLRP3 weakens the cell’s trans epithelial resistance by reducing the expression of claudin-1, occludin, and ZO-1, while increasing the expression of claudin-2 and repositioning occludin and ZO in Caco-2 monolayer. ⁷⁸ Likewise, the NLRP3 inflammasome was activated in antibiotic-exposed Caco-2 and mice, resulting in decreased expression of claudin and disrupted ZO-1 morphology. ⁷⁹ In addition to the destruction of AJC, abnormal death of intestinal epithelial cells is a major cause of intestinal mucosal mechanical barrier dysfunction. ⁸⁰ Autophagy has been shown to regulate cytokine-induced programmed cell death in intestinal epithelial cells, limiting intestinal inflammation. The inflammasome and autophagy are mutually regulated, and this bidirectional regulation provides the necessary checks and balances between host defenses against inflammatory responses and the prevention of excessive inflammation in organ and tissue damage and inflammatory diseases. NLRP6 deficiency results in defective autophagy in goblet cells and reduced mucin secretion in the lumen of the large intestine. ⁸¹ When NLRP6-deficient mice were treated with DSS, they became more susceptible to serious colitis due to a reduction in IL-18 expression by intestinal epithelial cells (IECs), and pathological damage increased compared to wild-type animals. ⁸² Akin to NLRP6, NLRP3 performs a protective function for intestinal epithelial cells. Compared to wild-type mice, NLRP3-deficient mice are more likely to suffer from C. rodentium infections. ⁸³ Simultaneously, activating the NLRP3 inflammasome can induce the compensatory proliferation of epithelial cells, protecting the integrity of the epithelial layer during DSS-induced colitis. ⁸⁴ NLRC4 is a double-edged sword. On the one hand, NLRC4 can restrict infections by S.typhimurium by activating pyroptosis and forcing infected intestinal epithelial cells to enter the lumen. ⁴³ On the other hand, excessive NLRC4 results in too much pyroptosis, which triggers small intestinal damage. ⁸⁵

Role of NLR in regulating mucosal immunity

The intestine serves as the largest immune organ in mammals, containing 90% of the immunocompetent cells. The intestinal mucosal immune barrier comprises gut-associated lymphoid tissues (such as Peyer’s patches and mesenteric lymph nodes), cells [such as innate immune cells (neutrophils, monocyte/macrophages, dendritic cells, mast cells, and innate lymphoid cells) and adaptive immune cells (B and T cells)], and molecules (such as immunoglobulins and AMPs). ²² Moreover, epithelial cells are increasingly viewed as an integral part of the immune barrier.

NLRP1 was the first pattern recognition receptor (PRR) discovered to form an inflammasome, and genome-wide association studies have identified NLRP1 mutations linked with IBD. ⁸⁶ Previous evidence showed that NLRP1b−/− mice showed a significant increase in the incidence of colitis, infections, and colitis-related tumorigenesis via the modulation of IL-1β and IL-18 levels. Moreover, the wound healing and defense responses in DSS-induced colitis and lipopolysaccharide (LPS)-stimulated cellular inflammation in mouse macrophages might be caused by the downregulation of NLRP1 expression. ⁴⁶ Similarly, gut-barrier defects and excessive intestine-related lymphoid tissue activation are features of NOD2 deficiency in humans and mice. ³⁸ Further investigations revealed that hyperresponsive macrophages and colitis promoted NOD2 signaling in IL-10-deficient mice, and a deficiency of NOD2 ameliorated chronic colitis in IL-10−/− mice. ⁸⁷ Additionally, NOD2−/− can lead to a steady-state imbalance, inactivated fibroblasts, and inflammatory macrophages, manifested by the increased expression of aberrant activation-related transcriptomes caused by the upregulation of upstream transcription factors such as WT1 and STAT3, and it causes the gp130 family to upregulate the expression of certain genes (IL-11, IL-6, OSM, etc), thereby increasing the risk of intestinal stenosis. ⁸⁸ Moreover, NLRP3 is the most widely studied immune regulator in NLRs, although its role is controversial. Chen et al. showed that the interaction of the NLRP3 inflammasome with NEK7 promotes IBD by mediating NF-κB signaling to regulate macrophage apoptosis. ⁴⁹ By contrast, NLRP3-inflammasome activation can facilitate the restoration and reproduction of the intestinal mucosa, which could be related to enhanced levels of caspase-1, IL-1β, and IL-18. ⁸⁹ IL-1β promotes the production of IL-22 and IL-10 to maintain epithelial integrity.^90,91 IL-18 and IL-1β were shown to promote compensatory epithelial cell proliferation and reduce DSS-induced inflammatory colon injury. ⁸³ Intriguingly, another study showed that uncontrollably active NLRP3 inflammasomes neutralize intestinal inflammation by inducing the production of Tregs and reshaping the gut microbiota in NLRP3-mutant mice. ⁵⁰ In addition to the above NLRs, NLRP6 is also an important regulator of IBD. NLRP6−/− mice showed an increase in the number of CD45+cells in the lamina propria, proliferation of colonic crypts, enlargement of Peyer’s patches, and formation of germinal centers. ⁵⁶ These mice exhibited increased mucosal permeability and were unable to recover from colitis.^82,92

In summary, NLRs play critical roles in immune cell development and function through multiple canonical immune regulatory mechanisms, including caspase-1, RIP2 kinase, mitogen-activated protein kinase (MAPK), and NF-κB signal pathway. However, with the deepening of the research on NLRs in IBD, the functions of these proteins have also become more diverse, but the exact roles of some NLRs in IBD are still controversial and deserve further study. In addition, the biochemical processes regulating NLRs activation and downstream functions remain to be further investigated.

Targeting NLRs with botanicals in IBD therapy

Many synthetic drugs and monoclonal antibodies are currently administered in the clinic to treat IBD, such as NF-κB inhibitors (e.g. sulfasalazine), TNF-α antagonists (e.g., infliximab), and glucocorticoids (e.g. prednisolone). However, the therapeutic effects of these drugs are unsatisfactory, as their long-term administration can produce severe side effects, leading to poor patient acceptance and compliance.^93–95 Therefore, exploring safe and effective drugs has become an urgent task for the treatment of IBD patients.

NLRs are important regulators of intestinal mucosal homeostasis and are promising therapeutic targets for IBD. NLRs have regulatory effects on multiple signaling molecules explicitly related to IBD diseases, such as NF-κB, ASC, caspase-1, MAPK, IL-1β, TNF-α, IL-6and IL-18 (Figure 2). The apparent breadth and magnitude of NLRs’ involvement illustrate the apparent centrality of this protein family. As of November 2019, 82 patents were pending for potential therapeutic NLRs modulators, and 22 patents had been granted. ⁹⁶ Additionally, there are multiple phase I clinical trials of NLRP3 inhibitors for CD that have been registered in public databases by Novartis. ⁹⁷

Figure 2.

The mechanism of NLRs in the pathogenesis of IBD.

NPs, including dietary compounds, herbal formulas, and their extracts, have been known to have unique curative effects in the management of chronic inflammatory diseases for thousands of years. Numerous experiments in vitro and in vivo have indicated that some active ingredients in botanicals and NPs perform anti-IBD functions by regulating NOD-like receptor signaling, with few toxic side effects compared to synthetic treatments. In this section, we review the current findings on targeting NLRs with botanicals in IBD intervention (Table 2).

Table 2.

Targeting NLRPs with botanicals in IBD therapy.

Agent	Experimental subjects	Outcomes	Potential mechanisms	References
Wogonoside	Female BALB/c mice (DSS), THP-1 cells	Inflammatory cytokines↓, inflammatory cell infiltration↓, colonic pathological damage↓	Blocking activation of NF-κB and NLRP3 inflammasome.	Sun et al. 98
Flavonoid VI-16	Male C57BL/6 J mice (DSS), Macrophages	Colonic pathological damage↓, inflammatory cytokines↓	Suppressing ROS/Txnip/NLRP3 signaling pathway.	Zhao et al. 99
Curcumin	Female C57BL/6 mice (DSS), Macrophages	DAI↓, inflammatory cytokines↓, colonic pathological damage↓	Blocking activation of NLRP3 inflammasome and ASC oligomerization.	Gong et al. 100
Procyanidin	Male C57BL/6 mice (DSS), Macrophages	Colonic pathological damage↓, inflammatory condition↓	Suppressing activation of MMP9, NF-κB, and NLRP3 inflammasome.	Chen et al. 101
Naringin	Male C57BL/6 mice (DSS), RAW264.7 cells	Inflammatory cytokines↓, disease severity↓, DAI↓	Activating PPARγ and suppressing NF-κB, MAPK, and NLRP3-inflammasome activation.	Cao et al. 102
Phloretin	Male C57BL/6 mice (DSS)	Escherichia coli and Lactobacillus levels rebalanced, gut epithelial barrier integrity↑	Upregulating ZO-1 and occludin expression, inhibiting NLRP3 inflammasome activation.	Zhang Z et al. 103
Genistein	C57BL6/J mice (DSS), Macrophages	Pro-inflammatory mediator production↓, disease severity↓, inflammatory cell infiltration↓	Inhibiting NLRP3 inflammasome by regulating TGR5-cAMP pathway.	Chen et al. 104
Cardamonin	Male C57BL/6 mice (DSS), Male BALB/c mice (TNBS:2,4,6-trinitrobenzenesulfonic acid)	Myeloperoxidase activity↑, DAI↓, colonic pathological damage↓	Activating AhR/Nrf2/NQO1 pathway and inhibiting NLRP3-inflammasome activation.	Wang et al. 105
Alpinetin	Female BALB/c mice (DSS), THP-1 cells	Diarrhea and colonic histological injury↓, inflammatory cytokines↓	Downregulating TLR4, NF-κB, and NLRP3-inflammasome expression.	He et al. 106
Formononetin	Male C57BL/6 mice (DSS), HCT-116 cells	Epithelial cell injury↓, disease severity↓	Increasing tight junction proteins expressions and inhibiting the NLRP3 pathway.	Liu et al. 107
Apigenin	Male C57BL/6 mice (DSS)	Inflammatory cytokines↓, colonic pathology damage↓.	Reprograming the gut microbiota through regulating NLRP6.	Radulovic et al. 108
Paeoniflorin	C57BL/6 mice (DSS), RAW264.7 cells	Gram-positive bacteria count and gram-positive bacterial infiltration↓, disease severity↓.	Inhibiting gram-positive bacteria-dependent MDP–NOD2 pathway.	Luo et al. 109
Toosendanin	Male C57BL/6 mice (DSS)	M1 macrophage polarization↓, disease severity↓	Upregulating HO-1/Nrf2 expression and inhibiting NLRP3 inflammasome activation.	Fan et al. 110
Asiatic acid	Female C57BL/6 mice (DSS), THP-1 cells	Inflammatory cytokines↓, disease severity↓	Suppressing mitochondria-mediated NLRP3-inflammasome activation.	Guo et al. 111
Dihydroartemisinin	Male C57BL/6 mice (DSS)	DAI↓, inflammatory cytokines↓, disease severity↓	Inhibiting the phosphorylation of NF-κB p65 and p38 MAPK, and NLRP3-inflammasome activation.	Liang et al. 112
Ginsenoside Rg1	C57BL/6 mice (DSS)	DAI↓, pro-inflammatory cytokines levels↓, colonic damage scores↓.	Activating TLR4-NLRP12-NF-κB pathway.	Zhu et al. 113
Celastrol	Male SD rats (DSS)	DAI and MDI↓, colonic histological damage↓, inflammatory cytokines↓	Modulating HSP90 and inducing autophagy to enhance NLRP3 sensitivity to CP-456773.	Saber et al. 114
Libertellenone M	Female C57/BL6 mice (DSS), RAW264.7 cells.	DAI↓, disease severity↓, inflammatory cytokines↓.	Inhibiting the nuclear translocation of NF-κB and the assembly of NLRP3 inflammasome	Fan et al. 115
Ginsenoside Rk3	male C57BL/6 mice (DSS)	Clinical symptoms↓, inflammatory cell infiltration↓, pro-inflammatory cytokines↓,	Inhibiting NLRP3-inflammasome activation	Tian et al. 116
Palmatine	Male BALB/C mice (DSS), THP-1 cells	DAI↓, colonic inflammation↓, disease severity↓.	Facilitating PINK1/Parkin-driven mitophagy-mediated NLRP3-inflammasome inactivation.	Mai et al. 117
Fumigaclavine C	female C57BL/6 mice (DSS), THP-1 cells	Disease severity↓, inflammatory cytokines↓, DAI↓.	Suppressing NF-kB, STAT3, STAT1, and NLRP3-inflammasome activation.	Guo et al. 118
Evodiamine	Male C57BL/6 mice (DSS)	DAI↓, Escherichia coli, and Lactobacillus levels rebalanced, colonic pathological damage↓.	Suppressing NF-κB and NLRP3-inflammasome activation.	Shen et al. 119
Carboxyamidotriazole	Female SD rats (TNBS)	Intestinal inflammatory and permeability↓, colonic pathological damage↓.	Inhibiting NLRP3-inflammasome and NF-κB activation.	Du et al. 120
Compound 1	Female C57BL/6 mice (DSS), THP-1 cells	Mucosal damage↓, inflammatory cell infiltration↓, CD11b+ infiltrating cells↓	A novel potent Nrf2/ARE inducer, inhibiting NLRP3-inflammasome activation.	Wang et al. 121
Chlorogenic acid	BALB/c mice (DSS) RAW264.7 cells.	Colonic inflammation↓, disease severity↓.	Suppressing miR-155/NF-κB/NLRP3 pathway.	Zeng et al. 122
Bergenin	Male Wistar rats (TNBS)	MPO↓, inflammatory cytokines↓, Disease severity↓.	Blocking pSTAT3, NF-κB canonical, and NLRP3/ASC pathways.	Lopes de Oliveira et al. 123
Brusatol	Male SD rats (TNBS), RAW264.7 cells.	Colonic pathology damage↓, oxidative stress↓, DAI↓.	Suppressing NF-κB and NLRP3 activation and enhancing Nrf2 expression	Zhou et al. 124
Fraxinellone	Male C57BL/6 mice (DSS), THP-1 cells	Macrophage infiltration↓, colonic pathology damage↓, inflammatory cytokines↓.	Suppressing NF-κB signaling and NLRP3 inflammasome activation.	Wu et al. 125
VitD3	Male C57BL/6 mice (DSS), Macrophages	Macrophage infiltration↓, colon inflammation↓, CD4+ T cells generation↓.	Inhibiting VDR/ NLRP3/ASC pathway and NLRP3-NEK7 interaction.	Cao et al. 126
MLB	Male C57BL/6 J mice (DSS).	Colonic pathology damage↓, inflammatory cytokines↓.	Inhibiting the activation of NRLP3/ASC/caspase-1 pathway.	Jiang et al. 127
Physalin B	Male BALB/c mice (DSS), RAW 264.7 cells	Colonic pathology damage↓, inflammatory cytokines↓, DAI↓.	Suppressing the activation of STAT3, β-arrestin1, and NLRP3 inflammasome.	Zhang et al. 128
Oryzanol	SD rats (DSS), Caco-2 cells	Gut-barrier damage of colitis rats↓abundances of Alloprevotella, Roseburia, Treponema, Muribaculaceae, and Ruminococcus↑.	SuppressingTLR4/NF-κB/NLRP3 signaling axis.	Xia et al. 129
Munronoid I	Female wild-type C57BL/6 mice, Mouse peritoneal macrophages, MODE-K cells	Colonic pathology damage↓, disease severity↓.	Promoting K48-linked ubiquitination and degradation of NLRP3	Ma et al. 130
Artemisinin analog SM934	Female C57BL/6 mice, HT-29 and Caco-2 cells.	Intestinal barrier function↑, intestinal epithelial cell apoptosis↓.	Suppressing NLRP3/NF-κB/MAPK signal axis	Shao et al. 131
Soy isoflavones	Male C57BL/6 mice (DSS), NCM460 cells	Pro-inflammatory cytokines↓, colonic pathology damage↓.	Inhibiting ERα/NLRP3 inflammasome activation.	Gao et al. 132
LEP	Male ICR mice	Oxidative stress damage↓, disease severity↓.	Regulating PPARγ/NF-κB and IL-6/STAT3 pathways and inhibiting NLRP3 inflammasome.	Zong S et al. 133
QCWZD	Male SD rats (DSS)	Butyricimonas, Blautia, and Odoribacter↑, disease severity↓.	Inhibiting the activity of the TLR4/Blimp-1 pathway to promote NLRP12 expression	Sun et al. 134
QRJPD	Male C57BL/6 J mice	Colonic pathology damage↓, disease severity↓.	Inhibit activation of the NLRP3 inflammasome	Zhang et al. 135
APS	Male C57BL/6 mice (DSS)	Colonic pathology damage↓, disease severity↓.	Inhibiting the NLRP3 signaling pathway	Tian et al. 136
LEM	Male C57BL/6 mice (DSS)	DAI↓, Inflammatory cell infiltration↓, intestinal barrier integrity↑.	Modulating NF-κB and MAPK pathways and Inhibiting NLRP3 inflammasome activation.	Zong et al. 137
RGE + EKN	C57BL/6 mice (DSS).	DAI↓, oxidative stress↓, pro-inflammatory cytokines↓, intestinal mucosal function↑.	Inhibiting TLR4/ NF-κB and NLRP3 inflammasome pathways	Saba et al. 138
CGB	C57BL/6 mice (DSS)	Inflammatory cell infiltration↓, colonic pathology damage↓.	Suppressing the NLRP3 signaling pathway and restores the expression of occludin and claudin-1.	Mahmoud et al. 139
Platycodon grandiflorum root	Macrophages, Intestinal epithelial cells, Mice (DSS)	Inflammatory cytokines↓,colonic pathology damage↓.	Inhibiting NF-κB signaling pathway and the expression of NLRP3 inflammasomes by activating AMP-activated protein kinase	Wang et al. 140

APS, astragalus polysaccharide; CGB, Canna × generalis L.H. Bailey; EKN, epimedium Koreanum Nakai; LEM, lycium ruthenicum Murray; LEP, lachnum polysaccharide; MAPK, mitogen-activated protein kinase; MLB; Magnesium lithospermate B; NF-κB, nuclear factor kappa B; RGE, Red ginseng extract; QCWZD, Qingchang Wenzhong decoction; QRJPD, Qingrejianpi decoction.

Flavonoids

Flavonoids have been reported to encompass multiple biological activities, including immune adjustment, scavenging free radicals, the changing of the microbiota constitution, antiapoptotic, and the restoring of damaged epithelial barriers. For this reason, flavonoids may be a promising approach for IBD management. For instance, wogonoside, a natural flavonoid isolated from Scutellaria baicalensis, attenuates DSS-induced colitis in mice through the inhibition of NF-κB and NLRP3 inflammasome. ⁹⁸ Similarly, Flavonoid VI-16 (a synthetic flavonoid compound), selectively inhibits Txnip-dependent activation of NLRP3 inflammasome to alleviate oxidative stress in colonic macrophages, thereby reducing DSS-induced colitis. ⁹⁹ Other flavonoids NLRP3 signaling inhibitors, such as curcumin, procyanidin, naringin, phloretin, cardamonin, alpinetin, genistein, and formononetin, have likewise been shown to be effective in the treatment of colitis.^{100 –107} Other flavonoid NLRs inhibitors also have a potentially preventive or therapeutic effect on colitis. Apigenin, a common dietary flavone, protects against gut inflammation by modulating the intestinal microbiota constituents through NLRP6 and C-type lectin Reg3b in mice. ¹⁰⁸

Terpenoids

Terpenoids constitute the largest class of NPs and are a rich reservoir of candidate compounds for drug discovery. Several preclinical studies have provided evidence that terpenoids are useful in IBD. Paeoniflorin, a natural monoterpenoid isolated from Paeonia lactiflora Pall, was proven to facilitate intestinal mucosal barrier protection in a DSS-induced colitis model. Detailed studies indicated that paeoniflorin ameliorates DSS-induced colitis in mice by decreasing the infiltration of gram-positive bacteria through the regulation of the gram-positive-bacterium-dependent MDP–NOD2 pathway. ¹⁰⁹ Toosendanin is derived from the fruits or bark of Melia toosendan Sieb. Et Zucc (Meliaceae), potently blocked NLRP3 inflammasome activation via Nrf2/HO-1 signaling in mouse IBD models. ¹¹⁰ Asiatic acid was demonstrated to block the ROS–NLRP3–caspase-1–IL-1β cascade in macrophages and experimental mouse colitis models. ¹¹¹ Dihydroartemisinin prevents DSS-induced colitis in mice by effectively blocking p38 MAPK signaling and NLRP3-inflammasome activation. ¹¹² Ginsenoside Rg1, a pharmacologically active component of ginseng, effectively blocked the expression of NLRP12 and NF-κB signaling and reduced the inflammation of DSS-induced colitis in mice. ¹¹³ Celastrol, a natural triterpenoid extracted from the root bark of Tripterygium wilfordii Hook. f. Celastrol, protected mice against colitis by inhibiting the activation of the NLRP3 inflammasome signaling. ¹¹⁴ Libertellenone M, a hippocampal diterpenoid from the marine-derived fungus Stibella fimetaris, inhibited NLRP3-inflammasome activation in a mouse model of IBD. ¹¹⁵ In addition, Ginsenoside Rk3, selectively blocked NLRP3 inflammasome activation, leading to reduced TNF-α, IL1β, and IL-6 production in a mouse model of IBD. ¹¹⁶

Alkaloids

An increasing number of recent studies have reported that alkaloids are effective in treating intestinal inflammatory disorders. Palmatine, originally isolated from medicinal rayon fibers, promotes PINK1/Parkin-mediated mitochondrial autophagy and inhibits NLRP3 inflammatory vesicle activation, leading to a decrease in IL-1β, thereby alleviating DSS-induced colitis in mice. ¹¹⁷ Fumigaclavine C, isolated from Aspergillus fumigatus cultures, inhibits the formation of the NLRP3 inflammasome to alleviate DSS-induced colitis. ¹¹⁸ Evodiamine, an alkaloidal compound isolated from the traditional Chinese medicine Evodia rutaecarpa (Juss.), exerts therapeutic activities in treating DSS-induced UC mice by regulating NF-κB signaling and the NLRP3 inflammasome and inhibiting the production of TNF-α, IL-1β, and IL-6. ¹¹⁹ Carboxyamidotriazole, an inhibitor of non-voltage-gated calcium channels, maintains intestinal integrity by inhibiting the expression of inflammatory cytokines, NF-κB signaling, and NLRP3-inflammasome activation in TNBS-induced mice. ¹²⁰ 3-(2-Oxo-2-phenylethylidene)−2,3,6,7-tetrahydro-1H-pyrazino[2,1-a]isoquinolin-4(1 1bH)-one (compound 1), a new potential Nrf2/ARE inducer, reduces DSS-induced inflammatory cell infiltration and colon pathological damage by activating Nrf2 to block the NLRP3 initiation step. ¹²¹

Phenylpropanoids

Phenylpropanoids are attractive molecules for the development of new drugs against a wide range of diseases, such as those related to inflammatory processes. In IBD, compounds from the plant phenylpropanoid metabolic pathway have yielded promising associations. Chlorogenic acid, a dietary polyphenol from the hydroxycinnamic acid family, widely exists in medicinal herbs and human diets. Zeng et al. found that chlorogenic acid can maintain intestinal microecology and immune balance by suppressing NLRP3-inflammasome-related signaling in DSS-induced mice. Furthermore, chlorogenic acid inhibits RAW264.7 cell activation by reducing the expression of p-NK-κB and NLRP3-inflammasome proteins, which depend on the downregulation of miR-155 expression. ¹²² Bergenin, a natural secondary metabolite abundant in Shiso Bergen, Mallotus japonicus, and Gabonese Shagroti, prevents non-classical and classical NLRP3/ASC inflammatory access by regulating pSTAT3 and NF-κB signaling to reverse TNBS-induced colon tissue damage in acute colitis in mice. ¹²³ Brusatol, a quassinoid compound isolated from Brucea javanica, has protective effects against TNBS-induced colitis that are closely related to the mechanism through inhibition of NF-κB and NLRP3-mediated inflammatory responses and regulation of Nrf2-mediated oxidative stress.^124,125

Other compounds

Apart from the NPs mentioned above, many other polysaccharides, proteins, amino acids, and other active NPs also exert anti-IBD biological activities by regulating NLR-mediated inflammatory responses. Fraxinellone, a product of limonoids originating from the root bark of Dictamnus dasycarpus, potently blocked the activation of NLRP3 inflammasome and NF-κB signaling in macrophage activation and alleviate intestinal inflammatory responses. ¹²⁶ 1,25-Dihydroxy Vitamin D3 (VitD3) is a steroid hormone with anti-proliferative/immunomodulatory effects, inhibited the oligomerization of the caspase recruitment domain (ASC) and specifically inhibits the activation of NLRP3. Subsequently, NLRP3 was found that NLRP3 mediates the inhibition of vitamin D receptors on caspase 1 activation and IL-1 secretion in DSS-induced UC mice. ¹²⁷ Magnesium lithospermate B is a salvianolic compound derived from Salvia miltiorrhizae radix, which protects the colonic mucosa from acute and chronic colitis by inhibiting the NLRP3/ASC/caspase-1 pathway in DSS-induced mice. ¹²⁸ Physalin B is a withanolide compound abundant in Physalis alkekengi L. var. franchetii (Mast.) Makino (a nutraceutical and Chinese herbal medicine), ameliorates general symptoms and colon pathological damage by inhibiting the activation of STAT3, β-arrestin1, and NLRP3 inflammasomes in DSS-induced colitis mice. ¹²⁹ Oryzanol, a nutrient component of cereal grains, attenuates intestinal barrier damage and inflammatory responses by modulating the DSS-induced intestinal microbiota-TLR4/NF-κB/NLRP3 signaling axis in UC rats. ¹³⁰ Monroxone I is a diterpenoid compound isolated and purified from the Meliaceae family that inhibited pro-inflammatory factor production and typical apoptosis of mouse peritoneal macrophages by promoting k48 ubiquitination and NLRP3 degradation in DSS-induced mice. ¹³¹ Artemisinin analog SM934, a new water-soluble artemisinin analog, protects mice with colitis from TNBS-induced intestinal barrier disruption by inhibiting epithelial cell apoptosis and focal death through regulation of the NLRP3/NF-κB/MAPK signaling axis. ¹³²

Plant extracts

Except for isolated phytochemicals, other botanicals and extracts may also have potential preventive or therapeutic effects on IBD driven by NLRs. Soy isoflavones (SIFs) are selective estrogen receptor modulators with anti-inflammatory activity. In IBD, SIFs ameliorate DSS-induced colitis by inhibiting ERα expression and the NLRP3-inflammasome pathway. ¹³³ Lachnum polysaccharide (LEP) alleviates DSS-induced colitis by suppressing NLRP3 activation and downregulating ASC, caspase-1, and IL-1β levels. ¹³⁴ Qingchang Wenzhong Decoction (QCWZD) is a traditional Chinese herbal formula widely used in clinical practice for the treatment of IBD. QCWZD protected against IBD by inhibiting TLR4/Blimp-1 pathway activity promotes NLRP12 expression and selectively ameliorates beneficial strains such as Odoribacterium, Braunschweiger, and Clostridium butyricum while reducing intestinal pathogenic bacteria, including Dora and Clostridium reaching. ¹³⁵ Qingrejianpi Decoction can reduce NLRP3 expression, reduce the secretion of inflammatory cytokines, and inhibit inflammatory infiltration of immune cells to inhibit the occurrence and development of IBD. ¹³⁶ Astragalus polysaccharide, the main active ingredient of Astragalus membranaceus, effectively attenuated colitis in mice by inhibiting the expression of apoptotic point proteins containing the C-terminal caspase collecting domain, caspase-1, NOD-like receptor protein 3, IL-18, and IL-1β. ¹³⁷ Lycium ruthenicum Murray is an edible ethnomedicinal and nutritional food that inhibits the NF-κB and MAPK pathways, suppresses NLRP3 inflammasome activation, and restores intestinal immune homeostasis by enhancing intestinal antioxidant defenses. ¹³⁸ Red ginseng extract (RGE) +Epimedium Koreanum Nakai (EKN) synergistically reduce the plasma and tissue levels of cytokines and pro-inflammatory mediators by inhibiting the expression of NF-κB of activated B cells and the nucleotide-binding domain of NLRP3 in DSS-induced colitic mice. In vitro, RGE + EKN can dramatically decrease the levels of nitric acid (NO), cytokines, and pro-inflammatory mediators by downregulating the expression of MAPK and NF-κB in RAW 264.7. ¹³⁹ Canna × generalis L.H. Bailey (CGB) is a traditional anti-inflammatory agent. Studies have confirmed that CGB rhizome extract restores DSS-induced intestinal mucosal dysfunction, reduces oxidative stress, and suppresses inflammatory responses in mice with colitis by inhibiting the NLRP3 inflammasome and TLR4/NF-κB pathways. ¹⁴⁰ Platycodon grandiflorus is a dual-use herb for medicinal and food purposes. The fermentation solution of Platycodon grandiflorus root reduces the level of inflammatory factors in serum and the expression of inflammatory factor mRNA in the intestine by inhibiting the AMPK/NF-κB/NLRP3 pathway, regulates the polarization of M1/M2 macrophages, and increases the expression of tight junction protein mRNA in intestinal epithelial cells in a mouse model of IBD. ¹⁴¹

Conclusions and perspectives

IBD has become a serious public health problem affecting human health and is becoming increasingly prevalent around the world. Due to its therapeutic difficulties and the need for long-term treatment, IBD has become a hot topic in the global medical field. The current consensus is that disruption of the intestinal mucosal barrier leading to the displacement of bacteria, which in turn causes a series of immune and inflammatory responses is thought to be the pathogenesis of IBD. Repairing the intestinal mucosal barrier is a highly coordinated process that involves the intestinal flora, TJs, inherent immune recognition, and self-renewal of epithelial cells. Several recent studies highlighted the important benefits of NLRs for gut health.

NLRs maintain the intestinal mucosal barrier’s integrity and homeostasis by controlling the diversity and stability of the gut microbiome, modulating inflammatory signaling events, and amplifying IEC crosstalk with immune cells. Experimental and clinical evidence indicates that NLRs mediate the establishment and homeostatic regulation of the intestinal mucosal barrier. NLRs have become an important target for the treatment of IBD and drug screening. However, diverse mechanisms may contribute to mucosal cures via NLRs. Consequently, there is a demand for further research to specifically illustrate the benefits of the various NLR-mediated signal-conduction mechanisms in the moderation of gut homeostasis and IBD.

This review focused on NPs that can modulate NLRs, making them promising candidates for the development of lead structures in anti-IBD drugs. However, most NPs targeting NLRs lack isoform selectivity, which is important for determining their potential use as therapeutic agents. Nevertheless, the structure types presented in this review offer a well-founded basis indicating that screening and chemical modifications of NPs will, in the future, identify not only more specific inhibitors with fewer adverse side effects but also important characteristics for the elucidation of NLRs’ functions concerning IBD treatment.

In the future, further studies are needed to explain the precise molecular modes of the functions of these botanicals in the activation of NLRs. In addition, the potential toxicities and drug-to-drug interactions of these botanicals in the human body should be explored. Furthermore, the anti-IBD effects of botanicals have not been clinically verified. Generally speaking, since the presence of NLRs is important in both the development and progression of IBD, addressing related NLR modulators is a promising starting point for developing new and potent anti-IBD drugs.