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. 2026 Apr 13;11(16):24269–24280. doi: 10.1021/acsomega.5c13226

Wheat Peptides Promote Recovery of the Enteric Nervous System via mAChR Activation to Relieve Constipation in Mice

Fei Shen †,§, Guanghua He †, Juan Du ∥, Haiying Cai †, Minjie Zhao §, Jing Wang §,∥, Haobin Zhao ∥, Lida Wang ⊥, Fengqin Feng §, Qianqian Wang ‡,§,*
PMCID: PMC13129869  PMID: 42077848

Abstract

Constipation represents a prevalent gastrointestinal disorder. This study demonstrates that wheat peptides (WP) effectively ameliorate loperamide-induced constipation in mice. Administration of WP orally at 0.5 mg/g body weight led to significant improvements in defecation, small intestinal motility, and the integrity of the intestinal barrier. WP further modulated inflammatory markers and enteric nervous system (ENS) regulators while correcting gut microbiota dysbiosis through restoration of microbial diversity and taxonomic composition. Crucially, colon transcriptomics revealed WP-mediated activation of calcium signaling pathway genes and the acetylcholine receptor gene. RT-qPCR validation confirmed the upregulation of key effectors, including muscarinic acetylcholine receptor Chrm1, inositol trisphosphate receptors (Itpr2/3), and calmodulin-like proteins (Calml3, Calm4). Three-dimensional imaging substantiated WP-induced augmentation of enteric neurons in constipated colons. Collectively, these results indicate that WP alleviates constipation through a multitarget mechanism involving muscarinic receptor activation, potentiation of calcium-dependent signaling, and promotion of enteric nervous system recovery, which collectively restore normal intestinal motility. WP thus emerges as a promising therapeutic candidate for constipation management.

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1. Introduction

Constipation is a common gastrointestinal condition defined by a symptom complex of difficult defecation, reduced stool frequency, prolonged colon transit, and hard feces. This disorder significantly compromises patient well-being by elevating health risks and contributing to diminished daily functioning and psychological stress. Current clinical management of constipation predominantly relies on osmotic orsecretory laxatives. However, their utility is constrained by adverse effects and patient responses, with approximately 50% of users reporting suboptimal dissatisfaction with over-the-counter (OTC) options. , Given this, developing safe and effective therapeutic agents for constipation is urgently needed. Natural bioactive substances have thus garnered considerable interest from consumers due to their favorable safety profile, characterized by low toxicity and minimal adverse effects.

The scientific focus for managing constipation has shifted toward natural bioactive substances. Notable examples such as probiotics, , saccharides, and fiber, are widely studied based on their capacity to stimulate gut motility. We previously found that WP at both 2 mg/g and 1 mg/g could significantly alleviate constipation in mice. , While patient preferences for dosage vary with symptom severity, a key treatment objective is to achieve relief at the lowest feasible dose, which also reduces costs. This research we chose a lower dose of 0.5 mg/g to know whether it has the anticonstipation ability.

In terms of mechanisms for allevating constipation, NBS may act through multiple pathways, including the regulation and repair of the enteric nervous system, gut microbiota and their metabolites, intestinal water absorption, and gut barrier and immune functions, all of which influence intestinal motility. ,, The enteric nervous system comprises enteric neurons, neurotransmitters, and their receptors. Most current research has primarily focused on how NBS regulates neurotransmitter to relieve constipation, while studies investigating the effects of bioactive peptides on the differentiation of enteric neurons are still quite rare. Our previous findings indicated that WP can modulate neurotransmitters, including acetylcholine. However, the underlying mechanisms of action and potential targets of WP for constipation relief remain unclear. In this study, we unexpectedly discovered through high-throughput transcriptomics that WP significantly upregulates acetylcholine receptors and promotes the development of enteric neurons, both of which are linked to constipation relief. This paper not only elucidates the biological activity of low-dose WP in alleviating constipation but also delves into the underlying mechanisms, providing new insights and theoretical support for the prevention and treatment of constipation.

2. Material and Methods

2.1. Materials

WP was obtained from Hangzhou Kangyuan Food Science and Technology Co., Ltd. (Hangzhou, China). Proximate analysis showed its basic chemical composition to contain 80.05% protein, 13.94% carbohydrate, 3.91% moisture, 3.54% ash, and 2.47% fat. The material was predominantly composed of low-molecular-weight peptides, with 75.19% being under 5000 Da. The analysis identified 18 amino acids with a total content of 76.66 g/100g. For the animal model, loperamide (Lop) was sourced by Xi’an Janssen Pharmaceutical Ltd., China. Activated carbon was supplied by Zhejiang Wanglin Biological Co. Ltd. (Hangzhou, China). Arabic gum was supplied by National Drug Reagents Co., Ltd. (Beijing, China).

2.2. Animals and Treatment

The protocol for this study received full ethical approval (code: 20220425–28) following evaluation by the Experimental Animal Ethics Committee of Zhejiang Chinese Medical University. After a one-week acclimation, during which mice were housed under a controlled 12-h light/dark cycle with ad libitum access to food and water, 36 male BALB/c mice (weighing 20 ± 2 g) were randomly assigned to three experimental groups (12 mice per group): control (CON), loperamide-induced constipation model (LOP), and wheat peptide-treated (WP). Throughout the experiment, each cage held four mice. Half an hour before the daily treatment, mice in LOP and WP groups were administered Lop at a dose of 10 mg/kg·bw to induce constipation, while the CON group received saline. The WP group was then daily treated with WP at a dose of 0.5 mg/g·bw, and the CON and LOP groups were given saline. Body mass and food consumption were monitored at 4-day intervals. After 16 days, following a 12-h fast, in accordance with the EU Directive, euthanasia was performed by cervical dislocation after mice were rendered fully unconscious through an intraperitoneal administration of 2% pentobarbital sodium at a dose of 40 mg/kg body weight.

2.3. Assessment of Intestinal Evacuation

2.3.1. Fecal Output Analysis

A 6 h fecal output test was conducted on day 13 to evaluate bowel function in mice. After a 16-h fast (with water permitted), the mice received their respective treatments the next morning: the LOP and WP groups were administered Lop at 10 mg/kg·bw, while the CON group received an equivalent volume of saline. After a 30 min interval, every mouse was given an activated carbon suspension (0.01 mL/g·bw) by oral gavage. They were then promptly moved to a clean cage supplied with food and water ad libitum. Several parameters were recorded, including the latency for the first appearance of a black stool, total stool pellet count, the wet and dry weights of feces, from which the water content of stool was calculated.

2.3.2. Assessment for Small Bowel Transit

This test was conducted on day 16 to determine the gastrointestinal (GI) transit rate. Following a 16-h fast (water provided), mice in LOP and WP groups were given Lop at a dose of 7.5 mg/kg·bw, while the CON group received an equivalent volume of saline. After a 30 min interval, all animals received an activated carbon suspension via oral gavage at a dose of 0.01 mL/g·bw. Following a 30 min interval after gavage, the mice were humanely sacrificed, and the complete gastrointestinal tract was collected for analysis. The rate of GI transit was determined by measuring the distance the carbon traveled along the small intestine and expressing it as a percentage of the total organ length.

2.4. Histological Analysis

Following fixation in 4% paraformaldehyde, ileum and colon specimens underwent standard paraffin embedding and were subsequently microtomed to obtain 5 μm-thick sections, and then followed by standard histological staining with hematoxylin and eosin (H&E). Stained ileal and colonic sections were randomly examined under a light microscope (Leica, Germany) at 4× and 10× magnifications, respectively. Villus height and crypt depth were quantified from the images using Image Pro 6.0 software (Media Cybernetics, USA). Image Pro 6.0 software (Media Cybernetics, USA) was used to measure the ileal crypt depth and villus height.

2.5. Biochemical Assays

According to the manufacturer’s instructions (Jiyinmei, Wuhan, China), the quantification was performed for substance P (SP), endothelin-1 (ET-1), somatostatin (SS), lipopolysaccharide (LPS), and motilin (MTL) in mouse serum. Each assay utilized 50 μL of serum and was conducted with specific commercial ELISA kits. For colon tissues, levels of tumor necrosis factor-alpha (TNF-α) and interleukin-10 (IL-10) were assessed. The method of biochemical assays according to our previous method. ,

2.6. Quantitative Real-Time PCR (qPCR) Analysis

The qPCR method refers to our previous method. Gene expression analysis in colon tissue began with RNA extraction using Invitrogen TRIzol (CA, USA). The RNA was then converted to cDNA using the Vazyme HiScript III RT SuperMix Kit from Nanjing, China. Quantitative PCR assays were conducted on a Roche LC480 system adhering to a published protocol. Expression levels were normalized to GAPDH and assessed via the 2–ΔΔCt method. All primers were shown in Table S1.

2.7. 16S rDNA Sequencing for Analysis of Intestinal Microbiota

Fecal samples collected on day 13 from three groups (CON, LOP, and WP, n = 6) were subjected to 16S rDNA sequencing for microbiota analysis. The extracted fecal DNA (TIANamp Stool DNA Kit, Beijing, China) was subjected to amplification of the V3–V4 region employing primers 341F/806R. The resulting amplicons were confirmed by gel electrophoresis, followed by library preparation and sequencing on an Illumina HiSeq PE250 system. Bioinformatic processing involved chimera removal and OTU clustering at 97% sequence identity using Usearch (V10). After normalization, OTUs were taxonomically classified in QIIME by comparing representative sequences to a reference database. Analyses of community structure and differential taxa were conducted with QIIME2 and R3.6.3.

2.8. Transcriptome Analysis

The transcriptomic profiling of colon tissues was conducted following a previously published protocol with minor adaptations. The samples were first processed for total RNA extraction. Subsequently, the RNA libraries were subjected to paired-end sequencing on an Illumina NovaSeq 6000 instrument by Shanghai Majorbio Biotechnology Co., Ltd. (Shanghai, China) to yield the sequencing data. To clean the raw data, adapter sequences and low-quality reads were trimmed using custom Perl scripts, and retained only high-quality clean data for all downstream analyses. Gene expression levels were estimated via the FPKM method. Differential expression analysis between the LOP vs CON and WP vs LOP groups was carried out using the DESeq R package (v1.10.1). Genes meeting the thresholds of |log2FoldChange| ≥ 1.5 and an adjusted P-value <0.05 (Benjamini-Hochberg method) were defined as differentially expressed genes (DEGs).

2.9. Analysis of SCFA and BCFA Levels in Cecal Samples

The concentrations of seven fatty acids in cecal contentsacetate, propionate, butyrate (SCFAs); isobutyrate, valerate, isovalerate, caproate (BCFAs)were determined. Briefly, a 50 mg aliquot was processed by homogenization in 250 μL sterile distilled water (5 min). Acidification with 10 μL of HCl was then performed, followed by vortexing (1 min) and room-temperature equilibration (5 min). An aliquot (200 μL) of the supernatant, obtained after centrifugation (12,000 rpm, 4 °C, 20 min), was mixed with 0.5 μL of 2-ethyl butyric acid (internal standard). This solution was then vortexed and subjected to a further 5 min centrifugation at the same speed and temperature. The final supernatant was analyzed on a Shimadzu GC-2014 capillary gas chromatograph equipped with an Agilent DB-FFAP column (Agilent Technologies, USA), with an injection volume of 1 μL.

2.10. 3D Visualization of Colonic Nerves

To visualize the architecture of colonic nerves, three-dimensional imaging was performed based on a published protocol. Following fixation in 4% paraformaldehyde, colon tissue segments were cleared using the CUBIC method. Briefly, the samples were first rinsed with PBS. They were then transferred through a series of solutions in sequential order: 50% ScaleCUBIC-1 reagent (1:1 mixture with water) for 24 h, pure CUBIC-1 reagent for 72 h, 50% ScaleCUBIC-2 reagent for 24 h, and finally pure CUBIC-2 reagent for 48 h. Following clearing, tissues were subjected to immunostaining with an antitubulin beta 3 antibody. This neuronal marker was allowed to incubate with the tissues for 3 days at room temperature. Ultimately, images of the prepared samples were acquired on a Nuohai LS18 tiling light sheet microscope, utilizing Bitplane’s Imaris 9.0 software for visualization and analysis.

2.11. Data Analysis

Data analysis was conducted with GraphPad Prism 6.0. For continuous variables, comparisons across multiple groups were performed via one-way ANOVA, followed by Tukey’s test for posthoc analysis. Data are presented as mean ± SEM, and statistical significance was defined at P < 0.05.

3. Results

3.1. WP Ameliorates Lop-Induced Constipation in Mice

As shown in Figure , compared to the CON group, mice in the LOP group exhibited a marked decrease in body weight gain (P < 0.001). This decrease was attenuated by subsequent WP administration (Figure B). Energy intake did not differ significantly across all groups, as shown in Figure C. In contrast to the CON group, constipated mice (LOP) exhibited a markedly prolonged the latency to the first appearance of a black stool (P < 0.0001, Figure D), alongside reductions in fecal pellet count, wet stool weight, water content, and GI transit rate (P < 0.05, Figure E and G-I). WP intervention (0.5 mg/g·bw) effectively counteracted these constipation phenotypes, except for fecal moisture level. Specifically, a 22.1% reduction in the time to first excretion was observed following WP administration (P < 0.01, Figure D). A corresponding 20.1% increase in the 6 h fecal pellet count was recorded (P < 0.05, Figure E). And a 37.5% increase in small intestinal transit rate was also recorded (P < 0.01, Figure I), compared to LOP group. Collectively, these data indicate that WP administration leads to the amelioration of constipation symptoms induced by Lop.

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Ameliorative effects of dietary WP on bowel function in constipated mice. (A) Schematic of the experimental design. (B) Body weight change and (C) energy consumption over the study period. (D) Latency to the first black stool defecation. (E) Fecal pellet count and (F) dry/(G) wet weight of feces collected over a 5 h period. (H) Calculated fecal moisture content. (I) Quantification of the small intestinal transit rate. (J) Representative photograph illustrating intestinal transit. Values are expressed as mean ± SEM (n = 12). Statistical differences among groups (P < 0.05) are indicated by different superscript letters.

3.2. WP Restores Intestinal Barrier Function in Constipated Mice

Histological examination revealed significant structural damage in the LOP group, including shortened ileal villi and a thinned colonic muscle layer compared to the CON group (Figure A-D). WP treatment effectively restored these architectural features to levels comparable to CON group (P > 0.05). The expression of colonic key tight junction proteins Claudin-1 and Zo-1 were significantly downregulated in the LOP group (P < 0.05, Figure E). WP administration markedly upregulated their expression (P < 0.05), although it did not significantly affect the mucin proteins Muc2 and Muc3 (P > 0.05). Collectively, these findings demonstrate that WP promotes the repair of the intestinal mechanical barrier in mice with constipation.

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Multifaceted restoration of intestinal homeostasis by WP in constipated mice. (A) Representative histology (H&E staining). Morphometric analysis of the ileum showing (B) villus height, (C) crypt depth, and (D) their ratio. (E) mRNA levels of key tight junction proteins in the colon. Systemic and local inflammatory markers: (F) serum LPS concentration, and colonic levels of (G) TNF-α and (H) IL-10. Expression of genes related to interstitial cells of Cajal: (I) c-Kit and (J) SCF. Serum concentrations of neuroendocrine peptides: (K) motilin (MTL), (L) substance P (SP), (M) somatostatin (SS), and (N) endothelin-1 (ET-1). Data are presented as mean ± SEM (n = 6). Distinct lowercase letters denote statistically significant differences among groups (P < 0.05).

3.3. WP Modulates Inflammatory Factor Levels in Constipated Mice

Given the observed histological indications of inflammation, we assessed the levels of key inflammatory cytokines. The presence of intestinal inflammation was confirmed, as serum LPS and colonic TNF-α levels were significantly elevated in the LOP group compared to the CON group (P < 0.05; Figure F–H). As shown in Figure G, WP treatment demonstrated a nonsignificant trend in reducing TNF-α levels (P > 0.05). Notably, IL-10, an anti-inflammatory cytokine was significantly suppressed in the LOP group (P < 0.05), and WP intervention effectively restored its level to a point statistically indistinguishable from that of controls (P > 0.05, Figure H). Taken together, these results demonstrate that WP helped restore the anti-inflammatory cytokine IL-10 and showed a nonsignificant trend in reducing the pro-inflammatory marker TNF-α in constipated mice.

3.4. WP Improves Smooth Muscle Pacing in Constipated Mice

Interstitial cells of Cajal (ICCs) play a pivotal role as the pacemaker for gastrointestinal motility. Their function is commonly assessed through the expression of c-Kit and its ligand, stem cell factor (SCF). Our analysis detected significantly suppressed colonic expression of both c-Kit and SCF genes in the LOP group relative to the CON group (c-Kit: P < 0.0001; SCF: P < 0.05; Figure I-J). Conversely, WP administration induced a pronounced upregulation of these transcripts relative to the LOP group (P < 0.0001 and P < 0.05). These results indicate that WP improves intestinal motility, at least in part, by restoring the transcriptional activity of key pacemaker genes in ICCs.

3.5. Effects of WP on Enteric Nervous System Activity in Constipated Mice

To assess the neuro-regulatory effect of WP, we measured key enteric nervous factors. In response to WP treatment, significant increases in motilin (MTL) and substance P (SP) (P < 0.01), along with decreases in somatostatin (SS) and endothelin-1 (ET-1) (P < 0.05), were observed in constipated mice, as detailed in Figure K–N. This coordinated regulation of excitatory and inhibitory factors indicates that WP helps restore the homeostatic balance of enteric nervous secretion.

3.6. WP Remoldels the Gut Microbial Composition in Constipated Mice

A total of 736 operational taxonomic units (OTUs) were identified across all samples via sequencing analysis. The OTU count varied across groups, with the maximum (674) observed in the WP group and the minimum (620) in the LOP group (Figure E). In terms of α-diversity, WP treatment significantly increased the Shannon index compared to the LOP group, restoring it to a level comparable to the CON group (P < 0.05, Figure A), although no significant statistical differences were detected in the Chao1, Ace, or Simpson indices (Figure B-D). Nonmetric multidimensional scaling (NMDS) of β-diversity revealed a distinct separation between the LOP and CON groups, with the WP group clustering intermediately (Figure F), indicating a partial normalization of community structure.

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WP ameliorated gut microbial dysbiosis in constipated mice. α-Diversity indices: (A) Shannon, (B) Simpson, (C) Chao1, and (D) Ace. (E) Venn diagram depicting unique and shared operational taxonomic units (OTUs). (F) Principal coordinate analysis (PCoA) plot showing β-diversity. Microbial composition displayed as (G) relative abundance bar plot at the phylum level and (H) clustered heatmap at the genus level. Data are mean ± SEM (n = 6). Statistical significance (P < 0.05) between groups is indicated by different letters.

Taxonomic analysis demonstrated that WP intervention reshaped the gut microbial community at the phylum and genus levels. Notably, WP reduced the Firmicutes/Bacteroidota ratio in constipated mice (Figure G). Heatmap visualization of the 30 most differentially abundant genera showed that WP treatment counteracted many of the microbial alterations induced by Lop (Figure H).

LEfSe analysis further identified specific bacterial taxa associated with WP intervention. Genera such as Jeotgalicoccus, Turicibacter, Oscillospiraceae, and Enterococcus were enriched in the WP group, while Peptococcaceae and Oscillospirales constituting the major abundant taxa in the CON group (Figure A). Notably, WP significantly increased the relative abundance of beneficial genera like Bifidobacterium, Jeotgalicoccus, and Turicibacter (P < 0.0001, Figure C-E), and concurrently suppressed the potentially harmful genus Helicobacter (P < 0.001, Figure F).

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Integrated analysis of gut microbiota restructuring and associated metabolic changes following WP intervention. (A) Cladogram from LEfSe analysis highlighting differentially abundant taxa. (B) Heatmap showing Spearman correlation coefficients between microbial genera and constipation-related physiological indexes. Relative abundances of selected genera: (C–F) Bifidobacterium, Jeotgalicoccus, Turicibacter, and Helicobacter. Concentrations of Main SCFAs: (G–I) acetate, propionate, and butyrate. (J) Total SCFAs. Individual BCFAs and others: (K–N) isobutyrate, valerate, isovalerate, and caproate. (O) Total BCFAs. Data are presented as mean ± SEM (A–F: n = 6; G–O: n = 8). Statistical differences (P < 0.05) are indicated by distinct letters above bars.

Spearman correlation analysis revealed significant relationships between these WP-modulated microbes and host physiological parameters (Figure B). The enrichment of Jeotgalicoccus was positively correlated with the expression levels of tight junction proteins (ZO-1 and Claudin-1), pacemaker cell markers (c-Kit, SCF), GI transit rate, and the anti-inflammatory cytokine IL-10. Turicibacter abundance was also positively associated with c-Kit and SCF. Conversely, Helicobacter showed a positive correlation with the time to first black stool and a negative correlation with GI transit rate. These results collectively suggest that WP alleviates constipation, in part, by beneficially remodeling the gut microbiota and restoring microbial-host interactions.

3.7. WP Modulates the SCFAs and BCFAs in the Cecum of Constipated Mice

Analysis of cecal contents revealed that WP intervention significantly altered the profile of microbial fermentation products. The total concentration of SCFAs in the WP group was markedly increased in contrast to the LOP group, whereas the total content of branched-chain fatty acids (BCFAs) was significantly decreased (P < 0.05; Figure J, O). Specifically, WP enhanced the levels of the beneficial SCFAs propionate and butyrate (P < 0.05; Figure H, I). Conversely, it suppressed the production of several BCFAs, including isobutyrate, valerate, and caproate (P < 0.05; Figure K, L, N).

3.8. Transcriptomic Analysis Reveals WP’s Impact on Colonic Gene Expression in Constipated Mice

A comparative transcriptome analysis was conducted to elucidate the genome-wide transcriptional changes underlying WP’s anticonstipation effect. Volcano plots and statistical summaries illustrated the differential gene expression between groups (Figure A, B, D). Relative to the CON group, a total of 569 genes were identified as differentially expressed in the LOP group (DEGs; 160 up, 409 down). WP intervention reversed this trend, modulating 416 DEGs (197 up, 219 down) relative to the LOP group. Heatmap visualization confirmed that WP treatment largely normalized the constipated transcriptomic profile toward that of the control (Figure ).

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Transcriptomic profiling reveals WP-mediated modulation of colonic gene expression. (A, B) Volcano plots displaying differentially expressed genes (DEGs) for the LOP vs CON and WP vs LOP comparisons, respectively. (C) Venn diagram identifying common and unique DEGs across comparisons. (D) Hierarchical clustering heatmap of DEG expression patterns (upregulated in red, downregulated in blue). (E) KEGG pathway enrichment scatter plot highlighting significantly altered signal transduction pathways. (n = 3).

Analysis of the top 20 enriched KEGG pathways highlighted the calcium signaling pathway as a key target, given its established role in intestinal motility (Table S2). RT-qPCR validation of seven selected genes from this pathway confirmed the transcriptomic data (Figure C, Table S2). WP significantly up-regulated key genes in the calcium signaling pathway, including the muscarinic acetylcholine receptor M1 (Chrm1, P < 0.05), inositol triphosphate receptors (Itpr2, P < 0.05), calmodulins (Calm4 and Calml3, P < 0.01), and the sodium/calcium exchanger (Slc8a3, P < 0.01) (Figure C).

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WP modulates mAChR signaling and enteric nerves to alleviate constipation. (A) Heatmap clustering of differentially expressed genes (DEGs) related to calcium signaling (WP vs LOP). (B) Serum acetylcholine (Ach) concentration. (C) Colonic mRNA levels of key calcium signaling components: Chrm1, Itpr2, Itpr3, Calml4, Calml3, and Slc8a3. (D) Representative images of colonic enteric neurons. (E) The quantitative measure of neuronal fluorescence intensity/area. (F) Schematic summary of the proposed mechanism of action for WP. Data are mean ± SEM (A: n = 3; B: n = 8; C: n = 6; D: n = 3). Statistical significance (P < 0.05) is denoted by different letters.

Since Chrm1 is activated by acetylcholine (Ach), we measured serum Ach levels. The LOP group exhibited a significant reduction in Ach, which was effectively restored by WP treatment (P < 0.05, Figure B). These results collectively demonstrate that WP upregulates the calcium signaling pathway, likely through enhancing Ach activation, thereby promoting intestinal motility and alleviating constipation.

3.9. WP Enhanced Recovery of Colonic Nerves in Constipated Mice

Representative images of colonic nerves are shown in Figure D. While the CON group displayed a robust network of enteric nerves, this structure was drastically diminished in the LOP group, complicating visualization under standardized staining. Treatment with WP partially reversed this effect, leading to a significant increase in observable nerves relative to the LOP model. To objectively quantify this restorative effect, we analyzed the neuronal fluorescence intensity/area across groups (n = 3). Quantitative analysis confirmed that the neuronal network signal was significantly reduced in the LOP group compared to the CON group (P < 0.01), and was significantly restored by WP treatment (P < 0.01 vs LOP group) (Figure E). These data provide quantitative support for the visual observation and demonstrate WP’s potential to mitigate constipation-induced damage to the enteric nervous system architecture.

4. Discussion

Previous studies established the efficacy of wheat peptides (WP) against constipation at doses of 1–2 mg/g. , This study demonstrates that a lower dose of 0.5 mg/g remains effective, significantly improving key defecation parameters such as the latency to first black stool, intestinal propulsion rate, and fecal pellet count. However, unlike higher doses, this low dose did not significantly alter fecal moisture content, implying that WP’s laxative mechanism extends beyond simple stool lubrication and may involve alternative pathways.

Lop-induced constipation is characterized by intestinal tissue damage, impaired barrier integrity, and low-grade inflammation, observations confirmed in our model. WP treatment effectively mitigated these pathological changes, restoring villus architecture, colonic muscle thickness, and the expression level of tight junction proteins (Claudin-1 and ZO-1), while rebalancing inflammatory cytokines (TNF-α and IL-10) (Figure ). This observation aligns with the work of Zhang et al., who demonstrated that Chitosan oligosaccharides also alleviate inflammation and protect intestinal barrier function in a Lop-induced murine constipation model. The high glutamine (Gln) content of WP may be a plausible contributor to these benefits. Gln deprivation is known to impair tight junctions via the PI3–K/Akt pathway, while Gln supplementation can preserve ZO-1 and Occludin expression and boost anti-inflammatory IL-10 in the gut, , aligning with our findings.

Our data further indicate that the gut microbiota is one of the interconnected mechanisms through which WP exerts its anticonstipative effect. WP administration increased α-diversity and shifted β-diversity and taxonomic composition toward a state resembling healthy controls. Notably, WP enriched beneficial Bifidobacterium and Jeotgalicoccus, the latter showing strong positive correlations with barrier proteins, anti-inflammatory markers, and intestinal motility factors (Figure B). Conversely, the constipated model exhibited a bloom of Helicobacter, which was suppressed by WP and correlated negatively with GI transit, consistent with other studies. The role of Turicibacter remains complex; while its increase has been ambiguously linked to constipation, its positive correlation with ICC markers (c-Kit, SCF) and the concurrent rise in cecal butyrate in our study suggest a potential beneficial role in this specific context.

The microbial remodeling by WP had functional consequences for metabolite profiles. WP significantly increased SCFAs, specifically propionate and butyrate, which are associated with faster colonic transit. , Concurrently, it reduced total BCFAs, including isobutyrate, valerate, and caproate, metabolites linked to prolonged transit time. Importantly, the observed microbial shifts and the associated increase in beneficial SCFAs like butyrate are likely not the sole driver but rather a component of a reciprocal relationship: the improved intestinal motility and healthier luminal environment fostered by WP may itself create conditions favorable for the growth of these bacteria, which in turn may further support gut function through metabolite production. Thus, the beneficial remodeling of the gut microbiota and its metabolic output may be presented as one of several parallel and mutually reinforcing pathways contributing to the overall amelioration of constipation by WP.

To elucidate the underlying molecular mechanisms, we employed transcriptomic analysis. KEGG enrichment pinpointed the calcium signaling pathway as critically involved. RT-qPCR validation confirmed that WP upregulates key genes in this pathway: the muscarinic acetylcholine receptor M1 (Chrm1), inositol trisphosphate receptors (Itpr2/3), calmodulins (Calm4/Calml3), and the sodium/calcium exchanger Slc8a3. As Chrm1 is activated by acetylcholine (Ach), the measured decrease in serum Ach in constipated mice and its restoration by WP (Figure B) provide a crucial link. Based on these integrated omics and biochemical findings, we propose a potential mechanistic hypothesis. In this model, WP may enhance ACh levels, which could activate the G-protein-coupled receptor Chrm1. This activation may trigger phospholipase C (PLC)-mediated production of IP3. IP3 binding to its receptors (IP3R, encoded by Itpr2/3) on the endoplasmic reticulum (ER) could lead to the release of Ca2+ into the cytosol. The subsequent depletion of ER Ca2+ stores may then stimulate store-operated calcium entry (SOCE), a process implicated in cellular responses including proliferation. The concomitant upregulation of calmodulin genes (Calm4/Calml3) is consistent with the activation of downstream Ca2+-sensitive pathways, such as MAPK, which are known to promote cell survival and growth. ,−

Supporting the functional relevance of this proposed signaling cascade, 3D imaging coupled with quantitative analysis revealed that WP significantly restored the Lop-induced loss of colonic enteric nerves (Figure D-E). While glutamine in WP is known to support cell proliferation, our findings point to a novel, complementary mechanism potentially involving ACh-mediated calcium signaling in enteric nervous system repair. While it is critical to distinguish our experimental observations from the hypothesized downstream sequence. We observed the upregulation of Chrm1, Itpr2/3, and related genes, along with increased serum ACh and nerve restoration, but the detailed signaling cascade from receptor activation to specific cellular outcomes remains to be fully experimentally validated. A key limitation of the current study is the lack of direct pharmacological intervention to establish causality. Future studies employing selective mAChR antagonists (e.g., pirenzepine) in WP-treated mice are essential to confirm the indispensable role of this receptor pathway in mediating the pro-motility and neuro-restorative effects of WP. Furthermore, the precise cellular outcomewhether true neurogenesis, neuroprotection, or enhanced neuronal maturationcannot be definitively distinguished by our current data. Future lineage-tracing or colocalization studies using proliferative markers (e.g., Ki67, BrdU) and mature neuronal markers (e.g., HuC/D) within the myenteric plexus are warranted to conclusively establish the nature of WP’s effect on the enteric neurons.

Taken together, our data support a model in which WP alleviates constipation through a multifaceted approach: restoring intestinal barrier integrity and anti-inflammatory balance, beneficially reshaping the gut microbiota and its metabolites, and potentially promoting enteric nervous system recovery via the upregulation of ACh and calcium signaling pathway components. The proposed mAChR/Ca2+ axis provides a testable mechanistic framework for future research. However, while our study indicated that WP exerts its prokinetic effects through the activation of mAChR signaling pathways, we acknowledge that direct evidence using specific mAChR antagonists or conditional knockout mice is necessary to definitively establish causality. Future studies employing such approaches will be crucial to fully elucidate the precise molecular mechanism underlying the therapeutic effects of WP.

5. Conclusions

In summary, this study demonstrates that a low dose of WP (0.5 mg/g·bw) effectively ameliorates Lop-induced constipation in mice. The therapeutic effects were mediated by multiple mechanisms: WP enhanced intestinal motility and barrier function, alleviated low-grade inflammation, and restored the balance of enteric nervous factors. Furthermore, WP remodeled the gut microbiota by promoting beneficial genera, including Bifidobacterium and butyrate producers. At the molecular level, our findings suggest that WP activates the muscarinic acetylcholine receptor to upregulate the calcium signaling pathway, which may promote enteric nervous system recovery and ultimately strengthen intestinal peristalsis.

Supplementary Material

ao5c13226_si_001.pdf (82.6KB, pdf)

Acknowledgments

This research was funded by the National Natural Science Foundation of China (32502183; 32372316; 321722141) and Zhejiang Provincial Leading Geese Plan Projects (2025C04040).

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.5c13226.

  • Sequences of primers and changes in gene transcript patterns in the calcium signaling pathway of mouse colon tissue among CON, LOP, and WP groups (PDF)

1.

F.S. and G.H. contributed equally to the work.

The authors declare no competing financial interest.

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