Capsaicin ameliorates irritable bowel syndrome in a mouse model by restoring intestinal barrier function and promoting gut microbial homeostasis

Irritable bowel syndrome (IBS) is a common gastrointestinal disorder characterized by recurrent abdominal pain and altered stool patterns, with diarrhea-predominant IBS (IBS-D) having the highest morbidity and increasing prevalence. The pathophysiological mechanisms of IBS-D remain unclear, hindering the development of effective treatments.[1] Capsaicin, the active compound in chili peppers, acts as a transient receptor potential vanilloid 1 (TRPV1) agonist. Although capsaicin has shown various beneficial effects, its potential for treating IBS remains understudied. A recent study published in Cell indicated that the absence of TRPV1 led to weight loss, colon shortening, and disruption of the gut barrier.[2] This study suggests that capsaicin, a TRPV1 agonist, may have the potential to treat IBS. Therefore, we aimed to investigate the therapeutic effects of capsaicin on IBS mice. The experimental design of the animal study is depicted in Figure 1. The experimental protocols were approved by the Ethics Committee of Soochow University (No. 202411A0339). Given the multifaceted nature of IBS, characterized by complex interactions among biological, psychological, and sociological factors, no single experimental model can fully replicate all its symptoms. To address this, our study established an IBS-D model in C57BL/6 mice by combining intrarectal administration of 2,4,6-trinitrobenzene sulfonic acid (TNBS) enemas with chronic unpredictable mild stress (CUMS) exposure, a method supported by previous research.[3] This approach seeks to replicate both the visceral pain and stress-related aspects of IBS-D, offering a more comprehensive model for investigating the disorder. To investigate the effects of capsaicin supplementation, model mice were fed a low-dose capsaicin diet (0.01%) during CUMS exposure. The dose of capsaicin was selected based on previous studies that demonstrated efficacy at low doses without notable toxicity.[4]Figure 1: Experimental design of the animal study. (A) Schedule for IBS modeling and analysis. (B) Protocol for chronic unpredictable mild stress (CUMS) to induce the irritable bowel syndrome (IBS) model. After seven days of rest following 2,4,6-trinitrobenzene sulfonic acid (TNBS) injection, the mice were subjected to seven stressors for three weeks in a random sequence: water deprivation (24 h), overnight illumination (12 h), exposure to a 4°C cold environment (5 min), food deprivation (24 h), exposure to a 40°C hot environment (5 min), damp bedding (4 h), and tail clamping (3 min). (C) Group assignment and drug treatment protocols. To investigate the effects of capsaicin supplementation, model mice were fed a low-dose capsaicin diet (0.01%) during CUMS exposure.Long-term capsaicin supplementation significantly alleviated abdominal pain in model mice, as evidenced by a reduction in abdominal withdrawal reflex (AWR) scores, and reduced both the frequency of diarrhea episodes and the fecal water content, indicating a therapeutic effect of capsaicin in alleviating IBS-like symptoms. In the sucrose preference test, IBS model mice presented significantly reduced sucrose intake, which is indicative of depression-like symptoms. However, capsaicin supplementation significantly increased sucrose intake in these mice. In the open field test, IBS model mice demonstrated reduced activity in the central area, as evidenced by a decreased distance traveled in the central area and a lower ratio of distance traveled in the central area to total distance traveled, further supporting the occurrence of anxiety-like behavior. Capsaicin treatment increased the distance traveled in the central area and the ratio of distance traveled in the central area to total distance traveled. Moreover, capsaicin-treated mice exhibited improved nesting behavior, which was impaired in IBS model mice, further supporting the positive effects of capsaicin on cognitive function and mood [Supplementary Figure 1, https://links.lww.com/CM9/C558]. The effects of capsaicin on the intestinal barrier were assessed by measuring intestinal permeability and the levels of tight junction (TJ) proteins, which play a pivotal role in maintaining barrier integrity [Supplementary Figure 2, https://links.lww.com/CM9/C558]. Capsaicin effectively reduced intestinal permeability in IBS model mice, as evidenced by decreased serum levels of diamine oxidase (DAO) and d-lactic acid, which are well-established biomarkers of intestinal barrier damage. Histological examination revealed that model mice presented defects in colonic epithelium, which were not observed in the control or capsaicin-treated group. Capsaicin supplementation increased the number of goblet cells in the colonic mucosa, although this difference was not statistically significant. Furthermore, capsaicin restored TJ integrity by increasing the expression of occludin and zonula occludens-1 (ZO-1) and reducing the expression of claudin-2. Moreover, capsaicin reduced TRPV1 expression in IBS mice, suggesting a potential denervating effect. These results suggest that dietary capsaicin intake restored TJ protein expression and aided in repairing the intestinal barrier in model mice. Given the pivotal role of gut microbiota dysbiosis in IBS, 16S rDNA analysis was performed; the findings revealed that dietary capsaicin intake increased microbial α-diversity (Shannon/Simpson indices) and β-diversity (operational taxonomic units [OTU]-level principal coordinates analysis [PCoA]) [Supplementary Figure 3A–C, https://links.lww.com/CM9/C558]. Capsaicin shifted the gut microbiota composition at the phylum level toward that observed in the control mice and restored critical IBS-associated taxonomic ratios (Actinobacteria/Proteobacteria, Bacteroidetes/Firmicutes) [Supplementary Figure 3D–F, https://links.lww.com/CM9/C558]. Microbiome analysis revealed significant alterations in composition at the genus level in IBS model mice compared with controls. Capsaicin mitigated these changes by enriching beneficial taxa (Muribaculum, Parabacteroides) while decreasing the abundance of dysbiotic genera (Clostridium) [Supplementary Figure 3G, https://links.lww.com/CM9/C558]. Linear discriminant analysis effect size (LEfSe) analysis confirmed capsaicin-driven enrichment of Ruminococcaceae and Anaerostipes, which correlated with the competitive exclusion of species of the IBS-associated phylum Firmicutes. Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) analysis further revealed that capsaicin increased the activity of pathways involved in secondary metabolite synthesis and energy metabolism, collectively indicating that gut microbiota modulation is central to capsaicin’s therapeutic effect [Supplementary Figure 4, https://links.lww.com/CM9/C558]. Given that an excess of fecal bile acids is thought to contribute to IBS-D, the concentrations of 29 bile acids, including 5 primary and 24 secondary bile acids, were quantified in the cecum [Supplementary Figure 5, https://links.lww.com/CM9/C558]. IBS-D model mice presented elevated total bile acid levels and primary/secondary bile acid ratios, reflecting excess excretion of bile acids. Capsaicin inhibited these perturbations by decreasing the levels of key primary bile acids (cholic acid, chenodeoxycholic acid) and secondary bile acids (ursodeoxycholic acid, deoxycholic acid, glycodeoxycholic acid, and taurohyodeoxycholic acid), which were elevated in model mice; this suggests that capsaicin alleviates IBS-D by regulating bile acid levels. Moreover, in vitro studies with Caco-2 cells confirmed that capsaicin treatment could reverse lipopolysaccharide (LPS)-induced intestinal barrier disruption. LPS caused a dose-dependent decrease in cell viability and a progressive reduction in occludin, claudin-1, and ZO-1 expression, whereas claudin-2 expression increased. Capsaicin alone caused a dose-dependent increase in occludin, claudin-1, and ZO-1. Thus, a concentration of 100 μg/ml LPS and 5 µmol/L capsaicin was used for further experiments [Supplementary Figure 6A–D, https://links.lww.com/CM9/C558]. We found that 5 µmol/L capsaicin partially reversed the disruption of TJ proteins caused by LPS, demonstrating its ability to repair the intestinal barrier at the cellular level [Supplementary Figure 6E and F, https://links.lww.com/CM9/C558]. To investigate the underlying mechanism, we examined the expression of AKT-mTOR-MLC2 signaling pathway components. LPS increased p-MLC2 expression, but capsaicin inhibited this effect, restoring MLC2 expression. In addition, capsaicin increased the phosphorylation of protein kinase B (AKT) and mammalian target of rapamycin (mTOR) [Supplementary Figure 6G, https://links.lww.com/CM9/C558]. In our study, we administered capsaicin orally through the diet to closely mimic natural consumption methods, thereby minimizing potential stress-related behavioral alterations associated with gavage feeding. Although capsaicin enemas have been used in certain post-inflammatory models of colonic hypersensitivity, we selected oral supplementation to preclude localized mucosal irritation inherent to rectal administration. This approach ensures a more natural and less invasive method of capsaicin delivery, aligning with our study’s objectives. Our study has several limitations. Only a single dose of capsaicin, an animal model, and a single cell line were used in this study. TRPV1 dependency was not confirmed through pharmacological inhibition or genetic knockdown models. The AKT-mTOR-MLC2 pathway remains unvalidated in vivo. Furthermore, the study did not address whether capsaicin can repair the epithelial barrier through modulation of the gut microbiota. Future studies should delineate the molecular interplay between capsaicin, gut microbial dynamics, and bile acid metabolism. Based on our study, we conclude that capsaicin supplementation exerts therapeutic effects in the IBS model mice by restoring the balance of gut microbiota and bile acid metabolism, as well as by enhancing the expression of tight junction proteins in the colon through the AKT-mTOR-MLC2 signaling pathway. These findings provide valuable insights for future IBS research and the development of novel treatment strategies. Funding This study was supported by grants from Science and Technology Plan Project of Suzhou City (No. SKY2023134), Natural Science Foundation of Jiangsu Provincial Higher Education Institutions (No. 23KJD320004), and the Jiangsu Provincial Department of Science and Technology (No. BE2023710). Conflicts of interest None.