Beneficial effect of butyrate-producing Lachnospiraceae on stress-induced visceral hypersensitivity in rats

Abstract

Background and aim: Emerging evidence indicates that psychological stress is involved in the pathogenesis of irritable bowel syndrome, which is characterized by visceral hypersensitivity and may be accompanied by gut dysbiosis. However, how such stress contributes to the development of visceral hypersensitivity is incompletely understood. Here, we aimed to investigate the influence that stress-induced microbial changes exert on visceral sensitivity, as well as the possible underlying mechanisms associated with this effect.

Methods: Male Sprague-Dawley rats underwent chronic water avoidance stress (WAS) to induce visceral hypersensitivity. Visceral sensitivity, colonic tight junction protein expression, and short-chain fatty acids of cecal contents were measured. Fecal samples were collected to characterize microbiota profiles. In a separate study, oral gavage of Roseburia in WAS rats was conducted to verify its potential role in the effectiveness on visceral hypersensitivity.

Results: Repeated WAS caused visceral hypersensitivity, altered fecal microbiota composition and function, and decreased occludin expression in the colon. Stressed rats exhibited reduced representation of pathways involved in the metabolism of butyrate and reduced abundance of several operational taxonomic units associated with butyrate-producing bacteria, such as Lachnospiraceae. Consistently, supplementation with Roseburia hominis, a species belonging to Lachnospiraceae, significantly increased cecal butyrate content. Moreover, Roseburia supplementation alleviated visceral hypersensitivity and prevented the decreased expression of occludin.

Conclusions: Reduction in the abundance of butyrate-producing Lachnospiraceae, which is beneficial for the intestinal barrier, was involved in the formation of visceral hypersensitivity. R. hominis is a potential probiotic for treating stress-induced visceral hypersensitivity.

Keywords: butyrate; intestinal barrier; microbiota; stress; visceral hypersensitivity.

Figure 1

Effect of chronic WAS on visceral sensitivity and intestinal tight junctions. (a) Abdominal withdrawal reflex scores in response to CRD; , WAS; , Sham. (b) Fecal pellet output numbers found in the containers after each 1‐h WAS; , WAS; , Sham. (c) Real‐time quantitative RT–PCR of colonic occludin mRNA normalized to expression of β‐actin. (d) Quantitative analysis of colonic occludin protein normalized to expression of GAPDH. (e) Representative image of occludin bands in western blotting experiments. *P < 0.05, **P < 0.01, compared with sham controls; n = 7 rats per group. WAS, water avoidance stress; GAPDH, glyceraldehyde phosphate dehydrogenase; CRD, colorectal distension.

Figure 2

Effect of WAS on microbial composition and function. (a) LEfSe analysis shows significantly different taxa between the two groups (left panel; , a: g_Candidatus_Soleaferrea; , b: f_Peptococcaceaae; , c: g_uncultured_f_Peptococcaceaae; , d: f_Veillonellaceae; , e: g_Megasphaera; , f: g_Diallister; , g: f_Prevotellaceae; , h: g_Prevotella_1; , i: c_Gammaproteobacteria; , j: o_Enterobacteriales; , k: f_Enterobacteriaceae; , l: o_Coriobacteriales; , m: o_Coriobacteriaceae; , n: g_Adlercreutzia; , o: o_norank_c_Cyanobacteria; , p: f_norank_c_Cyanobacteria; , q: g_norank_c_Cyanobacteria) and bacterial taxa with significantly different abundances (LDA scores > 2) (right panel; , Sham; , WAS). Labels beginning with c_ indicate class; o_, order; f_, family; g_, genus. WAS, water avoidance stress. LDA, linear discriminant analysis; LEfSe, LDA effect size. (b) Phylogenetic investigation of communities using reconstruction of unobserved states functional prediction shows significantly decreased butanoate metabolism pathways in the WAS group. *P < 0.05, compared with sham controls. (c) Heatmap showing correlations between altered microbial OTUs and disease phenotype parameters. Color intensity indicates the degree of correlation. Red, positive correlation; blue, negative correlation. n = 7 rats per group. *P < 0.05, **P < 0.01. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 3

Effect of Roseburia hominis oral gavage on visceral sensitivity. (a) Schematic diagram of the experiment. (b) Abdominal withdrawal reflex scores in response to CRD; , Sham + PBS; , WAS + PBS; , WAS + Rh. (c) Fecal pellet output numbers found in the containers after each 1‐h WAS; , Sham + PBS; , WAS + PBS; , WAS + Rh. *P < 0.05, **P < 0.01, *** P < 0.001, compared with sham controls; #P < 0.05, compared with WAS + Rh group. n = 6 rats per group. PBS, phosphate‐buffered saline. Rh, R. hominis; WAS, water avoidance stress; CRD, colorectal distension. [Color figure can be viewed at wileyonlinelibrary.com]

Figure 4

Effect of Roseburia hominis on HPA axis activity. (a) Relative weight of adrenal gland of rats. (b) Serum corticotropin‐releasing hormone level. **P < 0.01, compared with sham controls; #P < 0.05, compared with WAS + Rh group. n = 6 rats per group. HPA, hypothalamic–pituitary–adrenal; PBS, phosphate‐buffered saline; Rh, R. hominis; WAS, water avoidance stress.

Figure 5

Effect of Roseburia hominis oral gavage on intestinal barrier. (a) Real‐time quantitative RT–PCR of colonic occludin mRNA normalized to expression of β‐actin. (b) Quantitative analysis of colonic occludin protein normalized to expression of GAPDH. (c) Representative image of occludin bands in western blotting experiments. *P < 0.05, ***P < 0.001, compared with sham controls; #P < 0.05, compared with WAS +Rh group. n = 6 rats per group. PBS, phosphate buffered saline; GAPDH, glyceraldehyde phosphate dehydrogenase; Rh, R. hominis; WAS, water avoidance stress.

Figure 6

Effect of Roseburia hominis oral gavage on acetate (a), propionate (b), butyrate (c), and total SCFA concentration in cecal contents. *P < 0.05, compared with sham controls. ##P < 0.01, compared with WAS group. n = 6 rats per group. PBS, phosphate buffered saline; Rh, R. hominis; WAS, water avoidance stress; SCFAs, short‐chain fatty acids.