Berberine alleviates visceral hypersensitivity in rats by altering gut microbiome and suppressing spinal microglial activation

Abstract

Accumulating evidence shows that agents targeting gut dysbiosis are effective for improving symptoms of irritable bowel syndrome (IBS). However, the potential mechanisms remain unclear. In this study we investigated the effects of berberine on the microbiota-gut-brain axis in two rat models of visceral hypersensitivity, i.e., specific pathogen-free SD rats subjected to chronic water avoidance stress (WAS) and treated with berberine (200 mg· kg^-1 ·d^-1, ig, for 10 days) as well as germ-free (GF) rats subjected to fecal microbiota transplantation (FMT) from a patient with IBS (designated IBS-FMT) and treated with berberine (200 mg· kg^-1 ·d^-1, ig, for 2 weeks). Before the rats were sacrificed, visceral sensation and depressive behaviors were evaluated. Then colonic tryptase was measured and microglial activation in the dorsal lumbar spinal cord was assessed. The fecal microbiota was profiled using 16S rRNA sequencing, and short chain fatty acids (SCFAs) were measured. We showed that berberine treatment significantly alleviated chronic WAS-induced visceral hypersensitivity and activation of colonic mast cells and microglia in the dorsal lumbar spinal cord. Transfer of fecal samples from berberine-treated stressed donors to GF rats protected against acute WAS. FMT from a patient with IBS induced visceral hypersensitivity and pro-inflammatory phenotype in microglia, while berberine treatment reversed the microglial activation and altered microbial composition and function and SCFA profiles in stools of IBS-FMT rats. We demonstrated that berberine did not directly influence LPS-induced microglial activation in vitro. In both models, several SCFA-producing genera were enriched by berberine treatment, and positively correlated to the morphological parameters of microglia. In conclusion, activation of microglia in the dorsal lumbar spinal cord was involved in the pathogenesis of IBS caused by dysregulation of the microbiota-gut-brain axis, and the berberine-altered gut microbiome mediated the modulatory effects of the agent on microglial activation and visceral hypersensitivity, providing a potential option for the treatment of IBS.

Keywords: berberine; gut microbiome; microbiota-gut-brain axis; microglia; spinal cord; visceral hypersensitivity.

Fig. 1. Berberine prevented stress-induced visceral hypersensitivity and microglial activation.

a Design of the WAS and berberine treatment experiments. n = 10/group. b Fecal pellet output number in the container after each 1-h WAS. n = 10/group. c Abdominal withdrawal reflex scores in response to CRD. n = 10/group. d Levels of serum CRH. n = 7/group. e Number of tryptase-positive cells in the distal colonic mucosal tissues. f Immunohistochemistry showing tryptase-positive cells in the distal colon. n = 7/group. g Representative images of Iba-1-stained microglia in the dorsal lumbar spinal cord. h Number of Iba-1-positive cells in the dorsal lumbar spinal cord. i Representative three-dimensional reconstructions of Iba-1-stained microglia residing in the dorsal lumbar spinal cord. j Morphological parameters of microglia. n = 3/group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 2. Berberine altered the gut microbiota in stressed rats.

a Chao1 and Shannon indexes indicative of community richness and diversity. n = 10/group. b Principal coordinate analysis based on weighted UniFrac distances of the relative abundances of bacterial OTUs after berberine treatment. n = 10/group. c Taxonomic distributions of bacteria at the phylum level. n = 10/group. d The F/B ratio in each group. n = 10/group. e Relative abundance of Firmicutes in each group. n = 10/group. f Relative abundances of selected genera enriched by berberine. n = 10/group. g Design of the FMT experiment. n = 5 (GW); n = 4 (GWB). h Fecal pellet output number in the container after acute WAS. i Abdominal withdrawal reflex scores in response to CRD. *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 3. Transplantation of stool derived from an IBS patient induced visceral hypersensitivity, which was prevented by berberine.

a Design of the FMT experiment. n = 12 (GH); n = 14 (GI, GIBBR). b Abdominal withdrawal reflex scores in response to CRD. n = 12 (GH); n = 14 (GI, GIBBR). c Sucrose preference rates and d immobility time after bacterial colonization and berberine treatment. n = 6 (GH); n = 8 (GI, GIBBR). e Number of tryptase-positive cells in distal colonic mucosal tissues. f Immunohistochemistry showing tryptase-positive cells in the distal colon. n = 5 (GH); n = 6 (GI); n = 7 (GIBBR). *P < 0.05, **P < 0.01.

Fig. 4. The IBS-derived gut microbiota induced microglial activation, which was inhibited by berberine.

a Representative images of Iba-1-stained microglia in the dorsal lumbar spinal cord. b Number of Iba-1-positive cells in the dorsal lumbar spinal cord. c Representative three-dimensional reconstructions of Iba-1-stained microglia residing in the dorsal lumbar spinal cord. d Morphological parameters of microglia. n = 6/group. *P < 0.05, **P < 0.01.

Fig. 5. Berberine altered the structure and composition of the IBS-derived gut microbiota.

a Chao1 and Shannon indexes indicative of community richness and diversity. n = 12 (GH); n = 14 (GI, GIBBR). b Principal coordinate analysis based on weighted UniFrac distances of the relative abundances of bacterial OTUs after berberine treatment. n = 12 (GH); n = 14 (GI, GIBBR). c Weighted UniFrac principal coordinate analysis showing dynamic shifts after berberine or vehicle treatment. i = 5–7/group. d Comparison of F/B ratios. e Bacteria composition at the phylum level. n = 12 (GH); n = 14 (GI, GIBBR). f Dynamic alterations in different phyla in fecal samples during berberine or vehicle treatment. n = 5–7/group. g Relative abundances of selected genera enriched by berberine. n = 12 (GH); n = 14 (GI, GIBBR). h Heatmap showing the relationships between different genera and morphological parameters of microglia. n = 6/group. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. 1: Dendritic area; 2: dendritic length; 3: number of branch points; 4: number of segments; 5: number of terminal points.

Fig. 6. The functions and metabolites of the gut microbiota were altered after berberine treatment.

a Principal component analysis of the enrichment of KEGG pathways. n = 12 (GH); n = 14 (GI, GIBBR). b Pathway enrichment analysis of significantly altered Kyoto Encyclopedia of Genes and Genomes orthologies after berberine treatment. n = 12 (GH); n = 14 (GI, GIBBR). c Enrichment of pathways involved in propanoate and butanoate. n = 12 (GH); n = 14 (GI, GIBBR). d Fecal concentrations of SCFAs. n = 6 (GH); n = 8 (GI, GIBBR). *P < 0.05, **P < 0.01, ****P < 0.0001.

Fig. 7

Proposed model of the beneficial effects of berberine on gut dysbiosis and stress-induced brain–gut dysregulation.