Rifaximin-mediated gut microbiota regulation modulates the function of microglia and protects against CUMS-induced depression-like behaviors in adolescent rat

Abstract

Background: Chronic unpredictable mild stress (CUMS) can not only lead to depression-like behavior but also change the composition of the gut microbiome. Regulating the gut microbiome can have an antidepressant effect, but the mechanism by which it improves depressive symptoms is not clear. Short-chain fatty acids (SCFAs) are small molecular compounds produced by the fermentation of non-digestible carbohydrates. SFCAs are ubiquitous in intestinal endocrine and immune cells, making them important mediators of gut microbiome-regulated body functions. The balance between the pro- and anti-inflammatory microglia plays an important role in the occurrence and treatment of depression caused by chronic stress. Non-absorbable antibiotic rifaximin can regulate the structure of the gut microbiome. We hypothesized that rifaximin protects against stress-induced inflammation and depression-like behaviors by regulating the abundance of fecal microbial metabolites and the microglial functions.

Methods: We administered 150 mg/kg rifaximin intragastrically to rats exposed to CUMS for 4 weeks and investigated the composition of the fecal microbiome, the content of short-chain fatty acids in the serum and brain, the functional profiles of microglia and hippocampal neurogenesis.

Results: Our results show that rifaximin ameliorated depressive-like behavior induced by CUMS, as reflected by sucrose preference, the open field test and the Morris water maze. Rifaximin increased the relative abundance of Ruminococcaceae and Lachnospiraceae, which were significantly positively correlated with the high level of butyrate in the brain. Rifaximin increased the content of anti-inflammatory factors released by microglia, and prevented the neurogenic abnormalities caused by CUMS.

Conclusions: These results suggest that rifaximin can regulate the inflammatory function of microglia and play a protective role in pubertal neurodevelopment during CUMS by regulating the gut microbiome and short-chain fatty acids.

Keywords: Adolescence; Chronic unpredictable mild stress (CUMS); Gut microbiome; Microglia; Neurogenesis; Rifaximin; Short chain fatty acid (SCFA).

Fig. 1

Depressive-like behavior induced by CUMS and the protective effect of rifaximin. a Experimental flowchart. b Sucrose preference. c Frequency of crossing. d Number of rearing. e Duration in the center area. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the CON group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. the CUMS group

Fig. 2

Effects of rifaximin on the microbial composition and integrity of intestinal mucosa of CUMS rats. a Shannon index. b Simpson index. c Ace index. d Chao1 index. e Principal coordinate analysis. f Relative abundance of distinguishable phyla. g Relative abundance of distinguishable family. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the CON group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. the CUMS group. q values were verified by the Benjamini and Hochberg correction post hoc test

Fig. 3

Relevance between the short-chain fatty acid content in the brain and the gut microbiome. a Levels of SCFAs in the serum. b Levels of SCFAs in the brain. c Matrix is used to describe SCFA–microbe correlations. The depth of the color of the square indicates the magnitude of the correlation, where blue squares represent positive correlations and red squares represent negative correlations. White asterisks indicate the significance of the correlation (*q < 0.1, **q < 0.05). In a and b, black asterisks indicate the significance of the correlation *P < 0.05, **P < 0.01, ***P < 0.001 vs. the CON group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. the CUMS group

Fig. 4

Effects of rifaximin on microglia in the hippocampal DG of CUMS rats. a Iba-1 immunoreactivity of microglia in the DG. b Total number of microglia. c Sholl analysis. d Mean number of intersections. e Surface area of microglia. f Ramification length of microglia. g Soma volume of microglia. h Level of TNF-α in DG. i The level of IL-1β in DG. j Level of IL-10 in DG. k Level of IL-1ra in DG. l Proportion of Iba-1⁺/CD68⁺ cell. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the CON group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. the CUMS group

Fig. 5

Sodium butyrate regulates the microglia LPS response in vitro. a Level of TNF-α. b Level of IL-1β. c Level of IL-10. d Level of IL-1ra. e Median fluorescence intensity (MFI) of CD68. f Representative FACS plots showing CD68 expression gated on microglia treated with LPS or SB. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the CON group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. the LPS group

Fig. 6

Effects of rifaximin on neurogenesis and synaptic plasticity in the hippocampal DG of CUMS rats. a Immunofluorescence for DCX (red) and DAPI (blue) in the DG. b Length measurement of DCX⁺ cells. c Number of newborn neurons (DCX⁺). d Length of DCX⁺ cells. e Number of immature neurons (Ki67⁺/NeuN⁺). f Immunofluorescence for Ki67 (red) and NeuN (green) in the DG. g Immunofluorescence image depicting the migration of immature neurons (Ki67⁺/NeuN⁺). h Width of granule cell layer. W: the distance between the center of Ki67⁺/NeuN⁺ cell and the subgranular zone. h Width of the GCL. i Migration index of immature neurons. j Number of mature neurons (NeuN⁺). Values represent the mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001 vs. the CON group; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. the CUMS group