Clostridium butyricum regulates intestinal barrier function via trek1 to improve behavioral abnormalities in mice with autism spectrum disorder

Abstract

Background: Autism Spectrum Disorder (ASD) is a complex neurodevelopmental disorder that has been found to be associated with dysregulation of gastrointestinal functions and gut microbial homeostasis (the so-called "gut-brain axis"). ASD is often accompanied by poor performances in social interaction and repetitive behaviors. Studies on the gut-brain axis provide novel insights and candidate targets for ASD therapeutics and diagnosis. Based on the ASD mice model, this work aims to reveal the mechanisms behind the interaction of intestinal barrier function and probiotics in ASD mouse models.

Results: We found an altered intestinal barrier in both BTBR T⁺ Itpr3^tf/J (BTBR) and valproic acid (VPA) mice, including increased intestinal permeability, decreased expression of intestinal tight junction proteins (claudin1, claudin3, and occludin), and increased levels of IL-6, TNF-α, and IFN-γ. Based on intestinal microbial alternation, C. butyricum can drive reduced expression of histone deacetylases 1 (HDAC1) and enhanced intestinal barrier function, significantly promoting behavioral abnormalities of ASD in BTBR mice. In parallel, we confirmed that C. butyricum was involved in the regulation of intestinal function by the Trek1 channel, indicating that it is a target of C. butyricum/butyric acid to improve intestinal barrier function in ASD mice.

Conclusions: Our finding provides solid evidence for the gut microbiota involved in ASD through the brain-gut axis. In addition, the probiotics C. butyricum hold promise to improve gut health and ameliorate behavioral abnormalities associated with ASD.

Keywords: Clostridium butyricum; Autism spectrum disorder; Gut microbiota; Intestinal barrier function; Trek1.

Fig. 1

BTBR and VPA offspring display ASD-Related Behavioral Abnormalities. A Open field exploration test B Marble burying test C Self-grooming test D Social interaction test. E Adult ultrasonic vocalizations in a social encounter with a female. Significant differences are indicated by *p < 0.05, **p < 0.01 and ***p < 0.005. n = 10/group

Fig. 2

BTBR and VPA offspring display intestinal barrier dysfunction. A Intestinal permeability assay, measuring translocation of 4-kDa FITC-dextran from the intestinal lumen into the blood. B Colon protein levels of Trek1 and tight junction components relative to β-actin. C Immunohistochemistry staining for Trek1(400X). D Colon mRNA expression of cytokines relative to GAPDH. E Colon protein expression of cytokines. Data are normalized to controls. n = 10/group

Fig. 3

BTBR and VPA Offspring exhibit altered microbial beta diversity. A PCoA based on OTU level (abund_jaccard). B Bray_curtis based Hierarchical clustering tree on OTU level. C, D Mean relative abundances of taxa on phylum level. E, F Mean relative abundances of taxa on genus level. Significant differences are indicated by *p < 0.05, **p < 0.01 and ***p < 0.005. n = 8/group

Fig. 4

The intestinal barrier function of BTBR mice under Clostridium butyricum treatment. A The richness of Clostridium butyricum from fecal samples of control, BTBR, and BB mice. B The concentration of butyrate (ug/ml) from fecal samples of control, BTBR, and BB mice. C The PCoA based on OTU levels of ontrol, BTBR, and BB mice. D The wilcoxon rank-sum test on phylum level in BTBR mice with and without Clostridium butyricum treatments. E The wilcoxon rank-sum test on genus level in BTBR mice with and without Clostridium butyricum treatments. F The level of FITC in control, BTBR, and BB mice. G, H The expression level of tight junction proteins in control, BTBR, and BB mice. I The expression levels of pro-inflammatory cytokines in BTBR mice with and without Clostridium butyricum treatments. *p < 0.05 vs. BTBR group, ^#p < 0.05 vs. CON group and **p < 0.01 vs. BTBR group

Fig. 5

The intestinal barrier function of VPA mice under Clostridium butyricum treatment. A The richness of Clostridium butyricum from fecal samples of control, VPA, and VB mice. B The concentration of butyrate (ug/ml) from fecal samples of control, VPA, and VB mice. C The PCoA based on OTU levels of ontrol, VPA, and VB mice. D The wilcoxon rank-sum test on phylum level in VPA mice with and without Clostridium butyricum treatments. E The wilcoxon rank-sum test on genus level in VPA mice with and without Clostridium butyricum treatments. F The level of FITC in control, VPA, and VB mice. G, H The expression level of tight junction proteins in control, VPA, and VB mice. I The expression levels of pro-inflammatory cytokines in VPA mice with and without Clostridium butyricum treatments. *p < 0.05 vs. VPA group, ^#p < 0.05 vs. CON group, **p < 0.01 vs. VPA group and ***p < 0.001 vs. VPA group

Fig. 6

Behavioral abnormalities in BTBR offspring under Clostridium butyricum treatment. A The open field exploration performance of control, BTBR, and BB mice(A1: center entries; A2: call duration). B The marble buried of control, BTBR, and BB mice. C The sociability of control, BTBR, and BB mice. D The self grooming test of control, BTBR, and BB mice. E1 The number of calls under different hertz of control, BTBR, and BB mice. E2 The duration of calls under different hertz of control, BTBR, and BB mice. Significant differences are indicated by *p < 0.05 vs. BTBR group

Fig. 7

Behavioral abnormalities in VPA offspring under Clostridium butyricum treatment. A The open field exploration performance of control, VPA, and VB mice (A1: center entries; A2: call duration). B The marble buried of control, VPA, and VB mice. C The sociability of control, VPA, and VB mice. D The self grooming test of control, VPA, and VB mice. E1 The number of calls under different hertz of control, VPA, and VB mice. E2 The duration of calls under different hertz of control, VPA, and VB mice. Significant differences are indicated by *p < 0.05 vs. VPA group

Fig. 8

Clostridium butyricum modulates intestinal barrier function through Trek1 in BTBR and VPA offspring. A The experimental design outlook. B, C FITC in serum of different mice groups. D The relative quantity of protein claudin-1, claudin-3, and claudin of different mice groups. E The content of pro-inflammatory cytokines of different mice groups. Significant differences are indicated by *p < 0.05 and ^#p < 0.05 vs. Saline group

Fig. 9

The proposed mechanisms of Clostridium butyricum treatment in ASD mice