Xiaoyao San ameliorates maternal inflammation-induced neurobehavioral deficits by modulating the microbiota-gut-brain axis in offspring

Abstract

Background: XiaoYao San (XYS), a classical Traditional Chinese Medicine (TCM), has demonstrated efficacy in alleviating stress-related neuropsychiatric disorders. However, its therapeutic potential against maternal immune activation (MIA)-induced neurobehavioral impairments remains unexplored. This study aims to investigate the neuroprotective effects of XYS on MIA-related behavioral dysfunctions and elucidate its underlying mechanisms.

Results: Using a poly (I:C)-induced MIA mouse model, we demonstrated that XYS effectively ameliorates autism spectrum disorder (ASD) related behavioral phenotypes. Mechanistic investigations revealed that XYS exerts its therapeutic effects through: (1) Attenuation of core behavioral deficits including enhanced social interaction and reduced repetitive behaviors; (2) Downregulation of intestinal amino acid transporters; (3) Restoration of cerebral glutamate-GABA balance via modulation of glutamine pathway; (4) Structural remodeling of gut microbiota with specific enrichment of Bacteroides spp. Notably, B. uniformis was identified as a key microbial mediator capable of recapitulating XYS-mediated neurophysiological improvements through metabolic regulation.

Conclusion: This study elucidates XYS as a multi-target therapeutic agent that coordinately modulates gut microbial ecosystems, amino acid homeostasis, and neurotransmitter homeostasis. The findings provide novel insights into the gut-brain axis mechanisms of TCM formulations, offering a scientific foundation for developing microbiota-based intervention strategies for neurodevelopmental disorders.

Keywords: Xiaoyao san; amino acid transporter; gut microbiota; maternal immune activation (MIA); neurodevelopmental disorders.

FIGURE 1

XYS rescues ASD-like behaviors in MIA offspring. (A) The experimental timeline XYS treatment and behavioral tests. (B) Schematic of the three-chamber social interaction test and representative traces of test mice in this test. (C) The resident time in chambers of the test mice in the trial of habituation. (D) The resident time in chambers of the test mice in the trial of sociability. (E) The sociability index of the test mice, sociability index = (time in M1 – time in O)/(time in M1 + time in O). (F) The resident time in chambers of the test mice in the trial of social novelty. (G) The social novelty index of the test mice, social novelty index = (time in M2 – time in M1)/(time in M1 + time in M1). (H) Representative images of marble-burying test for the mice. (I) Quantification of the marble-burying index of test mice (n = 9–10 mice from different dams for each group). Graphs are mean ± SEM. Statistical details are provided in Supplementary Table S4.

FIGURE 2

XYS increases the relative abundance of Bacteroides in MIA offspring. (A–D) α diversity of fecal 16S rRNA sequencing data from MIA + Veh and MIA + XYS-H mice (n = 4 mice from different dams for each group). (E) PCA of fecal 16S rRNA sequencing data from MIA + Veh and MIA + XYS-H mice (n = 4 mice from different dams for each group). (F, G) Unweighted and weighted UniFrac PCoA of fecal 16S rRNA sequencing data from MIA + Veh and MIA + XYS-H mice (n = 4 mice from different dams for each group). (H–J) Relative abundances of the top 10 bacterial phylum, family, and genus from fecal 16S rRNA sequencing data (n = 4 mice from different dams for each group). (K) Cladogram showing the phylogenetic relationships of bacterial taxa was revealed by LEfSe (n = 4 mice from different dams for each group). (L) Bar chart showing the log-transformed LDA scores of bacterial taxa identified by LEfSe analysis (n = 4 mice from different dams for each group). (M) XYS increased the levels of Bacteroides in MIA offspring. Graphs are mean ± SEM. Statistical details are provided in Supplementary Table S4.

FIGURE 3

Bacteroides uniformis ameliorates the ASD-like behaviors in MIA offspring. (A) The experimental timeline Bacteroides uniformis treatment and behavioral tests. (B) Representative traces of test mice in three-chamber social interaction test. (C) The resident time in chambers of the test mice in the trial of habituation. (D) The resident time in chambers of the test mice in the trial of sociability. (E) The sociability index of the test mice, sociability index = (time in M1 – time in O)/(time in M1 + time in O). (F) The resident time in chambers of the test mice in the trial of social novelty. (G) The social novelty index of the test mice, social novelty index = (time in M2 – time in M1)/(time in M1 + time in M1). (H) Representative images of marble-burying test. (I) Quantification of the marble-burying index of the test mice (n = 12 mice from different dams for each group). Graphs are mean ± SEM. Statistical details are provided in Supplementary Table S4.

FIGURE 4

XYS alters the amino acid profile in the serum and brain of MIA offspring. (A) The experimental timeline for XYS treatment and serum untargeted metabolomic analysis. (B) PCA of the serum untargeted metabolomic data from MIA + Veh and MIA + XYS-H mice (n = 4 mice from different dams for each group). (C) Volcano plot of serum metabolites from MIA + Veh and MIA + XYS-H mice (n = 4 mice from different dams for each group). (D) Heat maps for clustering of serum differential metabolites between MIA + Veh and MIA + XYS-H mice (n = 4 mice from different dams for each group). (E) KEGG classification of the serum differential metabolites between MIA + Veh and MIA + XYS-H mice. (F) KEGG enrichment analysis of the serum differential metabolites between MIA + Veh and MIA + XYS-H mice. (G) Amino acid levels in MIA + XYS-H mice serum were normalized and shown as fold change (log₂ transformed) to levels in age-matched vehicle-treated controls. The amino acids with a fold change of >1.3 and P value <0.05 are in red and green. (H) Diagram of the mechanism of intestinal absorption of amino acids. (I) qPCR analysis of amino acids transporters' mRNA levels in the small intestine of the test mice (n = 4 mice from different dams for each group). (J) The amino acid and GABA levels in the brain of MIA + XYS-H mice were normalized and shown as fold change (log₂ transformed) to levels in age-matched vehicle-treated controls. The amino acids with a fold change of >1.3 and P value <0.05 are in red and green. Statistical details are provided in Supplementary Table S4.

FIGURE 5

Bacteroides uniformis restores Glu/GABA balance in MIA offspring. (A, B) The levels of Gln and Glu in the serum. (C–E) Gln, Glu, and GABA levels in the mPFC. (F) qPCR analysis of amino acids transporters' mRNA levels in the small intestine of the test mice (n = 5 mice from different dams for each group). Graphs are mean ± SEM. Statistical details are provided in Supplementary Table S4.

FIGURE 6

XYS upregulates GABAergic signaling-related genes and downregulates astrocyte differentiation-related genes in mPFC of MIA offspring. (A) Strategies for RNA-seq in the mPFC. (B) Heat maps for mPFC differentially expressed genes clustering between MIA + Veh and MIA + XYS-H mice (n = 4 mice from different dams for each group). (C) Volcano plot of mPFC differentially expressed genes between MIA + Veh and MIA + XYS-H mice (n = 4 mice from different dams for each group). (D, E) GO Enrichment analysis of the differentially expressed genes between MIA + Veh and MIA + XYS-H mice. GO terms for biological processes (top); GO terms for cellular components (middle); GO terms for molecular functions (bottom).

FIGURE 7

XYS reduces hyperactivation in the mPFC of MIA offspring. (A) Representative images illustrating GFAP (green) expression in the mPFC. Scale bars, 10 μm. (B) Representative images illustrating Iba1 (green) expression in the mPFC. Scale bars, 10 μm. (C) Representative images illustrating PV (green) expression in the mPFC. Scale bars, 10 μm. (D) Representative images illustrating c-Fos (green) expression in the mPFC. Scale bars, 10 μm. (E) Quantification of GFAP-expressing cells in the mPFC. (F) Quantification of Iba1-expressing cells in the mPFC. (G) Quantification of PV-expressing cells in the mPFC. (H) Quantification of c-Fos-expressing cells in the mPFC. n = 4 mice from different dams for each group. Graphs are mean ± SEM. Statistical details are provided in Supplementary Table S4.

FIGURE 8

Working model: XYS mitigates neurobehavioral abnormalities in MIA mice by regulating the gut microbiome, remarkably increasing Bacteroides, and modulating serum amino acids and brain neurotransmitters. It restores the excitation/inhibition balance, inhibits astrocyte differentiation, and enhances GABAergic signaling, alleviating autism-like behaviors.