Atractyloside-A ameliorates spleen deficiency diarrhea in mice via modulating Lactobacillus johnsonii-butyric acid-GPR43 axis and NF-κB -NLRP3 signaling pathway.

BACKGROUND: Spleen deficiency diarrhea (SDD) is regarded as a common gastrointestinal dysfunction in Traditional Chinese Medicine (TCM), which may lead to intestinal barrier damage and trigger intestinal inflammation. Previous studies have shown that Atractylenolide-A (AA) can effectively treat SDD by regulating intestinal flora. However, it remains uncertain whether AA can increase the levels of short-chain fatty acids (SCFAs) by restoring intestinal microbiota, thereby activating specific signaling pathways to regulate target protein and subsequently alleviate issues related to intestinal barrier function and inflammation.

PURPOSE: This study focused on examining the function of the signaling pathway involving microbiota, SCFAs, and G protein-coupled receptors (GPRs) in the anti-SDD effects of AA.

METHODS: The effects of AA on the Senna (SE) - induced SDD mouse model were assessed through various methods, including diarrhea scoring, H&E staining, qRT-PCR, and ELISA analysis. Subsequently, targeted metabolomics was employed to pinpoint essential metabolites that influence the intestinal microenvironment, while western blotting was utilized to measure the expression of GPRs and the NLRP3 inflammasome. Additionally, experiments involving dietary supplementation with SCFAs and AAV-shGPR43 were performed to determine whether the pharmacological effects of AA operate through SCFAs and rely on GPR43. Key bacterial species that play a role in AA's modulation of SCFAs' pharmacological effects were identified through metagenomic sequencing and single-strain experiments.

RESULTS: The findings of this research revealed that AA is capable of significantly reducing the intestinal inflammatory response, reversing damage to mucin synthesis, and alleviating the pathological symptoms linked to SDD. Furthermore, the use of Lactobacillus johnsonii, sodium butyrate (NaB), and SCFAs individually can lead to notable enhancements in various phenotypes related to SDD. In terms of mechanism, AA achieves its anti-SDD effects by elevating the levels of Lactobacillus johnsonii, facilitating the concentration of butyric acid, boosting GPR43 expression, and modulating the TLR4/NF-κB signaling pathway, which in turn inhibits the assembly of the NLRP3 inflammasome. Nonetheless, following the injection of AAV-shGPR43, the advantageous effects of both AA and NaB were negated, underscoring the significance of this target.

CONCLUSIONS: Gut microbiota-SCFAs-GPRs axis and NF-κB-NLRP3 pathway involve in the alleviation of diarrhea and inflammation in SDD mice intervened with AA, AA promotes the production of butyrate by influencing Lactobacillus johnsonii, stimulates GPR43, and suppresses the formation of the NLRP3 inflammasome via the regulation of the TLR4/NF-κB signaling pathway, which subsequently improves SDD in mice.