Chidan Tuihuang granule modulates gut microbiota to influence NOD1/RIPK2 pathway in cholestatic liver injury recovery

Abstract

Cholestatic liver injury (CLI), which occurs if bile acids are imbalanced and the liver becomes inflamed, is difficult to treat effectively OBJECTIVE: We investigated how the Chinese patent medicine Chidan Tuihuang granule (CDTH) ameliorates cholestatic liver injury with a focus on its effects on the NOD1/RIPK2 pathway and intestinal flora METHODS: We used an ANIT-induced SD rat model of CLI to evaluate the therapeutic effects of CDTH. The experimental design included control, model, UDCA (ursodeoxycholic acid) and CDTH treatment groups. UHPLC-Q-Orbitrap-HRMS was used to analyse the blood components of CDTH. The efficacy of CDTH was assessed by liver function tests, histopathological examination (HE and TUNEL staining), transmission electron microscopy, and ELISA to measure apoptosis and inflammatory markers. Mechanistic insights were obtained using transcriptomics and RT-qPCR, while alterations in the expression of key proteins were studied using western blotting, immunohistochemistry, and immunofluorescence. Furthermore, the impact of CDTH on the gut microbiota and its associated metabolite, meso-2,6-diaminopimelic acid (DAP), which is linked to NOD1 activation, was examined and confirmed through in vitro RESULTS: The experimental results demonstrated a notable elevation in serum levels of AST, ALT, ALP, TBA, TBIL, and DBIL in the rats belonging to the model group, accompanied by the infiltration of inflammatory cells, hepatocyte degeneration, and necrosis in the liver tissue. CDTH administration significantly improved liver function and cholestasis indicators. Transmission electron microscopy and TUNEL staining revealed a marked reduction in liver cell apoptosis with CDTH treatment. ELISA results showed that CDTH effectively reduced inflammatory markers. Transcriptomic analysis showed that CDTH inhibited the NOD1/RIPK2 pathway, resulting in a significant decrease in the expression of NOD1, RIPK2 and associated genes in liver tissue. Gut microbiota analysis demonstrated that CDTH regulated intestinal flora structure, reducing the abundance of DAP-producing Gram-negative bacteria such as lactobacilli. In vitro experiments confirmed that CDTH enhanced cell viability by downregulating the DAP-mediated NOD1/RIPK2 signaling pathway secreted by intestinal bacteria CONCLUSION: CDTH ameliorated liver damage in cholestatic rats by inhibiting the NOD1/RIPK2 signaling pathway through regulation of gut flora and downregulation of DAP metabolites.

Keywords: Chidan tuihuang granule; Cholestatic liver injury; Hepatology; Meso-2,6-diaminopimelic acid; NOD1/RIPK2 pathway; Phytotherapy.