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Cholestasis is a clinical condition resulting from impaired bile flow. Currently, patients with cholestasis face several barriers in seeking diagnosis and treatment. Zhuyu Pill (ZYP) is an ancient classic formula of the Coptis-Evodia herb couples (CEHC), and has been used for cholestasis treatment in the clinic, however, its underlying biological activity in cholestasis remain to be clarified. In this study, an α-naphthyl-isothiocyanate (ANIT, 50 mg/kg)-induced rat model of cholestasis was treated with ZYP. Serum biochemical indices and histopathological evaluation was performed, together with the metabolomics analyses of feces and 16S rDNA sequencing of the fecal microbiota. We evaluated the anti-cholestatic activity of ZYP and investigated the mechanisms underlying its correlation with fecal microbiota and fecal metabolite regulation. The relationships between biochemical indices and changes in gene expression associated with liver injury, levels fecal metabolites, and composition of fecal microbiota were analyzed. The results showed that both high (1.2 g/kg) and low (0.6 g/kg) doses of ZYP could effectively improve biochemical parameters in the blood of cholestasis-induced rat models; the intervention effect of high dose ZYP was superior to that that of lower dose ZYP. Based on a metabolomics test of fecal samples, significantly altered metabolites in the ANIT and ZYP treatment group were identified. In total, 734 metabolites were differentially expressed, and whose biological functions were mainly associated with amino acid metabolism, steroid hormone biosynthesis, and bile secretion. In addition, sequencing of the 16S rDNA unit in fecal samples revealed that the ZYP could improve the fecal microbiota dysbiosis that ANIT had induced. Therefore, we conclude that ANIT altering of blood biochemical and metabolic profiles and of fecal microbiota could effectively be alleviated with ZYP treatment. This study contributes to the “TCM wisdom” applied in clinical diagnosis and treatment of cholestasis.

Efficacy of Zhuyu Pill Intervention in a Cholestasis Rat Model: Mutual Effects on Fecal Metabolism and Microbial Diversity