Study On The Effect And Mechanism Of Paeoniflorin Treatment On ANIT-induced Cholestasis

Objective:To explore the effect and mechanism of paeoniflorin on a-naphthylisothiocyanate (ANIT) induced cholestasis.Method:We examined the serum alanine aminotransferase (ALT) and total bilirubin (TBIL) to explore the optimum dose of cholestatic model and the optimal time of collecting blood. We assessed the serum biochemical indices ALT, AST, alkaline phosphates (ALP), total bilirubin (TBil), direct bilirubin (DBil), total bile acid (TBA), gamma-glutamyl transpeptidase (y-GT) and pathological section to explore the treatment effects of paeoniflorin on ANIT-induced cholestasis. Serum metabolomic was designed to assess the effects and possible mechanisms of paeoniflorin on alpha-naphthylisothiocyanate (ANIT) induced cholestasis. Ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-Q-TOF/MS) combined with principle component analysis (PCA) and partial least squares discriminant analysis (OPLS-DA) were integrated to obtain differentiating metabolites for the pathways and find related biological pathways. This study was designed to examine the rats liver glutathione (GSH) coupled with the protein and RNA expression of Glutamate-cysteine ligase catalytic subunit (GCLc), Glutamate-cysteine ligase modifier subunit (GCLm), Protein kinase B (Akt), Nuclear factor-E2-related factor-2 (Nrf2), aiming to assess the antioxidant mechanism of paeoniflorin on ANIT-induced cholestasis.Results:(1) The results of the treatment effects of paeoniflorin on ANIT-induced cholestasisThe results demonstrated that the optimum dose of cholestatic model was 60 mg·kg-1ANIT and administration of ANIT after 36h was the optimal time of collecting blood. When compared with the control group, the data indicated that serum biochemical indices ALT, AST, ALP, TBil, DBil, TBA, y-GT were significantly higher (P<0.01) in model group. Moreover, paeoniflorin could remarkably downregulate serum biochemical indices and alleviate the histological damage of livertissue.(2) Serum metabolomic profiling reveals protective function of paeoniflorin on cholestasisThe results showed that there were 14 different expression metabolites in regulation of cholestasis in rats through analyzing the endogenous metabolites characteristic changes in control group, model group and the high dose paeoniflorin group. Then, we imported 14 different expression metabolites to MetaboAnalyst 3.0 database for pathway analysis and got 14 metabolic pathways. Finally, we imported the metabolites and metabolic pathways to KEGG to make correlation analysis. The results indicated that the protective mechanism of paeoniflorin mainly regulated the primary bile acid biosynthesis and affected the synthesis of bile acids through adjusting 14 characteristic biomarkers, which eventually treated ANIT-induced cholestasis.(3) The results of the antioxidant mechanism of paeoniflorin treatment on cholestasisThe results demonstrated that paeoniflorin significantly prevented ANIT-induced change in GSH and restored GSH and its related synthase GCLc and GCLm in all dose of paeoniflorin groups. Moreover, the mRNA and protein expression of Akt and Nrf2 were also activated by paeoniflorin in ANIT-induced rats. These findings indicated that paeoniflorin protected ANIT-induced cholestasis and increased GSH synthesis and its related synthase GCLc and GCLm by activating Nrf2 through PI3K/Akt-dependent pathway.Conclusion:With the methods of pharmacodynamics, metabolomics, molecular biology, this study has verified the protective effect of paeoniflorin on ANIT-induced cholestasis and elucidated the metabolites in regulation of cholestasis in rats, as well as the antioxidant mechanism of paeoniflorin on cholestasis. It provides a theoretical basis for its further application in the treatment of cholestasis.