Paeoniflorin,one of the primary bioactive components in Chi shao,are widely used in traditional Chinese medicine.A lot of evidences suggest that Paeoniflorin has potential anti-oxidant effects.However,whether Paeoniflorin plays roles in cholestasis is unclear.In this study,we examined the protective effect of Paeoniforin against alpha-naphthylisothicaynate(ANIT)-induced cholestasis in rats.Our data demonstrated that the high(0.2 g/kg body weight)and medium(0.lg/kg body weight)doses of Paeoniflorin significantly prevented ANIT-induced changes in bile flow and the serum levels of total bilirubin,direct bilirubin,total bile acid,γ-glutamyltranspeptidase,glutamate-pyruvate transaminase,glutarnate-oxaloacetic transaminase and alkaline phosphatase.Moreover,we also found that Paeoniflorin significantly inhibited nitric oxide and malondialdehyde production,and restored glutathione decrease induced by ANIT.EPR data further indicated that Paeoniflorin inhibited ANIT-mediated reactive oxygen species(ROS)production.The overexpression of NADPH oxidase 4 induced by ANIT were significantly reversed when treated with Paeoniflorin,suggesting that Paeoniflorin could scavenge ROS via inhibiting NADPH oxidase 4 expression.Paeoniflorin treatment could also relieve ANIT-induced liver pathological injuries as indicated by histological assay.These findings indicate that Paeoniflorin exerts a dose-dependent protective effect on ANIT-induced cholestatic liver injury in rats,and the mechanism of this activity is related to its attenuation of oxidative stress in liver tissue.