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The present investigation was designed to assess the effects and possible mechanisms of Paeoniflorin on α-naphthylisothioeyanate (ANIT)-induced liver injury with eholestasis.Paeoniflorin (0.2,0.1 or 0.05 g per kg body weight per day) was intragastrically (i.g.) administered to experimental rots six times before and four times after they were treated withANIT (50 mg/kg body weight daily i.g.),which caused liver injury with cholestasis.The serum levels of total bilirubin (TBIL),direct bilirubin (DBIL),total bile acid (TBA),glutamate-pyruvate transaminase (GPT),glutamate-oxaloacetic transaminase (GOT),alkaline phosphatase (ALP),γ-glutamyltranspeptidase (γ-GTP),and bile flow were measured to evaluate the protective effect of Paeoniflorin 36 h after the ANIT treatment.Meanwhile,indicators of reactive oxygen species (ROS),such as nitric oxide (NO),malondialdehyde (MDA),glutathione (GSH),and the NOX4 protein,were measured in rat livers to explore the potential mechanisms.The results demonstrated that the high (0.2 g/kg) and medium (0.1 g/kg) doses of Paeoniflorin significantly prevented ANIT-induced changes in bile flow (P<0.01) and the serum levels of TBtL,DBIL,TBA,γ-GTP,GPT,GOT and ALP (P<0.01).In addition,ANIT-induced increases in the levels of NO and MDA in the liver and GSH (P<0.01) were significantly reduced and the previously ANIT-suppressed NOX4 protein levels in the liver were significantly (P<0.01) elevated in the groups treated with 0.2 or 0.1 g/kg Paeoniflorin.The histopathology of the liver tissue showed that pathological injuries were relieved by the Paeoniflorin treatment.The results also showed that a low Paeoniflorin dose (0.05g/kg) exhibited protective effect on ANIT-induced liver injury through cholestasis by reversing a portion of the biochemical parameters and histopathological changes.These findings indicated that Paeoniflorin exerted 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.