Protective Effect Of Tectorigenin Against Acetaminophen-Induced Liver Injury

Acetaminophen?APAP?is a widely used analgesic and antipyretic drug,and APAP overdosing is likely to cause acute liver injury.In clinic,N-Acetyl-L-cysteine?NAC?is the only marketed drug for the treatment of APAP-induced injury.A number of natural products,such as caffeic acid,resveratrol,esculentoside A,flavone compounds isoquercitrin and silymarin,have been investigated for alleviating APAP toxicity.Tectorigenin?TG?,an isoflavonoid with anti-inflammatory and antioxidant activity,demonstrates protective effects on hepatic injury induced by hydrogen peroxide,carbon tetrachloride or tert-butyl hyperoxide.However,no research regarding the effect of TG on APAP-induced liver injury has been reported so far.In the present study,APAP induced liver injury model was established in mice.Moreover,effects of post-treatment and seven days'pretreatment of TG on APAP-induced liver injury as well as the possible mechanism of the liver protection were studied.Effects of different doses?200 mg/kg,400 mg/kg and 600 mg/kg?and fasted or fed condition on liver injury were investigated in mice.The results showed that alanine aminotransferase?ALT?and aspartate transaminase?AST?levels were significantly elevated 24 h after APAP treatment with an increasing dose of APAP,indicating a dose-dependent manner of liver injury.At the same dose of APAP,serum ALT and AST levels in fasted mice were both higher than that in fed group,suggesting that fasted condition may make mice more sensitive to APAP liver injury.Consequently,300 mg/kg APAP was chosen as appropriate dose in mice fasted overnight before intraperitoneal injection.Due to low solubility of TG,effects of common solvents or cosolvent on APAP induced hepatotoxicity should also be explored.Commonly used injection solutions include DMSO,ethanol,Tween 80,propylene glycol?PG?and Kolliphor?HS 15.It has been reported that both of DMSO and ethanol can inhibit CYP2E1,and they can attenuate APAP-induced injury.In addition,Tween 80 can cause hemolysis.As such,these three solvents are not suitable to be used as vehicle for APAP.Therefore,the effects of seven-day pretreatment of PG and HS 15 on APAP-induced liver injury were investigated,including evaluation of plasma levels of alanine aminotransferase and aspartate transaminase,hepatic glutathione levels,liver histological observation together with expression of CYP2E1 at designated time points after APAP dosing.Furthermore,inhibition of PG on the activity of mouse CYP2E1 was assessed in vitro with mouse liver microsomes.The results showed that HS 15 had no significant effect on APAP-induced liver injury model,while PG had protective effect on APAP liver injury.Based on western blot and enzyme inhibition results,it can be inferred that PG can attenuate APAP-induced liver injury via inhibiting the activity of CYP2E1.Therefore,10%HS 15 was finally selected as vehicle for TG.The study of TG's effect on APAP-induced liver injury was divided into two parts,including pretreatment and post-treatment of TG.The levels of serum transaminase and oxidative stress indicators,such as GSH content,superoxide dismutase activity,malondialdehyde content,ROS level and peroxidation hydrogenase activity,in liver were assayed at different time points?2 h,6 h and 24 h?.The results indicated that the activity of transaminase in the model group increased with the time,while 100 mg/kg TG treatment can reverse this increasment.Liver biochemical markers showed that pretreatment of TG may reduce the oxidative stress induced by APAP,and the catalase activity was not significantly altered.Furthermore,the IC₅₀ values of CYP2E1 in liver microsomes were determined and western blot and real-time quantitative PCR were used to evaluate expression levels of proteins and gene involved in APAP induced liver injury.The results showed that IC50 value of TG on CYP2E1 in mouse liver microsome is 196.6?M,indicating that TG had little inhibiton on CYP2E1.Western Blot showed that APAP-induced liver injury can cause up-regulation of MRP2 protein expression,down-regulation of CYP2E1 protein,and down-regulation of cell proliferation-associated protein P21 at 24 h after APAP treatment.Both TG pretreatment and post-treatment increased the expression of MRP2 and P21 while it did not alter protein expression of CYP2E1.The results of RT-PCR indicated that,APAP-induced liver injury can lead to down-regulation of expression levels of enzymes and transporters related with APAP metabolism and transport.In addition,expression levels of proteins and genes involved in p53/p21 and Nrf2/Keap1 pathway were altered in APAP induced liver injury.Pretreatment and post-treatment of TG both down-regulated expression of Ho-1 gene,which is mainly related to the antioxidant effect.TG post-treatment could affect p21 gene,which may be related to cell regeneration function.In summary,post-treatment and seven-day pretreatment of TG can both protect APAP-induced liver injury.TG may alter expression of the transporter MRP2,protein Ho-1 and P21 to protect the hepatocytes while it had little effect on CYP2E1 and GSH synthesis.