Study On Protective Effect And Anti-apoptotic Mechanism Of Total Flavonoids From Cyclocarya Paliurus(Bata1.)Iljinskaja Leaves On Acute Liver Failure In Mice

This study was conducted to investigate the protective effect and anti-apoptotic mechanism of total flavonoids of Cyclocarya paliurus leaves(CPF)on mice with acute liver failure(ALF)to provide scientifically theoretical basis for developing CPF as a natural hepatoprotective medicine.Crude CPF was extracted by microwave-assisted method,purified by polyamide resin,and characterized by UPLC-DAD-ESI-MS.The results showed that the purity of CPF was 72.56%,and the purified CPF was mainly consisted of five flavonoids including isoquercetin,quercetin-3-O-rhamnoside,kaempferol-3-O-rhamnoside,quercetin and kaempferol,with the percentage of 2.37%,1.94%,15.77%,4.86%and 2.64%,respectively.Sixty male Kunming mice were randomly divided into control group,model group,and CPF low,middle and high dose groups.CPF low,middle and high dose groups were given with 100,200 and 400 mg/kg CPF,respectively,and the control and model groups were given with equivalent volume of 0.5%CMC-Na.One week later,animals were intraperitoneally injected with LPS(50 ?g/kg)and D-GalN(800 mg/kg)to induce model of ALF.6 h later,blood and organs were collected,and the activities of ALT,AST,Caspase-3,GSH-PX and T-SOD and the levels of ROS and MDA were measured.HE staining and TUNEL were performed to observe the pathomorphology of liver and the apoptotic rate of hepatocytes.Besides,Elisa was applied to determine the serum content of TNF-?,IL-1? and IL-6,and RT-qPCR was used to measure the mRNA expression levels of TNF-?,IL-1?,IL-6 and Caspase-3 in liver.Finaly,immunohistochemistry(IHC)and western blot(WB)were used to determine the protein expression levels of CD 14,TNF-R1,Romo 1,Bcl-XL,Nrf2,HO-1,p-I?B?,p-IKKp and p-p65.Results showed as follow:(1)CPF significantly decreased the death rate of mice with ALF,and lowered the coefficients of liver and spleen(P<0.01).(2)CPF significantly suppressed the activities of ALT and AST in serum of mice with ALF(P<0.01),and lowered the contents of ROS and MDA in liver(P<0.01).(3)CPF significantly enhanced the activities of GSH-PX and T-SOD in liver of mice with ALF(P<0.01),while inhibition in Caspase-3 activity(P<0.01).(4)CPF significantly decreased the contents of TNF-?,IL-1? and IL-6 in serum of mice with ALF(P<0.01),and down-regulated mRNA expression levels of TNF-?,IL-1?,IL-6 and Caspase-3 in liver(P<0.01).(5)CPF significantly suppressed the protein expression levels of CD14,TNF-R1,Romo 1,p-I?B?,p-IKK? and p-p65(P<0.01),while increase in protein expression levels of Bcl-XL,Nrf2,and HO-1(P<0.01).(6)Results of HE staining and TUNEL showed that CPF significantly attenuated hepatocyte damage in liver of mice with ALF,and decreased the apoptotic rate of hepatocytes(P<0.01).