| Objective:To investigate the effects of astragaloside Ⅳ,calycosin separately glucoside,formononetin on proliferation,antioxidant ability and CYP2E1 expression in oxidative damaged Chang Liver cells.Methods:Chang Liver cells were used as the research object,bifendate was used as the positive control drug,then the cellular oxidative stress was induced by H2O2.At first,the total Chang Liver cell incubation time of a test cycle was determined by cell growth curve,which was drawn using MTT method.The safety concentrations of astragaloside IV,calycosin separately glucoside,formononetin,bifendate,and the inhibiting concentration of H2O2 were elected by cell viability,which were measured by MTT assay,too.So the Chang Liver cell oxidative damaged model was established.Cells were divided into 6 groups:blank control group,damaged model group,astragaloside IV group,calycosin separately glucoside group,formononetin group,and positive control group.Then,cell growth conditions of different treatment groups were observed by inverted microscope;proliferations of cells were evaluated by cell viability,cell cycle,apoptosis,which were measured by MTT method,flow cytometry,and DNA ladder assay,respectively;the degree of Chang Liver cells damage was evaluated by transaminase activity,which measured by microplate method;cellular antioxidant capacities were evaluated by endogenous antioxidant system related indexes,ROS,which were measured by microplate method,colorimetry,and DCFH-DA fluorescent probe method;the expressions of CYP2E1 were evaluated by different levels,including liver microsomes,mRNA,and protein,respectively with spectrophotometry,Real-time PCR method,and Western Blot technique.Results:Rate of cell growth was slow before 18 h,then the logarithmic phase was achieved at 24～36 h,but cell growth was inhibited after 36 h,which were showed by cell growth curve.Cell survival rates were not less than 90%,when the concentrations of four drugs were in range of 0.1～10 mg·L-1;furfthmore,cell viability was negatively correlated with the concentration of H2O2,it was(52.09±4.18)%when the concentration of H2O2 was at 250 μmol·L-1.Cell survival rate and antioxidant ability were decreased;apoptosis rate,transaminase activity,ROS level,and expression of CYP2E1 were increased;cells were blocked at G0/G1 phase in oxidative damaged Chang Liver cells which were induced by H2O2.The survival rates and antioxidant abilities of oxidative damaged cells were increased;apoptosis rate,transaminase activity,ROS level,and expression of CYP2E1 were decreased;retardation of cell cycle at G0/G1 phase was relieved with protections of the three components of Astragalus membranaceus(compared with damaged model group,P<0.05,or P<0.01),which taken as a whole had equivalent effects as the positive control group.Conclusion:Three Astragalus membranaceus ingredients(astragaloside IV,calycosin separately glucoside,formononetin)all had significant or extremely significant protective effects on oxidative damaged Chang Liver cells which were induced by H2O2,and the oxidative damage of Chang Liver cells had been relieved. |
