The Protective Effect Of Oxymatrine On Agomelatine-induced Hepatocyte Injury And Its Underling Mechanism

Objective: 1.To explore the injury effect of agomelatine and its mechanism on L02 cells.2.To evaluate the protective effect of oxymatrine on agomelatine-induced hepatocyte injury and its mechanism on L02 cells.Method: 1.To observe the injury effect of agomelatine on L02 cells.L02 cells were treated with agomelatine of different concentrations at different time points.(1)Using CCK-8 assays to evaluate the effect of agomelatine on the cell survival rate;(2)Using ALT and AST assays to access the influence of agomelatine on the degree of cellular damage;(3)Using flow cytometry to detect the effect of agomelatine on the apoptosis rate.2.To investigate the relationship between the agomelatine-induced hepatocyte injury and ER stress.(1)L02 cells were stimulated with 50 ?M agomelatine at different time points.Western blot was used to measure the protein level of BIP,p-IRE1?,ATF6,CHOP,p-e IF2? and p-AKT;(2)L02 cells were exposed to agomelatine in different concentrations for 24 h.Western blot was used to detect the protein level of BIP,p-IRE1?,ATF6,CHOP,pe IF2? and p-AKT;(3)L02 cells were pre-incubated with PERK pathway inhibitor 1 h,2.5,5,10 n M ISRIB,then L02 cells were treated with 50 ?M agomelatine for 24 h,and CCK-8 was used to access the cell viability.3.To observe the protective effect of oxymatrine on agomelatine-induced hepatocyte injury on L02 cells.L02 cells were pre-incubated with 0.5,1,2 ?M oxymatrine for 18 h,followed by treating with 50 ?M agomelatine for 24 h.(1)Using CCK-8 to measure the effect of oxymatrine on the cell viability for agomelatine-induced hepatocyte injury;(2)Using ALT and AST assays to access the degree of cellular injury for agomelatine-induced hepatocyte damage;(3)Using flow cytometry to evaluate the influence of oxymatrine on the apoptotic rate for agomelatine-induced hepatocyte damage;(4)Using TUNEL to detect the effect of oxymatrine on the apoptosis ratio for agomelatine-induced hepatocyte injury.4.To investigate the relationship between the protective effect of oxymatrine for agomelatine-induced hepatocyte injury and CHOP protein.(1)L02 cells were pre-incubated with 0.5,1,2 ?M oxymatrine for 18 h,followed by treatment with 50 ?M agomelatine for 24 h.Western blot was used to evaluate the protein level of BIP,p-IRE1?,ATF6,CHOP and p-AMPK?;(2)The thermal stability of oxymatrine with AKT protein was detected by cell thermal transfer assay;(3)SURFLEX-DOCK software was used to calculate the molecular docking scores of oxymatrine and AMPK protein;(4)L02 cells were administered with 1 ?M MG132,25 ?M CQ and 50 n M Baf A1 for 1 h.Western blot was used to detect the protein level of CHOP;(5)L02 cells were pre-incubated with 0.5,1,2 ?M oxymatrine for 18 h,then incubated with 1 ?M MG132 for 1 h before treatment with agomelatine.Western blot was used to measure the protein level of CHOP.Result: 1.To observe the injury effect of agomelatine on L02 cells.L02 cells were treated with agomelatine of different concentrations at different time points.(1)CCK-8 results showed that the cell viability was significantly decreased,compared with the control group.(2)ALT and AST results showed that the absolute value of ALT and AST was significantly increased,compared with the control group.(3)Flow cytometry results showed that the apoptosis rate was significantly increased,compared with the control group.2.To investigate the relationship between the agomelatine-induced hepatocyte injury and ER stress.(1)L02 cells were stimulated with 50 ?M agomelatine at different time points.Western blot results showed that the protein level of BIP,p-IRE1?,ATF6 and CHOP was significantly elevated,the protein level of p-AKT was significantly decreased,and there was no significant change for p-e IF2? protein,compared with the control group.(2)L02 cells were treated with agomelatine of different concentrations for 24 h.Western blot results showed that the protein level of BIP,p-IRE1?,ATF6 and CHOP was significantly elevated,the protein level of p-AKT was significantly decreased,and there was no significant change for p-e IF2? protein,compared with the control group.(3)CCK-8 results showed that there was no significant change after treatment with ISRIB,compared with the agomelatine-treated group.3.To observe the protective effect of oxymatrine on agomelatine-induced hepatocyte injury on L02 cells.(1)CCK-8 results showed that the cell viability was significantly increased after treatment with 0.5,1,2 ?M oxymatrine,compared with the model group.(2)ALT and AST results suggested that the absolute values of ALT and AST was significantly reduced after given 0.5,1,2 ?M oxymatrine,compared with the model group.(3)Flow cytometry results showed that the apoptotic rate was significantly reduced after treatment with 2 ?M oxymatrine,compared with the model group.(4)TUNEL results showed that the apoptosis rate was significantly decreased after treatment with 1,2 ?M oxymatrine,compared with the model group.4.To investigate the relationship between the protective effect of oxymatrine for agomelatine-induced hepatocyte injury and CHOP protein.(1)Western blot analysis showed that CHOP protein level was significantly reduced but BIP,p-IRE1? and ATF6 protein level did not change after given oxymatrine,compared with the model group.(2)Cell thermal migration results showed that oxymatrine did not maintain the thermal stability of AKT protein.(3)SURFLEXDOCK results showed that the binding capacity of oxymatrine and AMPK protein was much lower than A-769662 and PKA.(4)Western blot results showed that MG132 significantly increased CHOP protein levels but CQ and Baf A1 did not change,compared with control group.(5)Western blot results showed that the protein level of CHOP was significantly decreased after given oxymatrine,compared with the model group.However,MG132 reversed the protein level of CHOP,compared with oxymatrine-treated group.Conclusion: 1.Agomelatine induced hepatocyte injury on L02 cells.2.Agomelatine induced hepatocyte injury through activating the signal pathway of IRE1?-CHOP and ATF6-CHOP.3.Oxymatrine ameliorated agomelatine-induced hepatocyte injury on L02 cells.4.Oxymatrine ameliorated agomelatine-induced hepatocyte injury through promoting proteasome-mediated CHOP degradation.