Protective Effect Of Luteolin On Palmitic Acid-induced H9c2 Cardiomyocyte Injur

Objective:In this study,we used 0.2mmol·L^-1palmitate acid（PA）to damage H9c2 cardiomyocytes to establish a model of lipotoxic myocardial injury at the cellular level,and to investigate the relationship between lignocaine mitigation of myocardial lipotoxic damage and the AMPK/m TOR/p70S6K pathway.Methods:H9c2 cardiomyocytes were stimulated with 0.2 mmol/L PA for 24 h,pretreated with PA for 1h and then treated with 15 mmol/L LU for 24 h.The experimental group was divided into normal group,PA group and luteolin group according to this method.Cell viability was assessed by CCK8 method.Oxidative stress levels in cells were tested with kits,and the superoxide dismutase（SOD）activity and malonaldehyde（MDA）content were determined.DHE and Annexin V-FITC/PI staining were used to detect intracellular reactive oxygen species(reactive oxygen species,ROS level and cell apoptosis,and the changes of mitochondrial membrane potential（MMP）were determined by JC-1.The expression of AMPK/m TOR/p70S6K pathway protein,Bax,Bcl-2,cleaved caspase-3 and cleaved Caspase-3 were detected by Western blot.The m RNA expression levels of AMPK,m TOR and p70S6K were detected by real-time quantitative PCR.Results:（1）.Compared with normal group,H9c2 cardiomyocytes were stimulated with 0.2 mmol·L-1 PA for 24 h,and cell viability decreased significantly（P<0.01）;Intracellular SOD level decreased and MDA level increased significantly（P<0.05）,the intracellular ROS level increased（P<0.01）,apoptosis was obvious and mitochondrial membrane potential decreased（P<0.05）;Expression of apoptosis-related proteins Bax and cleaved caspase-3 increased,and expression of Bcl-2 decreased（P<0.05 or P<0.01）;AMPK m RNA and protein expression decreased,while m TOR and p70S6K m RNA and protein expression increased（P<0.05 or P<0.01）.（2）.After pretreatment with 10μmol·L^-1,15μmol·L^-1and 20μmol·L^-1LU for 1 h,the cell survival rate was increased compared with PA group.After treatment with 15μmol·L^-1LU,the cell viability increased most obviously（P<0.01）;LU treatment reversed PA-induced cell damage,decreased intracellular MDA activity,increased SOD level,and significantly increased MMP（P<0.05 or P<0.01）,inhibit cell apoptosis;Intracellular Bax and cleaved caspase-3 expression were down-regulated,and Bcl-2 protein expression was up-regulated（P<0.05）Activation of AMPK increased m RNA level and protein expression,and inhibited m RNA and protein expression of m TOR and p70S6K（P<0.05 or P<0.01）.Conclusion:LU can relieve H9c2 cell damage induced by 0.2 mmol·L^-1PA,reduce oxidative stress and inhibit cell apoptosis,and its protective effect may be related to AMPK/m TOR/p70S6K signaling pathway.