Luteoloside Attenuates Anoxia-induced H9c2 Cells Injury Via Regulating Autophagy Mediated By 14-3-3η

Objective:To elaborate the protective effect of Lut against anoxia injury of H9c2 cells by up-regulating 14-3-3η,and regulating autophagy activity via AMPKα-mTOR/ULK1 signaling pathway.Method:In our study,H9c2 cells were pretreated with Lut and underwent in anoxia injury model.H9c2 cells were optionally divided into 6 groups,the experiments were repeated three times;(1)Control group;(2)Anoxia group;(3)20 μM Lut+Anoxia group;(4)20μM Lut+AD 14-3-3 ηRNAi+Anoxia group;(5)20 μM Lut+Rapamycin+Anoxia group;(6)20 μM Lut+3-MA+Anoxia group.Cell viability was measured with CCK-8;spectrophotometric was used to determine the lactate dehydrogenase(LDH),superoxide dismutase(SOD),glutathione peroxide enzyme(GSH-Px),catalase(CAT)activities,malondialdehyde(MDA)contents,the level of ATP,and degree of opening of mitochondrial permeability transition pores(mPTP);the expression of 14-3-3η,P62,LC3 and AMPKα,p-AMPKα,mTOR,p-mTOR,ULK1 and p-ULK1 were assayed by Western blotting.The activity of autolysosomes was observed under fluorescent inverted microscope;the intracellular reactive oxygen species(ROS)and mitochondrial membrane potential(MMP)were detected by flow cytometry.Results:H9c2 cells in all groups were analyzed after Anoxia treatment:1.Compared with the Control group,when H9c2 cells were underwent in anoxia treatment,the cell viability decreased,the activity of LDH increased,the expression of 14-3-3η decreased and the fluorscence intensity of autolysosomes increased.2.Compared with Anoxia group,20 μM Lut pretreatment significangtly increased cell viability,decreased LDH activity,up-regulated 14-3-3η,decreased P62 protein expression,increased LC3-Ⅱ/β-actin ratio,and further increased the fluorescence intensity of autolysosomes,the ratio of p-AMPKα/AMPKα and p-ULK1/ULK1 increased,the ratio of p-mTOR/mTOR decreased.Additionally,the activities of SOD,GSH-Px and CAT improved,the content of MDA reduced,the level of ATP recovered,intracellular ROS generation decreased,MMP was stabilized,and the opening of mPTP inhibited.These effects were similar to Rapamycin(mTOR inhibitor).3.After H9c2 cells were infected with AD14-3-3ηRNAi,the above-mentioned effects of Lut were weakened.And compared with AD 14-3-3ηRNAi+Lut+Anoxia group,co-pretreatment with Lut and 3-MA(autophagy inhibitor)had the similar effects.Conclusion:Lut pretreatment could up-regulate the expression of 14-3-3η through AMPKα-mTOR/ULK1 signaling pathway,regulate autophagy activity,alleviate intracellular oxidative stress,maintain mitochondrial function,and protect H9c2 cells from anoxia injury...