14-3-3η Mediates The Regulative Effect On Autophagy Of Astragaloside Ⅳ Against A/R Injury In H9c2 Cells

Objective:To investigate the autophagy regulation of Astragaloside Ⅳ(As-Ⅳ)against A/R injury in H9c2 cells and its relationship with 14-3-3η protein up-regulated by As-Ⅳ.Method:H9c2 cardiomyocyte-like cells were used.H9c2 cells were pretreated with As-Ⅳ for nutritional preconditioning and were used to establish hypoxia-reoxygenation injury model in this study.H9c2 cells were randomly divided into 6 groups:(1)Control group;(2)A/R group;(3)50 μM As-Ⅳ+A/R group;(4)50 μM As-Ⅳ+AD1433ηRNAi+A/R group;(5)50 μM As-Ⅳ+ADBeclin1RNAi+A/R group;(6)50 μM As-Ⅳ+3-MA+A/R group,each group repeated 3 times.Cell viability was measured with CCK-8;the level of leakage of LDH was measured by microplate reader;the expression of 1433η,P62,LC3 and beclin1 protein were assayed by western blotting;LysoTracker staining was used for lysosomal acidification analysis;to measure intracellular reactive oxygen species(ROS),mitochondrial membrane potential(MMP)and the opening of mitochondrial permeability transition pores(mPTP)via flow cytometry;apoptosis was detected by Annexin V-FITC/PI double staining or TUNEL.Results:H9c2 cells in all groups were analyzed after A/R treatment: 1.As-Ⅳ pretreatment increased the cell viability,decreased the activity of LDH and increased the expression of 14-3-3η protein in H9c2 cell suffered from A/R injury.2.Down-regulation of 14-3-3η eliminated the protective effects of As-Ⅳ pretreatment on cell viability and LDH activity in H9c2 cells.3.As-Ⅳ pretreatment up-regulated P62,reduced the LC3-II/I ratio and decreased beclin1 protein expression.While infected AD1433ηRNAi down-regulated 1433η expression,the above effects were reversed.LysoTracker Red staining revealed that H9c2 cells increased fluorescence intensity following A/R injury;the fluorescence intensity was attenuated afer pretreatment with As-Ⅳ.After infection with AD1433ηRNAi,As-Ⅳ pretreatmentcaused the pH of the lysosome to remain low and obviously increased the cell fluorescence intensity.4.The As-Ⅳ pretreatment group,the As-Ⅳ pretreatment group after ADBeclin1 RNAi infection or the addition 3-MA group after As-Ⅳ pretreatment significantly reduced ROS production,stabilized cell membrane potential,inhibited mPTP opening,and finally reduced cell apoptosis rate;these protective effects of As-Ⅳ pretreatment were obviously abolished by AD14-3-3ηRNAi.There results were all statistically significant(p < 0.01).Conclusion:As-Ⅳ pretreatment regulats the beclin1 dependent autophagy pathway by up-regulating the 14-3-3η protein,thereby reducing autophagy and exerting protective effects on H9c2 cells against A/R injury.