Ginsenoside Rb1 Attenuates HR Injury Of H9c2 Cells Through Mitochondrial ATP-sensitive Potassium Channels

ObjectiveTo investigate whether ginsenoside Rb1 can attenuate hypoxia-reoxygenation induced cardiomyocyte injury by activating mitochondrial ATP-dependent potassium channels(Mito K_ATP).MethodsH9c2 cells derived from embryonic rat hearts treated with hypoxia and reoxygenation（HR）in a Anaero Pack system and sugar-free medium to simulate myocardial ischemia-reperfusion injury.The cells were divided into 5 groups:group A was the control group,group B was the hypoxia reoxygenation group,group C was the ginsenoside Rb1 pretreatment group,and group D was the ginsenoside Rb1 and Mito K_ATP specific blocker 5-hydroxydecanoate（5-HD）co-pretreatment group,the E group was the Mito K_ATPspecific agonist Diazoxide pretreatment group,and the other groups except the control group were subjected to hypoxia/reoxygenation treatment after different treatments（hypoxia for 3 hours,reoxygenation for 3 hours）.The effects of different concentrations of ginsenoside Rb1 on the cell viability of H9c2 at different concentrations were detected by CCK-8method.After the treatment of hypoxia-reoxygenation,the ATP content in the cells and the content of lactate dehydrogenase（LDH）in the supernatant were detected.The mitochondrial membrane potential（MMP）intensity of each group was detected by Rhodamine 123（Rh123）,the ROS production of each group was detected by DFCH-DA,and the mitochondrial membrane permeability of each group was detected by Calcein-AM/Co Cl₂.The apoptotic rate was detected by Hoechst 33342method and TUNEL/DAPI method.The mitochondria were extracted by gradient centrifugation and the cytoplasm was separated.The apoptosis-related proteins in mitochondria and cytoplasm were detected by Western blot.ResultsGinsenoside Rb1 at a concentration of 0μM-200μM was not toxic to H9c9 cells.After hypoxia-reoxygenation treatment,the ATP content of the HR group was significantly lower than that of the control group,the leakage of LDH was significantly increased,the mitochondrial membrane potential intensity was significantly decreased,and the ROS production and The degree of openness of m PTP was significantly increased.The amount of cytochrome c to cytoplasmic transfer in mitochondria was significantly increased,the expression of cleaved-caspase3 was increased,and the apoptotic rate was significantly increased.Indicators of the ginsenoside Rb1 pretreatment group and the diazoxide pretreatment group have improved significantly compared with the HR group,and the protective effect was obvious.However,the ginsenoside Rb1 plus 5-HD pretreatment group had significant differences compared with the ginsenoside Rb1pretreatment group,and the protective effect almost disappeared.ConclusionsIn the process of hypoxia and reoxygenation of H9c2 cells,ginsenoside Rb1can maintain mitochondrial membrane potential by activating mitochondrial ATP-sensitive potassium channels,increase ATP content and reduce ROS production,thereby reducing the opening of mitochondrial membrane permeability transition pores.Mitochondrial protection reduces the release of cytochrome c into the cytosol from mitochondria,thereby attenuating apoptosis induced by hypoxia-reoxygenation.This experiment provides a theoretical basis for the treatment of myocardial ischemia-reperfusion injury with ginsenoside Rb1,and also provides a basis for ginsenoside Rb1 as a mitochondrial protective agent for other diseases.