Effects Of Kappa-opioid Receptor Agonist On Autophagy Induced By Hypoxia/Reoxygenation Injury In H9c2 Cardiomyocytes

ObjectiveTo observe the effects of different degrees of hypoxia/reoxgenation injury on autophagy and the changes of the cell survival rate using cultured H9c2 rat cardiomyocytes in vitro.To explore the effects of activation of kappa opioid receptor on autophagy of H9c2 myocardial cells induced by hypoxia/reoxgenation injury.MethodsH9c2 rat cardiomyocytes were cultured in hypoxic incubator with 0.5%FBS combined with high glucose medium?glucose-free medium and PBS for 3 hours differently.MTT assay was used to detect cell survival rate,flow cytometry was used to detect cell autophagy rate by MDC fluorescence staining,real-time qPCR was used to detect the expression of Atg5 mRNA.Cultured H9c2 myocardial cells were precondi tioned by U50488H at concentrations of 0.1 u mol/L,1 ?mol/L and 10 ?mol/L for 24 hours respectively.Preconditioned cells were used to perform the established hypoxia/reoxgenation injury model for further testing the same as above.ResultsThe survival rate of H9c2 cardiomyocytes decreased in all H/R model groups compared with the control group,and gradually decreased in 0.5%FBS combined with high glucose medium-?glucose-free medium-and PBS-treated H/R groups respectively.There were significant differences in the autophagy rate of H9c2 cardiomyocytes and the mRNA expression levels of Atg5 in H/R groups compared with the control group.The autophagy rate and mRNA expression levels of Atg5 gradually decreased in 0.5%FBS combined with high glucose medium-?glucose-free medium-and PBS-treated H/R groups,and increased in 0.5%FBS combined with high glucose medium-and glucose-free medium-treated H/R groups compared with the control group,however decreased in PBS-treated H/R group.The survival rate of H9c2 cardiomyocytes decreased in H/R group and all U50488H preconditioning groups compared with the control group.There was no significant difference of the survival rate among U50488H preconditioning groups and H/R group.The results of flow cytometry and real-time qPCR showed that the autophagy rate and the expression of Atg5 mRNA were up-regulated in H/R group and U50488H preconditioning groups compared with the control group.There was no significant difference in autophagy rate in U50488H preconditioning group with both dose of 0.1?mol/L and 1 ? mol/L compared with H/R group,while there was significant difference in both the autophagy rate and the experession of Atg5 mRNA in 10 ? mol/L group.ConclusionsThe hypoxia/reoxygenation model for studying autophagy of H9c2 cardiomyocytes can be established by using 0.5%FBS combined with high glucose medium or glucose-free medium in hypoxic incubator.The level of autophagy of cardiomyocyte is related to the degree of hypoxia/reoxygenation injury.Moderate ischemia and hypoxia can activate autophagy,while excessive ischemia and hypoxia can inhibit autophagy.Preconditioning with U50488H,a selective kappa-opioid receptor agonist,increased the level of autophagy of H9c2 cardiomyocytes in a concentration-dependent manner,but had no significant effect on the cell survival rate during hypoxia/reoxygenation injury,which suggested that the cell survival may be affected by many factors,which need further study.