Study On The Activity And Mechanism Of Plantamajoside Against Hypoxia Reoxygenation Injury In H9c2 Myocardial Cells

Myocardial ischemia is closely related to the development of various heart diseases,and it is an acute cause and aggravating factor for myocardial infarction and malignant arrhythmia.Restoring blood supply to the ischemic part of the heart is currently the main treatment for ischemic heart disease,but blood reperfusion can lead to Myocardial Ischemia Reperfusion Injury(MIRI)which cause the patients' condition into deteriorate and affect the prognosis of patients.Prevention and treatment of myocardial ischemia-reperfusion injury is still the main problem faced by cardiovascular doctors.The pathogenesis of MIRI is complex,and the increase of oxygen free radicals,myocardial energy metabolism disorder and calcium overload are closely related to the occurrence and development of MIRI,and the interaction eventually leads to myocardial cell necrosis and apoptosis.Increased oxygen free radicals cause mitochondrial dysfunction,which activates the mitochondrial apoptotic signaling pathway and leads to apoptosis through a number of signal transductions.It is also a hot research topic to inhibit the increase of oxygen free radicals and activate the relevant pathways that inhibit apoptosis.The Plantamajoside has anti-oxidation,anti-apoptosis and anti-inflammatory effects.The previous study of our group confirmed that the Plantamajoside has effect of anti-cardiomyocyte hypertrophy,However,the research on the myocardial ischemia reperfusion injury of the Plantamajoside has been reported rarely.Therefore,this study establishes a model of hypoxia-reoxygenation injury of H9c2 cardiomyocytes in vivo to simulate vitro myocardial ischemia-reperfusion injury.To study protection of Plantamajoside against MIRI and its possible mechanisms.At the same time,there are studies have revealed that the activation of ILK/Akt/c-Src signaling pathway is closely related to the pathophysiological process of MIRI.ILK overexpression can reduce the number of cardiomyocyte apoptosis during reperfusion injury.This study explored the possible role of this pathway in the inhibition of myocardial ischemia reperfusion by the PMS,and elucidated its related mechanisms.ObjectiveTo study the effect of plantamajoside on hypoxia-reoxygenation injury of H9c2 cardiomyocytes,and to explore the protective effect of plantamajoside on myocardial ischemia-reperfusion injury.Methods(1)MTT assay to determine the effect of hypoxia-reoxygenation injury on H9c2 cardiomyocyte survival rate,to establish H9c2 cell hypoxia reoxygenation injury model;(2)MTT assay to determine different concentrations(5-120?M)of PMS for H9c2 cardiomyocytes Survival rate;(3)Activity verification of the plantamajoside by lactate dehydrogenase release test,Hoechst 33342 fluorescence stain test,malondialdehyde release test,superoxide dismutase activity test and ROS release test;(4)Annexin V/PI double staining assay for the inhibition activity of apoptosis of H9c2 cells by plantamajoside;(5)Western blotting was used to determine the effect of PMS on the mitochondrial apoptosis signal-ing pathway and ILK/Akt/c-Src signaling pathway protein expression products in H9c2 cells against hypoxia-reoxygenation injury related pathways.(6)RT-PCR assay to detect the effect of PMS on gene expression in H9c2 cells against hypoxia-reoxygenation-related pathways.Results(1)Hypoxia for 7 hours and reoxygenation for 6 hours can significantly reduce the survival rate of H9c2 cells to about 50%;(2)There is not cytotoxicity of the PMS in the concentration range of 5-40 ?M,the PMS(10,20,40 ?M)increased cell survival induced by hypoxiareoxygenation in a concentration-dependent way and is treated cells alone does not affect proliferation and growth;(3)Different concentrations of PMS(10,20,40 ?M)compared with hypoxia reoxygenation injury(H/R)group,The PMS can significantly reduce the release of LDH,MDA and ROS,and increase the activity of SOD enzyme in a concentration-dependent way;(4)Compared with the H/R injury group,different concentrations PMS(10,20,40?M)can significantly reduce cell apoptosis in a concentration-dependent way;(5)The PMS can promote the expression of ILK,p-Akt,Bcl-2 protein,and reduce the expression of Bax,Caspase-3,p-c-Src and Cytochromec in a concentration-dependent way.(6)RT-PCR was used to detect the effect of PMS on mitochondrial apoptosis signaling pathway and ILK/Akt/c-Src signaling pathway gene expression products in H9c2 cells against hypoxia-reoxygenation injury-related pathways.ConclusionsThe Plantamajoside has protective effects on hypoxia reoxygenation injury of H9c2 cells,and its protective mechanism may be related to ROS clearance,inhibition of mitochondrial apoptosis pathway and activation of ILK/Akt/c-Src signaling pathway.