Protective Effect And Mechanism Of Tetrandrine On Cardiomyocyte Injury Induced By Hypoxia/Reoxygenation Of H9c2 Cells

Acute myocardial infarction is a high incidence in the clinic,and the mortality of patients with this disease is in the forefront of cardiovascular disease.At present,the effective method of diagnosis and treatment for patients with acute myocardial infarction is to implement cardiovascular dredging in the first time.Percutaneous coronary intervention(PCI)is the main method to rescue patients with acute myocardial infarction(AMI),but this method often leads to myocardial ischemia/reperfusion injury,which seriously affects the clinical treatment effect.Therefore,it is of great clinical significance to find an effective method to improve the myocardial ischemia/reperfusion injury after PCI.Tetrandrine is an alkaloid extracted from the dry root of Stephania tetrandra S.Moore.Studies have shown that tetrandrine has many biological activities,such as anti-inflammatory,anti-cancer,antihypertensive,immunosuppressive and cytoprotective effects.However,the regulation of tetrandrine on the energy metabolism of myocardial ischemia reperfusion injury is not clear.The purpose of this study was to observe the protective effects of tetrandrine at different concentrations on hypoxia/reoxygenated cardiomyocytes,and to investigate the changes of energy metabolism and the expression of related signal molecules,eg.Akt,HIF-α and STAT.In addition,the signal mechanism for the protection of hypoxia/reoxygenated myocardial cells was further clarified by interfering with the JAK/STAT signaling pathway.Part one Effects of tetrandrine at different concentrations on the viability and energy metabolism of hypoxia/reoxygenation H9c2 cardiomyocytesObjective: To observe the effects of different concentrations of tetrandrine on the viability and energy metabolism of H9c2 cells after hypoxia /reoxygenation.Methods: The viability of myocardial cells was determined by MTT experiment,the survival rate of cardiac myocytes was measured by Hochest assay,and the energy metabolism of cardiac myocytes was evaluated by detecting glucose consumption and lactate level.Results: Tetrandrine could attenuate the myocardial injury induced by hypoxia/reoxygenation which was concentration-dependent.There was significant difference between the middle and high dose groups and the model group(cell survival rate: 66.1±6.5%;76.2±5.1%,model group,42.7 ±5.9g,P < 0.05).Redue the content of lactate dehydrogenase(High dose group: 142.8±25.1 U/I;model group:323.8±32.8 U/I,P<0.05).Reduce apoptosis and increase glycolysis,restore cell energy supply,increase ATP content in a concentration dependent manner(High dose group: 18.65±2.02 nm/mg,model group: 6.24±1.24 nm/mg,P < 0.05).Summary: tetrandrine can protect the myocardial ischemia / reperfusion(MI/R)damage by improving the glycolysis level of cardiac myocytes.Part two Tetrandrine attenuates hypoxia/reoxygenated H9c2 cardiomyocyte damage by regulating STAT pathwayObjective: To investigate the effects of tetrandrine on the changes of Akt,HIF-α and STAT signal molecules in hypoxia/reoxygenated cardiomyocytes.Methods: The protein expression of Akt,HIF-α,STAT and hexokinase were measured by Western blot.Results: The protein expression of hexokinase could be significantly up-regulated by tetrandrine.It has regulatory effect on upstream regulatory protein STAT,but does not on PI3K/Akt and HIF-α.Summary: The STAT pathway plays an important role in alleviating hypoxia/reoxygenation of myocardial cells.Part three The mechanism of tetrandrine regulate STAT signaling pathwayObjective: Tetrandrine has been found to protect hypoxia/reoxygenated myocardium injury by regulating STAT,but the mechanism remains unclear.Methods: This study investigated the activity of H9c2 myocardial cells,lactate dehydrogenase activity,caspase 3 activity and energy metabolism by combining the powders with different JAK inhibitors.Results: The effect on cell viability,lactate dehydrogenase level and glycolysis level have no significantly diffrecent in single tetrandrine group between combinded with JAK1/2 inhibitor,but afer combinded with JAK3,the protected effect on hypoxia/reoxygenation cells by tetrandrine was sinificently decreased.Summary: Tetrandrine can protect the hypoxia/reoxygenation myocardium by JAK3/STAT pathway.Conclusion: Tetrandrine could improve the injury of hypoxic/ reoxygenated cardiomyocytes in a concentration-dependent manner,reduce cell apoptosis,restore the level of glycolysis,and up-regulate the protein expression of glycolytic rate-limiting enzyme hexokinase.Tetrandrine could regulate the upstream regulatory protein STAT,but had no effect on PI3K/Akt and HIF-α.When Tetrandrine and JAK3 inhibitor were used together,the protective effect of Tetrandrine on cells was significantly decreased,and the regulation of glycolysis was obviously disappeared,which was significantly different from that of Tetrandrine alone.However,when Tetrandrine and JAK1/2 inhibitor were used together,the protective effect of Tetrandrine on the cells was not significant,but there was no significant difference in the regulation of glycolysis between the two groups.