The Protective Effect And Mechanism Of Preconditioning With Jiawei Danshen Decoction On H9C2 Cardiomyocytes During Hypoxia/reoxygenation Injury

[Objective] To investigate the protective effect of Yiqi Huoxue Prescription of Jiawei Danshen Decoction on H9C2 cardiomyocytes during hypoxia / reoxygenation injury and its infuluence on autophagy,speculateing the anti myocardial ischemia reperfusion injury mechanism,to provide experimental basis for clinical application of Jiawei Danshen Decoction.[Methods] The blank and drug-containing serum were made with serum pharmacology method,and the final working concentration of medicated serum were established by four methylthiazolyl tetrazolium(MTT)assay and lactate dehydrogenase(LDH)kit.H9C2 cardiomyocytes cutured in vitro were used to establish model of hypoxia/reoxygenation injury and randomly divided into six groups.They were serum control group(CG),hypoxia / reoxygenation group(HRG),Jiawei Danshen Decoction group(JDG),JDG +3-methyladenine(a inhibitor of autophagy)group(JIG)and JDG +Rapamycin(RAPA)(an agonists of autophagy)(JAG).The growth and morphological changes of myocardial cells in each experimental group were observed by inverted microscope;The cell viability of H9C2 cardiomyocytes were measured by MTT assay.The LDH leakage rate and CK activity of cell culture supernatant were measured by colorimetric method.Transmission electron microscope was applied to observe the changes of autophagosome formation in cardiomyocytes.The real-time fluorescence quantitative PCR and Western blot(Western blot)methods were used respectively to detect the expression of autophagy related genes Atg5,Beclin1 m RNA and autophagy related protein Beclin1 LC3.[Results] The cell survival rate increased compared with 5%,20%,25% and 30% contained serum group(P<0.05)after cutured with15%(v/v)serum of Jiawei Danshen Decoction 48 hours,however,the LDH leakage rate and CK activity in culture supernatant decreased(P<0.05);Compared with the CG group,the degree of myocardial cell degeneration and necrosis in HRG group rised under the inverted microscope,the cell survival rate decreased(P<0.01),LDHleakage rate and CK activity increased in the cell culture supernatant,autophagosomes increased under electron microscope,the expression level of Atg5 and Beclin1gene up-regulated,Beclin1 and LC3 protein expression was increased;Compared with HRG group,the degree of myocardial cell degeneration in JDG and JAG group both reduced,meanwhile,the cell survival rate significantly increased(P<0.01),LDH leakage rate and CK activity in culture supernatant decreased,the autophagosome formation in cardiomyocytes increased under electron microscope,the expression of Atg5,Beclin1 m RNA further increased(P<0.05),the expression of Beclin1 and LC3 protein increased;Compared with HPG,the morphological changes and growth conditions in JDG and JAG group were similar under inverted microscope,and the LDH leakage rate and CK activity in the supernatant were no significant difference,but the autophagic vacuoles increased significantly,and the expression of Atg5,Beclin1 m RNA increased,the expression of Beclin1,LC3 protein increased,too.(P<0.05).[Conclusion] The final working concentration of medicated serum was 15%;Preconditioning with Jiawei Danshen Decoction has a delayed protective effect on H9C2 cardiomyocytes during Hypoxia/Reoxygenation Injury,and its mechanism may be by up-regulating autophagy.Both medicated serum pretreatment group and hypoxia preconditioning group can simulate ischemia preconditioning protection,but the mechanisms may be different,the degree of autophagy in medicated serum group was more apparent.The protective effect of hypoxia preconditioning may be mediated by up-regulating autophagy partly.