Effect Of Salvianolate On The PI3K/Akt Pathway And MitoKATP And Its Protection In Hypoxia/Reoxygenation Cardiomyocytes

Objective To investigate the effect of salvianolate on PI3K/Akt pathway,mitoKATP of hypoxia/reoxygenation H9C2 cardiomyocytes and its role in cardiomyocyte apoptosis,and to study the protective effect of salvianolate on hypoxia/reoxygenation cardiomyocytes.Methods Experiments were executed in four parts.Part1:To culture H9C2 cardiomyocytes,establish myocardial ischemia reperfusion injury model by hypoxia/reoxygenation method and observe cell morphology under inverted microscope.Part2:To investigate the optimal concentration of salvianolate on myocardial H/R injury.H9C2 cardiomyocytes were randomly assigned to six groups:control,H/R,H/R+salvianolate25mg/L,H/R+salvianolate50mg/L,H/R+salvianolate100mg/L,H/R+salvianolate200mg/L.Part3:The protective effect of salvianolate on hypoxic-reoxygenated cardiomyocytes by mitochondrial ATP sensitive potassium channel and its mechanism.Cardiac myocytes were randomly assigned to five experimental groups:control,H/R,H/R+salvianolate,H/R+salvianolate+5-HD,H/R+5-HD.After treatment in each experimental group,the activity of cardiomyocytes in each group was detected by CCK-8 method.Part4: To investigate the mechanism of salvianolate in the process of PI3K/Akt and mitoKATP pathway mediating the apoptosis of myocardial cells induced by hypoxia/reoxygenation.H9C2 cardiomyocytes were randomly divided into four groups: control,H/R,H/R+salvianolate,H/R+salvianolate+5-HD.MRNA levels of P53,Bcl-2,Bax,PI3K and Akt in each group were determined by RT-PCR.Results(1)Under an inverted microscope,normal cardiomyocytes showed fusiform adherent growth,and the damaged cells were spherical and not adherent,suspended in the medium.(2)The results of CCK-8 method showed that the cell viability of the H/R group was significantly lower than that of the control group(P<0.001);Compared with the H/R group and other salvianolate groups,when the salvianolate was at a concentration of100mg/L,the OD value of cardiomyocytes was the largest and the cell viability was the best.Compared with the H/R group,the cell survival rate of the salvianolate group at100mg/L was significantly increased(P<0.001).Therefore 100mg/L is the optimal protective drug concentration for salvianolate,and this concentration is used in subsequent experiments.(3)After the application of 5-HD,CCK-8 test results showed that compared with the H/R+salvianolate group,the activity of myocardial cells in the H/R+salvianolate+5-HD group was significantly reduced(P<0.05).However,5-HD alone had no effect on the activity of H/R cardiomyocytes.(4)Compared with the control group,the m RNA expression levels of P53,Bax,PI3K and Akt in the H/R group were increased(P<0.05).Compared with the H/R group,the m RNA expression levels of pro-apoptotic genes P53 and Bax in the H/R+salvianolate group were significantly decreased(P<0.001),while the expression level of anti-apoptotic genes Bcl-2 was significantly increased(P<0.001),and the expressions of PI3K and Akt were also increased(P<0.01).Compared with the H/R+ salvianolate group,the expressions of P53 and Bax in the H/R+salvianolate+5-HD group were increased(P<0.01),while the expressions of Bcl-2,PI3K and Akt were decreased(P<0.05).Conclusions Salvianolate pretreatment can inhibit myocardial cell apoptosis,improve the activity and survival rate of myocardial cells,and reduce ischemia-reperfusion injury.This protective effect may depend on the activation of PI3K/Akt and mitoKATP pathways,which open a new direction for the application of traditional Chinese medicine in the cardiovascular field.