The Mechanism Of 14-3-3? Mediating Gastrodin Against Cardiomyocytes Anoxia/Reoxygenation Injury

Objective: Gastrodin is the highest effective monomer from traditional Chinese medicine gastrodia,and has been widely used in cardiovascular applications in recent years.In this study,H9c2 cells were used to establish the model of Anoxia/Reoxygenation(A/R)injury,and then the formed 14-3-3?-siRNA adenovirus was mediated to further investigate the mechanism of how 14-3-3? works in the process that gastrodin protects against cardiomyocytes from A/R damage.Methods: H9c2 myocardial cells were randomly divided into 5 groups after culture and repeated 6 times for each group.H9c2 cardiomyocytes were divided as following groups:(1)Control group;(2)(A/R)group;(3)GAS + A/R group;(4)GAS + negative control + A/R group;(5)GAS + siRNA14-3-3?+ A/R group.The expression of 14-3-3? protein was detected by Western Blotting;the MTS assay was used to measure the cell survival rate of each group;the flow cytometry was used to test each group's apoptosis,reactive oxygen species(ROS)and mitochondrial membrane potential(??m).And the content of creatine phosphokinase(CPK),the activity of lactate dehydrogenase(LDH),casepase 3 and the opening of mitochondrial permeability transition pore(mPTP)in each group were also determined.Results: 1.Comparing the A/R group and gastrodin pretreatment group of different concentrations,we found that gastrodin concentration of 20mg/L 14-3-3? protein expression significantly increased,and the cell survival rate was the most obvious,the difference has statistical significance(P<0.01).2.Compared with the A/R group,the GAS pretreatment group with the concentration of 20 mg/L could significantly reduce the generation of ROS,maintain the stability of mitochondrial membrane potential,decrease the activity of LDH and CPK,depress the activity of casepase 3 and the openning of mPTP(P<0.01),eventually reduce cell apoptosis,the difference has statistical significance(P<0.01).Compared with GAS + A/R group,when transfected with adenovirus siRNA14-3-3?,The anti-myocardial A/R damage effect of gastrodin was reversed.The results showed that the protective effect of GAS on myocardial cells were correlated with 14-3-3 protein.Conclusion: Gastrodin has a protective effect on H9c2 cardiomyocyte A/R injury,which may be related with inhibition of oxidative injury by up-regulating 14-3-3 ? protein expression.