Effect And Mechanism Of Protein Kinase D Inhibitor On Hydrogen Peroxide-induced H9c2 Cardiomyocytes Oxidative Stress Injury

Background:During acute myocardial infarction,the major damage to myocardial tissue is caused by initial ischemia and subsequent reperfusion injury.There are various explanations for the pathology of myocardial ischemia-reperfusion injury,but oxidative stress has been widely accepted as one of the important factors.Active oxygen radicals play a major role in oxidative stress.Excessive production first destroys mitochondrial function and directly affects energy metabolism,eventually leading to loss of myocardial function.However,studies in recent years have confirmed that the production of proper amount of reactive oxygen species can activate protein kinase D and alleviate myocardial ischemia-reperfusion injury,but the specific mechanism of action is still unclear.Objective:To elucidate the role of protein kinase D in mitochondrial oxidative stress levels in H9c2 cardiomyocyte apoptosis.Methods:1.H9c2 cardiomyocytes were treated with H₂O₂?25,50,100,200,400umol/L?at different concentrations.CCK-8 assay was used to detect cell proliferation activity,cell survival rate was calculated,and H₂O₂concentration with a cell survival rate of about 60%was selected to establish an oxidative stress injury model.2.All H9c2 cardiomyocytes were randomly divided into four groups:Control group;H₂O₂?Hydrogen peroxide?model group;CRT0066101?5umol/L?preconditioning+H₂O₂ model group;CRT0066101?5umol/L?treatment group;3.The ROS production of H9c2 cardiomyocytes was detected by reactive oxygen species detection kit and fluorescence microscope.The changes of mitochondrial membrane potential were observed by Rhodamine123 fluorescence staining.The levels of reactive oxygen species in each group were detected by DCFH-DA and flow cytometry.Annexin V-FITC/PI double staining flow cytometry for detecting apoptosis of H9c2 cardiomyocytes in each group.4.The expression of p-PKD,PKD,Bax,Bcl-2 and Cleaved Caspase-3 protein in cells was detected by Western blot.Results:1.The effect of different concentrations of H₂O₂ on cell viability was detected by CCK-8 method.The results showed that there was no significant effect on cell viability except H₂O₂ concentration of 25umol/L.The other concentrations increased with H₂O₂ concentration on H9c2cardiomyocytes.The inhibition also increased gradually,and the apoptosis increased.When the concentration of H₂O₂ was 200umol/L,the cell survival rate was about 62.36%,which was close to 60%.Therefore,the oxidative stress model of H9c2 cardiomyocytes was prepared by using 200umol/L H₂O₂.2.The ROS production of H9c2 myocardial cells in each group was detected by reactive oxygen assay kit and fluorescence microscope.The results showed that compared with the Control group,the fluorescence intensity of the H₂O₂ model group and the CRT0066101 pretreatment+H₂O₂ model group were significantly increased,and the fluorescence intensity of the CRT0066101 treatment group was not significantly changed.Compared with the H₂O₂ model group,the fluorescence intensity in the CRT0066101 pretreatment+H₂O₂ group increased more significantly.3.The changes of mitochondrial membrane potential in each group were observed by fluorescent staining with Rhodamine 123.The results showed that the fluorescence intensity of H₂O₂ model group and CRT0066101 pretreatment+H₂O₂ model group were significantly lower than that of Control group.The fluorescence intensity of CRT0066101treatment group was not obvious.Compared with the H₂O₂ model group,the CRT0066101 pretreatment+H₂O₂ group showed a more significant decrease in fluorescence intensity.4.DCFH-DA and flow cytometry were used to detect the ROS levels in each group.The results showed that compared with the Control group,the ROS levels in the H₂O₂ model group and the CRT0066101pretreatment group and the H₂O₂ model group were all increased,while the levels in the CRT0066101 treatment group were not significantly changed.Compared with the H₂O₂ model group,the reactive oxygen level in the CRT0066101 pretreatment+H₂O₂ model group increased more significantly.5.Annexin V-FITC/PI double staining flow cytometry was used to detect the apoptosis rate of H9c2 myocardial cells in each group.The results showed that compared with the Control group,the apoptosis rate of H₂O₂ model group and the CRT0066101 pretreatment+H₂O₂ model group was significantly increased,while the apoptosis rate of the CRT0066101 treatment group was not significantly changed.However,compared with the H₂O₂ model group,the apoptosis rate increased more significantly in the CRT0066101 pretreatment+H₂O₂ group.6.In Western blot experiments,the protein expression levels of p-PKD?Bax?Cleaved caspase-3 in H9c2 myocardial cells in the H₂O₂model group and the CRT0066101 pretreatment+H₂O₂ model group were significantly increased compared with the Control group,and the protein expression levels of Bcl-2 were decreased.In the CRT0066101 treatment group,the expression of p-PKD was significantly decreased,but the expression of other proteins was not significantly changed compared with the Control group.However,the protein expressions of p-PKD and Bcl-2in the CRT0066101 pretreatment+H₂O₂ group were significantly down-regulated compared with the H₂O₂ model group,and the protein expressions of Bax and Cleaved caspase-3 were increased.PKD expression in each group showed no significant change.Conclusions:1.Rat H9c2 myocardial cells were exposed to 200umol/L H₂O₂solution for 2h,which could induce oxidative stress injury model of myocardial cells.2.Pretreatment with protein kinase D inhibitor CRT0066101significantly reduced H9c2 cardiomyocyte activity and increased apoptosis after H₂O₂ stimulation.3.Protein kinase D regulates H₂O₂-induced apoptosis induced by oxidative stress in H9c2 cardiomyocytes via the mitochondrial pathway.