| Objective:To investigate the relationship between the amelioration of myocardial ischemia-reperfusion injury（MIRI）by Hyperoside（Hyp）and PKC（Protein kinase C）/mitoKATP（mitochondrial ATP channel）signaling pathway in the myocardium.Methods:1.In vivo experiments:MIRI rat model was established by ligating the left anterior descending branch of the coronary artery.After one week of adaptive feeding,72 healthy male SD rats were randomly divided into 9 groups（n=8 for each）,i.e.,Sham,model（MIRI）,Hyp（50 mg/kg）,Hyp+PKCαblocker BisI（50 mg/kg+2.5mg/kg）,Hyp+PKCεblocker CHE（50 mg/kg+8 mg/kg）,Hyp+mitoKATP blocker5-HD（50 mg/kg+10 mg/kg）,PKCαblocker BisI（2.5 mg/kg）,PKCεblocker CHE（8mg/kg）,mitoKATP blocker 5-HD（10 mg/kg）.Rats were given 0.9%normal saline（NS）by gavage in Sham and MIRI group,while rats in Hyp group and Hyp-treatment combined with inhibitor group were given Hyp（50 mg/kg）by gavage for 10 days.At the end of intragastric administration on the 10th day,rats were intraperitoneally injected with each corresponding inhibitor 1 hour before the operation in Hyp-treatment combined with inhibitor group and each corresponding inhibitor group.Electrocardiograms of rats before ischemia,30 minutes after ischemia and 2 hours after reperfusion were recorded by the BL-420 biological function acquisition system recorder.After reperfusion,blood was collected from the abdominal aorta and serum was centrifuged.The levels of CK-MB,MDA,SOD,and ATP were monitored by ELISA.HE,TTC and TUNEL staining were used to observe the myocardial histopathological changes and apoptosis rate.Western blot was utilized to observe the effect of Hyp and inhibitors of PKCα,PKCε,and mitoKATP signal pathway on the protein expression of Nrf2,Caspase-3,PKCε,and Kir6.2 in myocardial tissue of rats with myocardial ischemia-reperfusion injury.2.In vitro experiment:Hypoxia reoxygenation H9c2 cardiomyocytes were randomly assigned to Normal group,Hypoxia reoxygenation（Model）group,Hyp（50μmol/L）group,Hyp+PKCαblocker BisI（50μmol/L+1μmol/L）group,Hyp+PKCεblocker CHE（50μmol/L+4μmol/L）group,Hyp+mitoKATP blocker GB（50μmol/L+3 nmol/L）group,BisI（1μmol/L）group,CHE（4μmol/L）group,and mitoKATP blocker GB（3 nmol/L）group.ELISA was used to measure the content of ATP and MDA and activity of SOD and CK-MB in the supernatant of H9c2 cardiomyocytes.Western blotting was used to observe the effects of Hyp and inhibitors of PKCα,PKCεand mitoKATP signal pathway on Nrf2,Caspase-3,PKCεand Kir6.2 protein levels in H9c2 cardiomyocytes.Flow cytometry and laser confocal technique were used to detect the effect of Hyp and signal pathway blockers on the apoptosis rate and Ca2+concentration in H9c2 cardiomyocytes.Results:1.In vivo experiments:（1）Compared with those of the Sham group,ST segment of ECG in MIRI group was significantly elevated as compared with those of the Sham group（P<0.01）,and the ventricular wall in ligation area was gray-white with naked eyes.After reperfusion,the ST segment gradually fell back,which proved that the model was successful.（2）The results of ELISA showed that the MDA content and CK-MB activity in the serum of rats with MIRI group were significantly increased,and SOD activity and ATP content were significantly decreased as compared with those of the Sham group（P<0.01）.Compared with those of the MIRI group,Hyp obviously reduced MDA content and CK-MB activity,and increased SOD activity and ATP content（P<0.01）.Compared to those in the Hyp group,the MDA content in the serum of MIRI rats was increased,the SOD activity and ATP content were significantly decreased in the Hyp-treatment together with inhibitors group（P<0.05,P<0.01）.Compared with those of the corresponding inhibitor groups,MDA content and CK-MB activity decreased,and SOD activity and ATP content increased in the Hyp-treatment combined with inhibitor groups（P<0.01）.（3）The results of HE staining showed that the anterior wall of areas in the anterior wall of myocardium in MIRI group,with obvious eosinophilic changes,disordered cell arrangement,hyperemia of cytoplasm and light staining;The results of TUNEL staining showed that the apoptosis index of cardiomyocytes increased significantly in MIRI groups;TTC staining showed that the infarct area of MIRI group was larger.In Hyp group and Hyp-treatment combined with the inhibitor group,the pathological damage of myocardium was significantly improved,and the white area was evidently reduced as compared with those in the Sham group.The pathological damage of rats in the Hyp-treatment combined with inhibitor group was still obvious as compared to those in the Hyp group.（4）Western blot results show that,compared with those of the Sham group,Nrf2 and PKCεprotein expression in MIRI group significantly decreased（P<0.01）,Kir6.2 protein expression was not apparently different（P>0.05）,and Caspase-3 protein expression markedly increased（P<0.01）.Compared with those in the MIRI group,the expression levels of Nrf2,PKCεand Kir6.2 proteins in Hyp group were significantly increased（P<0.01）,and Caspase-3 protein expression was obviously decreased（P<0.01）.The expression of Nrf2,PKCεand Kir6.2 proteins was down-regulated to some extent（P<0.05,P<0.01）,and Caspase-3 protein was up-regulated to some extent as compared with those in the Hyp group（P<0.05）.However,the upregulatory effect of Hyp on the protein expression of Nrf2,PKCε,Kir6.2,and Caspase-3 was significantly inhibited by PKCαblocker BisI,or PKCεblocker CHE,or mitoKATP blocker 5-HD（P<0.05,P<0.01）.2.In vitro experiment:（5）The results of ELISA showed that,compared with those in the Normal group,MDA content and CK-MB activity were significantly increased,and SOD activity and ATP content were significantly decreased in the supernatant of H9c2cardiomyocytes in the Model group（P<0.01）.On the contrary,Hyp significantly reduced the MDA content and CK-MB activity and increased the SOD activity and ATP content in the supernatant of H9c2 cardiomyocytes following hypoxia/reoxygenation injury.In contrast with those of the Hyp group,ATP content significantly was decreased in the supernatant of H9c2 cardiomyocytes in Hyp-treatment combined with inhibitor group（P<0.05）.MDA content and CK-MB activity were apparently decreased,and SOD activity and ATP content were markedly increased in Hyp-combined with inhibitor group as compared with those of the corresponding inhibitor groups（P<0.01）.（6）In contrast with those of the Normal group,Nrf2 and PKCεprotein expression was significantly decreased in the supernatant of H9c2 cells in the Model group（P<0.01）,Kir6.2 protein expression was not significantly different（P>0.05）,while Caspase-3 protein expression was increased（P<0.01）.Hyp evidently increased the expression of Nrf2,PKCεand Kir6.2 proteins,and decreased the expression of Caspase-3 protein in H9c2cardiomyocytes following hypoxia/reoxygenation injury（P<0.01）.The expression of Nrf2,PKCε,Kir6.2 proteins was down-regulated（P<0.05,P<0.01）,and the expression of Caspase-3 protein was up-regulated in the Hyp-treatment combined with inhibitor group in contrast with those of the Hyp group（P<0.01）.The protein levels of Nrf2,PKCε,and Kir6.2 were increased significantly（P<0.05,P<0.01）,and Caspase-3 protein expression was decreased notably in Hyp-treatment combined with inhibitor groups as compared with those in the corresponding inhibitor groups（P<0.01）.（7）Compared with those of the Normal group,the apoptosis rate of H9c2cardiomyocytes was increased significantly in Model group（P<0.01）.Hyp markedly decreased the apoptotic rate of H9c2 cardiomyocytes following hypoxia/reoxygenation injury（P<0.01）.The apoptotic rate of H9c2 cardiomyocytes in Hyp-treatment combined with inhibitor groups was increased obviously in contrast with those of the Hyp group（P<0.05,P<0.01）.Compared with those in the corresponding inhibitor groups,the apoptotic rate of H9c2 cardiomyocytes was significantly lower in Hyp-treatment combined with inhibitor group（P<0.05,P<0.01）.（8）The results of laser confocal detection show that Ca2+fluorescence intensity of Model group increased obviously as compared with those of the Normal group（P<0.01）.Hyp significantly decreased Ca2+fluorescence intensity of H9c2cardiomyocytes following hypoxia/reoxygenation injury（P<0.01）.Ca2+fluorescence intensity of H9c2 cardiac myocytes in Hyp-treatment combined with inhibitor groups was increased as compared with those in the Hyp group（P<0.05）,which was significantly decreased in contrast with those of the corresponding inhibitor groups（P<0.05,P<0.01）.Conclusion:Hyp has beneficial effects on alleviating myocardial ischemia-reperfusion injury in rats.The mechanism may be related to activation of the PKC/mitoKATP signaling pathway in the myocardium of rat.Further research indicates that Hyp may not only induce protective effect against myocardial ischemia-reperfusion injury via activating PKCαdirectly,but also opening mitoKATP channel through the activation of PKCε,then regulate the structure and function of many kinds of myocardial subcellular cells,and remove oxygen free radicals,and maintain the function of mitochondria,and restore energy metabolism,and inhibit Ca2+flow into the cell,and reduce cell apoptosis. |
