Protective Effects Of Hydroxy Safflower Yellow A In Hypoxia/Reoxygenation Induced H9c2 Cardiomyocytes By PI3K/Akt/Hexokinase Ⅱ Pathway

Part one Effects of hydroxy safflower yellow A on the viability of H9c2 myocardial cellsObjective: With the wide application of coronary interventional therapy,coronary artery bypass and coronary artery thrombolytic therapy,myocardial ischemia / reperfusion(myocardial ischemia/reperfusion,MI /R)caused two times injury is a common clinical problem,seriously affect the health of patients.Hydroxysafflor yellow A(Hydroxysaffloryellow A HSYA)is traditional Chinese herbal medicine for circulation disease,the study shows that HSYA has obvious protective effect on vascular tissue injury,but hydroxysafflor yellowA in ischemia reperfusion injury in remains to be studied.Methods: This experiment used H9c2 myocardial cell culture to establishment of hypoxia / reoxygenation model,through the MTT experiment examine the hydroxysafflor yellow A on the survival rate of the myocardial cell effect;detection of lactate dehydrogenase(Lactate cells dehydrogenase,LDH),superoxide dismutase(Superoxide dismutase,SOD)(Methane dicarboxylic,aldehyde MDA,MDA)index to evaluated the effects of hydroxysafflor yellow A on oxidative stress;at the same time,PI3 K,hexokinase,ERK inhibitors treated with hydroxysafflor yellow A in hypoxia/oxygen H9c2 model.Result:1 Compared with the normal group,hypoxia / reoxygenation cell model group cell survival rate decreased significantly in the model cell.In HSYA groups,with the increase of the dose showed a significant protective effect.2 Compared with the model group,when combinded with PI3 K inhibitorLY294002 or hexokinase inhibitor 3-BrPA,HSYA protective effect decreased significantly,and had no significant differences to model groep,In ERK inhibitors group,alough,the hydroxysafflor yellow A myocardial cell protection effect is weak,but no significant difference with HSYA group.3 Compared with the normal group,LDH increased in model group significantly,indicating that cells mitochondria injury increased significantly.to the hydroxysafflor yellow A could significantly decreased LDH lever,and has significant differences with the model group.But when given to PI3 K inhibitor LY294002 or hexokinase inhibitor 3-BrP,the protective effect of HSYA was significantly reduced,and in ERK inhibitors,the HSYA myocardial cell protection also decreased,but with no significant difference to the single drug group.4 Compared with the normal group,hypoxia / reoxygenation model group SOD significantly decreased,MDA significantly increased.The results showed that the oxidative stress of cells was increased significantly,and in HSYA group,MDA significantly increased,and the content of SOD was significantly higher than model group.But when combinded with PI3 K inhibitor LY294002 and hexokinase inhibitor 3-BrPA,the cellular response to oxidative stress improved by HSYA effect was significantly decreased,while for ERK inhibition,the HSYA myocardial cell protection also decreased,but with no significant difference to the HSYA group.Part two Effects of hydroxy safflower yellow A on mitochondria and energy metabolism of H9c2 myocardial cellsObjective: Mitochondria play a key role in MIRI.Ischemia reperfusion injury,oxidative stress can lead to mitochondrial dysfunction,affecting normal mitochondrial energy supply,reduce the synthesis of ATP,influence cell function,and oxidative stress which induced by ischemia reperfusion also can cause the release of cytochrome C from mitochondria,leading to activation of caspase 3 that trigger cell apoptosis.HSYA has a certain protective effect can improve oxidative stress in myocardial cells in hypoxia reoxygenation model,for mitochondrial function whether it has protective effect to be investigated.Methods: This study tested ATP content,cytochrome C in cytoplasm and mitochondria in hypoxia / reoxygenation in H9c2 cell.At the same time,evaluation of protection the effect on cell apoptosis in HSYA by using Annexin-V-FITC double staining and caspase 3.Result:1 Compared with the normal group,ATP decreased significantly in hypoxia / reoxygenation cells;HSYA could significantly increased ATP in model cells and with significant difference compared with the model group.At the same time,PI3 K inhibitor LY294002 or HK inhibitor 3-BrPA group,ATP decreased significantly,but in ERK inhibitors group,has no significant difference to HSYA group.2 Compared with the normal group,Content of cytochrome C in cytoplasm of model group was significantly increased,decreased significantly in mitochondria.HSYA can significantly reduce the cytochrome C content in cytoplasm,increased mitochondrial content showed a significant protective effect.To the PI3 K inhibitor LY294002 or HK inhibitor 3-BrPA,the protective effect of HSYA was significantly reduced,cytosolic cytochrome C content was significantly higher than single group,and decreased the contents of cytochrome C in mitochondria,However,there was no significant difference between the two groups after treatment with ERK inhibitors.3 Compared with the normal group,Content of Caspase3 increased significantly in model cells;HSYA decreased the content of Caspase3 and has significant difference with the model group.To PI3 K inhibitor LY294002 or HK inhibitor 3-BrPA,the protective effect of HSYA significantly decreased,Caspase3 content increased significantly with the single drug groups have significant differences;To ERK inhibitors has no significant difference to HSYA groep.4 Compared with the normal group,Cell apoptosis increased significantly in the model group;Treatment with HSYA has significant difference with model group.To PI3 K inhibitor LY294002 or HK inhibitor 3-BrPA,apoptosiswas significantly increased,the inhibition effect of apoptosis by HSYA was significantly reduced.Part three Effects of hydroxy safflower yellow A on Akt,GSk-3 beta,Hexokinase Ⅱ in H9c2 cellsObjective: The phosphatidylinositol-3-kinase(phosphatidylinositol-3-OH,kinase,PI3K)and extracellular signal regulated kinase(extracellular signal-regulated protein kinase,ERK)two signaling pathways called reperfusion injury salvage kinase signal pathway,RISK signal pathway.Both PI3K/Akt and ERK can promote the phosphorylation of GSK-3,which can reduce the consumption of mitochondrial ATP and reduce the production of ROS.HK II is an important target protein of PI3K/Akt signaling pathway,which plays an important role in Akt mediated myocardial mitochondrial protection.Activated Akt is raised to mitochondria and induces increased mitochondrial interaction with HKII.We found that the protective effect of HSYA on ischemia reperfusion injury,but in the PI3 K,ERK,HK II inhibitor under different protective effects.In this study,we investigated the effect of HSYA on PI3 K,ERK,HK II in cells.Methods: In this study,through the establishment of hypoxia /reoxygenation in H9c2 cells,gaven the HSYA and different inhibitors,cells were detected by Western blot p-Akt,p-GSK-3 beta,HK II protein content.Result:1 Compared with the normal group,p-Akt in the cytoplasm were increased in hypoxia / reoxygen model cell group;HSYA could increased the content of p-Akt cells with significant difference compared with the model group.At the same time,the content of p-Akt was significantly decreased after PI3 K inhibitor.2 Compared with the normal group,the levels of p-GSK-3 in the cytoplasm of the hypoxia / reoxygenation model group increased,and the content of GSK-3 in the cells increased after HYSA,which was significantly different from that in the model group.However,the activation of Akt wassignificantly weaker than that of PB98059,and the content of GSK-3 was significantly decreased after ERK inhibitor,ERK inhibitor PB98059 significantly reduced the induction of p-GSK-3 by the HYSA.3 Compared with the normal group,the expression of HK II in the hypoxia / reoxygenation model group was significantly decreased,but the content of HK II in the cells was significantly higher than that in the model group and the blank group after HYSA.At the same time,the content of HK II was significantly reduced after HK II inhibitor 3-BrPA,and the effect of HK II inhibitor 3-BrPA on the induction of HK II was significantly decreased.Conclusions:1 In the hypoxia / reoxygenation model,the protective effect of HSYA on cells was dependent on concentration manner.2 HSYA can significantly reduce the level of cellular oxidative stress,reduce the level of LDH,caspase 3 and apoptosis in cells,and restore mitochondrial energy metabolism.3 PI3 K inhibitor pretreatment(LY294002)or hexokinase Ⅱ inhibitor(3-BrPA),the protective effect of HSYA decreased significantly.In contrast,pretreatment with ERK inhibitor(PD98059)had no significant effect on the protective effect of HSYA on myocardial cells.4 ERK through the phosphorylation of GSK3-beta to protect the myocardium,but the effect of HSYA on GSK3-beta phosphorylation is weak.Hydroxy safflower yellow can significantly up regulate Akt and hexokinase Ⅱ.At the same time,the levels of GSK3-beta phosphorylation were significantly decreased after the treatment with ERK5 This study showed that the protective effect of HSYA was mainly through PI3K/AKT/Hexokinase Ⅱ pathway.