The Effect Of HSYA On Anti-myocardial Ischemia-reperfusion Injury By Regulating Mitochondrion

Objective：To construct rat models of myocardial ischemia-reperfusion injury bysurgical methods, and to explore the role and mechanism of HSYA anti-myocardialischemia-reperfusion injury.Methods：(1) Rat models of myocardial ischemia-reperfusion injury wereestablished by surgical methods; using biochemical reagent kits to detect CK, LDH,SOD and GSH-Px activities, and the contents of MDA in serum, H2O2and NO;calcium concentration was measured by flow cytometry with fluo-3fluorescenceprobe; apoptosis was measured by TUNEL and flow cytometry double staining withAnnexin Ⅴ-FITC kits, respectively; LC3protein expression was measured withWestern blotting, LC3Ⅱ/LC3Ⅰindicated the autophagy;(2) Cyt c and gap junctionprotein in mitochondia, Cyt c in cytoplasma, apoptosis related proteins Bcl-2, Bax andcaspase-3in cells, autophagy related proteins Beclin1, Agt5and pAkt in cells, weremeasured by Western blotting assay, respectively; and eNOS in tissue homogenateswas measured by enzyme-linked immunosorbent assay (ELISA).Results：(1) After myocardial ischemia-reperfusion lasting for1h and24h, ascompared with sham group, CK and LDH in serum release increased, the contents ofMDA in serum, H2O2and NO in tissue homogenates increased significantly, butSOD and GSH-Px activities in serum decreased, and calcium ion concentration incells increased significantly, resuls of flow cytometry and TUNEL assay showed thatapoptotic cells increased, and then, Western blotting result showed that LLC3Ⅱ/LC3Ⅰratio increased, it indicated autophagy increasing, these indicators were moreobvious in groups myocardial ischemia-reperfusion lasting for24h;(2) Aftermyocardial ischemia-reperfusion lasting for1h and24h, Western blotting resultsshowed, as compared with sham group, Cyt c expression in myocardial mitochondriareduced, and that in cytoplasma increased, and Bcl-2expression in cells decreased,but Bax expression increased, and caspase-3was activated; plasma membranedCa2+-ATPase (PMCA) and sarco/endoplasmic reticulum Ca2+ATPase (SERCA)expression decreased, autophagy related proteins of Beclin1and Atg5all increased,and phosphorylated Akt and eNOS activity all decreased, but Akt expression did not change obviously, the chages of these protein expressions was more obvious in24hgroup.(3) After HSYA treatment for myocardial ischemia-reperfusion model rats,serum CK and LDH release decreased, the contents of serum MDA and H2O2inhomogenates all decreased, serum SOD and GSH-Px all increased, but NO content inhomogenates increased, calcium ion concentration decreased in cells, apoptosis andautophagy all decreased.(4) Western blotting results showed that after HSYAtreatment for myocardial ischemia-reperfusion moded rats, mitochondrial Cyt cexpression increased, but cytoplasma Cyt c decreased, Bcl-2expression increased,Bax expression decreased, and caspase-3activation decreased, SERCA expressionincreased, but PMCA expression changes was not obvious, Beclin1and Atg5alldecreased, pAkt and eNOS all increased, but Akt did not showed obvious change.Conclusion：(1) Myocardial ischemia-reperfusion injury can cause CK and LDHactivity increasing in serum, and enhance oxidative damage on myocardial cells,reduce antioxidant capability, induce calcium ion overload, increase apoptosis andautophagy, and it was more serious at24h of reperfusion injury.(2) HYSA can playagainst myocardial ischemia-reperfusion injury role by reducing these indicators asdecribed above;(3) HSYA can play role against calcium overload induced bymyocardial ischemia-reperfusion, and the role can be regulated by SERCA, but not byPMCA.(4) Anti-myocardial ischemia-reperfusion injury of HSYA may be related toreducing Cx43, thereby inhibiting the generation of reactive oxygen species in thecells.(5) The inhibitory effects of HSYA on apoptosis induced by myocardialischemia-reperfusion injury involved the activation of the mitochondrial pathway, andwas regulated by Bcl-2protein expression decreasing and Bax protein expressionincreasing;(6) The inhibitory effects of HSYA on autophagy induced by myocardialischemia-reperfusion injury was related to Beclin1and Agt5expression decreasingand activation of pAkt-eNOS pathway.