| AIM:Ischemic heart disease, especially in myocardial ischemia-reperfusion injury, has a highmorbidity and mortality, which threats to human life. Adiponectin is one of the cytokines offat cells, which can protect the myocardiac cells, It can reduced MI/R injury significantly byinhibit myocardial apoptosis. The research of the mechanism in myocardial protection ofadiponectin in recent decades has been ongoing, but the exact mechanism of adiponectinmyocardial protection is not yet clear. The recently studies have found that adiponectin canprotect the myocardium by calreticulin (CRT)/CD91. This experiment aims to study therole of CRT/CD91in adiponectin against myocardial ischemia reperfusion injury and thespecific mechanisms of CRT/CD91in adiponectin myocardial protection, so as to be thepromotion of the research progress of adiponectin myocardial protection.METHODS:1. To build the model of myocardial ischemia-reperfusion injury in mice through the use of the CRT/CD91Stealth RNAi gene silencing processing, Then divided all70C57BL/6j (20~25g) into7groups according to the principle of random: Operation controlgroup (sham); Operation control+Scramble SiRNA group (sham+Scramble SiRNA);Myocardial ischemia-reperfusion injury group (MI/R+Scramble SiRNA); Myocardialischemia-reperfusion injury+CRT genome inhibiting group (MI/R+Calreticulin SiRNA);Myocardial ischemia-reperfusion injury+LRP-1inhibit genome group (MI/R+SiRNALRP-1); Myocardial ischemia-reperfusion injury suppression++CRT genome APN intreatment group (MI/R+Calreticulin SiRNA+gAPN); Myocardial ischemia-reperfusioninjury+genome inhibit LRP-1+APN in treatment group (MI/R+SiRNA LRP-1+gAPN). At last, Using the western blotting to detect myocardial CRT and CD91(LRP-1)expression level, detecting left ventricular ejection fraction with ultrasonic, detectingmyocardial infarction area with myocardial Evan’s blue/TTC staining, and the PI3K, andAkt, the expression level of eNOS phosphorylation with western blotting.2. To establish the myocardial hypoxia reoxygenation reperfusion injury model, Thendivided all SD rats into7groups according to the principle of random: The control group(control);Control+Scramble SiRNA group (control+Scramble SiRNA);Myocardialischemia-reperfusion injury group (MI/R+Scramble SiRNA);Myocardialischemia-reperfusion injury+CRT genome inhibiting group (MI/R+CalreticulinSiRNA);Myocardial ischemia-reperfusion injury+LRP-1inhibit genome group (MI/R+SiRNA LRP-1);Myocardial ischemia-reperfusion injury suppression++CRT genomeAPN in treatment group (MI/R+Calreticulin SiRNA+gAPN);Myocardialischemia-reperfusion injury+genome inhibit LRP-1+APN in treatment group (MI/R+SiRNA LRP-1+gAPN).At last, Detecting the expression level of CRT and LRP-1, theusing TUNEL apoptosis dyeing, Caspase3activity detection, LDH release quantity todetect the cell apoptosis. downstream molecular PI3K, and Akt, eNOS phosphorylationexpression level with western blotting.RESULTS:1. The myocardial ischemia-reperfusion injury in mice in vivo:1) Western blotting showed the expression level of CRT and LRP-1was rose significantly (p <0.01) in myocardial ischemia-reperfusion injury in mice, thecardiac function was declined, the myocardial infarction area was increased.2) After the Si RNA of CRT and CD91process, the cardiac function was declined,the myocardial infarction area was increased.3) In the treatment group with adiponectin, the myocardial infarction size and cardiacfunction improved significantly than the siRNA processing group in mice, theinfarction area reduced significantly, cardiac function recovered significantly (p <0.01). All the result ecplain the CRT and LRP-1play a role in adiponectin protectthe myocardium against ischemia-reperfusion injury.4) Western blotting results was shown that the expression of PI3k was increased, Akt,eNOS phosphorylation has been increase significantly after ischemia-reperfusion;After the Si RNA of CRT and CD91process, the expression of PI3k was decreased,Akt, eNOS phosphorylation has been decreased; after treatment in adiponectin, theexpression of PI3k was increased, Akt, eNOS phosphorylation has been increasesignificantly (p <0.01). All the result ecplain the CRT and LRP-1play a role inadiponectin in myocardial ischemia-reperfusion injury of protection, which throughPI3K/Akt/eNOS way.2. In vitro myocardial cell ischemia-reperfusion injury in mice experiment:1) Western blotting showed the expression level of CRT and LRP-1was rosesignificantly (p <0.01) in myocardial cell reoxygenation and the release of LDHand caspase-3activity was increased significantly2) After the Si RNA of CRT and D91process, the cell apoptosis and the release ofLDH and caspase-3activity was increased significantly (p <0.05).3) In the treatment group with adiponectin, the apoptosis of myocardial cellsdecreased significantly and the release of LDH quantity and caspase-3activitysignificantly reduced (p <0.01). All the result explain that the CRT and LRP-1playa role in adiponectin protect the myocardium against ischemia-reperfusion injury4) Western blotting results was shown that after reoxygenation,the expression of PI3k wasincreased, Akt, eNOS phosphorylation has been increased; After the Si RNA of CRTand D91process, the expression of PI3k was decreased, Akt, eNOS phosphorylation has been decreased; After treatment in adiponectin, the expression of PI3k wasincreased, Akt, eNOS phosphorylation has been increase significantly (p <0.01). Allthe result explain that the CRT and LRP-1play a role in adiponectin in myocardialischemia-reperfusion injury of protection, which through PI3K/Akt/eNOS wayCONCLUSION:This topic in myocardial ischemia-reperfusion injury model in mice and rats myocardialhypoxia reoxygenation model by using Stealth RNAi gene silencing processing to blockingCRT and CD91, the findings was enumerate as following:1. In the myocardial ischemia-reperfusion injury in mice and rats myocardial cellhypoxia reoxygenation, the expression level of CRT and CD91(LRP-1) was increasedsignificantly, the myocardial cell injury and apoptosis significantly increased; After the SiRNA of CRT and D91process, the myocardial cell injury increased, which proved that theCRT and CD91play a role in the protection in the MI/R; After the application ofadiponectin myocardial cell injury and apoptosis significantly reduced, and the cardiacfunction improved significantly, suggests that adiponectin can significantly improve themyocardial ischemia-reperfusion injury. The result above illustrating that the importantpotential of the CRT and CD91in protecting myocardial ischemia-reperfusion injury ofadiponectin.2. The downstream molecular of CRT and CD91: PI3K, and Akt, eNOSphosphorylation level was increased in myocardial ischemia-reperfusion injury in vivo andrats obviously up-regulated after myocardial cell hypoxia reoxygenation, after the use ofStealth RNAi to block the CRT/CD91gene expression, the PI3K, and Akt, eNOSphosphorylation levels were significantly lower. Howerver, after the application ofadiponectin the PI3K, and Akt, eNOS phosphorylation levels significantly raised. Prove thatthe CRT and CD91play a role in the protection of adiponectin through PI3K/Akt/eNOSpathways. |
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