Research Of Insulin Inhibition Of Cardiac Icrovascular Endothelial Cells Against Ischemia/Reperfusion Injury: Through Akt/FoxO/SVV Pathways

BackgroundAs a common clinical syndrome, acute myocardial infarction means the necrosis ofmyocardium caused by the sudden occlusion of coronary artery. Revascularization therapyis crucial for those patient,however often accompanied with futhur undesiable injurydefined as ischemia/reperfusion injury(I/RI).Which may cause the deterioration of thedesease even death.For now,we don’t have effective treatment strategies towards I/RIwhich make it an improtant area for reaseachers. Previously,study reported that theapoptosis of cardiac microvascular endothelial cell occurred earlier than thecardiomyocytes at the early stage of I/RI[1], which might suggest the inhibation of theapoptosis of CMECs could reduce the early injury of I/RI. Insulin,one of the improtantcompotent of GIK solution,which has been proved the inhibiation effect of apoptosis ofcardiomyocytes or endothelial cell throguh activetion of “Akt/eNOS/NO” survival signalpathway. However, whether insulin could protect CMECs against I/RI and the possiablemechniasm are still not sure.Both FoxO1and FoxO3a are recently discovered moleculars of FoxO family proteins which regulate apoptosis through phosphorylation. However, whether insulin couldactivate FoxO1or FoxO3a during the I/R is still unclear. As a recently discoveredapoptosis inhibitory protein,survivin played an important role in the such as repair ofvascular injury,the regulation of smooth muscle cell proliferation and so on.We previouslyhave confirmed that insulin could up regulate the expression of survivin to inhibitedapoptosis of CMECs induced by ischemia/reperfusion injury CMECs.The exact mechanism is still not clear whether FoxO1or FoxO3a are related.In thisstudy, we established the simulated ischemia/reperfusion model of CMECs and observedthe protection effect of insulin. Furthurmore, we try to test the roles of Akt/FoxO1/SVVand Akt/FoxO3a/SVV during I/RI.ObjectiveTo explore the effect and mechanisms of insulin protection effect of cardiacmicrovascular endothelial cells against ischemia/reperfusion injury.MethodsCMECs were isolated from the adult Sprague-Dawley rats hearts. Establish thesimulated ischemia/reperfusion model. Cells were devided randomly into five groups:1) simulated ischemia/reperfusion;2）SI/R+Insulin;3) SI/R+Insulin+Wortmanningroup;4)SI/R+Insulin+FoxO1siRNA;5）SI/R+Insulin+FoxO3a siRNA.The apoptosis ofCMECs was detected by TUNEL,Transwell was used to detect cell migration,theexpression or the phosphorylation of Akt,p-Akt,FoxO1,p-FoxO1,FoxO3a,p-FoxO3a andSVV protein was analyzed by Western blot.Results1. Cardiac microvascular endothelial cells were isolated and cultured successfullyand then the ischemia/reperfusion model was established.2. Both the capase-3and apoptosis index were increased after SI/R compared withSI/R+Ins (P<0.05). While administration of FoxO1si RNA and FoxO3asi RNAduringreperfusion dramatically increased apoptosis index and capase-3when comparedwith SI/R+Ins (P<0.05).3. Western blot analysis showed that when compared with SI/R, insulin protein cansignificantly increase SVV protein level, after added with PI3K specific inhibitorwortmannin, SVV protein expression decreased (P<0.05) compared with SI/R+Ins,At the same time when added with FoxO1/3a siRNA,SVV protein expressiondecreased,moreover SI/R+Ins+FoxO3a siRNA Significantly lower than SI/R+Ins+FoxO1siRNA.ConclusionInsulin could exert protective effect of CMECs against ischemia/reperfusion injurythrough activation of Akt/FoxO1and FoxO3a/SVV pathways, survivin are the downstream molecus of both pathways, however phosphorylation of FoxO3a may play animportant role.