Ischemia Postconditioning Protects Cardiomyocyte Against Ischemia Reperfusion Injury Through HIF-1α/iNOS Pathway

Backgrounds Amute myocardial infarction(AMI)has been the worldwide main cause of mortality these years.Although percutaneous transcoronary angioplasty(PTCA)can open the blocked vessls in time and reflow the ischemia myocardium,the following ischemia reperfusion(I/R)injury brings more problem.Various cardioprotection strategy against I/R injury show limited effect,despite plenty of work focusing on them.The pharmacological treatment has been reported to be difficult to match the target selectively.Though the cardioprotection of ishcmeia preconditioning(IPC)is significantly effective,meanwhile it is limited in clinical application by the unpredictability of the ischemia.Then ischemia postconditioning(IPostC)attracts wide concern as its nice clinical adaptive and excellent cardioprotective effects.However,the main mechanism of IPostC is still not confirmed.Hypoxia induced factor(HIF),as the transcriptional regulator of various genes,can trigger a series of reaction as its natural degration is inhibited by the dcrease of oxygen and oxidation reaction under ischemia pathological condition.Many results evidenced that the increase of HIF-1α expression shows significant cardioprotection,which can enhance the tolerance of cardiomyocyte against ischemia and limit the infarct size.Furthermore,it has been reported that overexpression of HIF-1αcanattenuate the I/R injury,and the increase of HIF-1α by IPostC can inhibit the infarction of the heart.Multifarious of downstream protein of HIF-1α,such as VEGF,Glut-4,Glut-1 and iNOS,have been revealed to be cardioprotective.But it’s still not cear whether HIF-1α takes a part in the cardioprotection of IPostC.And which downstream protein makes the main contribution and trggers more signalpathways to inprocve the beneficial effect of IPostC?What’s more,the protective effect of hypoxia postconditioning in H9C2has been reported to be connected with the increase of iNOS recently.According to the activity of NO/cGMP and ROS,which have close ties with iNOS,in the mechanism of postconditioning,we speculate that iNOS palys an important role in the postconditioning process.And we think that IPostC protecting the heart against I/R injury increases the expression of iNOS,which is the downstream protein of HIF-1α,by upregulating the HIF-1α.Objective:To investigate the cardioprotective against I/R injury by IPostC through HIF-1α/iNOS pathway.Methods:Establish mice ischemia reperfusion model and cardiomyocyte hypoxia reoxygenation model.Select C57/B6 male mice randomly for operation and inject 100mg/kg 2-MeOE2(HIF-α inhibitor)or 2mg/kg 1400W(iNOS inhibitor)15min before reperfusion.Extract tissue protein from Control,I/R,IPostC,IPostC+2-MeOE2 and IPostC+1400W groups.Culture H9C2 cardiomyocyte and add 2-MeOE2(to 5μmol/L)or 1400W(to 20μmol/L)on the onset of reperfusion.Extract cell protein from groups including Control,H/R,HPostC,HPostC+2MeOE2 and IPostC+1400W.We used siRNA to inhibit the expression of HIF-1α,which consisted of 4 groups like Control+NS-siRNA,H/R+NS-siRNA,HPostC+NS-siRNA and IPostC+HIF-1α-siRNA.We used 1%triphenyltetrazolium chloride staining to measure the infarct size.Cell apoptosis was detected by Annexin V-FITC staining with the flow-cytometer and CCK-8.The level of HIF-1α protein and iNOS protein in the samples frome tissues and cells were tested by Western Blot.Real-Time PCR was used to detect the expression of mRNA of iNOS in the samples of tissues and cells.Results:1)TTC staining evidenced IPostC can reduce the infarct size,meanwhile 2-MeOE2 and 1400W can inhibit this beneficial effect respectively.2)The cardioprotection against apoptosis of IPostC,which was detected by Annexin V-FITC staining and CCK-8,can also be attenuated by 2-MeOE2 and 1400W respectively in H9C2 cells.3)The expression of HIF-1α and iNOS was upregulated by IPostC,which was detected by Western Blot and RT-PCR.4)The siRNA inhibiting HIF-1αwas found limiting the protection of IPostC by CCK-8.5)We found that the inhibition of HIF-1α can attenuate the cardioprotection of IPostC and decrease the expression of iNOS.Conclusion:IPostC can upregulate the expression of HIF-1α and iNOS to improve the myocardium function against I/R injury.Inhibition of iNOS and HIF-1αrespectively can neutralize the cardioprotectionof postconditioning.HIF-1α can regulate iNOS as the upstream protein in the mechanism of postconditioning.We concluded that IPostC can attenuate T/R injury through HIG-1α/iNOS pathway.