| [Objectives]During recent years,the occurance rate of ST-elevated Myocardial Infarction(STEMI)kept increasing in China.Although reperfusion therapy has been widely conducted,its clinical effect is still being interfered by many factors.In the aspect of basic research,the existence of ischemia/reperfusion(I/R)injury tremendously damage the effect of reperfusion therapy and can still not be satisfactorily prevented or treated.Thus,there is no standard prevention or treatment of myocardial I/R injury mentioned in the current guidelines.The objectives of current study are to explore feasible optimizing strategy for STEMI using basic research methods.Try to use All-trans retinoic acid(ATRA)as a preventional treatment of myocardial I/R injury,and to clarify the mechanism of ATRA in I/R injury as well as the correlation with the effect of Receptor for Advanced Glycation Endproducts(RAGE)during I/R injury.Specifically,the current study was designed to use myocardial I/R model in both cardiaomyocyte and animal level and detect whether ATRA could effectively reduce I/R injury;to explore whether this effect is majorly obtained through antagonizing RAGE effect and other possible mechanism,and moreover,to explore and conclude feasible strategy of antagonizing myocardial I/R injury,thus presenting a theoretical foundation and interventional strategy for reducing I/R injury.[Methods](1)Establish a hypoxia/re-oxygenation injury model of H9C2 cells to mimic I/R injury.Compare cell survival rate under different ATRA concentration(10nM-100uM)interference,thus confirming the effective concentration and toxic concentration of ATRA.Use Annexin V and Flow Cytometry method to detect anti-apoptotic effect of ATRA with different concentration on cardiomyocyte.Use TUNEL staining method to to detect anti-apoptotic effect of ATRA.Use Western-blots method to detect apoptotic related signal Capase-3(cysteinyl aspartate specific proteinase-3),BCL-2(B-cell lymphoma-2),p38MAPK(p38 mitogen-activated protein kinase),JNK(c-Jun N-terminal kinase)and ERK(extracellular regulated protein kinases)expression with or without ATRA interference.(2)Under the platform of hypoxia/re-oxygenation injury model of H9C2 cells which mimics I/R injury,use Western-blots method to detect expression difference of A disintegrin and metalloproteinase 10(ADAM10)and RAGE with different concentration of ATRA interference.Repeat the experiments mentioned above while using ADAM10-shRNA interference and detect apoptotic signal Capase-3,BCL-2,p38 MAPK,JNK and ERK expression with different concentration of ATRA interference.Use Carboxy-H2 DCFDA reagent to detect oxdative stress with different concentration of ATRA interference.(3)Use mice to establish a model of acute myocardial infarction and reperusion by left anterior descending artery(LAD)ligation surgery.Use TTC staining method to detect infarcted area difference while using ATRA to interfere both wild type mice and RAGE gene knock out mice.Use echocardiogram method to detect left ventricular function difference while using ATRA interference.Use TUNEL staining method to detect difference of cardiomyocyte apoptosis while using ATRA interference.Use Western-blots method to detect apoptotic signal Capase-3,BCL-2,p38 MAPK,JNK,ERK expression with or without ATRA interference.Use Immunohistochemical staining method to confirm expression of apoptotic signal p38 MAPK,JNK,ERK,as well as ADAM10 and RAGE expression in ventricular tissue slice with or without ATRA interference.[Results](1)In the model of H9C2 cells hypoxia/re-oxygenation injury which mimics I/R injury,cell survival rate was significantly higher using ATRA concentration 1nM to 10?M compared with DMSO control(p<0.01).However,cell survial rate was grately decreased using ATRA concentration at 100?M(p<0.01).The amount of Annexin positive cell which represents apoptotic cell was significantly decreased(p<0.01)while using ATRA interference between concentration 1nM to 10?M and this was found in a concentration dependent pattern.TUNEL staining results showed that with ATRA concentration 10 nM to 10?M,the amount of TUNEL positive cell which represents apoptotic cell was significantly decreased(p<0.01)and was also in a concentration dependent pattern.Western blots showed that with ATRA concentration 1nM to 10?M,the expression of caspase-3 which straightly related with apoptosis was significantly decreased and the expression of BCL-2 which was a protective marker of apoptosis was significantly increased(both p<0.01).Moreover,the expression of phosphorylate-activated apoptotic signal p38 MAPK,JNK and ERK were significantly decreased(all p<0.01)and this also presented in a concentration dependent pattern.(2)In H9c2 cells hypoxia/re-oxygenation injury model,the expression of ADAM10 was significantly increased when incubated with ATRA concentration 10 nM to 10?M while RAGE expression significantly decreased(both p<0.01),and this was found in a positive and negative correlation pattern separately.ADAM10-shRNA effectively silence the expression of ADAM10(p<0.01)and significantly increased RAGE expression(p<0.01).Use ATRA1?M did not restore ADAM10 expression and over-expressed RAGE signal(p>0.05).While ADAM10 was silenced by using ADAM10-shRNA,the expression of caspase-3 was significantly increased(p<0.01)and BCL-2 significantly decreased(p<0.01).While H9c2 cells were incubated with both ATRA1?M and ADAM10-shRNA,the expression of caspase-3 was significantly decreased comparing with using ADAM10-shRNA alone(p<0.01),but was significantly increased comparing with using ATRA1?M alone(p<0.01),and the expression of BCL-2 was significantly increased comparing with using ADAM10-shRNA alone(p<0.01),but was significantly decreased comparing with using ATRA1?M alone(p<0.01).While ADAM10 was silenced by using ADAM10-shRNA,the expression of phosphorylate-activated apoptotic signal p38 MAPK,JNK and ERK were significantly decreased(all p<0.01).While H9c2 cells were incubated with both ATRA1?M and ADAM10-shRNA,the expression of phosphorylate-activated apoptotic signal p38 MAPK,JNK and ERK were significantly decreased comparing with using ADAM10-shRNA alone(all p<0.01),but was significantly increased comparing with using ATRA1?M alone(p<0.01).After incubated with ATRA concentration 10 nM to 10?M,ROS production detected by Carboxy-H2 DCFDA reagent was significantly decreased(p<0.01)and this was found in a concentration dependent pattern.(3)TTC staining relusts showed that infarcted area in wild type mice with left anterior descending artery(LAD)ligation and reperfusion model was significantly decreased while using ATRA pre-treatment(p<0.05).The same result was found in RAGE gene knock out mice comparing with wild type mice with or without ATRA pre-treatment(both p<0.05).Echocardiogram revealed that cardiac output,left ventricular end-diastolic volume and ejection fraction of wild type mice with ATRA pre-treatment and received LAD ligation and reperfusion was significantly decreased(all p<0.05)comparing with sham mice,but the cardiac output and left ventricular ejection fraction was still significantly higher than those did not receive ATRA pre-treatment(both p<0.05).TUNEL stating of myocardium slice showed that the amount of apoptotic cells was significantly decreased with ATRA pre-treatment(p<0.05).The expression of apoptotic protective signal BCL-2 in ventricular cardiomyocytes was significantly increased while caspase-3 was significantly decreased with ATRA pre-treatment(both p<0.05).The expression of phosphorylate-activated apoptotic signal p38 MAPK,JNK and ERK were significantly decreased ATRA pre-treatment(all p<0.05).Similarly,Immunohistochemical staining results showed significantly decreased phosphorylate-activated apoptotic signal p38 MAPK,JNK and ERK(all p<0.05),significantly increased ADAM10 expression and significantly decreased RAGE expression(both p<0.05).[Conclusion]Our study revealed that ATRA treatment could significantly prevent myocardial I/R injury subsequent to reperfuion therapy after STEMI.1)ATRA could effectively prevent I/R injury after reperfuion,decrease myocardial apoptosis and infarct area.2)The effect of ATRA in myocardial I/R injury prevention is majorly due to elevated expression of ADAM10 level,thus increasing RAGE cut which leads to myocardial apoptosis.3)ATRA is also effective in reducing oxidative stress by down-regulation of reactive oxygen species(ROS)which is also correlated with myocardial I/R injury. |
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