| Objective:Ischemic heart disease is a common chronic non-communicable disease that induces myocardial ischemia,hypoxia and even necrosis,and it is the primary cause of heart failure.Ischemic heart disease has become a leading cause of death which seriously threatens human health.At present,the main methods for clinical treatment of ischemic heart disease include thrombolytic therapy and arterial bypass surgery.These therapies can effectively reestablish coronary blood flow in a short period of time and improve myocardial ischemic injury to some extent.However,myocardial ischemia/reperfusion(I/R)may cause more severe damage to myocardial structure and function,which was defined as myocardial I/R injury.Currently,several mechanisms of myocardial I/R injury have been identified,including oxygen free radical aggregation,neutrophil-endotheliocyte interaction,intracellular calcium overload,cardiomyocyte apoptosis,abnormal energy metabolism,and etc.Since myocardial I/R is a complicated pathological process which involves multiple mechanisms,no rapid and effective methods have been applied for improvements of myocardial damage caused by myocardial I/R.Ischemic preconditioning and postconditioning have been widely applied for treatments of myocardial I/R injury.However,the applicability of ischaemic preconditioning is limited by the unpredictable nature of ischaemic events during the operative period.Ischemic postconditioning includes local ischemic postconditioning and remote ischemic postconditioning(RIPostC).Although it has been proved that local ischemic postconditioning can effectively reduce infarct size and preserve endothelial function in a myocardial IR model,the local ischemic postconditioning strategy involves repetitive trauma to the artery that may contribute to restenosis,which limits its clinical application in organs that have suffered life-threatening ischemia.RIPostC is an effective,feasible and non-invasive method to preserve ischemic heart through repeated occlusion/release cycles in the lateral femoral artery.A large number of animal experiments and clinical studies have shown that RIPostC can reduce myocardial damage caused by myocardial I/R.However,little is known about the molecular mechanisms underlying cardioprotection induced by RIPostC.Aaldehyde dehydrogenase 2(ALDH2)is one of the key enzymes involved in alcohol metabolism in mitochondria.ALDH2,closely related to oxidative stress,can reduce the generation of reactive oxygen species(ROS);resulting in inhibition of cell injury.ROS generation is an important mechanism for myocardial I/R injury,and mitochondria are important sources of ROS.Therefore,ALDH2 located in mitochondria plays an extremely important role in regulating oxidative stress,which makes us hypothesize that the cardioprotective effects of RIPostC may be associated with ALDH2.However,the relevant researches are limited at present.In this study,we explored the effects of RIPostC treatment on myocardial I/R injury both in vivo and in vitro.In the meantime,the effect of RIPostC treatment on the expression level of ALDH2 in mouse myocardial tissues and rat H9c2 cells was also examined.The purpose of this study was to analyze the relationship between cardioprotective role of RIPostC and the expression of ALDH2,and to provide a theoretical basis for elucidating the cardioprotection mechanism of RIPostC.Methods:Chamber One.RIPostC alleviates myocardial I/R-induced injury and up-regulates ALDH2 expression in miceA total of 30 male C57BL/6 mice were randomly assigned into three groups(n =10 per group)including the Sham group,I/R group and I/R + RIPostC group.Mice in the I/R group received tracheotomy,and mice were ventilated with mixed atmosphere consisting of air and oxygen.After a left thoracotomy,an 8-0 silk suture with a slipknot was ligated around the left anterior descending coronary artery(LCA).The ischemia lasted for 45 min,and the following reperfusion(2 h)was established through releasing the slipknot.Mice in the Sham group received same surgical procedures without I/R.Mice in the I/R + RIPostC group received I/R for 45 min,and the RIPostC treatment induced by three cycles of left femoral artery occlusion(5 min)with a microvascular clamp under an operating microscope and reperfusion(5 min),during the reperfusion period.After that,the atriums and the right ventricle were removed,and the left ventricle was stained by 2%Evans blue dye and 2,3,5-triphenyltetrazolium chloride solution(TTC;2%).The percentage of myocardial infarct size/area at risk(AAR)was analyzed.The ALDH2 mRNA and protein expression levels in the left ventricle tissues were measured by reverse transcription-quantitative PCR(RT-qPCR)and Western blot analysis,respectively.Chamber Two,RIPostC attenuates oxygen-glucose deprivation(OGD)/re-oxygenation(OGD/R)-induced cell injury through up-regulating ALDH2 expressionRIPostC attenuates OGD/R-induced H9c2 cell injuryCells were divided into three groups including the Control group(cells were kept in normoxic culture for 5 h),the OGD/R group(cells received OGD for 3 h followed by re-oxygenation for 2 h).and the OGD/R + RIPostC group(OGD for 3 h,followed by three cycles of 5 min re-oxygenation and 5 min OGD,then re-oxygenation for 90 min).Cell viability,percentage of apoptotic cells,ROS generation,expression of p53 protein and proteins related to apoptosis were testified by CCK-8 assay,flow cytometry,DCFH-DA staining and Western blot analysis,respectively.RIPostC up-regulates ALDH2 expressionH9c2 cells were divided into three groups including the Control group,the OGD/R group and the OGD/R+RIPostC group,and ALDH2 protein expression was measured by Western blot analysis.RIPostC alleviates OGD/R-induced cell injury through up-regulating ALDH2 expressionH9c2 cells were transfected with sh-NC or sh-ALDH2.and ALDH2 protein expression was determined by Western blot analysis.H9c2 cells were divided into five groups including the Control group,the OGD/R group,the OGD/R + RIPostC group,the OGD/R + RIPostC + sh-NC group and the OGD/R + RIPostC +sh-ALDH2 group.Then,cell viability,percentage of apoptotic cells,ROS generation,expression of p53 protein and proteins related to apoptosis were all measured.Chamber Three,RIPostC up-regulates ALDH2 expression via the PI3K/mTOR pathway,and thereby attenuates OGD/R-induced cell injuryH9c2 cells were incubated in DMEM containing PI3K inhibitor(LY294002)or mTOR inhibitor(Rapamycin)for 0,6,12 and 24 h,and expression of PI3K,phospho(p)-PI3K,mTOR and p-mTOR was determined by Western blot analysis.Subsequently,H9c2 cells were divided into four groups including the Control group,the OGD/R group,the OGD/R + RIPostC group and the OGD/R + RIPostC +LY294002/Rapamycin group.Expression of PI3K,p-PI3K,mTOR and p-mTOR was determined.Results:Chapter One,RIPostC alleviates myocardial I/R-induced injury and up-regulates ALDH2 expression in micePercentage of infarct size/AAR in left ventricle tissues from the I/R group was 56.9%± 2.9%while that from the I/R + RIPostC group was 27.9%± 4.1%.The percentage of infarct size/AAR in the I/R + RIPostC group was significantly lower than the I/R group(P<0.01),indicating that RIPostC could attenuate myocardial I/R-induced myocardial infarct and improve the outcome of myocardial I/R injury.The relative expression of ALDH2 mRNA in the left ventricle tissues from the Sham group,the I/R group and the I/R + RIPostC group was 0.99± 0.05,0.46 ± 0.09 and 0.80 ± 0.04.The relative expression of ALDH2 protein in the Sham group,the I/R group and the I/R + RIPostC group was 1.00 ± 0.05.0.37±0.07 and 0.79±0.08.Both mRNA and protein expression levels of ALDH2 in the I/R group were significantly lower than the Sham group(both P<0.01).Moreover,mRNA and protein expression levels of ALDH2 in the I/R + RIPostC group were markedly higher than the I/R group(both P<0.05).Results indicated that myocardial I/R could down-regulate ALDH2 expression,and the down-regulation could be mitigated by RIPostC treatmentsChapter Two,RIPostC attenuates OGD/R-induced cell injury through up-regulating ALDH2 expressionRIPostC attenuates OGD/R-induced H9c2 cell injuryCompared with the Control group,OGD/R induced significant reduction of cell viability(P<0.01).up-regulation of p53 protein expression(P<0.01).increase of apoptotic cells(P<0.001),up-regulation of pro-apoptotic Bax and cleaved caspase-3 as well as down-regulation of anti-apoptotic Bcl-2,and enhancement of ROS generation(P<0.01).In the meantime,we found those effects of OGD/R on H9c2 cells were all significantly mitigated by RIPostC,presenting significant increase of cell viability(P<0.01).down-regulation of p53 protein expression(P<0.05).decrease of apoptotic cells(P<0.05),down-regulation of Bax and cleaved caspase-3 as well as up-regulation of Bcl-2,and reduction of ROS generation(P<0.05),when compared with the OGD/R group.Those results collectively indicated that RIPostC could alleviate OGD/R-induced H9c2 cell injury.RIPostC up-regulates ALDH2 expressionOGD/R prominently down-regulated ALDH2 protein expression relative to the Control group(P<0.01),and the down-regulation was bated by RIPostC relative to the OGD/R group(P<0.05).Results suggested the possible involvements of ALDH2 in the protective role of RIPostC against OGD/R injury.RIPostC alleviates OGD/R-induced cell injury through up-regulating ALDH2 expressionAfter lentiviral infection,ALDH2 protein levels in cells transfected with sh-ALDH2 were dramatically lower than that in sh-NC-transfected cells(P<0.001),presenting that ALDH2 was successfully silenced after lentiviral infection.Then,transfected and untransfected cells received OGD/R with or without RIPostC,and cell viability,p53 expression,cell apoptosis and ROS generation were all testified.Results showed that effects of RIPostC on OGD/R-treated cells were all remarkably reversed by ALDH2 knockdown,as evidenced by decreased cell viability(P<0.01),elevated p53 protein level(P<0.01),enhanced apoptotic cells(P<0.001),up-regulation of Bax and cleaved caspase-3 as well as down-regulation of Bcl-2,and increased ROS level(P<0.01).Results indicated that the protective effects of RIPostC on OGD/R-treated cells were reversed by ALDH2 knockdown successfully.In another word,RIPostC might protect H9c2 cells against OGD/R injury through up-regulating ALDH2 expression.Chapter Three.RIPostC up-regulates ALDH2 expression via the PI3K/mTOR pathway,and thereby attenuates OGD/R-induced cell injuryAfter stimulation with PI3K inhibitor(LY294002)for 24 h,phosphorylated levels of PI3K and mTOR were observably decreased with time while total levels of PI3K and mTOR remained unchangeable.Under stimulation with mTOR inhibitor(Rapamycin),phosphorylated level of mTOR was observably decreased with time while phosphorylated level of PI3K and total levels of PI3K and mTOR remained unchangeable.Results indicated that LY294002 could inhibit phosphorylation of PI3K and mTOR,and Rapamycin could repress phosphorylation of mTOR in H9c2 cells.Afterwards,we found effects of RIPostC on expression of ALDH2 in OGD/R-treated H9c2 cells were significantly abrogated by either LY294002(P<0.01)or Rapamycin(P<0.01),when compared to the OGD/R + RIPostC group.Those results suggested that inhibition of the PI3K/mTOR pathway could reverse RIPostC-induced up-regulation of ALDH2 expression in H9c2 cells.Conclusions:Herein,the protective effects of RIPostC on myocardial I/R injury were verified both in vivo and in vitro.The effects of ALDH2 knockdown on the protective role of RIPostC in myocardial cells were analyzed via measurements of ALDH2 expression after RIPostC treatments.Meanwhile,the effects of the PI3K/mTOR pathway on ALDH2 expression regulated by RIPostC were examined.This study deeply explored the underlying mechanism of RIPostC in myocardial I/R injury,and results showed that:(1)In mice,RIPostC reduced myocardial infarct size and up-regulated ALDH2 expression.(2)In H9c2 cells,OGD/R reduced cell viability,up-regulated p53 protein expression,elevated percentage of apoptotic cells,up-regulated pro-apoptotic Bax and active caspase-3 expression as well as down-regulated anti-apoptotic Bcl-2 expression,and enhanced ROS generation.In the meantime,we also found that RIPostC could attenuate OGD/R-induced H9c2 cell injury.(3)In H9c2 cells,OGD/R down-regulated ALDH2 protein expression,whereas the down-regulation was reversed by RIPostC.(4)In H9c2 cells,ALDH2 knockdown reversed the protective effects of RIPostC on OGD/R-treated cells,showing decreased cell viability,up-regulated p53 protein expression,elevated percentage of apoptotic cells,up-regulated Bax and active caspase-3 expression as well as down-regulated Bcl-2 expression,and enhanced ROS generation.(5)In H9c2 cells,inhibition of PI3K or mTOR could reverse RIPostC-induced up-regulation of ALDH2 protein expression. |
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