Resveratrol Inhibits Hypoxia/reoxygenation Induced Apoptosis Of Cardiac Microvascular Endothelial Cells And Its Mechanism

Background：In recent years, the incidence of coronary heart disease (CHD) has increased rapidly,although many effective treatment strategies has been developed and rescued many dyingpatients. myocardial ischemia/reperfusion injury (MI/RI) always occurs after successfulreperfusion therapy after acute myocardial infarction. The mechanisms of I/RI werecomplicated and relevant to cell necrosis, apoptosis and autophagy. Previously, studieshave already proved that cardiomyocytes and endothelial cell injured by I/RI wasimportant. However, we and other groups has found recently that the cardiacmicrovascular endothelial cells (CMECs) also played crucial role during I/RI, cause theCMECs apoptosis induced by I/RI was occurred early that cardiomyocytes[1]and alongwith obvious pathological alteration morphological and functional related. Studies also proved that protect CMECs also improve heart function after I/R. Hence, CMECsprotection becomes a novel strategy of reducing I/RI.Resveratrol is widely found in grapes, veratrum, polygonum cuspidatum, a phenoliccompound in plants, has anti-inflammatory, antioxidant, inhibiting platelet aggregationand regulation of blood lipid metabolism and antitumor role[2]. Recent years studies foundthat Resveratrol can provide a significant inhibition of ischemia/reperfusion-inducedapoptosis of myocardial cells, improving heart function. Paper reported that the effectiveprotection of PI3K/Akt signaling pathway can take part in the process in the CMECsinduced by hypoxia/reoxygenation injury, and regulate cell proliferation, differentiationand glucose metabolism of effects[3]; HIF-1α is widespread in the body’s tissues andmammalian transcriptional regulation in vivo survival factorin in the hypoxic environment;It can adjust a series of hypoxic gene expression, transfer oxygen, inhibition of apoptosisand survival stress and energy metabolism in maintaining the body’s oxygen plays animportant physiological role in the steady state.Previously, our studies found that Resveratrol reduces myocardial cell apoptosisinduced by hypoxia-reoxygenation[4], but resveratrol inhibits hypoxia/reoxygenationinduced apoptosis of myocardial microvascular endothelial cells is not seen through thePI3K/Akt/HIF-1α signaling pathway-related literature reports.Objective1. Observe the cell viability and apoptosis of cultured myocardial microvascularendothelial cells in in hypoxia/reoxygenation model and pretreated with differentconvention of Resveratrol.2. Explore whether the mechanism of Resveratrol protection is related to PI3K/Akt/HIF-1α signaling pathways activation.MethodsPart I: Influence of resveratrol on hypoxia/reoxygenation-induced apoptosis and reducedcell viability of myocardial microvascular endothelial cells in vitro.1. Preparation of SD rat left ventricle of the heart, using enzyme digestion method for isolating CMECs, containing20%of fetal calf serum (FBS) culture in DMEM culturefluid to maintain. Cells was identified with Immunofluorescence double staining withDAPI and Dil-Ac-LDL.2. Establish hypoxia model of myocardial microvascular endothelial cells. All cells weredivided into several groups: control group, hypoxia group, different concentrations ofResveratrol group (5,10,20,30,40μmol/L).Myocardial microvascular endothelial cellviability was detected with MTT colorimetric assay, cell apoptosis was measured withTUNEL staining.Part II: Molecular mechanism exploration.Preparation of2-3generation of CMEC and establish hypoxia/reoxygenation injurymodel. All cells were divided into four groups:1. control group (control)2. hypoxia/reoxygenation Group (H/R)3.H/R+20μmol/L resveratrol groups4.H/R+20μmol/LLY294002+20μmol/L resveratrol groups. Myocardial microvascular endothelial cellviability was detected with MTT colorimetric assay. Migration capabilities of CMECs wasdetected with Transwell; for apoptosis of endothelial cells were detected withPI-AnnexinV and TUNEL staining. And expression of p-Akt and HIF-1α protein wasdetected with Western blot.Results1. Cell identification: cytoplasmic Dil-Ac-LDL-positive (red fluorescence), DAPIpositive expression of nucleus (blue fluorescence), confirmed that the cultured cellswere myocardial microvascular endothelial cell (CMEC).2. MTT, TUNEL respectively detection indicates that: different concentration (5,10,20,30,40μmol/L) resveratrol Reed alcohol could improve cell viability, inhibit cellapoptosis and which were dose dependence. Concentration of20μmol/L appearedoptimal and obviously protective effect (P<0.05).3. Results of WB indicated that, expression of p-Akt and HIF-1α protein in H/R group ishigher than it in control group,20μmol/L resveratrol pretreatment could furtherincreased p-Akt and HIF-1α protein expression (P<0.01) which was significantly reduced when treated with PI3K/Akt specific inhibitor LY294002. Cell apoptosisnumber, cell viability and migration capability of CMECs were all reduced inLY294002group when compared with H/R+Res group (P<0.05).Conclusions1. Different concentrations of Resveratrol could improve the viability and reduceapoptosis of cardiac microvascular endothelial cell during hypoxia-reoxygenation.The protective effect was dose dependent, and the optimal concentration was alsodetermined.2. The mechanism of the protective effect of Resveratiol was partially related toPI3K/Akt signaling pathway activation and of HIF-1α protein up-regulation.