Protective Mechanisms Of Asiatic Acid On Cardiomyocytes Apoptosis And Heart Failure In Mice

Section I The effect of asiatic acid on lactate-induced cardiomyocytes apoptosisObjective:To determine the effect and molecular mechanisms of asiatic acid(AA)on lactate-induced cardiomyocytes apoptosis.Methods:1.Neonatal rat cardiomyocytes were treated with various concentrations of AA(0?5?10?20?30 ?mol/L)for 24 h or/and exposure to various concentrations of lactate(0?2.5?5?10?20?40 mmol/L)for 24 h,and cell viability was determined by a CCK-8 assay.2.There were four experimental groups:i)Control,ii)AA,iii)Lactate and iv)Lactate+AA.The cells were incubated with AA(20 ?mol/L)for 24 h prior to lactate(20 mmol/L)stimulation for 24 h.3.The levels of intracellular ROS were measured by flow cytometry using DCFH-DA as the probe.4.The mitochondrial membrane potential(??m)was determined by flow cytometry and visualized by fluorescence microscopy using the fluorescent probe JC-1.5.Cell apoptosis was evaluated by flow cytometry using an Annexin V-FITC/P1 staining kit.6.Cardiomyocytes were stained by the terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick-end labeling(TUNEL)technique using an in situ cell death detection kit,and visualized under a fluorescence microscope.7.The expression of mitochondrial MCT1 and cytoplasmic cytochrome c,cleaved caspase-9 and-3 were assayed by Western blot.Results:1.Lactate stimulation significantly increased intracellular ROS levels and activated mitochondria-dependent apoptosis,including reduction of ??m,release of cytochrome c,and upregulation of cleaved caspase-9 and-3 expression.2.AA pretreatment significantly inhibited lactate-induced apoptosis,intracellular ROS generation,and loss of ??m.AA also reduced the expression of cytoplasmic cytochrome c,cleaved caspase-9 and-3 in lactate-stimulated cardiomyocytes.3.The expression of mitochondrial MCT1 was significantly increased in lactate-stimulated cardiomyocytes.AA pretreatment could further increase the expression of mitochondrial MCT1 induced by lactate.Conclusions:Our data demonstrate for the first time that AA exerts a cytoprotective role in lactate-induced apoptosis,including inhibition of oxidative stress and mitochondria-dependent caspase activation,as well as upregulation of mitochondrial MCT1 expression.Section? The effect of asiatic acid on the development of heart failure induced by aortic constriction in miceObjective:To determine the effect and molecular mechanisms of asiatic acid(AA)on heart failure(HF)induced by aortic constriction.Methods:1.Animal models were created by transverse aortic coarctation(TAC).All mice underwent echocardiography after 8 weeks post-surgery.The mice were then euthanized,the hearts were harvested and the left ventricular weight/body weight(LVW/BW)was calculated.2.There were three experimental groups:i)Sham group;ii)TAC group;iii)TAC +AA group,AA oral gavage mice with 100mg/kg/day from fifth week to eighth week.3.Intracellular lactate and MDA concentrations were measured by colorimetric.4.Cardiomyocytes were stained by the terminal deoxynucleotidyl transferase(TdT)-mediated dUTP nick-end labeling(TUNEL)technique using an in situ cell death detection kit,and visualized under a fluorescence microscope.5.The expression of mitochondrial MCT1 and cytoplasmic cytochrome c,cleaved caspase-9 and-3 were assayed by Western blot.Results:1.TAC mice displayed significantly increased LVW/BW and heart failure post-surgery 8 weeks.Treatment with AA significantly reduced LVW/BW and ameliorated cardiac dysfunction and failure.2.The levels of lactate and mitochondrial MCT1 was significantly increased in TAC mice.AA treatment significantly reduced lactate level and further increased the expression of mitochondrial MCT1 in TAC mice.3.AA treatment significantly inhibited TAC-induced apoptosis and intracellular MDA generation.AA also reduced the expression of cytoplasmic cytochrome c,cleaved caspase-9 and-3 in TAC mice.Conclusions:AA exerts a cardioprotective role in TAC-induced heart failure,including inhibition of oxidative stress and mitochondria-dependent caspase activation,as well as upregulation of mitochondrial MCT1 expression.