Protection Effects Of CVB-D On Cardiac Myocytes Apoptosis Induced By Aldosterone Via Crosstalk Of MAPKs And Mitochondrial Function In Vitro

Objective: To reproduce the cardiac myocytes apoptosis model induced by aldosterone(ALD)and explore the crosstalk of MAPKs signal and mitochandoria function,further investigate the protective effects of CVB-D on cardiac myocytes apoptosis induced by ALD via MAPKs signaling and mitochondrial function crosstalk in vitro,and provide a novel therapeutic targets for cardiovascular diseases induced by cardiac myocytes apoptosis.Methods: The primary cardiac myocytes were digested by 0.08% trypsin from 1~3 day SD neonal rat,and then was purified by differential adhension,and further block the proliferation of cardiac fibroblasts by treated with chemical inhibitors(0.1 mmol/L 5-BrdU).The morphology was observed by inverted microscope,and cardiac myocytes were identified by immunocytochemical methods with cardiac troponinand c-TNT.The dose-toxicity of ALD on cardiac myocytes were determined by MTT assay for designating 0.01μM、0.1μM、1μM、10μM、100μM.Finally,the optimal concentration and time of ALD were chosen exposed with 10μM and 36 h.The optimal conditions for ALD were assayed with MTT methods for designating 0.01 μM,0.1 μM,1 μM,10 μM and 100 μM,cardiac myocytes were pretreated with different concentrations of CVB-D for 1 h before exposure to ALD.Then the optimal concentration of CVB-D was determined as 10 μM and 1 μM.The experiment was divided into two parts as following:(1)Protective effect of CVB-D on ALD-induced cardiac myocytes apoptosis: the five groups was designated as following: control group,explosure to ALD alone group(10μM),CVB-D high-dose group(CVB-D H,10μM)and low-dose group(CVB-D L,1μM)with ALD and Spironolactone(Spiro,10μM)with ALD for up to 36 h.The cell viability was evaluated by MTT value and LDH leakage;The morphology was measured by Giemsa staining and HE staining.Flow cytometry and TUNEL staining were used to detect the apoptosis of cardiac myocytes.(2)The protective mechanism of CVB-D on ALD induced cardiac myocytes apoptosis via MAPKs signal and mitochondria function: the 8 groups were designed as following: normal control group(Ctrl.),model group(ALD,10μM),spiro group(10μM),CVB-D(H)group(10μM),CVB-D(L)group(10μM),P38 inhibitor group(SB203580,10 μmol / L),JNK inhibitor group(SP600125,5 μmol / L)and mitochondrial mPTP pore inhibitor group(Bongkrekic acid solution,20 μmol / L).MTT was used to determine the cell viability.The protein expression of p-P38,p-JNK,P38,JNK was evaluated by Western blotting.JC-1 fluorescence staining was used to detect mitochondrial membrane potential.Results: The primary cultured neonatal cardiac myocytes began to appear pseudopodia and beat after cultured 48 h which the cell get with trypsin digesting and differential adhension,and then the cell was synchronous beated contenuing cultured 72 h.The cardic myocytes were more than 92% by immunocytochemical method with troponin which was brown yellow particles in the cytoplasm.After exposed to ALD alone,the MTT value of cardiac myocytes decrease and LDH releasing increase(P<0.01),which indicated that ALD induced the primary cultured cardiac myocytes injury.Pretreated with CVB-D and Spironolactone,MTT value and LDH leakage significantly ameliorated compared with model group(P<0.01).The pathological morphology was also alleviated ALD-induced primary cultured neonatal rat cardiac myocytes injury detecting by Giemsa staining and HE staining.Flow cytometry confirmed that ALD could induce apoptosis of cardiac myocytes,and the apoptosis rate was attenuated by CVB-D and spironolactone.Western blot results confirmed that the expression of caspase-3,p-P38 and p-JNK protein was up-regulated by ALD,which could be reversed by CVB-D and spironolactone in some degree.The mitochondrial mPTP pore inhibitor could decrease the expression of p-P38 and p-JNK induced by ALD.JC-1 fluorescence staining showed that P38 inhibitor and JNK inhibitor reversed ALD-induced mitochondrial membrane potential decreased.Conclusion: ALD could induce cardiac myocytes apoptosis,the mechanism is related to the up-regulation of p-P38 and p-JNK protein expression and down-regulation of cardiac myocytes mitochondrial membrane potential.There is crosstalk between MAPK signaling pathway and mitochondria function in the apoptotic process of cardiac myocytes,and CVB-D protect cardiac myocytes against ALD-induced apoptosis via regulation crosstalk between MAPK signaling pathway and mitochondria function.