| Objective:1.To investigate the role of calpain signal ?ANXA5 and MAPKs signal transduction system in ALD-induced cardiomyocyte apoptosis,and elucidate the mechanism of ALD-induced cardiomyocyte apoptosis,so as to find the target of action of cardiomyocyte apoptosis.2.To analyze the protective effect of OMT on myocardial apoptosis induced by ALD,and to explain its exact mechanism.3.The left ventricular hypertrophy(LVH)is a common event in the essentialhypertension(EH).We evaluated the ANXA5 promoter polymorphism in a cross-sectional study in EH patients.Method:1.In the first chapter,the primary cultured cells of SD neonatal rat cardiomyocytes were isolated and purified by 0.06% trypsin digestion and differential adherence method.Morphological changes were observed by inverted microscope,and cardiomyocytes and their purity were identified by cardiomyocyte specific troponin and actin immunocytochemistry.MTT assay was used to observe the effect of different concentrations of ALD on cardiomyocytes,and to explore the relationship between the optimal concentration and effect of ALD on cardiomyocytes.ALD-induced cardiomyocyte apoptosis model was established and verified by MTT assay and flow cytometry.There were control group,ALD group,ALD +anti-calpain-1 group,ALD +anti-calpain-2 group,ALD +anti-P38 group,ALD +anti-ERK group and ALD +anti-JNK group.The expressions of Calpain-1,Calpain-2,p38,JNK,ERK1/2 mRNA and protein were detected by Real-time PCR and Western-blotting.There was ALD+ anti-ANXA5 group,The expressions of Calpain-1,Calpain-2,p38 mRNA and protein were detected by Real-time PCR and Western-blotting.2.In the second chapter,the experiment was divided into control group,ALD group,ALD +L-OMT group,ALD +H-OMT group,ALD +aspirin group and ALD +spironolactone group.The cell morphology was observed Giemsa staining and HE staining,apoptosis were did by MTT assay,cytotoxicity was detected by LDH,and the expression of calpain?p38 and ANXA5 were detected by Real-time PCR and Western-blotting.3.A total of 850 EH patients including 337 with LVH were testified.Genotyping of ANXA5 promoter SNPs were conducted by SNaPshot assays.Result: 1.Cardiom-yocytes were successfully isolated and cultured,anti-cTnIantibody detectionmethod cTnI identified by HE staining.Different concentrations of ALD exerted different apoptosis rate on cardiomyocytes,in a dose-dependent manner.Combined with the results of the previous laboratory and related literature to determine the final ALD experimental concentration of 10-5 mol / L.The model of myocardial cells was modeled by using ALD experimental concentration of 10-5 mol / L and 48 h for 48 h.The expression of Gene in cardiomyocyte injury induced by ALD wit mass-spectrometer measurement,ALD group VS controls group,The Gene expression of ANXA5 and p 38?calpain1?calpain2 increased.Flow cytometry was used to observe the degree of apoptosis of myocardium.Flow cytometry showed that the apoptosis rate was 37.8% in ALD-induced myocardial apoptosis model.Calpain protein and mRNA were detected by ALD-induced cardiomyocyte cell injury model.Real-time PCR results showed that the mRNA transcription of Calpain-1 and calpain-2 was significantly increased after ALD cardiomyocyte interference(p <0.05).Western-blotting results showed a significant increase in Calpain-1 and Calpain-2 protein levels in ALD mode(p <0.05).Real-time PCR results showed that p38 mRNA transcription was significantly increased in ALD model(p <0.05).Western blotting showed that the expression of p38 and p-p38 in ALD-induced cardiomyocyte injury model was significantly up-regulated(p <0.05).Although JNK,p-JNK,ERK1/2 and p-ERK1/2 were up-regulated,the difference was not statistically significant(p <0.05),while the expression of Caspase-3 was not significantly different(p > 0.05).5.The levels of calpain-1,calpain-2 and p-38 mRNA were significantly decreased after anti-calpain-1 intervention.Calpain-2 and p-38 mRNA levels were significantly decreased(p <0.05)after calpain-2 intervention.The levels of p-38,JNK and ERK1/2 were significantly decreased after anti-p-38,anti-JNK and anti-ERK intervention respectively(p <0.05).The apoptosis rate of myocardium induced by ALD was significantly decreased after anti-calorie-1,anti-calpain-2,anti-JNK,anti-ERK and anti-p38,but still higher than the control group(p <0.05).2.OD value of ALD group was significantly lower than that of control group(p <0.05).ALD +L-OMT group(25 ?g / ml)and ALD +H-OMT group(50 ?g / ml)could significantly inhibit the apoptosis and increased the OD of MTT(p<0.05).The OD of MTT in ALD +aspirin group and ALD +spironolactone group was significantly higher than that in ALD group(p<0.05).LDH release rate in ALD group was significantly lower than that of control group(p<0.05).LDH release rate in ALD +L-OMT group(25 ?g / ml)and ALD +H-OMT group(50)were significantly lower than ALD group(p<0.05).The LDH leakage rate of ALD +aspirin group and ALD +spironolactone group was significantly lower than that of ALD group(p<0.05).Cardiomyocytes were stained with Giemsa,the nuclei were stained with blue and purple,and the cytoplasm was dyed red.ALD +L-OMT group(25 ?g / ml),ALD +H-OMT group(50 ?g / ml)were significantly higher than those in ALD group(p<0.05)/ Ml),ALD +aspirin group and ALD +spironolactone group cell surface area decreased,the shape close to normal.After myocardial staining by HE,the nucleus was stained with blue and purple,and the cytoplasm was dyed red.ALD +L-OMT group(25 ?g / ml),ALD +H-OMT group(50 ?g / ml)were significantly higher than those in ALD group(p <0.05),and the ALD +50 ?g / ml),the cell surface area of ALD +aspirin group and ALD +spironolactone group was decreased and the morphology was close to normal.The expression of calpain-1,calpain-2 and p38 mRNA in ALD group was significantly higher than that in control group(p <0.05).The expression level in ALD +L-OMT group(25 ?g / ml),ALD +H-OMT group(50 ?g / ml)group was significantly lower than that in ALD group(p<0.05).ALD +aspirin group and ALD +spironolactone group could significantly inhibit the upregulation of calpain-1,calpain-2 and p38 mRNA induced by ALD(p <0.05).The expression of calpain-1,calpain-2 and p38 protein in ALD group was significantly higher than that in control group(p <0.05).The expression level in ALD +L-OMT group(25 ?g / ml),ALD +H-OMT group(50 ?g / ml)group was significantly lower than that in ALD group(p<0.05).ALD +aspirin group and ALD +spironolactone group could significantly inhibit the upregulation of calpain-1,calpain-2 and p38 protein induced by ALD(p<0.05).3.All of selected SNPs displayed their risky significance in LVH,and rs1050606 revealed its remarkable value(p= 0.008 in dominant and p= 0.006 in co-dominant models).Further haplotype of M1 and M2 also confirmed their hazardous effects in the onset of LVH in EH patients(p =0.022 and 0.032).Conclusion:1.ALD may upregulate calpain-1,calpain-2 and p-38?ANXA5 expression levels by activating calpain p38 and ANXA5,activating caspase-3 to induce cardiomyocyte apoptosis.There may be some up-to-down between calpain and ANXA5?p38.2.OMT has an inhibitory effect on ALD-induced cardiomyocyte apoptosis,and the myocardial protective effect of OMT may be achieved by its anti-apoptotic effect.OMT can significantly improve the myocardial injury induced by ALD,and its mechanism may inhibit calpain-1,calpain-2 and p38 ?ANXA5expression by calpain p38 and ANXA5 pathway,thus improving the apoptosis of cardiomyocytes.3.This results identified the ANXA5 rs1050606,M1 and M2 haplotypes were significant associated with the LVH in guizhou EH patients. |
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