钙激活中性蛋白酶Calpain-1在犬酒精性心肌病心肌细胞凋亡中的表达及不同药物对其的干预作用

目的 观察钙激活中性蛋白酶(Calpain-1)和凋亡相关蛋白在酒精性心肌病犬心肌细胞凋亡中的表达及缬沙坦和肉毒碱对其的干预作用.方法 实验犬28只,随机分为酒精组、缬沙坦+酒精组、肉毒碱+酒精组和正常对照组,每组7只动物,4组给予相同饮食,前3组通过采取逐渐增加饮用酒精浓度并长期定量摄入的方法建立酒精性心肌病模型,缬沙坦+酒精组、肉毒碱+酒精组分别给予缬沙坦及肉毒碱进行干预.6个月后病理观察各组犬心肌纤维化程度,电镜观察肌丝排列、润盘连接、细胞核、染色质、细胞器等超微结构、检测心功能和心肌细胞凋亡情况,Western blot法测定Calpain-1蛋白的表达,免疫组织化学法测定促凋亡蛋白(Bad)和抗凋亡蛋白(Bcl-xl)的表达.结果 酒精组、缬沙坦+酒精组和肉毒碱+酒精组左心窜射血分数和左心室短轴缩短率均低于正常对照组(P均<0.01),左心室舒张末期内径和左心室收缩末期内径则高于正常对照组(P均<0.01).缬沙坦+酒精组左心室射血分数和左心室短轴缩短率高于酒精组(P均<0.01),左心室舒张末期内径和左心室收缩末期内径则低于正常对照组(P均<0.01).肉毒碱+酒精组只有左心室射血分数和左心室短轴缩短率高于酒精组(P均<0.01).酒精组心肌细胞凋亡数、Bad和Calpain-1蛋白表达均明显高于正常对照组,而Bcl-xl蛋白明显低于正常对照组(P均<0.05).缬沙坦+酒精组和肉毒碱+酒精组心肌细胞凋亡数、Bad和Calpain-1蛋白表达明显低于酒精组,而Bcl-xl蛋白表达则明显高于酒精组(P均<0.05).结论 酒精加剧心肌细胞凋亡、心脏结构和功能恶化,缬沙坦和肉毒碱通过不同机制下调Calpain-1和Bad的蛋白表达,卜调Bcl-xl的蛋白表达,缬沙坦的干预效果优于肉毒碱.

Abstract：

Objective To evaluate the effects of valsartan and carnitine on cardiomyocyte Calpain-1and Bcl-xl expressions of dogs with chronic alcohol intake-induced cardiomyopathy.Methods Dogs were randomly assigned into 4 groups(n=7 each):(1)alcohol fed(free access to 5%,1st week;10%2nd week;500 ml 25%bolus plus free access to 5%from 3 to 24 weeks,A);(2)alcohol+valsartan (5 mg·kg-1·d-1,B);(3)alcohol+carnitine(300 mg·kg-1·d-1,C);(4)Control(D).After six months,all animals were assessed for left ventricular(LV)function by echocardiography.The Bad and Bcl-xl protein expressions were evaluated by immunohistochemistry.The expression of Calpain-1 protein was determined with Western blot.Myocardial morphology was quantified on HE stained slices and under electron microscopy.The terminal deoxynucleotidyl transferase-mediated iotin-dUTP nick end-labeling(TUNEL)was performed for apoptosis analysis.Results Compared with group D.LVEDD and LVESD were significantly increased while EF and FS significantly decreased in group A.In alcohol fed group,expressions of Bad and Calpain-1 protein were significantly increased while Bcl-xl protein expression was downregulated,all changes could be significantly attenuated by intervention with valsartan and carnitine(all P<0.05).Conclusion These data suggest that alcohol could promote cardiac myocyte apoptosis,reduce cardiac function and aggravate myocardial remodeling which valsartan and carnitine could reduce alcoholic cardiomyopathy by downregulating Calpain-1 and Bad protein expression and upregulating expression of Bcl-xl protein.

Effect of valsartan and carnitine on cardiomyocyte Calpain-1 and Bcl-xl expressions of dogs with chronic alcohol intake-induced cardiomyopathy

Objective To evaluate the effects of valsartan and carnitine on cardiomyocyte Calpain-1and Bcl-xl expressions of dogs with chronic alcohol intake-induced cardiomyopathy.Methods Dogs were randomly assigned into 4 groups(n=7 each):(1)alcohol fed(free access to 5%,1st week;10%2nd week;500 ml 25%bolus plus free access to 5%from 3 to 24 weeks,A);(2)alcohol+valsartan (5 mg·kg-1·d-1,B);(3)alcohol+carnitine(300 mg·kg-1·d-1,C);(4)Control(D).After six months,all animals were assessed for left ventricular(LV)function by echocardiography.The Bad and Bcl-xl protein expressions were evaluated by immunohistochemistry.The expression of Calpain-1 protein was determined with Western blot.Myocardial morphology was quantified on HE stained slices and under electron microscopy.The terminal deoxynucleotidyl transferase-mediated iotin-dUTP nick end-labeling(TUNEL)was performed for apoptosis analysis.Results Compared with group D.LVEDD and LVESD were significantly increased while EF and FS significantly decreased in group A.In alcohol fed group,expressions of Bad and Calpain-1 protein were significantly increased while Bcl-xl protein expression was downregulated,all changes could be significantly attenuated by intervention with valsartan and carnitine(all P<0.05).Conclusion These data suggest that alcohol could promote cardiac myocyte apoptosis,reduce cardiac function and aggravate myocardial remodeling which valsartan and carnitine could reduce alcoholic cardiomyopathy by downregulating Calpain-1 and Bad protein expression and upregulating expression of Bcl-xl protein.