黄芪注射液对阿霉素性心肌细胞凋亡、内质网应激与缝隙连接蛋白表达的影响

Effect of Astragalus injection on cardiomyocyte apoptosis,endoplasmic reticulum stress and expression of connexin in cardiomyopathy rats induced by adriamycin

目的:研究黄芪注射液对阿霉素(ADR)所致心肌病大鼠心肌细胞凋亡、内质网应激与缝隙连接蛋白表达的影响。方法:36只Wistar雄性大鼠随机分为3组(n=12):对照组、ADR组及黄芪注射液组。对照组腹腔注射0.9%Nacl(10 ml/kg体重);ADR组腹腔注射ADR 2 mg/kg体重;黄芪注射液组在每次腹腔注射ADR 2 mg/kg体重的同时,注射黄芪注射液10 g/kg体重,每周注射1次,共注射3次。实验第7周末,3组大鼠行心脏彩超检测左室舒张末期内径、左室收缩末期内径及左室射血分数;处死大鼠后取左心室组织行HE、Masson、醋酸铀及柠檬酸铅染色,于光镜及透射电镜下观察心肌病理及超微结构改变;采用TUNEL法检测大鼠心肌细胞凋亡,用免疫组化技术检测大鼠心肌细胞缝隙连接蛋白Cx43及p-Cx43表达,采用real time PCR检测大鼠心肌细胞内质网应激伴侣蛋白Grp78,ATF-4及CHOP表达。结果:与对照组比较,ADR组大鼠LVEDD、LVESD增大,LVEF减少;心肌纤维排列紊乱,心肌纤维间质水肿,大量淋巴细胞浸润;线粒体肿胀、破坏,呈空泡样;心肌细胞凋亡数明显增多(P<0.01);内质网应激相关蛋白Grp78、ATF-4及CHOP表达明显增高(P<0.01);缝隙连接蛋白Cx43表达减少,而pCx43表达增多。与ADR组比较,黄芪注射液组大鼠LVEDD、LVESD减少,LVEF增加;心肌病理及超微结构明显改善,同时心肌细胞凋亡数明显减少(P<0.01);内质网应激伴侣蛋白Grp78、ATF-4及CHOP表达明显减少(P<0.01);缝隙连接蛋白Cx43表达增多,而p-Cx43减少。结论:黄芪注射液可有效改善阿霉素导致的心肌损伤,其机制可能与黄芪注射液抑制ADR诱导的内质网应激及缝隙连接蛋白磷酸化有关。

Objective： To investigate the effect of Astragalus injection on cardiomyocyte apoptosis,endoplasmic reticulum stress and connexin protein in cardiomyopathy rats induced by adriamycin. Methods： Thirty-six male Wister rats were randomly divided into control group（ n = 12）,adriamycin（ ADR） group（ n = 12） and Astragalus group（ n = 12）. The normal saline（ 10 ml/kg body weight） was injected intraperitoneally in control group rats,ADR（ 2 mg/kg body weight） was injected intraperitoneally in ADR group rats,ADR（ 10 ml/kg body weight） and Astragalus injection（ 10 ml/kg body weight） were injected intraperitoneally in rats of astragalus group,one time a week,totle 3 times. By the end of the 7 thweek,the left ventricular end-diastolic diameter（ LVEDD）,left ventricular end-systolic diameter（ LVESD） and left ventricular ejection fraction（ LVEF） were measured by echocardiography. Then the rats in the three groups were sacrificed and the left ventricle section was stained by HE,Masson,uranyl acetate/lead citrate respectively,the cardiomyopathy and ultrastructural changes were observed under light microscope and transmission electron microscope. The apoptosis of rat cardiomyocyte were analyzed by TUNEL. The expression of connexin Cx43 and p-Cx43 was detected by immunohistochemistry. The expression of glucose-regulated protein 78（ Grp78）,activating transcription factor 4（ ATF-4） and C/EBP homologous protein（ CHOP） were detected by real time PCR. Results： Compared with control group,LVEDD,LVESD increased and LVEF decreased,myocardial fibers were disordered and edematous,infiltrated by lymphocytes,the mitochondria were destroyed and vacuolized,and the number of cardiomyocyte apoptosis was increased（ P〈0. 01） in ADR group. The expression of Grp78,ATF-4,CHOP and p-Cx43 were increased,and the expression of Cx43 was decreased in ADR group. However,compared with ADR group,LVEDD,LVESD decreased and LVEF increased,the cardiomyopathy and ultrastructural changes were significantly improved,the number of cardiomyocyte apoptosis was significantly decreased（ P〈0. 01）; the expression of Grp78,ATF-4,CHOP and p-Cx43 decreased（ P〈0.01）; the expression of Cx43 increased in Astragalus group（ P〈0. 01）. Conclusion： Astragalus injection may effectively improve the myocardial damage induced by adriamycin,its mechanism may be related to the inhibition of endoplasmic reticulum stress（ ERS） and the decrease of phosphorylation of CX43 in cardiomyopathy rats induced by adriamycin.