摘要
目的:探讨美他嗪对心肌缺血再灌注损伤大鼠钙蛋白酶(Calpain)的调控及其对细胞凋亡的作用机制。方法:采用随机数字表法将30只SD大鼠分为假手术组(Sham组)、模型组和曲美他嗪干预组,每组10只。Sham组大鼠仅置缝线但不结扎冠状动脉,其余两组大鼠均采用手术结扎大鼠心肌冠状动脉左前降支(LAD)进行造模,造模期间,Sham组和模型组大鼠给予0.9%氯化钠溶液灌胃处理,曲美他嗪干预组大鼠给予曲美他嗪20 mg/kg灌胃处理。采用苏木精-伊红染色法观察各组大鼠心肌组织情况,以速率法对肌酸激酶(CK)和乳酸脱氢酶(LDH)等心肌酶指标水平进行测定;采用DNA原位末端标记(TUNEL)法检测心肌细胞凋亡情况,并应用免疫组织化学法检测心肌组织中Caspase-3、B细胞淋巴瘤2(Bcl-2)蛋白表达水平;通过荧光分光光度计测定荧光强度来体现Calpain活性,并采用蛋白质印迹法测定焦亡相关蛋白表达水平[核苷酸结合寡聚化结构域样受体蛋白3(NLRP3)、Caspase-1 P10蛋白、凋亡相关斑点样蛋白(ASC)和白细胞介素1β(IL-1β)]。结果:Sham组大鼠心肌细胞核清晰可辨,心肌纤维整齐排列,胞浆均匀着色;模型组大鼠心肌细胞核浓缩,心肌纤维排列混乱,细胞变性呈空泡状,且可伴有炎症细胞浸润;曲美他嗪干预组大鼠心肌细胞间质呈轻度水肿,心肌纤维排列无异常。Sham组大鼠未发生心肌梗死;曲美他嗪干预组大鼠心肌梗死面积较模型组显著缩小(P<0.05);模型组和曲美他嗪干预组大鼠心肌酶CK、LDH水平均明显高于Sham组,其中曲美他嗪干预组各项心肌酶指标水平均低于模型组(P<0.05),差异均有统计学意义。Sham组中可见少量心肌细胞凋亡,模型组中心肌细胞呈弥漫分布,凋亡数量明显增多;经曲美他嗪干预后,心肌细胞凋亡明显减少。与Sham组相比,模型组大鼠Caspase-3蛋白表达水平明显升高,Bal-2蛋白水平明显降低(P<0.05);与模型组相比,曲美他嗪干预组大鼠Caspase-3蛋白表达水平明显降低,Bal-2蛋白表达水平明显升高(P<0.05),差异均有统计学意义。模型组大鼠缺血/再灌注(I/R)心肌细胞Calpain活性明显高于Sham组和曲美他嗪干预组,差异有统计学意义(P<0.05),Sham组与曲美他嗪干预组大鼠I/R心肌细胞Calpain活性的差异无统计学意义(P>0.05)。模型组和曲美他嗪干预组大鼠NLRP3、IL-1β、ASC和Caspase-1 P10蛋白表达水平均明显高于Sham组,曲美他嗪干预组各项焦亡蛋白相关水平均低于模型组,差异均有统计学意义(P<0.05)。结论:曲美他嗪可通过抑制Calpain活性来减轻细胞凋亡,进而起到减轻心肌缺血再灌注损伤大鼠心脏损伤的作用。
OBJECTIVE:To probe into the regulation of trimetazidine on calpain in rats with myocardial ischemia-reperfusion injury and its mechanism of action on cell apoptosis.METHODS:Thirty SD rats were randomly divided into the Sham operation group(Sham group),model group,and trimetazidine intervention group via the random number table method,with 10 rats in each group.Rats in the Sham group were only sutured but not subjected to coronary artery ligation.Rats in the remaining two groups were all modeled by surgical ligation of the left anterior descending coronary artery(LAD)of the rat myocardium.During the modeling,the Sham group and model group were treated with intragastric administration of 0.9%normal saline,while the trimetazidine intervention group was treated with intragastric administration of 20 mg/kg trimetazidine.Myocardial tissue of rats in each group was observed by Hematoxylin and Eeosin staining,and the levels of myocardial enzymes such as creatine kinase(CK)and lactate dehydrogenase(LDH)were measured by rate method.The apoptotic status of myocardial cells was detected by DNA in situ end labeling(TUNEL)method,and the expression levels of Caspase-3 and B-cell lymphoma 2(Bcl-2)proteins in myocardial tissues were detected by immunohistochemistry.Calpain activity was demonstrated by fluorescence intensity determination with a fluorescence spectrophotometer and expression levels of pyrophosphate-related proteins[nucleotide binding oligomerization domain-like receptor protein 3(NLRP3),Caspase-1 P10 protein,apoptosis-related macular protein(ASC),and interleukin-1β(IL-1β)]by Western blotting.RESULTS:In the Sham group,the nuclei of myocardial cells were clear and distinguishable,the myocardial fibers were arranged neatly,and the cytoplasm was stained evenly.In the model group,the nuclei of myocardial cells were condensed,and the myocardial fibers were disorganized;the cells became denatured and became vacuolar,which could be accompanied by inflammatory cell infiltration.In the trimetazidine intervention group,myocardial intercellular substance showed mild edema,and there was no abnormal arrangement of myocardial fibers.No myocardial infarction was observed in the Sham group.The myocardial infarct size of rats in the trimetazidine intervention group was significantly smaller than that of model group(P<0.05);the levels of myocardial enzyme CK and LDH in the model group and the trimetazidine intervention group were significantly higher than those in Sham group,and the myocardial enzyme indicators in the trimetazidine intervention group were lower than those in the model group(P<0.05),with statistically significant differences.A small amount of cardiomyocyte apoptosis was observed in Sham group,while the cardiomyocytes in the model group were distributed diffusely,and the number of apoptosis increased significantly.After trimetazidine intervention,myocardial cell apoptosis decreased significantly.The expression level of Caspase-3 protein in the model group was significantly higher than that in the Sham group,and the level of Bal-2 protein decreased significantly(P<0.05);compared with the model group,the expression level of Caspase-3 protein in the trimetazidine intervention group was lower,and the expression level of Bal-2 protein was higher(P<0.05),with statistically significant differences.Calpain activity in ischemia/reperfusion(I/R)myocardial cells of rats in the model group was significantly higher than that in the Sham group and trimetazidine intervention group,with statistically significant differences(P<0.05).There was no significant difference in Calpain activity in I/R myocardial cells of rats between the Sham group and trimetazidine intervention group(P>0.05).The expression levels of NLRP3,IL-1β,ASC and Caspase-1 P10 protein in the model group and trimetazidine intervention group were significantly higher than those in the Sham group,and the levels of pyroptosis-related proteins in the trimetazidine intervention group were lower than those in the model group,the differences were statistically significant(P<0.05).CONCLUSIONS:Trimetazidine can reduce cell apoptosis by inhibiting Calpain activity,and then play a role in alleviating the cardiac injury of rats with myocardial ischemia-reperfusion injury.
作者
何文凤
薛成
郑健康
帅壮
岳荣川
HE Wenfeng;XUE Cheng;ZHENG Jiankang;SHUAI Zhuang;YUE Rongchuan(Laboratory of Cardiovascular Diseases,Dept.of Cardiology,Affiliated Hospital of North Sichuan Medical College,Sichuan Nanchong 637000,China)
出处
《中国医院用药评价与分析》
2023年第1期16-20,共5页
Evaluation and Analysis of Drug-use in Hospitals of China
基金
国家自然科学基金项目(No.81600232)
川北医学院附属医院院级课题(No.2019IC036)。
