尼古丁促进高糖高脂诱导的心肌细胞凋亡的机制研究  

The mechanism underlying the facilitation of nicotine on highglucose-and high-fat-induced cardiomyocyte apoptosis

作　　者：赵静 郭蕊[1] 孟芝君 刘彩红 谢耀丽 刘晶[3] 曹济民 王亚静 Zhao Jing;Guo Rui;Meng Zhi-jun;Liu Cai-hong;Xie Yao-li;Liu Jing;Cao Ji-min;Wang Ya-jing(College of Basic Medicine,Shanxi Medical University,Taiyuan,Shanxi 030000,China;Department of Clinical Laboratory,People's Hospital Affiliated to Shanxi Medical University,Taiyuan,Shanxi 030012,China;Department of Endocrinology,The Second Hospital of Shanxi Medical University,Taiyuan,Shanxi 030001,China)

机构地区：[1]山西医科大学基础医学院,山西太原030000 [2]山西医科大学附属人民医院检验科,山西太原030012 [3]山西医科大学第二医院内分泌科,山西太原030001

出　　处：《中国现代医学杂志》2023年第2期25-34,共10页China Journal of Modern Medicine

基　　金：国家自然科学基金(No:81670278);国家自然科学基金青年科学基金(No:82000799);山西省应用基础研究项目青年科技研究基金(No:201801D221397)。

摘　　要：目的 探究尼古丁对心肌细胞凋亡的影响及与吸烟密切相关细胞色素酶P4501A1(Cyp1a1)和神经元乙酰胆碱受体β4亚基(Chrnb4)基因在尼古丁诱导心肌细胞凋亡中的作用机制。方法 根据不同方法将细胞分为对照组、尼古丁组、高糖/高脂模型组、尼古丁+高糖高脂模型组、shRNA-NC组、shRNA-Cyp1a1组、AMPK抑制剂组、shRNA-Cyp1a1+AMPK抑制剂组、shRNA-Chrnb4组、shRNA-Chrnb4+AMPK抑制剂组。制备H9C2细胞高糖/高脂模型,转染Cyp1a1或Chrnb4干扰质粒,采用尼古丁或AMPK抑制剂处理。流式细胞术检测细胞凋亡、活性氧、线粒体膜电位,ELISA法检测细胞内超氧化物歧化酶活性和微量丙二醛含量,Western blotting检测细胞中Cyp1a1、Chrnb4、Caspase-2、Caspase-3、Caspase-9、p-AMPK的表达。结果 尼古丁组、高糖/高脂模型组、尼古丁+高糖/高脂模型组SOD较对照组降低(P <0.05),尼古丁+高糖/高脂模型组较高糖/高脂模型组降低(P <0.05)。尼古丁+高糖/高脂模型组MDA较对照组和高糖/高脂模型组升高(P <0.05)。高糖/高脂模型组、尼古丁+高糖/高脂模型组ROS含量较对照组升高(P <0.05),尼古丁+高糖/高脂模型组较高糖/高脂模型组升高(P <0.05)。尼古丁组、高糖/高脂模型组、尼古丁+高糖/高脂模型组线粒体膜电位较对照组降低(P <0.05),尼古丁+高糖/高脂模型组较高糖/高脂模型组降低(P <0.05)。尼古丁组、高糖/高脂模型组、尼古丁+高糖/高脂模型组Cyp1a1和Chrnb4 mRNA相对表达量较对照组升高(P <0.05),尼古丁+高糖/高脂模型组较高糖/高脂模型组升高(P <0.05)。尼古丁组和尼古丁+高糖/高脂模型组Caspase-2、Caspase-3、Caspase-9、Cyp1a1、Chrnb4蛋白相对表达量较对照组升高(P <0.05),p-AMPK蛋白相对表达量较对照组降低(P <0.05),高糖/高脂模型组p-AMPK蛋白相对表达量较对照组降低(P <0.05),Caspase-9、Cyp1a1、Chrnb4蛋白相对表达量较对照组升高(P <0.05),尼古丁+高糖Objective To explore the effects of nicotine on cardiomyocyte apoptosis and the roles of Cytochrome P450 Family 1 Subfamily A Member 1(Cyp1a1) and Cholinergic Receptor Nicotinic Beta 4 Subunit(Chrnb4) genes in nicotine-induced cardiomyocyte apoptosis. Methods The cells were divided into control group,nicotine group, high-glucose and high-fat model group, nicotine + high-glucose and high-fat model group, shRNANC group, sh RNA-Cyp1a1 group, AMPK inhibitor group, shRNA-Cyp1a1 + AMPK inhibitor group, shRNAChrnb4 group, and shRNA-Chrnb4 + AMPK inhibitor group. The high-glucose and high-fat model of H9C2 cells was constructed, transfected with Cyp1a1 or Chrnb4 interference plasmids, and treated with nicotine or AMPK inhibitor. Apoptosis, reactive oxygen species(ROS) and mitochondrial membrane potential were detected by flow cytometry. The intracellular superoxide dismutase(SOD) activity and malondialdehyde(MDA) content were detected by ELISA. Besides, Western blotting was used to detect the expressions of Cyp1a1, Chrnb4, Caspase-2,Caspase-3, Caspase-9, and p-AMPK in cells. Results Compared with the control group, the SOD activity was lower in nicotine group, high-glucose and high-fat model group, and nicotine + high-glucose and high-fat model group(P < 0.05). The SOD activity was even lower in the nicotine + high-glucose and high-fat model group than that in the high-glucose and high-fat model group(P < 0.05). The MDA content in the nicotine + high-glucose and high-fat model group was higher than that in the high-glucose and high-fat model group(P < 0.05). The ROS level in the high-glucose and high-fat model group and nicotine + high-glucose and high-fat model group was higher than that in the control group(P < 0.05), and that in the nicotine + high-glucose and high-fat model group was even higher compared with the high-glucose and high-fat model group(P < 0.05). The mitochondrial membrane potential was lower in the nicotine group, high-glucose and high-fat model group, and nicotine + high-glucose and high-fat model gr

关 键 词：糖尿病 心肌病 尼古丁 心肌细胞凋亡 CYP1A1 Chrnb4 

分 类 号：R587.1[医药卫生—内分泌]