AMPK/NRF2/NQO1信号通路与SD大鼠酒精性心肌病的相关性研究

Relationship between AMPK/NRF2/NQO1 signaling pathway and alcoholic cardiomyopathy in SD rats

目的明确单磷酸腺苷依赖的蛋白激酶/核因子E2相关因子2/还原型烟酰胺腺嘌呤二核苷酸磷酸脱氢酶醌1(Adenosine monophosphate dependent protein kinase/nuclear factor E2-related factor 2/NADPH dehydrogenase quinone 1,AMPK/NRF2/NQO1)信号通路与SD大鼠的酒精性心肌病(alcoholic myocardiopathy,ACM)的关系。方法选用健康的8周龄SD大鼠构建ACM模型,采用随机数字表法分成2组,全价颗粒饲料+饮水组(对照组n=24只),全价颗粒饲料+饮酒组(实验组n=24只)。实验组大鼠于每日10:00、17:00给予定量[20 g/(kg·d)]20%浓度酒精喂养(灌胃);正常对照组同时给予等量蒸馏水(灌胃)。从造模开始,实验组以20%酒精浓度持续喂养12周。记录大鼠一般情况。分别于第4周、第8周、第12周实施超声心动图与病理分析。马松染色(Masson)检测大鼠心肌纤维化,免疫组化及RT-qpcr法检测大鼠心肌组织中AMPK、NRF2、NQO1蛋白及基因的表达,分析其关系,从蛋白及基因水平探讨ACM的发病机制。结果心脏彩超:实验组大鼠与对照组相比左室射血分数(left ventricular ejection fraction,LVEF)左室短轴缩短率(left ventricular fractional shortening,LVFS)均下降(P<0.05);病理:对照组HE染色无异常,实验组心肌细胞出现充血水肿等变性;Masson染色:与对照组相比,实验组大鼠心肌纤维化加重;免疫组化:与对照组相比,实验组大鼠心肌AMPK、NRF2、NQO1蛋白表达减弱,染色变浅;RT-qpcr:与对照组相比,实验组大鼠心肌AMPK、NRF2、NQO1基因表达减弱(P<0.05)。结论大鼠心肌纤维化及损伤程度与饮酒时间及浓度积累呈正相关,随着饮酒时间的延长,酒精在体内蓄积,心肌纤维化及损伤程度更严重,抑制AMPK/NRF2/NQO1信号通路可能是分子机制。

Objective Identify the relationship between Adenosine monophosphate dependent protein kinase/nuclear factor 1(Adenosine monophosphate dependent protein kinase/nuclear factor 1 E2-related factor 2/NADPH dehydrogenase quinone 1,AMPK/NRF2/NQO1)signaling pathway and alcoholic myocardiopathy(ACM)in SD rats.Methods Healthy 8-week-old SD rats were selected to construct ACM model and divided into 2 groups by random number table method:full grain feed+drinking water group(control group n=24 rats)and full grain feed+drinking alcohol group(experimental group n=24 rats).Rats in the experimental group were given quantitative(20 g/(kg.d))20%concentration alcohol feeding(gavage)at 10:00 and 17:00 every day;The normal control group was also given the same amount of sterile water(gavage).Starting from modeling,the experimental group was fed at 20%alcohol for 12 weeks.The general condition of the rats was recorded.Echocardiography and pathological analysis were performed at 4,8 and 12 weeks respectively.Masson staining was used to detect myocardial fibrosis,immunohistochemistry and RT-qPCR were used to detect the expression of AMPK,NRF2 and NQO1 proteins and genes in myocardial tissue of rats.The relationship between AMPK,NRF2 and NQO1 proteins and genes was analyzed,and the pathogenesis of ACM was discussed from the level of proteins and mRNA.Results Compared with the control group,left ventricular ejection fraction(LVEF)left ventricular ejection fraction(LVEF)Left ventricular fractional shortening,LVFS were all decreased(P<0.05).There was no abnormality in the control group,but hyperemia and edema happened in the experimental group in HE staining.Compared with the control group,the myocardial fibrosis of the experimental group was aggravated.Compared with the control group,the expression of AMPK,NRF2 and NQO1 in the myocardium of experimental group was decreased,and the staining was shallow.Compared with the control group,the expressions of AMPK,NRF2 and NQO1 mRNA in the experimental group were decreased(P<0.05).Conclusion The degree of myocardial fibrosis and injury in rats is positively correlated with the duration and concentration accumulation of alcohol drinking.With the extension of alcohol drinking time,myocardial fibrosis and injury become more serious.Inhibition of the AMPK/NRF2/NQO1 signaling pathway may be the molecular mechanism.