糖皮质激素受体激活对心肌细胞损伤后影响的实验研究

Experimental study on the effect of glucocorticoid receptor activation on cardiomyocytes after injury

目的观察糖皮质激素受体(GR)激活对小鼠心梗(MI)后心功能和心肌细胞缺氧刺激的影响及其机制。方法将80只C57B/L6背景的野生雄性小鼠,按照随机数字表法随机分为生理盐水(盐水)组和地塞米松(地米)组,再分别分为假手术组和手术组(MI),即盐水+假手术组(20只)、地米+假手术组(20只)、盐水+MI组(20只)和地米+MI组(20只)。术前24h分别注入生理盐水(盐水)或地塞米松(地米,20 mg/kg),MI组结扎小鼠左冠状动脉前降支制作MI模型,假手术组只绕线不结扎,在术后第1、7天用小动物超声仪检测心功能指标;于术后1天取心脏组织行免疫组织化学染色及蛋白印迹法观察GR磷酸化水平、TUNEL染色观察细胞凋亡水平;于术后7天取心脏组织行Masson染色观察梗死面积、多因子试剂盒及实时荧光定量PCR检测脂联素蛋白和mRNA水平;并体外培养H9c2大鼠心肌细胞系,高内涵扫描实验观察地米对心肌细胞缺氧刺激后的凋亡和坏死的影响。结果与盐水+MI组比较,地米+MI组的磷酸化GR水平明显升高(1.75倍),小鼠MI后左室射血分数、舒张末期、收缩末期内径和短轴缩短率等心功能指标均明显改善(均为P<0.05),心脏损伤减轻,梗死面积减小(34.8%±5.8%比44.1%±3.6%,P<0.05),脂联素的蛋白(2.82倍)和mRNA(6.45倍)水平均明显升高(均为P<0.05),且凋亡细胞减少(P<0.05)。细胞实验显示与对照组相比,地米可显著减少缺氧刺激导致的H9c2心肌细胞凋亡和坏死(均为P<0.05)。结论GR激活对小鼠MI后的心肌细胞损伤具有保护作用,这一作用可能与其抗炎和抗细胞凋亡、坏死作用有关。

Objective To observe the effect and mechanism of glucocorticoid receptor(GR)activation on cardiac function after myocardial infarction(MI)in mice and on myocardial cell hypoxia stimulation.Methods Eighty wild male mice with C57B/L6 background were randomly divided into saline(SAL)group and dexamethasone(DEX)group according to random number table method,and then divided into sham operation group(SHA)and operation group(MI),that is,SAL+SHA group(n=20),DEX+SHA group(n=20),SAL+MI group(n=20)and DEX+MI group(n=20).SAL or DEX(20 mg/kg)was injected in 24 h before surgery.The mice in the MI group were ligated the anterior descending branch of the left coronary artery to make the MI model.The mice in the SHA group were only wound the wire without ligation.On the 1st and 7th day after the operation,cardiac function indexes were detected with a small animal ultrasound system;heart tissue was taken for immunohistochemical staining and Western blotting to observe GR phosphorylation level and TUNEL staining to observe the level of apoptosis on the postoperative day.On the 7th day after operation,heart tissues were taken for Masson staining to observe the infarct size,multi-factor kit and real-time fluorescence quantitative PCR to detect adiponectin protein and mRNA levels;and cultured H9c2 rat cardiomyocyte cell line in vitro,high content scanning experiment to observe the effect of rice on cardiomyocytes Effects of apoptosis and necrosis after hypoxia stimulation.Results Compared with the SAL+MI group,the DEX+MI group was associated with significantly increased phosphorylated GR level(1.75 times),improved cardiac function indexes,such as the left ventricular ejection fraction,end diastolic,end systolic diameter and short axis shortening rate(all P<0.05),reduced heart damage,less infarct size(34.8%±5.8%vs.44.1%±3.6%,P<0.05),increased levels of adiponectin protein(2.82 times)and mRNA(6.45 times),and decreased apoptotic cells(P<0.05).The cell experiment showed that compared with the control group,DEX could significantly reduce the apoptosis and necrosis of H9c2 cardiomyocytes caused by hypoxia stimulation(both P<0.05).Conclusions GR activation has a protective effect on myocardial cell injury after MI,which may be related to its anti-inflammatory,anti-apoptotic and anti-necrotic effects.