Vinexin-β基因敲除在小鼠心肌梗死后心脏重构中的作用

The role of Vinexin-β gene knockout in cardiac remodeling after myocardial infarction in mice

目的采用野生型(WT)小鼠和Vinexin-β基因敲除(KO)小鼠建立心肌梗死(MI)模型,研究Vinexin-β在心肌梗死后心脏重构中的作用及机制。方法分别以WT和KO小鼠为实验对象,采取结扎左冠状动脉前降支的方法建立小鼠MI模型(WT-MI组,n=34;KO-MI组,n=27);只在前降支处穿过一支短丝线不做结扎的假手术组(WT-Sham组,n=12;KO-Sham组,n=15)。术后7 d采用HE染色评价MI面积,超声心动图检测心功能,生物信号采集处理系统监测血液动力学指标。原位末端标记(TUNEL)染色法检测MI周边区心肌细胞凋亡,Western blot法检测MI周边区抗凋亡基因Bcl-2、caspase-3和促凋亡基因Bax、caspase-3 cleavage的蛋白表达水平及核转录因子(NF)-κB通路中IκBα和p65蛋白磷酸化(p-IκBα,p-p65)及其总蛋白(T-IκBα,T-p65)水平;免疫荧光实验检测MI周边区的T淋巴细胞(CD3^+)、中性粒细胞(LY6G+)及巨噬细胞(MAC1+)等炎症细胞浸润程度。从小鼠存活率、心功能、病理分析及分子生物学等途径来评价Vinexin-β在MI后心脏重构中的作用。结果与WT-MI组相比,KO-MI组可提高小鼠的存活率(81.48%vs 50.00%,P<0.05),减小MI面积[(7.62±1.08)%vs(15.48±1.24)%,P<0.05]和改善心功能。TUNEL染色显示,KO-MI组梗死周边区的凋亡细胞较WT-MI组减少[(0.75±0.12)%vs(1.07±0.48)%,P<0.05]。Western blot法显示,KO-MI组梗死周边区抗凋亡基因Bcl-2和caspase-3的蛋白表达水平较WT-MI组升高(P均<0.05);促凋亡基因Bax和caspase-3 cleavage蛋白表达水平较WT-MI组降低(P均<0.05);KO-MI组的p-IκBα、T-p65和p-p65蛋白表达水平较WT-MI组显著降低,T-IκBα水平显著升高(P均<0.05),提示IκBα和p65的磷酸化水平受到抑制。免疫荧光染色显示,KO-MI组梗死周边区的T淋巴细胞、中性粒细胞及巨噬细胞等炎症细胞数量较WT-MI组减少(P均<0.05)。结论 Vinexin-β基因缺失可减轻小鼠MI周边区的炎症和凋亡反应,从而减小MI面积和改善心功能,因此可提高小鼠MI后的存活率。

Objective To investigate the effect of Vinexin-β gene knockout on cardiac remodeling following myocardial infarction in mice. Methods Taking the mice of wild type（ WT） and Vinexin-β knockout（ KO） as experimental objects,the myocardial infarction（ MI） mice models were established by ligating left anterior descending branch of coronary artery（ WT-MI group,n = 34; KO-MI group,n = 27）. In addition,the sham operation groups（ only a short silk thread was inserted through the anterior descending branch without ligation） were established as controls（ WT-Sham group,n = 12; KO-Sham group,n = 15）. Seven days after operation,HE staining was used to evaluate the infarction area. Biological signal acquisition and processing system was used to monitor the haemodynamic indexes. TUNEL staining method was used to detect the myocardial cell apoptosis in the border zone of infarcted lesion. Western blot method was used to detect the expressions of Bcl-2,caspase-3（ anti-apoptosis genes） and Bax,caspase-3 cleavage（ apoptosis gene） proteins in the border zone of infarcted lesion,as well as the expression levels of phosphorylated and total nuclear factor-κB（ NFκB） inhibitory proteins α（ IκBα） and p65 proteins（ p-IκBα,T-IκBα,p-p65,T-p65） in NFκB pathway. Immunofluorescence assay was used to detect the infiltration degree of inflammatory cells including T lymphocytes（ CD3^＋）,neutrophils（ LY6 G＋） and macrophages（ MAC1＋） in the border zone of infarcted lesion. The effect of Vinexin-β on cardiac remodeling after myocardial infarction was evaluated by survival rate of mice,cardiac functions,pathological analysis and molecular biology.Results Compared with WT-MI group,the survival rate of mice and cardiac functions improved in KO-MI group,and MI area decrease in KO-MI group（ all P〈0. 05）. TUNEL staining showed that apoptotic cells of the border zone of infarcted lesion in KO-MI group were significantly less than those in WT-MI group（ P〈0. 05）. Western blot showed that（ 1） the protein levels of anti-apoptotic genes increased,and the protein levels of pro-apoptotic genes decreased at the border zone of infarcted lesion in KO-MI group compared with WT-MI group（ all P〈0. 05）;（ 2） compared with WT-MI group,the phosphorylation levels of p65 and IκBα proteins（ p-p65,T-p65,p-IκBα） of NF-κB pathway in KO-MI group decreased,TIκBα increased（ all P〈0. 05）. Immunofluorescence assay showed that T lymphocytes,neutrophils and macrophages at the border zone of infarcted lesion in KO-MI group were significantly less than those in WT-MI group（ all P〈0. 05）.Conclusion Vinexin-β gene deficiency can alleviate the inflammation and apoptosis reactions at the border zone of infarcted lesion,and thus reduce the area of myocardial infarction and improve the heart functions,therefore improve the survival rate after myocardial infarction in mice.