NOD2通过调控细胞外基质活性及炎性因子水平介导心肌梗死后心室重构

NOD2 mediates cardiac remodeling after myocardial infarction by regulating the activity of extracellular matrix and inflammation

目的探讨细胞内模式识别受体——核苷酸结合寡聚化结构域(NOD)蛋白家族中NOD2蛋白是否参与心肌梗死(MI)后心室重构及相关机制。方法结扎小鼠冠状动脉左前降支建立MI模型。实时荧光定量PCR检测不同梗死时间假手术组、手术组梗死区、手术组非梗死区NOD2的m RNA表达水平,免疫组化染色和Western blot测定梗死28 d心肌组织NOD2的蛋白表达。采用NOD2-/-基因敲除小鼠建立MI模型,实验分为4组:野生/假手术组(WT/Sham组)、NOD2基因敲除/假手术组(NOD2-/-/Sham组)、野生/MI组(WT/MI组)和NOD2基因敲除/MI(NOD2-/-/MI组)。小动物超声心动图测定心室重构指数并判断心功能,Masson和Tunel染色观察小鼠心梗后心肌纤维化程度及细胞凋亡情况,酶联免疫吸附法(ELISA)测定梗死心肌组织的促炎细胞因子水平,Western blot和明胶酶谱法检测心肌组织裂解液基质金属蛋白酶9(MMP-9)蛋白及活性变化,并用免疫组化染色观察MI后炎性细胞浸润和心脏成纤维细胞转分化成心肌成纤维细胞情况。结果与WT/Sham组相比,WT/MI组非梗死区和梗死区NOD2 m RNA表达水平均升高(均为P <0. 05),其中梗死区NOD2的m RNA表达及蛋白表达最高。超声心动图提示,NOD2缺失能减轻MI后心功能障碍和心室重构的程度; Tunel和Masson染色显示,NOD2-/-/MI组的细胞凋亡和心肌纤维化程度均明显降低(均为P <0. 05)。NOD2缺失还能降低MI区炎性因子水平、炎性细胞的浸润及MMP-9的活性(均为P <0. 05)。结论 NOD2通过调控MMP-9蛋白及活性、炎性介质水平和炎性细胞浸润来介导MI后心室重构过程。

Objective To investigate whether nucleotide-binding oligomerization domain containing 2(NOD2)protein participate in the process of cardiac remodeling after myocardial infarction(MI)and the related mechanism of NOD2 regulating cardiac remodeling. Methods Mouse MI model was established by ligation of the left anterior descending coronary artery in mice.Sham operated ones underwent the same procedure but without ligation.Both infarct and remote myocardium from sham and post-MI 3,7,14 and 28 d were homogenized to evaluate mRNA expression of NOD2 by quantitative real-time polymerase chain reaction(qPCR),and its protein level was detected by Western blot and immunohistochemistry 28 days post-MI.To further investigate the role of NOD2 in post-MI cardiac remodeling process,NOD2 gene knockout mouse were used to establish myocardial infarction model.The experiment was divided into 4 groups:wild sham operation group,NOD2-/-knocking mouse sham operation group,wild model group and NOD2-/-knocking mouse model group.Left ventricular internal diameter and cardiac function 28 days post-MI were acquired by small animal echocardiography;Masson staining and Tunel staining were used to observe the degree of myocardial fibrosis and apoptosis after MI;the tissue proinflammatory factor levels were measured by enzyme-linked immunosorbent assay(ELISA);the protein level and activity changes of matrix metalloproteinase 9(MMP-9)of cardiac tissue lysates were detected by Western blot and gelatin zymography.The inflammatory cells infiltration after myocardial infarction and cardiac fibroblasts(cardiac fibroblast)trans-differentiation into cardiomyocytes fibroblasts were observed by immunohistochemical method. Results Compared with sham operation group,the expression of NOD2 mRNA in the non-infarct area and infarct area were all increased in MI group(P<0.05),both mRNA expression and protein expression of NOD2 in infarction area were increased most significantly.NOD2 deficiency exhibited improvements in cardiac dysfunction and remodeling after MI.Furthermore,TUNEL staining and Masson trichrome staining indicated that NOD2 deficiency protected against ischemia-induced cell death and cardiac fibrosis(both P<0.05).Also deletion of NOD2 reduced the levels of inflammatory mediators,inflammatory cell infiltration and MMP-9 activity after MI(all P<0.05). Conclusions NOD2 mediates cardiac remodeling after myocardial infarction by regulating the levels of MMP-9 protein and activity,inflammatory mediators production and inflammatory cell infiltration.