血管紧张素转换酶2过表达对大鼠急性心肌梗死后心室重构的影响及其分子机制

Effect of overexpresssion of angiotensin converting enzyme 2 on ventricular remodeling in rats after myocardial infarction

目的探讨血管紧张素转换酶2(angiotensin converting enzyme 2,ACE2)过表达对大鼠急性心肌梗死(acute myocardial infarction,AMI)后心室重构的影响及其可能的分子机制。方法采用开胸结扎冠状动脉法建立SD大鼠AMI模型,将模型大鼠随机分为MI、MI+(NS)、MI+AdEGFP、MI+AdACE2组,另设SHAM(假手术)组,每组15只,MI+NS、MI+AdEGFP和MI+AdACE2组沿心肌梗死周边区选取5个点,通过直接心肌内注射方式分别注射NS、AdEGFP和AdACE2,SHAM和MI组不注射。术后4周末,采用Western blot法检测大鼠心肌梗死周边区心肌组织中ACE2、α-SMA、MKP-1、ERK1/2、p-ERK1/2、P38、TGF-β1蛋白的表达;HE染色后于普通显微镜下观察心肌组织结构及细胞炎症变化,天狼猩红-饱和苦味酸染色后于普通显微镜及偏振光显微镜下分别观察心肌胶原表达分布情况;SP免疫组化染色法检测大鼠心肌梗死周边区心肌组织中AngⅡ、Ang(1-7)、α-SMA蛋白的表达,同时采用ELISA法检测Ang(1-7)蛋白的表达;使用羟脯氨酸试剂盒检测大鼠心肌梗死周边区心肌组织中羟脯氨酸含量。结果术后4周末,MI+AdACE2组大鼠心肌梗死周边区心肌组织中ACE2蛋白的表达量显著高于其他各组,同时Ang(1-7)蛋白的表达量也显著升高(P<0.05);与SHAM组相比,MI、MI+NS、MI+AdEGFP组大鼠心肌梗死周边区心肌组织中AngⅡ、Ang(1-7)、α-SMA蛋白的表达量显著升高(P<0.05),MI+AdACE2组与MI、MI+NS、MI+AdEGFP组相比,AngⅡ、α-SMA蛋白表达水平降低,Ang(1-7)蛋白表达水平升高更显著;与MI、MI+NS、MI+AdEGFP各组相比,MI+AdACE2组大鼠心肌梗死周边区心肌组织中MKP-1蛋白的表达水平明显增加(P<0.05),p-ERK、P38、TGF-β1蛋白的表达水平降低,羟脯氨酸含量显著降低(P<0.05);HE染色和天狼猩红-饱和苦味酸染色证实,ACE2对AMI后梗死周边区的炎症反应和胶原重塑均有明显的改善作用。结论 ACE2在心肌过表达能有效改善大鼠AMI后心肌梗死周边区心肌纤维化进程,缓解心室重构,其机制可能与ACE2调节肾素-血管紧张素系统(rennin-angiotensin system,RAS)、平衡丝裂原活化蛋白激酶(mitogen activated protein kinases,MAPK)活性相关。

Objective To investigate the effect of adenovirus-angiotensin convertin enzyme 2（AdACE2） on ventricular re- modeling in rats after acute myocardial infarction （AMI） as well as the possible molecular mechanism. Methods Seven- ty-five SD rats were randomly divided into MI, MI ＋ normal saline （NS）, MI ＋ adenovirns-EGFP （AdEGFP）, MI＋ AdACE2 and sham operation （SHAM） groups, fifteen for each. AMI model was established by left anterior descending coronary artery ligation through a lateral thoracotomy in rats. Rats in MI＋NS, MI ＋ AdEGFP and MI ＋ AdACE2 groups received direct intramyocardial injection with NS, AdEGFP and AdACE2 respectively, while those in MI and SHAM groups received no injection. Four weeks later, the expressions of ACE2, α-SMA, MKP-1, ERK1/2, p-ERK1 /2, P38 and TGF-β1 in cardiac tissues around infarction zone were determined by Western blot. The structure of cardiac tissue and inflammation in cells were observed under common microscope after HE staining. The expression and distribution ofcardiac collagen were observed under common microscope and polarizing microscope after picrosirius red staining. The ex- pressions of Ang Ⅱ and α-SMA in eardiac tissues around infarction zone were determined by immunohistochemical assay with SP staining, while that of Ang （1-7） by both immunohistochemical assay and ELISA, and hydroxyproline content by hydroxyproline determination kit. Results Tile expression level of ACE2 in cardtiac tissues around infarction zone of rats in MI ＋ AdACE2 group 4 weeks after operation was significantly higher than those in other groups, while that of Ang （1-7） increased significantly （P 〈 0. 05）. The expression levels of Aug Ⅱ , Ang （1-7） and ct-SMA in MI, MI ＋ NS and MI ＋ AdEGFP groups were significantly higher than those in SHAM group （P 〈 0. 05）. The expression levels of Ang Ⅱand ct- SMA were significantly lower, while that of Ang （1-7） was renmrkably higher, in MI＋AdACE2 group than in MI, MI ＋ NS and MI ＋ AdEGFP groups. As compared with those in MI, MI ＋ NS and MI ＋ AdEGFP groups, the expression level of MKP-1 in M1 ＋ AdACE2 group ineased significantly （P 〈 0. 05）, while those of p-ERK, P38 and TGF-β1 decreased, and hydroxyproline content decreased significantly （P 〈 0. 05）. HE staining and picrosirius red staining proved that ACE2 improved the inflammatory reaction and collagen modeling in cardiac tissues around infarction zone significantly. Conclu- sion ACE2 overexpression improved the fibrosis of cardiac tissues around infarction zone and ameliorated the ventricular remodeling of rats after AMI by a possible mechanism associated with regulating reninin-angiotensin system （RAS） and mitogen activated protein kinase （MAPK） activities.