Ghrelin对易损斑块内血管新生和心肌梗死后血管新生的影响及机制研究

Effects and mechanisms of ghrelin on angiogenesis in vulnerable plaque and myocardial infarction

目的:探讨ghrelin对易损斑块内血管新生和心肌梗死后血管新生的影响及机制。方法:将ApoE^(-/-)小鼠分为易损斑块组、易损斑块+心梗双模型组和ghrelin干预组。所有ApoE^(-/-)小鼠均高脂饮食喂养12周诱导动脉粥样硬化易损斑块,构建的动脉粥样硬化易损斑块模型即为易损斑块组。遗传背景相同的C57BL76J小鼠正常饮食喂养12周,分为正常对照组和单纯心梗组。第8周时,单纯心梗组、易损斑块+心梗双模型组和ghrelin干预组结扎冠状动脉左前降支构建急性心梗模型。急性心梗造模后,ghrelin干预组腹腔注射ghrelin(100 μg/kg,2次/d),直至第12周实验结束。超声心动图检测各组左室射血分数(left ventricular ejection fraction,LVEF)和左室短轴缩短率(left ventricular fraction shortening,LVFS),油红O染色观察主动脉寨粥样斑块面积的百分比,CD31免疫组化染色检测易损斑块内和心梗周边区新生血管生成密度(microvascular density,MVD),Masson染色检测心肌梗死面积。Real-time PCR和Western blot分别检测各组血管内皮细胞生长因子(vascular endothelial growth factor,VEGF)、血管生成素(angiopoietins,Ang)-1/2和络氨酸激酶受体(tyrosine-protein kinase receptor,Tie)-2的表达。结果:①与单纯心梗组相比,易损斑块+心梗双模型组[(65.518±4.160)%,P=0.000]心梗面积明显增加,LVEF[(58.560±11.900)%,P=0.012]和LVFS[(27.182±7.807)%,P=0.013]降低,心梗周边区MVD(5.800±0.837,P=0.000)减低,VEGF(1.870±0.122,P=0.001)、Ang-1(1.830±0.056,P=0.007)、Ang-2(1.660±0.217,P=0.006)和Tie-2(1.660±0.115,P=0.020)表达降低,ghrelin干预后可明显缩小心梗面积[(39.751±3.039)%,P=0.000],增加易损斑块+心梗双模型组LVEF[(74.679±6.535)%,P=0.013]和LVFS[(37.507±5.210)%,P=0.020],增加心梗周边区MVD(9.857±1.345,P=0.000),升高VEGF(3.503±0.384,P=0.004)、Ang-1(3.277±0.186,P=0.017)、Ang-2(3.450±0.187,P=0.000)和Tie-2(3.133±0.139,P=0.009)的表达;②易损斑块组、易损斑块+心梗双模型组和ghrelin干预组主动脉窦粥样斑块面积的百分比、斑块内MVD、斑块内VEGF、Ang-1、Ang-2和Tie-2的基因与表达在3组间无统计学差异,ghrelin对易损斑块合并心梗小鼠斑块内血管新生无影响。结论:Ghrelin可通过上调VEGF、Ang-1、Ang-2和Tie-2的基因与蛋白表达,促进易损斑块+心梗双模型组小鼠心梗后血管新生,减少心肌梗死面积,稳定和缩小易损斑块。Ghrelin对易损斑块+心梗双模型组小鼠斑块内血管新生无影响。

Objective:To investigate the effects and mechanisms of ghrelin on angiogenesis in vulnerable plaque and myocardial infarction.Methods:The ApoE^(-/-)mice were divided into vulnerable plaque group,vulnerable plaque+myocardial infarction group and ghrelin-intervened group.All ApoE^(-/-)mice were fed with a high-fat diet for 12 weeks to induce atherosclerotic vulnerable plaques.The induced-atherosclerotic vulnerable plaque model was established in vulnerable plaque group.The C57BL/6J mice were fed for 12 weeks on a normal diet and divided into control group and myocardial infarction group.At the 8th week of this study,myocardial infarction group,vulnerable plaque+myocardial infarction group and ghrelin group were subjected to acute myocardial infarction model.After this model,the ghrelin group was administered ghrelin(100 fxg/kg,bid)until the end of the 12th week.The left ventricular ejection fraction(LVEF)and left ventricular fraction shortening(LVFS)were detected by echocardiography and the percentage of atherosclerotic plaque area in aortic sinus was observed by Oil red 0 staining.The microvascular density(MVD)in vulnerable plaque and surrounding area of myocardial infarction were measured by CD31 immunohistochemistry and Masson staining,respectively.The mRNA and protein expression of vascular endothelial growth factor(VEGF),angiopoietins(Ang)-1/2 and tyrosine-protein kinase receptor(Tie)-2 were respectively examined by real-time PCR and Western blot.Results:①Compared with myocardial infarction group,the myocardial infarct size in vulnerable plaque+myocardial infarction group[(65.518±4.160)%,P=0.000] was increased,whereas the LVEF[(58.560±11.900)%,P=0.012]and LVFS[(27.182±7.807)%,P=0.013],the MVD(5.800±0.837,P=0.000)in the peripheral area of myocardial infarction,and the expression of VEGF(1.870±0.122,P=0.001),Ang-1(1.830±0.217,P=0.006)and Tie-2(1.660±0.115,P=0.020)were decreased,significantly.Additionally,the ghrelin intervention had significantly reduced myocardial infarct size[(39.751±3.039)%,P=0.000],and improved LVEF[(74.679±6.535)%,P=0.013].LVFS[(37.507±5.210)%,P=0.020],the MVD(9.857±1.345,P=0.000)in the peripheral area of myocardial infarction,and the expression of VEGF(3.503±0.384,P=0.004)Ang-1(3.277±0.186,P=0.017),Ang-2(3.450±0.187,P=0.000)and Tie-2(3.133±0.139,P=0.009)of the vulnerable plaque+myocardial infarction group.②There were no significant differences in the percentage of aortic sinus plaque area,the MVD in vulnerable plaque,and the expression of VEGF,Ang-1,Ang-2 and Tie-2 among the vulnerable plaque group,the vulnerable plaque+myocardial infarction group and the ghrelin-intervened group.Regrettably,ghrelin had no effect on angiogenesis in vulnerable plaque+myocardial infarction mice.Conclusion:Ghrelin can promote angiogenesis in ischemic myocardium,reduce myocardial infarct size,and stabilize vulnerable plaque by increasing the mRNA and protein expression of VEGF,Ang-1,Ang-2 and Tie-2.Ghrelin had no effect on angiogenesis in vulnerable plaque of vulnerable plaque+myocardial infarction group.