血管过氧化物酶1对心肌梗死后心肌纤维化机制的影响

Effect of vascular peroxidase 1 on the mechanism of myocardial fibrosis after myocardial infarction

目的探讨血管过氧化物酶1在心肌梗死(MI)后心肌纤维化中的作用,及其对心肌纤维化机制的影响。方法建立心肌梗死SD大鼠模型,于造模后7、14、28 d采用Masson染色法观察心肌胶原纤维的表达,免疫荧光法观察心肌组织中MPO、VPO1、α-SMA的表达及定位,免疫组织化学法观察心肌组织中3-Cl Tyr和Ki67的表达情况,Western blot检测心肌成纤维细胞中MPO、VPO1、α-SMA和CollagenⅠ的蛋白表达水平,RNAi技术沉默心肌成纤维细胞中VPO1的表达,并通过Western blot、Masson染色和CCK-8细胞增殖检测来探究VPO1对心肌纤维化机制的影响。结果在MI大鼠模型中,心肌间质纤维化程度较重;MPO、VPO1和α-SMA的荧光染色结果均呈阳性;随MI后时间的推移,VPO1、α-SMA和Collagen I的表达均显著升高(P<0.01),MPO在MI后7 d表达量很高(P<0.01),但在28 d与sham组差异无统计学意义(P>0.05);免疫组化染色后发现,MI组均有大量棕褐色着色细胞,在7、14、28 d时,3-Cl Tyr和Ki67表达量均高于sham组(P<0.05或P<0.01)。通过对大鼠尾静脉注射siRNA抑制VPO1表达,心肌纤维化程度显著下降(P<0.01),大鼠存活率相对提高(P<0.05)。同时,si-VPO1处理后,心肌梗死的心肌成纤维细胞中α-SMA和CollagenⅠ表达受到显著抑制(P<0.01),Ki67阳性细胞增殖数量也显著减少(P<0.01)。结论VPO1是心肌梗死后心肌纤维化的关键调节因子之一,可能通过调节心肌成纤维细胞增殖分化以及胶原蛋白I型合成来介导心肌纤维化的发展,VPO1有望成为缺血性心肌病的治疗靶点。

Objective To investigate the role of vascular peroxidase 1 in myocardial fibrosis after myocardial infarction and its effect on the mechanism of myocardial fibrosis.Methods The rat model of myocardial infarction was established.The expression of myocardial collagen fibers was observed by Masson staining on the 7 th,14 th and 28 th day after modeling.The expression and location of MPO,VPO1 and α-SMA in myocardial tissue was observed by immunofluorescence staining.The expression of 3-Cl Tyr and Ki67 in myocardial tissue was observed by immunohistochemical staining.The protein levels of MPO,VPO1,α-SMA and CollagenⅠwere detected by Western blot,and the expression of VPO1 in cardiac fibroblasts was silenced by RNAi technique.Then,the effects of VPO1 on the mechanism of myocardial fibrosis were investigated by Western blot,Masson staining and CCK-8 cell proliferation assay.Results In the MI rat model,the degree of myocardial interstitial fibrosis was heavier;the fluorescence staining results of MPO,VPO1 andα-SMA were positive;the expression of VPO1,α-SMA and CollagenⅠsignificantly increased with the time after MI(P<0.01).The expression of MPO was high on the 7th day after MI(P<0.01),but there was no difference between the 28th day and the sham group(P>0.05).Immunohistochemical staining revealed that there were a large number of brown-colored cells in the MI group.At 7th,14th,and 28th day,the expression levels of 3-Cl Tyr and Ki67 were higher than those in the sham group(P<0.05 or P<0.01).After inhibiting the expression of VPO1 by injecting siRNA into the tail vein of rats,it was found that the degree of myocardial fibrosis was significantly decreased(P<0.01),and the survival rate of the rats was relatively increased(P<0.05).At the same time,the expression ofα-SMA and CollagenⅠin myocardial infarcted myocardium fibroblasts was significantly inhibited after si-VPO1 treatment(P<0.01),and the number of Ki67 positive cells was also significantly decreased(P<0.01).Conclusion VPO1 is one of the key regulators of myocardial fibrosis after myocardial infarction.It may mediate the development of myocardial fibrosis by regulating the proliferation and differentiation of cardiac fibroblasts and collagen type I synthesis.VPO1 is expected to be a therapeutic target for cardiomyopathy.