摘要
目的探究PKM2拟素化(neddylation)修饰对肺动脉高压大鼠右心室纤维化的影响。方法将SD大鼠随机分为对照组、模型组和MLN4924组,HE染色法检测肺动脉,Masson染色检测右心室纤维化程度。提取大鼠原代心脏成纤维细胞,免疫荧光法检测α-平滑肌肌动蛋白(α-SMA)表达,使用si-NC、si-PKM2、pcDNA3.1-PKM2转染原代心脏成纤维细胞后,蛋白质免疫印迹法检测PKM2及纤维化相关Ⅰ型胶原蛋白(Collagen Ⅰ)、Ⅲ型胶原蛋白(Collagen Ⅲ)、基质金属蛋白酶2(MMP2)、基质金属蛋白酶9(MMP9)等蛋白水平。蛋白质免疫共沉淀法检测PKM2 neddylation修饰。实时荧光定量PCR法检测大鼠心脏组织中PKM2的mRNA表达水平。蛋白质免疫印迹法检测PKM2蛋白质降解时间。结果HE染色结果显示,模型组肺小动脉(纤维层)和内膜(内转运蛋白)之间的距离显著加宽,中间层的厚度增加,Masson染色显示,与对照组相比,模型组显示更多的胶原沉积,纤维化更严重的。模型组的PKM2蛋白表达水平高于对照组,而mRNA水平无显著性差异。PKM2在大鼠肺动脉高压模型中右心室组织存在neddylation修饰。在心脏成纤维细胞中敲低Nedd8或用MLN4924抑制neddylation修饰可下调PKM2及纤维化相关蛋白Collagen Ⅰ、Collagen Ⅲ、MMP2、MMP9等蛋白水平,促进PKM2蛋白质降解速率[(3.03±0.23)~(11.97±0.66)h,t=-12.82,P<0.001],过表达Nedd8则提高PKM2蛋白表达。MLN4924组大鼠右心室纤维化程度及α-SMA蛋白表达水平低于模型组。结论在肺动脉高压大鼠模型中,neddylation修饰增强了PKM2的蛋白质稳定性进而促进右心室纤维化过程。
Objective Exploring the effect of PKM2 neddylation modification on myocardial fibrosis in pulmonary hypertension rats.Methods SD rats were randomly divided into 3 groups:control group,model group,and MLN4924 group.Pulmonary arteries were detected by HE staining,and right ventricular fibrosis was detected by Masson staining.Primary cardiac fibroblasts were isolated from rats and the expression of α-Smooth muscle actin(α-SMA)was measured by immunofluorescence assay.The si-NC,si-PKM2,and pcDNA3.1-PKM2 were transfected into the primary cardiac fibroblasts and the expression levels of PKM2,fibrosis related proteins Collagen Ⅰ,Collagen Ⅲ,MMP2,and MMP9 were detected by Western blotting.Immunoprecipitation assay was used to detect the PKM2 neddylation modification.Real time fluorescence quantitative PCR method was used to detect the mRNA expression level of PKM2 in the rat heart tissues.The degradation of PKM2 protein was detected by Western blotting,and the half-life of PKM2 was determined.Results The HE staining results showed that the space between pulmonary arterioles(fibrous layer)and intima(inner transport protein)in the model group was significantly widened and the intermediate layer were thickened.Masson staining showed that the collagen deposition was increased and the fibrosis was more severe in the model group when compared with those in the control group.The expression level of PKM2 protein in the model group was higher than that in the control group,while there was no significant difference in the mRNA expression level.PKM2 underwent neddylation modification in the right ventricular tissues of pulmonary hypertension rats.Knocking down Nedd8 in cardiac fibroblasts or inhibiting neddylation modification with MLN4924 could downregulate the expression levels of PKM2 and fibrosis related proteins Collagen Ⅰ,Collagen Ⅲ,MMP2,MMP9,etc.,promoting the degradation of PKM2 protein[(3.03±0.23)-(11.97±0.66)h,t=-12.82,P<0.001].However,the overexpression of Nedd8 could increase the expression level of PKM2 prote
作者
郭文昀
王丽霞
王金琳
GUO Wenyun;WANG Lixia;WANG Jinlin(Department of Cardiovascular Medicine,Joint Support Force 940th Hospital,Lanzhou,730050,China)
出处
《医学分子生物学杂志》
CAS
2024年第2期108-114,共7页
Journal of Medical Molecular Biology
基金
甘肃省自然科学基金(No.22JR5RA012)
联勤保障部队第940医院院内项目(No.2021yxky080)。
关键词
肺动脉高压
PKM2
拟素化修饰
右心室纤维化
pulmonary arterial hypertension
PKM2
neddylation
right ventricular fibrosis
