医学分子生物学杂志2024，Vol.21Issue(2)：108-114,7.DOI:10.3870/j.issn.1672-8009.2024.02.003
肺动脉高压大鼠模型中PKM2 Neddylation修饰促进右心室纤维化的研究
Effect of PKM2 Neddylation Modification on Right Ventricular Fibro-sis in Pulmonary Hypertension Rats
摘要
Abstract
Objective Exploring the effect of PKM2 neddylation modification on myocardial fi-brosis in pulmonary hypertension rats.Methods SD rats were randomly divided into 3 groups:con-trol 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 isola-ted from rats and the expression of α-Smooth muscle actin(α-SMA)was measured by immunofluo-rescence assay.The si-NC,si-PKM2,and pcDNA3.1-PKM2 were transfected into the primary car-diac 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 pro-tein 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(in-ner 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 pro-tein.The degree of right ventricular fibrosis and the expression level of α-SMA protein in the MLN4924 group were lower than those in the model group.Conclusion Neddylation modification enhances the protein stability of PKM2,thereby promoting the process of right ventricular fibrosis in the rat model of pulmonary arterial hypertension.关键词
肺动脉高压/PKM2/拟素化修饰/右心室纤维化Key words
pulmonary arterial hypertension/PKM2/neddylation/right ventricular fibrosis分类
医药卫生引用本文复制引用
郭文昀,王丽霞,王金琳..肺动脉高压大鼠模型中PKM2 Neddylation修饰促进右心室纤维化的研究[J].医学分子生物学杂志,2024,21(2):108-114,7.基金项目
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) This work was supported by grants from the Natural Science Foundation of Gansu Province(No.22JR5RA012),the Project of Joint Support Force 940 Hospital(No.2021yxky080) （No.22JR5RA012）
