摘要
目的:观察体外过表达胶原三螺旋重复蛋白1(CTHRC1)对血管紧张素Ⅱ(AngⅡ)诱导的病理性心肌重构的影响及相关机制。方法:将体外培养大鼠H9c2心肌细胞分为GFP组(转染阴性对照GFP-AAV9型基因腺病毒4.8×10^(10)PFU/mL)及CTHRC1组(转染CTHRC1-AAV9型基因腺病毒2.4×10^(10)PFU/mL)。再将GFP组分为Vehicle-GFP组(转染阴性对照GFP-AAV9型基因腺病毒4.8×10^(10)PFU/mL)和GFP-AngⅡ组(转染阴性对照GFP-AAV9型基因腺病毒4.8×10^(10)PFU/mL,AngⅡ10μmol/L);将CTHRC1组分为Vehicle-CTHRC1组(转染CTHRC1-AAV9型基因腺病毒2.4×10^(10)PFU/mL)、CTHRC1-AngⅡ组(转染CTHRC1-AAV9型基因腺病毒2.4×10^(10)PFU/mL,AngⅡ10μmol/L)。采用蛋白免疫印迹(Western Blot)法检测CTHRC1蛋白表达;鬼笔环肽染色评价H9c2细胞表面积。10%十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)后对凝胶进行考马斯亮蓝染色,切胶并利用高分辨率质谱进行蛋白鉴定,对差异基因进行基因本体(GO)、基因集富集分析(GSEA)功能富集分析。试剂盒检测超氧化物歧化酶(SOD)、过氧化氢酶(CAT)及丙二醛(MDA)活性。结果:与Vehicle-GFP组比较,GFP-AngⅡ组H9c2细胞表面积增大(P<0.001);与Vehicle-CTHRC1组比较,CTHRC1-AngⅡ组H9c2细胞表面积增大(P<0.001);与GFP-AngⅡ组比较,CTHRC1-AngⅡ组比较H9c2细胞表面积增大(P<0.001)。与Vehicle组比较,AngⅡ组H9c2细胞表达水平在75 kDa附近降低。与GFP组比较,CTHRC1组有31个蛋白显著下调、30个蛋白显著上调(P<0.05);对显著下调蛋白进行GO分析,谷胱甘肽代谢、蛋白质稳定等信号通路被显著富集;对显著下调蛋白进行GSEA分析,谷胱甘肽代谢通路被显著富集。与GFP-AngⅡ组比较,Vehicle-GFP组H9c2细胞中SOD和CAT活性降低,MDA活性增高(P<0.05或P<0.001);与Vehicle-CTHRC1组比较,CTHRC1-AngⅡ组H9c2细胞中SOD和CAT活性降低,MDA活性增高(P<0.01或P<0.001);与GFP-AngⅡ组比较,CTHRC1-AngⅡ组H9c2细胞中SOD和CAT活性降低,MDA活性增高(P<0.05或P<0.001)。结论:体外过表达CTHRC1可加重AngⅡ诱导的病理性心肌重构,其功能与抑制谷胱甘肽代谢通路有关。
Objective:To observe the effect of overexpression of collagen triple helix repeat containing-1(CTHRC1)in vitro on pathological myocardial remodeling induced by angiotensinⅡ(AngⅡ)and the related mechanisms.Methods:The rat H9c2 cardiomyocytes cultured in vitro were divided into the GFP group(transfected with negative control GFP-AAV9 type gene adenovirus 4.8×10^(10) PFU/mL)and the CTHRC1 group(transfected with CTHRC1-AAV9 type gene adenovirus 2.4×10^(10) PFU/mL).The GFP group was further divided into the Vehicle-GFP group(transfected with negative control GFP-AAV9 type gene adenovirus 4.8×10^(10) PFU/mL)and the GFP-AngⅡgroup(transfected with negative control GFP-AAV9 type gene adenovirus 4.8×10^(10) PFU/mL,AngⅡ10μmol/L).the CTHRC1 group was divided into the Vehicle-CTHRC1 group(transfected with CTHRC1-AAV9 type gene adenovirus 2.4×10^(10) PFU/mL)and the CTHRC1-AngⅡgroup(transfected with CTHRC1-AAV9 type gene adenovirus 2.4×10^(10) PFU/mL,AngⅡ10μmol/L).The expression of CTHRC1 protein was detected by Western Blot.The surface area of H9c2 cells was evaluated by phalloidin staining.After 10%sodium dodecyl sulfatepolyacrylamide gel electrophoresis(SDS-PAGE),the gel was stained with Coomassie Brilliant blue,cut,and protein identification was performed using high-resolution mass spectrometry.Gene Ontology(GO)gene,and set enrichment analysis(GSEA)functional enrichment analysis was conducted for the differentially expressed genes.The activities of superoxide dismutase(SOD),catalase(CAT),and malondialdehyde(MDA)were detected using kits.Results:Compared with the Vehicle-GFP group,the surface area of H9c2 cells in the GFP-AngⅡgroup increased(P<0.001).Compared with the Vehicle-CTHRC1 group,the surface area of H9c2 cells in the CTHRC1-AngⅡgroup increased(P<0.001).Compared with the GFP-AngⅡgroup,the surface area of H9c2 cells in the CTHRC1-AngⅡgroup increased(P<0.001).Compared with the Vehicle group,the expression of H9c2 cardiomyocytes in the AngⅡgroup decreased around 75 kDa.Compared with the GFP group,the CTHRC1 group showed 31 proteins significantly downregulated and 30 proteins significantly upregulated(P<0.05).For the significantly downregulated proteins,the GO analysis showed that signaling pathways such as glutathione metabolism and protein stability were significantly enriched.For the significantly downregulated proteins,the GSEA analysis showed that the glutathione metabolism pathway was significantly enriched.Compared with the GFP-AngⅡgroup,the H9c2 cardiomyocytes in the Vehicle-GFP group showed lower SOD and CAT activities and higher MDA activity(P<0.05 or P<0.001).Compared with the Vehicle-CTHRC1 group,the H9c2 cardiomyocytes in the CTHRC1-AngⅡgroup showed lower SOD and CAT activities and higher MDA activity(P<0.01 or P<0.001).Compared with the GFP-AngⅡgroup,the H9c2 cardiomyocytes in the CTHRC1-AngⅡgroup showed lower SOD and CAT activities and higher MDA activity(P<0.05 or P<0.001).Conclusion:Overexpression of CTHRC1 in vitro could aggravate pathological myocardial remodeling induced by AngⅡ,and its function was related to the inhibition of the glutathione metabolism pathway.
作者
李楠
付贺青
初士棋
张姝
聂慧娟
董玉
刘天龙
LI Nan;FU Heqing;CHU Shiqi;ZHANG Shu;NIE Huijuan;DONG Yu;LIU Tianlong(The Affiliated Hospital of Inner Mongolia Medical University,Hohhot 010059,Inner Mongolia,China;Inner Mongolia Medical University,Hohhot 010110,Inner Mongolia,China)
出处
《中西医结合心脑血管病杂志》
2026年第6期869-877,共9页
Chinese Journal of Integrative Medicine on Cardio-Cerebrovascular Disease
基金
国家自然科学基金项目(No.82160058)
内蒙古自治区医师协会临床医学研究和临床新技术推广项目(No.YSXH2024KYF049)
内蒙古医科大学教育教学改革研究与实践项目(No.NYJXGGSJ2024024)。
关键词
心肌重构
胶原三螺旋重复蛋白1
氧化应激
富集分析
谷胱甘肽代谢
实验研究
myocardial remodeling
collagen triple helix repeat containing-1
oxidative stress
enrichment analysis
glutathione metabolism
experimental study
