摘要
目的探讨PKM2促进ox-LDL刺激的血管平滑肌细胞增殖和糖代谢紊乱的机制。方法首先观察ApoE^(-/-)小鼠主动脉斑块血管平滑肌细胞PKM2表达。然后以人主动脉血管平滑肌细胞为研究对象,利用MMT法观察PKM2表达对细胞增殖的影响,蛋白质印迹法检测NF-κB p65、pEGFR、活性β-ctaenin、cyclin D1、GLUT1、LDHA蛋白表达,定量PCR法检测PKM2 mRNA表达,免疫共沉淀检测PKM2与EGFR之间相互作用,最后利用分光光度法检测培养基葡萄糖消耗量和乳酸生成量。结果PKM2蛋白在ApoE^(-/-)小鼠主动脉斑块血管平滑肌细胞中表达增强;PKM2蛋白和mRNA表达与ox-LDL刺激呈现时间和剂量依赖性正相关,并且受ox-LDL诱导的NF-κB p65蛋白调控;PKM2上调pEGFR、活性β-catenin、cyclin D1、GLUT1和LDHA表达和促进细胞增殖,使细胞葡萄糖消耗量和乳酸产生量增加;免疫共沉淀显示PKM2与EGFR之间存在相互作用;分别抑制PKM2、EGFR和β-catenin表达可抑制ox-LDL诱导的细胞增殖。结论PKM2介导的EGFR活性增强促进ox-LDL诱导的血管平滑肌细胞增殖和Warburg效应。
Objective To investigate the molecular mechanism of PKM2 in the proliferation and glucose metabolism disorder of vascular smooth muscle cells stimulated by ox-LDL.Methods The expression of PKM2 in vascular smooth muscle cells in aortic lesions of ApoE^(-/-)mice was detected by IHC.PKM2 levels of ox-LDL-stimulated VSMC were measured by MMT.The protein expressions of NF-κB p65,pEGFR,activeβ-ctaenin,cyclin D1,GLUT1 and LDHA were detected by Western blotting,and PKM2 mRNA was detected by fluorescent quantitative PCR.The interaction between PKM2 and EGFR was detected by co-immunoprecipitation.Finally,the glucose consumption and lactate production were measured by spectrophotometry.Results The PKM2 level was enhanced in VSMCs of ApoE^(-/-)mice.The PKM2 protein and mRNA were positively correlated in a time and dose-dependent manner upon ox-LDL stimulation.NF-κB p65 was responsible for upregulated PKM2 level in VSMCs induced by ox-LDL.PKM2 up-regulated pEGFR,activeβ-catenin,cyclin D1,GLUT1 and LDHA expression and promoted cell proliferation,and increased glucose consumption and lactate production.Co-immunoprecipitation showed that there was interaction between PKM2 and EGFR.Inhibition of PKM2,pEGFR andβ-catenin protein levels could prevent ox-LDL-induced proliferation of VSMCs respectively.Conclusion PKM2-mediated EGFR activation promotes proliferation and the Warburg effect in ox-LDL-induced VSMCs.
作者
钟智强
吴书玲
张航
孙明洪
姜雨晴
ZHONG Zhi-qiang;WU Shu-ling;ZHANG Hang;SUN Ming-hong;JIANG Yu-qing(不详;Department of Clinical Laboratory,the First Affiliated Hospital of Zhejiang Chinese Medical University,Hangzhou,Zhejiang 310018,China)
出处
《中国卫生检验杂志》
CAS
2021年第24期2961-2967,共7页
Chinese Journal of Health Laboratory Technology
基金
浙江省中医药科学研究基金项目(2021ZA060)。
