摘要
目的 探究PI3K/Akt/mTOR信号通路在同型半胱氨酸(homocysteine,Hcy)诱导的人血管平滑肌细胞(human vascular smooth muscle cells,hVSMCs)增殖、迁移和表型转化中的作用。方法 体外培养hVSMCs,给予100μmol·L-1Hcy刺激48 h,复制高Hcy血症细胞模型。采用MTT法检测hVSMCs增殖,细胞划痕实验检测hVSMCs迁移;RT-qPCR和Western blot法检测hVSMCs表型标志蛋白α-SMA、SM22α和OPN的mRNA表达和蛋白表达;Western blot法检测PI3K/Akt/mTOR信号通路相关蛋白的表达。给予PI3K信号通路的抑制剂LY294002和激动剂740Y-P刺激hVSMCs,检测对Hcy所致hVSMCs增殖、迁移变化和表型转化的影响。结果 Hcy刺激可以诱导hVSMCs发生增殖、迁移及表型转化,并且hVSMCs中PI3K、Akt、mTOR、p-PI3K和p-Akt的蛋白表达均升高(P均<0.05);给予PI3K信号通路抑制剂LY294002干预后,与Hcy组比较,hVSMCs增殖降低(P<0.05);hVSMCs划痕面积增大(P<0.05);α-SMA和SM22α mRNA和蛋白表达量均增加(P均<0.05),OPN的mRNA和蛋白表达量均降低(P均<0.05)。给予PI3K信号通路激动剂740Y-P干预后,与Hcy组比较则得到了相反的结果。结论 Hcy通过激活PI3K/Akt/mTOR信号通路,诱导hVSMCs增殖、迁移及由收缩表型向合成表型转化,抑制PI3K信号通路对Hcy诱导的hVSMCs增殖、迁移及表型转化起到保护作用。
Objective To explore the role of PI3K/Akt/mTOR signaling pathway in the proliferation,migration and phenotypic switch of human vascular smooth muscle cells(hVSMCs)induced by homocysteine(Hcy).Methods hVSMCs were cultured in vitro and stimulated with 100μmol·L^(-1) Hcy for 48 h to replicate the hyperhomocysteinemia cell model.The proliferation of hVSMCs was detected by MTT assay.The migration of hVSMCs was detected by cell scratch assay.The mRNA and protein expressions ofα-SMA,SM22αand OPN were detected by RT-qPCR and Western blot.The expression of PI3K/Akt/mTOR signaling pathway related proteins were detected by Western blot.hVSMCs were stimulated by LY294002,an inhibitor of PI3K signaling pathway,and 740Y-P,an agonist of PI3K signaling pathway and the effects on proliferation,migration and phenotypic switch of hVSMCs induced by Hcy were detected.Results Hcy stimulation could induce the proliferation,migration and phenotypic switch of hVSMCs,and the protein expressions of PI3K,Akt,mTOR,p-PI3K and p-Akt in hVSMCs were significantly increased(Pall<0.05).After stimulation with,the PI3K signaling pathway inhibitor LY294002,compared with Hcy group,the proliferation of hVSMCs was significantly decreased(P<0.05).The scratch area of hVSMCs increased(P<0.05).The mRNA and protein expressions ofα-SMA and SM22αwere increased(P all<0.05),the expression of OPN mRNA and protein were significantly decreased(Pall<0.05).Compared with the Hcy group,the PI3K signaling pathway agonist 740Y-P had the opposite result.Conclusion Hcy could induce hVSMCs proliferation,migration and phenotypic switch from contractile phenotype to synthetic phenotype by activating PI3K/Akt/mTOR signaling pathway.Targeted inhibition of PI3K signaling pathway could protect hVSMCs proliferation,migration and phenotypic switch induced by Hcy.
作者
潘泓乐
王秀玉
曾玥
马星
徐灵博
张鸣号
PAN Hongyue;WANG Xiuyu;ZENG Yue;MA Xing;XU Lingbo;ZHANG Minghao(Ningxia Medical University,Yinchuan 750004,China;NHC Key Laboratory of Metabolic Cardiovascular Diseases Research,Yinchuan 750004,China;Ningxia Key Laboratory of Vascular Injury and Repair Research,Ningxia Medical University,Yinchuan 750004,China)
出处
《宁夏医科大学学报》
2023年第7期660-667,共8页
Journal of Ningxia Medical University
基金
宁夏自然科学基金项目(2021AAC03122)
宁夏回族自治区重点研发计划项目(2021BEG03093)
宁夏医科大学校级科研项目(XT2022028)
宁夏回族自治区大学生创新创业训练计划项目(S202210752012)
宁夏医科大学大学生创新创业训练计划项目(X202210752047)。
