吲哚-3-甲醇通过抑制mTOR/HIF-1α信号通路减弱低氧诱导的肺动脉平滑肌细胞增殖  

Indole-3-carbinol inhibits the proliferation of hypoxia-induced pulmonary arterial smooth muscle cells by blocking the mTOR/HIF-1αpathway

作　　者：陈昌贵 易春峰 余志华[1] 张帆[1] 李佐民[1] 贺立群[1] CHEN Chang-gui;YI Chun-feng;YU Zhi-hua;ZHANG Fan;LI Zuo-min;HE Li-qun(Department of Cardiology,Wuhan No.1 Hospital,Wuhan 430022)

机构地区：[1]武汉市第一医院心血管内科,武汉430022

出　　处：《中南药学》2024年第9期2300-2306,共7页Central South Pharmacy

基　　金：湖北省卫生健康委科研项目(No.WJ2023F043)。

摘　　要：目的观察吲哚-3-甲醇(I3C)对低氧诱导的肺动脉平滑肌细胞(PASMCs)增殖的作用。方法采用0.2%Ⅰ型胶原酶消化分离SD大鼠PASMCs,以低氧(1%O2)作为诱导剂,建立PASMCs增殖的细胞模型,以不同浓度的I3C(25、50、100、200μmol·L^(-1))干预24 h,检测细胞增殖及存活率;设置哺乳动物雷帕霉素靶蛋白(mTOR)抑制剂rapamycin或缺氧诱导因子-1α(HIF-1α)抑制剂LW6为阳性对照组,以100μmol·L^(-1 )I3C,HIF-1α稳定剂DMOG或mTOR激活剂MHY1485干预24 h后,CCK-8试剂盒检测细胞增殖;流式细胞仪检测细胞周期;Western blot检测HIF-1α、总的mTOR及磷酸化的mTOR和细胞周期调控相关蛋白的表达,确定I3C抑制PASMCs增殖的作用机制。结果25~100μmol·L^(-1 )I3C抑制低氧诱导的PASMCs增殖的作用具有浓度依赖性(P<0.05),而100μmol·L^(-1)与200μmol·L^(-1 )I3C抑制细胞增殖的作用无明显差异,且I3C无明显细胞毒性作用。I3C(100μmol·L^(-1))与LW6均可抑制低氧诱导的PASMCs增殖,阻滞细胞周期于G0/G1期,抑制HIF-1α、细胞周期蛋白(Cyclin)D1、Cyclin E、细胞周期蛋白依赖性激酶(CDK)2、CDK4和CDK6的表达(P<0.05),且DMOG能够逆转I3C的上述作用(P<0.05)。另外I3C与rapamycin在抑制低氧诱导的PASMCs增殖与mTOR/HIF-1α信号通路活化方面作用相似,且MHY1485能够逆转I3C抑制细胞增殖及mTOR/HIF-1α信号通路活化的作用(P<0.05)。结论I3C可通过抑制mTOR/HIF-1α信号通路减弱低氧诱导的PASMCs增殖。Objective To determine the effect of indole-3-carbinol(I3C)on the proliferation of hypoxiainduced pulmonary artery smooth muscle cells(PASMCs).Methods PASMCs were isolated from the pulmonary arteries of SD rats and digested with 0.2%collagenaseⅠ.PASMCs were treated with hypoxia(1%O2)to stimulate the proliferation,then intervened with different concentrations of I3C(25,50,100,and 200μmol·L^(-1))for 24 h to determine the cell proliferation and viability.To explore the molecular mechanisms of how I3C suppressed the cell proliferation under hypoxic conditions,PASMCs were treated with 100μmol·L^(-1 )I3C,mammalian target of rapamycin(mTOR)agonist MHY1485 or hypoxia-inducible factor 1α(HIF-1α)stabilizer DMOG for 24 h,and groups treated with mTOR inhibitor rapamycin or HIF-1αinhibitor LW6,were used as positive controls.Cell proliferation was analyzed by the CCK-8 kit.The cell cycle progression was determined by flow cytometry.The expressions of HIF-1α,total and phosphorylated mTOR,and cell cycle regulatory proteins were examined by Western blot.Results The proliferation of hypoxia-induced PASMCs was inhibited with I3C(25~100μmol·L^(-1))in a concentration-dependent manner(P<0.05).There was no obvious difference in the inhibition of hypoxia-induced PASMC proliferation treated with 100 and 200μmol·L^(-1 )I3C,and I3C was not cytotoxic at the experimental concentration.Both I3C(100μmol·L^(-1))and LW6 blocked the proliferation of PASMCs,arrested cell cycle progression in G0/G1 phase,downregulated the expression of HIF-1α,Cyclin D1,Cyclin E,Cyclin-dependent kinase(CDK)2,CDK4 and CDK6(P<0.05),while DMOG reversed all the above effect of I3C on hypoxia-induced PASMCs(P<0.05).In addition,I3C(100μmol·L^(-1))exerted similar effects with rapamycin in inhibiting the proliferation and inactivated the mTOR/HIF-1αpathway in hypoxia-induced PASMCs.More specifically,MHY1485 reversed the effect of I3C on blocking cell proliferation and inhibiting the mTOR/HIF-1αpathway in hypoxia-induced PASMCs(P<0.05).Conclusion I3C may

关 键 词：低氧 肺动脉平滑肌细胞 吲哚-3-甲醇 增殖 哺乳动物雷帕霉素靶蛋白/缺氧诱导因子1α 

分 类 号：R96[医药卫生—药理学]