GW501516对低氧条件下肺动脉平滑肌细胞增殖的作用及其机制  

Effects of GW501516 on the proliferation of pulmonary artery smooth muscle cells induced by hypoxia and its mechanisms

作　　者：陈昌贵 贺立群[1] 田立群[1] 易春峰 CHEN Chang-gui;HE Li-qun;TIAN Li-qun;YI Chun-feng(Department of Cardiology,Wuhan No.1 Hospital,Wuhan 430022,China)

机构地区：[1]武汉市第一医院心血管内科,湖北武汉430022

出　　处：《中国应用生理学杂志》2022年第2期119-125,共7页Chinese Journal of Applied Physiology

基　　金：湖北省自然科学基金资助项目(2018CFC801)。

摘　　要：目的:观察过氧化物酶体增殖物激活受体δ(PPARδ)激动剂GW501516对低氧原代大鼠肺动脉平滑肌细胞(PASMCs)增殖的影响,并探讨其可能机制,为低氧肺血管重构的防治寻找新靶点。方法:对照组PASMCs采用21%氧气培养,低氧组采用3%氧气诱导PASMCs增殖,通过不同浓度的GW501516(10、30、100 nmol/L)低氧条件下孵育PASMCs 12、24、48 h筛选GW501516抑制低氧PASMCs增殖的最适浓度;选择100 nmol/L GW501516和(或)蛋白激酶B(AKT)激动剂SC79在低氧条件下孵育PASMCs 24 h,探讨GW501516抑制PASMCs增殖可能机制,通过CCK-8与BrdU试剂盒检测细胞增殖与DNA的合成,流式细胞仪分析细胞周期,实时定量PCR(RT-PCR)检测细胞周期蛋白(Cyclin)D1,细胞周期蛋白激酶抑制蛋白p27(p27)mRNA的表达,Western blot检测PPARδ、总的和磷酸化蛋白激酶B(AKT)与糖原合酶激酶3β(GSK3β)的表达。结果:与低氧组相比,不同浓度的GW501516(10、30、100 nmol/L)干预12、24、48 h后能够抑制低氧条件下PASMCs增殖与DNA的合成,且100 nmol/L GW501516抑制作用最强(P<0.05或P<0.01);与对照组相比,100 nmol/L GW501516干预PASMCs 24 h能够显著上调PPARδ的表达,而低氧可显著下调PPARδ的表达(P<0.01);与低氧组相比,100 nmol/L GW501516干预24 h后能够显著抑制PASMCs增殖与DNA的合成(P<0.01),增加处于G0/G1期的PASMCs比例,明显减少S期和G2/M期的PASMCs比例(P<0.05或P<0.01),显著抑制Cyclin D1 mRNA的表达并促进p27 mRNA的表达(P<0.01),显著抑制AKT与GSK3β磷酸化(P<0.01),而与100 nmol/L GW501516低氧组相比,AKT激动剂SC79能够逆转100 nmol/L GW501516上述作用(P<0.05或P<0.01)。结论:GW501516通过抑制AKT/GSK3β信号通路抑制低氧条件下PASMCs增殖。Objective:To investigate the effects of the peroxisome proliferator-activated receptorδ(PPARδ)agonist GW501516 on the proliferation of primary rat proliferation of pulmonary artery smooth muscle cells(PASMCs)induced by hypoxia,in order to discover new drugs for the treatment and prevention of pulmonary vascular remodeling.Methods:The PASMCs in the control group were cultured with 21%oxygen,while the PASMCs in the hypoxia group were cultured with 3%oxygen to induce cell proliferation.PASMCs were incubated with GW501516 at the concentrations of 10,30 and 100 nmol/L under hypoxic conditions for different time points(12,24,and 48 h)to find out the appropriate concentrations of GW501516 for inhibition the proliferation.PASMCs were incubated with 100 nmol/L GW501516 and(or)protein kinase B(AKT)agonist SC79 for 24 h to explore related mechanisms of GW501516 in regulating the proliferation.The proliferation and DNA synthesis were determined by CCK-8 and BrdU kit.The cell cycle progression was analyzed by flow cytometry.The mRNA expressions of Cyclin D1 and the cyclin kinase inhibitor p27(p27)were measured by quantitative real-time PCR(RT-PCR).The expressions of PPARδ,total and phosphorylated forms AKT and glycogen synthase kinase 3β(GSK3β)were detected by Western blot.Results:Compared with the hypoxia group,PASMCs incubated with different concentrations of GW501516(10,30,100 nmol/L)for 12,24,48 h under hypoxic conditions could inhibit the proliferation and DNA synthesis,and the greatest level of suppression of proliferation was induced by GW501516 at the concentration of 100 nmol/L(P<0.05 or P<0.01).Compared with the control group,the expression of PPARδwas upregulated markedly in PASMCs incubated with 100 nmol/L GW501516 for 24 h,while hypoxia could downregulate the expression of PPARδsignificantly(P<0.01).Compared with the hypoxia group,100 nmol/L GW501516 blocked the proliferation and DNA synthesis of PASMCs significantly(P<0.01),increased the proportion of PASMCs in G0/G1 phase while decreased the proportion o

关 键 词：低氧 肺动脉平滑肌细胞 GW501516 过氧化物酶体增殖剂激活受体δ 增殖 蛋白激酶B/糖原合酶激酶3β 

分 类 号：R34[医药卫生—基础医学]