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

Objective: To investigate the effects of the peroxisome proliferator-activated receptor δ (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 PPARδ was upregulated markedly in PASMCs incubated with 100 nmol/L GW501516 for 24 h,while hypoxia could downregulate the expression of PPARδ significantly(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 of PASMCs in S phase and G2 /M phase(P＜0.05 or P＜0.01), markedly downregulated the mRNA expression of cyclin D1 and upregulated the mRNA expression of p27(P＜0.01), significantly inhibited the protein expressions of phosphorylated AKT and GSK3β(P＜0.01). Compared with the 100 nmol/L GW501516 hypoxia group, AKT agonist SC79 reversed all the above effects of 100 nmol/L GW501516 on hypoxia stimulated PASMCs(P＜0.05 or P＜0.01). Conclusion: GW501516 inhibits hypoxia induced proliferation in PASMCs via inactivating AKT/GSK3β signaling pathway.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。.