GW501516对低氧条件下肺动脉平滑肌细胞增殖的作用及其机制*

doi:10.12047/j.cjap.6211.2022.020

摘要/Abstract

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。

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

Abstract: 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.

Key words: hypoxia, pulmonary artery smooth muscle cells, GW501516, peroxisome proliferator-activated receptor δ, proliferation, protein kinase B/glycogen synthase kinase 3β

中图分类号:

陈昌贵, 贺立群, 田立群, 易春峰. GW501516对低氧条件下肺动脉平滑肌细胞增殖的作用及其机制^*[J]. 中国应用生理学杂志, 2022, 38(2): 119-125.

CHEN Chang-gui, HE Li-qun, TIAN Li-qun, YI Chun-feng. Effects of GW501516 on the proliferation of pulmonary artery smooth muscle cells induced by hypoxia and its mechanisms[J]. CJAP, 2022, 38(2): 119-125.

参考文献

[1] Chen TJ, Zhou QY, Tang HY, et al. miR-17/20 controls prolyl hydroxylase 2(PHD2)/hypoxia‐inducible factor 1(HIF1) to regulate pulmonary artery smooth muscle cell proliferation[J]. J Am Heart Assoc, 2016, 5(12): e004510.
[2] Parra V, Bravo-Sagua R, Norambuena-Soto, et al. Inhibition of mitochondrial fission prevents hypoxia-induced metabolic shift and cellular proliferation of pulmonary arterial smooth muscle cells [J]. Biochim Biophys Acta Mol Basis Dis, 2017, 1863(11): 2891-2903.
[3] Degueurce G, D'Errico I, Pich C, et al. Identification of a novel PPARbeta/delta/miR-21-3p axis in UV-induced skin inflammation[J]. EMBO Mol Med, 2016, 8(8): 919-936.
[4] Cheang WS, Wong WT, Zhao L, et al. PPAR delta is required for exercise to attenuate endoplasmic reticulum stress and endothelial dysfunction in diabetic mice[J]. Diabetes, 2017, 66(2): 519-528.
[5] Kintz P, Ameline A, Gheddar L, et al. Testing for GW501516(cardarine) in human hair using LC/MS-MS and confirmation by LC/HRMS [J]. Drug Test Anal, 2020, 12(7): 980-986.
[6] Weihrauch M, Handschin C. Pharmacological targeting of exercise adaptations in skeletal muscle: Benefits and pitfalls [J]. Biochem Pharmacol, 2018, 147: 211-220.
[7] Hwang JS, Ham SA, Yoo T, et al. Sirtuin 1 mediates the actions of peroxisome proliferator-activated receptorδ on the oxidized low-density lipoprotein-triggered migration and proliferation of vascular smooth muscle cells[J]. Mol Pharmacol, 2016, 90(5): 522-529.
[8] Kang ES, Hwang JS, Lee WJ, et al. Ligand-activated PPARdelta inhibits angiotensin II-stimulated hypertrophy of vascular smooth muscle cells by targeting ROS [J]. PLoS One, 2019, 14(1): e0210482.
[9] 何丽囡, 兰钰茹, 贺光明, 等. 白藜芦醇通过调控HIF-1α/NOX4/ROS通路抑制低氧诱导的大鼠肺动脉平滑肌细胞氧化应激与增殖[J]. 生理学报, 2020, 72(5): 551-558.
[10] Wei L, Deng W, Cheng Z, et al. Effects of hesperetin on platelet-derived growth factor-BB-induced pulmonary artery smooth muscle cell proliferation [J]. Mol Med Rep, 2016, 13(1): 955-960.
[11] Dai L, Zhou J, Li T, et al. STRIP2 silencing inhibits vascular smooth muscle cell proliferation and migration via P38-AKT-MMP-2 signaling pathway [J]. J Cell Physiol, 2019, 234(12): 22463-22476.
[12] 黄林静, 张聪聪, 赵美平, 等. MAPK信号通路对大鼠低氧性PASMCs增殖、凋亡的调控[J]. 中国应用生理学杂志, 2017, 33(3): 226-230.
[13] Parikh V, Bhardwaj A, Nair A. Pharmacotherapy for pulmonary arterial hypertension [J]. J Thorac Dis, 2019, 11(Suppl 14): S1767-S1781.
[14] Kikuchi N, Satoh K, Kurosawa R, et al. Selenoprotein P promotes the development of pulmonary arterial hypertension: Possible novel therapeutic target[J]. Circulation, 2018, 138(6): 600-623.
[15] 卜宝英, 徐喜媛, 韩俊, 等. 低氧对人肺动脉平滑肌和人肺动脉内皮细胞中HMGB1及相关炎症因子表达的影响[J]. 中国应用生理学杂志, 2019, 35(5): 406-410.
[16] 郝家乐, 徐立聪, 黄天鹏, 等. 载脂蛋白E对低氧诱导小鼠肺动脉平滑肌细胞增殖的影响及其机制[J]. 中国应用生理学杂志, 2019, 35(5): 414-417.
[17] Tian D, Teng X, Jin S, et al. Endogenous hydrogen sulfide improves vascular remodeling through PPARdelta/SOCS3 signaling[J]. J Adv Res, 2020, 27: 115-125.
[18] Hytönen J, Leppänen O, Braesen JH, et al. Activation of peroxisome proliferator-activated receptor-delta as novel therapeutic strategy to prevent in-stent restenosis and stent thrombosis[J]. Arterioscler Thromb Vasc Biol, 2016, 36(8): 1534-1548.
[19] Chen MJ, Cheng AC, Lee MF, et al. Simvastatin induces G(1) arrest by up-regulating GSK3β and down-regulating CDK4/cyclin D1 and CDK2/cyclin E1 in human primary colorectal cancer cells[J]. J Cell Physiol, 2018 , 233(6): 4618-4625.
[20] Wang J, Li XM, Bai Z, et al. Curcumol induces cell cycle arrest in colon cancer cells via reactive oxygen species and Akt/ GSK3β/cyclin D1 pathway[J]. J Ethnopharmacol, 2018, 210: 1-9.
[21] Liu G, Li X, Li Y, et al. PPARdelta agonist GW501516 inhibits PDGF-stimulated pulmonary arterial smooth muscle cell function related to pathological vascular remodeling [J]. Biomed Res Int, 2013, 2013: 903947.
[22] Shun L, Hao L, Lihe L, et al. WISP1 overexpression promotes proliferation and migration of human vascular smooth muscle cells via AKT signaling pathway[J]. Eur J Pharmacol, 2016, 788: 90-97.
[23] Li S, Zhai C, Shi W, et al. Leukotriene B(4) induces proliferation of rat pulmonary arterial smooth muscle cells via modulating GSK-3beta/beta-catenin pathway[J]. Eur J Pharmacol, 2020, 867: 172823.
[24] Li B, Zhu Y, Sun Q, et al. Reversal of the Warburg effect with DCA in PDGF-treated human PASMC is potentiated by pyruvate dehydrogenase kinase-1 inhibition mediated through blocking Akt/GSK-3beta signalling[J]. Int J Mol Med, 2018, 42(3): 1391-1400.
[25] Lin Z, Zhichen P, Junsong W, et al. Baicalin inhibits hypoxia-induced pulmonary artery smooth muscle cell proliferation via the AKT/HIF-1alpha/p27-associated pathway[J]. Int J Mol Sci, 2014,15(5): 8153-8168.
[26] Tseng AS, Engel FB, Keating MT. The GSK-3 inhibitor BIO promotes proliferation in mammalian cardiomyocytes[J]. Chem Biol, 2006, 13(9): 957-963.
[27] Zhang H, Jiang L, Guo Z, et al. PPARbeta/delta, a novel regulator for vascular smooth muscle cells phenotypic modulation and vascular remodeling after subarachnoid hemorrhage in rats[J]. Sci Rep, 2017, 7: 45234.

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

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