全反式维甲酸调控AMPK/mTOR通路抑制血管平滑肌细胞增殖的研究
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摘要
目的探讨AMP依赖的蛋白激酶(AMPK)在全反式维甲酸(ATRA)抑制血管平滑肌细胞(VSMC)增殖中的作用。方法采用MTS实验检测VSMC的增殖情况并进行细胞活力测定;Western blot测定AMPK活化后pAMPKα和AMPKα的表达水平及哺乳动物雷帕霉素靶蛋白(mTOR)信号通路激活后p-mTOR和mTOR的表达水平;利用AMPK抑制剂(CC)通过Western blot测定p-AMPKα和AMPKα的表达水平及A7r5细胞活力进一步确定AMPK在ATRA抗VSMC增殖中的作用。结果 ATRA可以剂量和时间依赖性方式抑制A7R5细胞的增殖(P均<0.05);ATRA可以剂量依赖性显著地上调Thr172处AMPKα的磷酸化水平(P<0.05),而不改变AMPKα的总水平;ATRA可以剂量依赖性抑制mTOR的磷酸化水平(P<0.05),而不改变mTOR的总水平。AMPK抑制剂(CC)通过抑制AMPKα,部分抵消ATRA介导的对VSMC增殖的抑制作用。ATRA(4μmol/L)和CC共刺激A7R5细胞可显著下调AMPKα的磷酸化水平(P<0.05);与此同时,与单独ATRA(4μmol/L)处理相比,ATRA(4μmol/L)与CC共同处理,可显著缓解ATRA抑制A7r5细胞的增殖作用(P<0.05)。结论 ATRA可能通过激活AMPK和抑制mTOR信号通路抑制VSMC的增殖。
Objective This study was to investigate the role of AMP-dependent protein kinase(AMPK)in the inhibition of vascular smooth muscle cell(VSMC)proliferation by all-trans retinoic acid(ATRA). Methods MTS was used to detect VSMC proliferation and cell viability. Western blot was used to determine the p-AMPK and AMPK expression after AMPK activation and expression of p-mTOR and mTOR after activation of rapamycin target protein(mTOR)signaling pathway. To further determine the role of AMPK in ATRA′s anti-proliferation of VSMC by AMPK inhibitor(CC),the expression of pAMPK and AMPK expression and the viability of A7 r5 cells were determined by Western blot. Results ATRA significantly reduced the cell proliferation of A7 R5 in a dose-and time-dependent manner. ATRA significantly enhanced the phosphorylation level of AMPKα(Thr172)and reduced the phosphorylation levels of mTOR in a dose-independent manner.Inhibition of AMPKα by CC significantly negated the protective effect of ATRA on VSMC proliferation. Conclusion Our results demonstrated that ATRA might inhibit VSMC proliferation by direct activation of AMPK and inhibition of mTOR signaling pathway.
Objective This study was to investigate the role of AMP-dependent protein kinase(AMPK)in the inhibition of vascular smooth muscle cell(VSMC)proliferation by all-trans retinoic acid(ATRA). Methods MTS was used to detect VSMC proliferation and cell viability. Western blot was used to determine the p-AMPK and AMPK expression after AMPK activation and expression of p-mTOR and mTOR after activation of rapamycin target protein(mTOR)signaling pathway. To further determine the role of AMPK in ATRA′s anti-proliferation of VSMC by AMPK inhibitor(CC),the expression of pAMPK and AMPK expression and the viability of A7 r5 cells were determined by Western blot. Results ATRA significantly reduced the cell proliferation of A7 R5 in a dose-and time-dependent manner. ATRA significantly enhanced the phosphorylation level of AMPKα(Thr172)and reduced the phosphorylation levels of mTOR in a dose-independent manner.Inhibition of AMPKα by CC significantly negated the protective effect of ATRA on VSMC proliferation. Conclusion Our results demonstrated that ATRA might inhibit VSMC proliferation by direct activation of AMPK and inhibition of mTOR signaling pathway.
引文
[1]Bennett MR,Sinha S,Owens GK.Vascular smooth muscle cells in atherosclerosis[J].Circ Res,2016,118(4):692-702.
[2]Di Pietro N,Formoso G,Pandolfi A.Pandolfi.Physiology and pathophysiology of oxLDL uptake by vascular wall cells in atherosclerosis[J].Vascul Pharmacol,2016,84:1-7.
[3]Day EA,Ford RJ,Steinberg GR.AMPK as a therapeutic target for treating metabolic diseases[J].Trends Endocrinol Metab,2017,28(8):545-560.
[4]Viollet B,Horman S,Leclerc J,et al.AMPK inhibition in health and disease[J].Crit Rev Biochem Mol Biol,2010,45(4):276-295.
[5]Wang J,Ma A,Zhao M,et al.AMPK activation reduces the number of atheromata macrophages in ApoE deficient mice[J].Atherosclerosis,2017,258:97-107.
[6]Jung CH,Ro SH,Cao J,et al.mTOR regulation of autophagy[J].FEBS Lett,2010,584(7):1287-1295.
[7]傅国华,龚勋,吴赛珠,等.雷帕霉素联用阿托伐他汀减轻大鼠血管平滑肌细胞氧化应激损伤[J].热带医学杂志,2013,13(5):538-541,576.
[8]Rui K,Lu L,Yanting Z,et al.Knockdown of AMPKalpha2promotes pulmonary arterial smooth muscle cells proliferation via mTOR/Skp2/p27(Kip1)signaling pathway[J].Int J Mol Sci,2016,17(6).pii:E844.
[9]Naoki I,Keita K,Hiroki S,et al.Activation of AMP-activated protein kinase by retinoic acid sensitizes hepatocellular carcinoma cells to apoptosis induced by sorafenib[J].Cancer Sci,2015,106(5):567-575.
[10]Le Y,Lin S,Peixian G,et al.Effect of AMPK signal pathway on pathogenesis of abdominal aortic aneurysms[J].Oncotarget,2017,8(54):92827-92840.
[11]Sunaga H,Matsui H,Anjo S,et al.Elongation of long-chain fatty acid family member 6(Elovl6)-driven fatty acid metabolism regulates vascular smooth muscle cell phenotype through AMP-activated protein kinase/kruppel-like factor 4(AMPK/KLF4)signaling[J].J Am Heart Assoc,2016,5(12).pii:e004014.
[12]Ronan TS,Tino S,Sven S,et al.Inhibition of the PI3K/AKT/mTOR Pathway Leads to Down-Regulation of c-Myc and Overcomes Resistance to ATRA in Acute Myeloid Leukemia[J].Blood,2015,126:1363.
[13]Ke R,Xu Q,Li C,et al.Mechanisms of AMPK in the maintenance of ATP balance during energy metabolism[J].Cell Biol Int,2018,42(4):384-392.
[14]Hardie DG,Alessi DR.LKB1 and AMPK and the cancermetabolism link-ten years after[J].BMC Biol,2013,11:36.2019-03-07
[2]Di Pietro N,Formoso G,Pandolfi A.Pandolfi.Physiology and pathophysiology of oxLDL uptake by vascular wall cells in atherosclerosis[J].Vascul Pharmacol,2016,84:1-7.
[3]Day EA,Ford RJ,Steinberg GR.AMPK as a therapeutic target for treating metabolic diseases[J].Trends Endocrinol Metab,2017,28(8):545-560.
[4]Viollet B,Horman S,Leclerc J,et al.AMPK inhibition in health and disease[J].Crit Rev Biochem Mol Biol,2010,45(4):276-295.
[5]Wang J,Ma A,Zhao M,et al.AMPK activation reduces the number of atheromata macrophages in ApoE deficient mice[J].Atherosclerosis,2017,258:97-107.
[6]Jung CH,Ro SH,Cao J,et al.mTOR regulation of autophagy[J].FEBS Lett,2010,584(7):1287-1295.
[7]傅国华,龚勋,吴赛珠,等.雷帕霉素联用阿托伐他汀减轻大鼠血管平滑肌细胞氧化应激损伤[J].热带医学杂志,2013,13(5):538-541,576.
[8]Rui K,Lu L,Yanting Z,et al.Knockdown of AMPKalpha2promotes pulmonary arterial smooth muscle cells proliferation via mTOR/Skp2/p27(Kip1)signaling pathway[J].Int J Mol Sci,2016,17(6).pii:E844.
[9]Naoki I,Keita K,Hiroki S,et al.Activation of AMP-activated protein kinase by retinoic acid sensitizes hepatocellular carcinoma cells to apoptosis induced by sorafenib[J].Cancer Sci,2015,106(5):567-575.
[10]Le Y,Lin S,Peixian G,et al.Effect of AMPK signal pathway on pathogenesis of abdominal aortic aneurysms[J].Oncotarget,2017,8(54):92827-92840.
[11]Sunaga H,Matsui H,Anjo S,et al.Elongation of long-chain fatty acid family member 6(Elovl6)-driven fatty acid metabolism regulates vascular smooth muscle cell phenotype through AMP-activated protein kinase/kruppel-like factor 4(AMPK/KLF4)signaling[J].J Am Heart Assoc,2016,5(12).pii:e004014.
[12]Ronan TS,Tino S,Sven S,et al.Inhibition of the PI3K/AKT/mTOR Pathway Leads to Down-Regulation of c-Myc and Overcomes Resistance to ATRA in Acute Myeloid Leukemia[J].Blood,2015,126:1363.
[13]Ke R,Xu Q,Li C,et al.Mechanisms of AMPK in the maintenance of ATP balance during energy metabolism[J].Cell Biol Int,2018,42(4):384-392.
[14]Hardie DG,Alessi DR.LKB1 and AMPK and the cancermetabolism link-ten years after[J].BMC Biol,2013,11:36.2019-03-07