AMPK激活剂抑制PDGF诱导的大鼠血管平滑肌细胞增殖的机制研究

Proliferative effect of PDGF and anti-proliferative activity of AMPK on vascular smooth muscle cells

目的:观察血小板源性生长因子(PDGF)及一磷酸腺苷激活的蛋白激酶(AMPK)激活剂5-氨基咪唑-4-甲酰胺核糖核苷(AICAR)干预后血管平滑肌细胞(VSMCs)的增殖变化,探讨PDGF对平滑肌细胞的促增殖效应及激活AMPK抑制增殖机制。方法:SD大鼠主动脉血管平滑肌细胞经PDGF及AICAR干预24 h、48 h、72 h后,分为A组(AICAR)、P组(PDGF)、A+P组(AICAR+PDGF)和对照组,4个组用MTT法测量细胞的增殖情况;并检测不同AICAR作用时间下(30 min、1 h、3 h、6 h、12 h)AMPK的活化情况和mTOR的蛋白活性,以及上述各组中AMPK活化和mTOR蛋白活性。结果:(1)与对照组相比,PDGF干预组的MTT值显著增加(P<0.05),AMPK激活组能显著抑制PDGF诱导的MTT值的增加效应(P<0.05);(2)AICAR可诱导细胞AMPK的磷酸化水平增加(P<0.05),AICAR的诱导效应有随药物干预时间增加而逐渐增强的趋势;(3)与对照组相比,AICAR干预后p-mTOR表达活性显著减弱(P<0.05),随着药物干预时间延长,p-mTOR表达也呈逐渐减弱的趋势;(4)各组干预12 h后分别检测p-AMPK表达强度,与对照组比较,P组显著减弱(P<0.01),A+P组显著增强(P<0.01),而A+P组与P组比较,A+P组强于P组(P<0.01),A组较对照组显著增强(P<0.01);检测p-mTOR表达强度,与对照组比较,P组显著增强(P<0.05),A+P组较低(P<0.05),A+P组低于P组(P<0.05),A组低于对照组(P<0.05)。结论:PDGF刺激能促进VSMCs增殖,该促增殖效应可被AMPK激活剂AICAR所抑制;细胞mTOR活性下调可能参与AMPK活化诱导的抑制VSMCs增殖的作用。

AIM： To investigate the proliferative effect of platelet-derived growth factor（PDGF） and anti-proliferative activity of AMP-activated protein kinase（AMPK） on vascular smooth muscle cells（VSMCs）.METHODS： The proliferation of VSMCs cultured with PDGF and activation of AMPK were observed.VSMCs were divided in 4 groups： control group;PDGF group;5-aminoimidazole-4-carboxamide-1-β-D-riboside（AICAR） group and AICAR＋PDGF group.The time course of AMPK activation was determined.The protein level of mTOR was also measured.RESULTS： Compared with control group,the proliferative rate in PDGF group was significantly increased.The growth of VSMCs was inhibited in a time-dependent manner and the activity of p-mTOR was significantly decreased in AICAR group.Compared with control group,the expression of p-AMPK in PDGF group was significantly decreased,and that in AICAR group and AICAR＋PDGF group was significantly increased.The expression of p-AMPK in AICAR＋PDGF group was higher than that in PDGF group.The activity of p-mTOR in PDGF group was significantly higher than that in control group,while that of AICAR group and AICAR＋PDGF group was significantly decreased.The expression of p-mTOR in AICAR＋PDGF group was lower than that in PDGF group.CONCLUSION： Stimulation of VSMCs with PDGF promotes the cell proliferation,which can be inhibited by AICAR.The proliferation of VSMCs activated by AMPK is probably correlated with the down-regulation of mTOR expression.