运动干预高血压小动脉平滑肌细胞表型转换中miR-143/145对蛋白激酶B信号的调节作用

摘要

目的:探讨运动对高血压小动脉平滑肌细胞(VSMC)表型转换的调节作用,并探索在VSMC表型转换过程中miR-143/145对蛋白激酶B(Akt)信号通路的调节机制.方法:3月龄正常大鼠(Wistar Kyoto rats,WKY)和原发性高血压大鼠(Spontaneously hypertensive rats,SHR)各分为安静对照组(WKY-C组和SHR-C组)和运动干预组(WKY-E组和SHR-E组).运动组进行8周的正式有氧跑台训练(速度为20m/min,坡度为0,每周5天,每天60 min),并监测血压.于末次干预后48 h对肠系膜动脉的形态、VSMC表型标志蛋白、miR-143/145表达及Akt信号的激活状态进行检测.离体实验运用脂质体转染干扰miR-145在分离培养VSMC中的表达,对VSMC表型、miR-145表达、Akt信号活性及该通路上下游胰岛素样生长因子Ⅰ受体(IGF-1R)、胰岛素受体底物(IRS-1)和p70核蛋白体S6激酶(p70s6K)的mRNA进行定量.结果:SHR-E组血压显著低于SHR-C组(P＜0.05),SHR-E组动脉壁厚显著小于SHR-C组(P＜0.05);SHR-E组收缩表型标志蛋白钙调蛋白(calponin)表达显著高于SHR-C组(P＜0.05),而其合成表型标志蛋白骨桥蛋白(Osteoppontin,OPN)表达显著低于SHR-C组(P＜0.05);SHR-E组miR-145表达显著高于SHR-C组(P＜0.01),而miR-143未有明显变化;SHR-C组Akt磷酸化程度较WKY-C被显著抑制(P＜0.01),SHR-E组Akt磷酸化程度与SHR-C比有显著性差异(P＜0.05).离体实验中,转染miR-145 mimic使VMSC中收缩表型标志蛋白α-actin在转染后表达显著高于NC (negative control)组(P＜0.05),细胞形态呈长梭形;miR-145 inhibitor使VSMC在转染后α-actin表达显著低于NC组(P＜0.05).Akt磷酸化程度在VSMC转染miR-145 mimic后较NC组被显著抑制(P＜0.05),下游IGF-1R和IRS-1 mRNA亦被显著抑制(P＜0.05);细胞经转染miR-145 inhibitor后Akt磷酸化程度显著升高(P＜0.05),miR-145 inhibitor使IGF-1R和IRS-1 mRNA在转染后表达显著高于NC组(P＜0.05);miR-145干扰对Akt下游p70s6K mRNA靶向抑制作用不显著.结论:运动改善高血压大鼠血压症状的同时使小动脉结构发生适应性变化,有助于维持小动脉中VSMC收缩表型;miR-145参与了VSMC表型转换过程中Akt信号通路的反馈调节,但运动后Akt的激活并不是miR-145过表达导致的,运动可能通过其它途径激活Akt信号并促进VSMC向收缩表型转换.%Objective To explore the effect of exercise on vascular smooth muscle cell(VSMC) phenotype switching in hypertensive arteries and to figure out the regulatory mechanisms of mircroRNA (miR)-143/145 on Akt signaling during the process.Methods Three-month old(Wistar Kyoto rats) WKY and (spontaneously hypertensive rats) SHR were divided into 4 groups:WKY-C,SHR-C,WKY-E,and SHR-E,which were subjected to 8wk moderate treadmill training (E) or sedentary as control (C) with blood pressure being monitored.After the last bout of exercise,mesenteric arteries were isolated to determine VSMC phenotypic marker,miR143/145 expression and Akt phosphorylation.In transfection experiment in vitro,miR-145 mimic and miR-145 inhibitor were transfected into cultured VSMC,and given immunofluorescent staining using α-actin to detect the cell morphology.VSMC phenotype marker,Akt phosphorylation,and mRNA expressions of the insulin-like growth factor Ⅰ receptor (IGF-IR),Insulin receptor substrate 1 antibody(IRS-1),and p70S6K were determined.Results The blood pressure of SHR-E reduced significantly compared with that of SHR-C(P＜0.05),and the arterial thicknessof SHR-E decreased significantly (P＜0.05).The VSMC contractile marker calponin in SHR-E increased significantly when compared with SHR-C(P＜0.05),while the proliferative marker osteoppontin (OPN) in SHR-E reduced significantly than that in SHR-C(P＜0.05).The miR-145 of SHR-E was significantly enhanced(P＜0.05),while there was no significant difference in the miR-143.The Akt of SHR-E was activated more significantly than SHR-C(P＜0.05).The miR-145 overexpression by transfecting miR-145 mimic into VSMC increased α-actin significantly(P＜0.05),while miR-145 inhibitor made α-actin a decrease.Akt activation was significantly inhibited by miR-145 mimic and enhanced by miR-145 inhibitor(P＜0.05).The miR-145 significantly inhibited IRS-1 and IGF-1R mRNA(P＜ 0.05),but the targeting effects were not significant on p70S6K mRNA.Conclusions Exercise ameliorates the high blood pressure and remodels arterioles,which may rely on its regulatory role on VSMC switching from proliferative to contractile phenotype,and miR-145 is involved in this process.However,the Akt activation is not caused by the overexpression of miR-145,but through other means to promote the above VSMC switching.