二甲双胍激活AMPK/mTOR信号通路抑制大鼠腹主动脉瘤形成

Metformin inhibiting abdominal aortic aneurysm formation in rats by activating AMPK/mTOR signaling pathway

目的:研究二甲双胍(MET)通过AMPK/mTOR信号通路抑制人腹主动脉瘤(AAA)的发展机制。方法:40只雄性SD大鼠,体重(200±20)g,被分成4组:正常对照组(Control)、MET+AAA组、AAA组和C.C+AAA组,每组10只。通过腹主动脉腔内灌注猪胰弹性蛋白酶建立大鼠AAA模型。AMPK激活剂MET及抑制剂Compound C(C.C)通过灌胃及腹腔注射给药。免疫组织化学染色观察各组腹主动脉管壁巨噬细胞、新生血管及血管平滑肌细胞(VSMC)的变化。Western blot分析p-AMPK、p-mTOR、MMP2/9、VSMC表型转换相关蛋白α-SMA和OPN的表达。结果:MET+AAA组的腹主动脉直径明显小于AAA组[(2.51±0.04)mm比(2.89±0.10)mm,P<0.05],但C.C+AAA组腹主动脉直径明显大于AAA组[(3.51±0.05)mm比(2.89±0.10)mm,P<0.05]。免疫组织化学染色结果:与AAA组相比,MET+AAA组中巨噬细胞和新生血管明显减少,VSMC数量明显增多;C.C+AAA组中巨噬细胞和新生血管均明显增加,VSMC数量明显减少(P<0.05)。Western blot结果显示MET+AAA组的p-AMPK、α-SMA表达水平明显高于AAA组,p-mTOR、MMP2/9和OPN的表达水平明显低于AAA组(P<0.05);而C.C+AAA组的结果与MET+AAA组结果相反。结论:MET可能通过AMPK/mTOR信号通路增强AMPK活性,抑制下游的p-mTOR及MMP-2/9的表达,抑制巨噬细胞浸润和新生血管形成,调节VSMC向收缩表型转化,从而抑制AAA的形成和发展。以AMPK/mTOR信号通路为干预靶点进行干预可以为临床防治AAA提供新的途径和思路。

Objectives:To study the mechanism of the metformin(MET)inhibiting AAA development based on AMPK/mTOR signal pathway.Methods:AAA models were induced by intraluminal porcine pancreatic elastase(PPE)infusion.Forty male SD rats weighing(200±20)g were divided into control group,MET+AAA group,AAA group and C.C+AAA group(n=10).AAA models were induced by intraluminal porcine pancreatic elastase(PPE)infusion.Rats in MET+AAA group and C.C+AAA group received injections with compound C(C.C)and MET therapy respectively.Immunohistochemistry analysis of the contens of macrophages,neovasculars and VSMCs in these different groups.The expression of p-AMPK,p-mTOR,MMP2/9,α-SMA and OPN were analyzed by Western blot.Results:Compared with AAA group,MET significantly inhibits the expansion of abdominal aorta in MET+AAA group(2.51±0.04 mm VS 2.89±0.10mm,P<0.05),but this effect was inhibited by C.C in C.C+AAA group(3.51±0.05 mm VS 2.89±0.10 mm,P<0.05).Compared with AAA group,VSMCs were relatively preserved in MET+AAA group.MET treatment also reduced mural macrophage density and neoangiogenesis.Using western blot,we found that p-AMPK andα-SMA levels were higher in MET+AAA group compared with AAA group,p-mTOR,MMP2/9 and OPN levels were significantly lower in MET+AAA group(P<0.05).But in C.C+AAA group,we came to the opposite results.Conclusion:Through AMPK/mTOR signal pathway,MET could inhibit the formation and debelopment of AAA by enhancing the activity of AMPK and inhibiting the expression of p-mTOR and MMP2/9,further inhibiting macrophage infiltration and neovascularization,regulating the transformation of VSMC to contractile phenotype.The AMPK/mTOR signal pathway as the intervention target can provide a new way and thinking for clinical prevention and treatment of AAA.