二甲双胍通过激活AMPK/STAT3通路调控巨噬细胞分化抑制小鼠动脉粥样硬化形成

Metformin regulates macrophage differentiation and inhibits formation of atherosclerosis by activating AMPK/STAT3 pathway in mice

目的探究二甲双胍(Met)通过腺苷酸活化蛋白激酶(AMPK)/信号转导及转录激活因子3(STAT3)信号通路调控巨噬细胞表型对小鼠动脉粥样硬化(As)形成的影响。方法体外培养RAW264.7巨噬细胞,使用脂多糖促进巨噬细胞分化,同时使用Met刺激细胞。流式细胞术检测不同表型(CD86、CD206)巨噬细胞比例。Western blot检测相关信号通路蛋白诱导型一氧化氮合酶(iNOS)、精氨酸酶1(Arg-1)、AMPK、磷酸化AMPK(pAMPK)、STAT3、磷酸化STAT3(pSTAT3)蛋白表达水平。高脂饲料喂养ApoE-/-小鼠构建As模型。实验组(Met组)小鼠予Met灌胃干预,对照组(CTL组)小鼠给予同体积生理盐水灌胃干预。3个月后,提取小鼠大动脉全长(头臂干至双侧髂动脉)及主动脉根部,分别行油红O染色和免疫荧光染色,评价主动脉脂质沉积情况和脂质斑块内巨噬细胞不同表型比例。提取大动脉蛋白,Western blot验证相关信号通路的AMPK、pAMPK、STAT3、pSTAT3蛋白表达水平。结果细胞实验表明,Met刺激后M1巨噬细胞比例明显减少,M2巨噬细胞比例明显增加(P<0.05),AMPK活性明显增加,而STAT3活性明显下降。动物实验表明,在造模3个月后,油红O染色示Met组小鼠大动脉全长和主动脉瓣膜处脂质沉积均较CTL组明显减少(P<0.05);免疫荧光染色示Met组脂质斑块内M1巨噬细胞比例较CTL组明显减少,而M2巨噬细胞比例较CTL组明显增多(P<0.05)。Western blot实验显示,与CTL组相比,Met组AMPK活性明显增加,而STAT3活性明显降低(P<0.05)。结论Met通过激活AMPK抑制STAT3活性,调控斑块内巨噬细胞表型分化,抑制小鼠As形成。

Aim To explore the effect of metformin(Met)regulating macrophage phenotype through adenosine monophosphate-activated protein kinase(AMPK)/signal transducer and activator of transcription 3(STAT3)signal pathway on the formation of atherosclerosis(As)in mice.Methods RAW264.7 macrophages were cultured in vitro,lipopolysaccharide was used to promote macrophage differentiation,and Met was used to stimulate cells.The proportion of macrophages with different phenotypes(CD86,CD206)was detected by flow cytometry.The protein expression levels of inducible nitric oxide synthase(iNOS),arginase-1(Arg-1),AMPK,phosphorylated AMPK(pAMPK),STAT3 and phosphorylated STAT3(pSTAT3)were detected by Western blot.ApoE-/-mice were fed with high-fat diet to construct As model.Mice in the experimental group(Met group)were given Met intragastric intervention,and mice in the control group(CTL group)were given the same volume of normal saline intragastric intervention.After 3 months,the whole length of the mouse aorta(from the brachiocephalic trunk to the bilateral iliac arteries)and the aortic root were extracted and stained with oil red O and immunofluorescence respectively to evaluate the lipid deposition of the aorta and the different phenotypic proportion of macrophages in the lipid plaque.The large artery proteins were extracted,and the expression levels of AMPK,pAMPK,STAT3 and pSTAT3 in related signal pathways were verified by Western blot.Results Cell experiments showed that after Met stimulation,the proportion of M1 macrophages decreased significantly,the proportion of M2 macrophages increased significantly(P<0.05),AMPK activity increased significantly,and STAT3 activity decreased significantly.Animal experiments showed that three months after modeling,oil red O staining showed that the lipid depositions in the whole length of large artery and aortic valve in Met group were significantly lower than those in CTL group(P<0.05);immunofluorescence staining showed that the proportion of M1 macrophages in lipid plaque in Met group was significantly lower than that in CTL group,while the proportion of M2 macrophages was significantly higher than that in CTL group(P<0.05).Western blot experiment showed that AMPK activity increased significantly and STAT3 activity decreased significantly in Met group compared with the CTL group(P<0.05).Conclusion Met inhibits the activity of STAT3 by activating AMPK,regulates the phenotypic differentiation of macrophages in plaque and inhibits the formation of As in mice.