沙格列汀激活AMPK-PPARα途径促进巨噬细胞向M2型极化减轻动脉粥样硬化的作用研究

Saxagliptin activating the AMPK-PPARαpathway to promote M2 polarization of macrophages to reduce atherosclerosis

目的探讨沙格列汀对巨噬细胞极化的影响,以及沙格列汀减轻动脉粥样硬化的可能机制。方法ApoE-/-小鼠给予高脂饲料喂养,建立动脉粥样硬化模型,以C57BL/6小鼠作为对照组,喂养8周后,以15 mg/kg沙格列汀灌胃给药,1次/d,给药12周后,全自动生化仪分析小鼠血脂水平变化,HE染色观察主动脉组织形态学变化,油红O染色检测主动脉斑块面积变化,免疫组织化学染色检测主动脉中iNOS和Arg-1表达,Western blot检测巨噬细胞极化相关蛋白与AMPK-PPARα途径相关蛋白表达。将RAW264.7细胞随机分为对照组、诱导组(1μg/ml LPS和20 ng/ml IFN-γ诱导24 h)、沙格列汀组(细胞诱导后,使用含1μmol/L沙格列汀的培养液培养24 h)、AMPK抑制剂组(10μmol/L AMPK抑制剂Compound C预处理细胞2 h,再进行诱导和沙格列汀处理),各组对应处理后收集细胞,实时荧光定量PCR检测巨噬细胞极化相关基因表达情况,免疫荧光染色检测M1巨噬细胞和M2巨噬细胞表达变化。结果与对照组比较,模型组小鼠血清中TG、TC和LDL-C水平升高,HDL-C水平降低(P<0.05),主动脉内膜增厚,形成明显的斑块(P<0.05);与模型组比较,经过沙格列汀治疗后,小鼠血脂水平及斑块形成得到明显改善(P<0.05)。与对照组比较,模型组主动脉iNOS表达升高,Arg-1及p-AMPK、PPARα蛋白表达均下降(P<0.05);与模型组比较,沙格列汀组主动脉iNOS表达下降,Arg-1及p-AMPK、PPARα蛋白表达升高(P<0.05)。与对照组比较,诱导组RAW264.7细胞中PPAR-α、TGF-β、Arg-1 mRNA表达水平下调,TNF-α、IL-6 mRNA表达水平上调(P<0.05),M1型标志物iNOS阳性比例升高,M2型标志物CD206阳性比例下降(P<0.05);与诱导组比较,沙格列汀组PPAR-α、TGF-β、Arg-1 mRNA表达水平上调,TNF-α、IL-6 mRNA表达水平下调(P<0.05),M1型标志物iNOS阳性比例下降,M2型标志物CD206阳性比例升高(P<0.05);与沙格列汀组比较,AMPK抑制剂组PPAR-α、TGF-β、Arg-1 mRNA表达水平下调,TNF-α、IL-6 mRNA表达水平上调(P<0.05),同时,M1型标志物iNOS阳性比例升高,M2型标志物CD206阳性比例下降(P<0.05)。结论沙格列汀可促进巨噬细胞向M2型极化,改善动脉粥样硬化,其作用机制可能与激活AMPK-PPARα途径相关。

Objective To investigate the effect of saxagliptin on macrophage polarization and study the possible mechanism of saxagliptin to reduce atherosclerosis.Methods The atherosclerosis model of ApoE-/-mice were established by high-fat diet feeding.C57BL/6 mice were used as control group.After 8 weeks of feeding,15 mg/kg saxagliptin was administered by gavage,once a day.After 12 weeks of administration,the changes in blood lipid levels in mice were analyzed by automatic biochemical analyzer;the morphological changes in the aorta were observed by HE staining;the changes in aortic plaque area were detected by oil red O staining;the expression of iNOS and Arg-1 in the aorta was detected by immunohistochemical staining;the expression of macrophage polarization-related proteins and AMPK-PPARαpathway-related proteins was detected by Western blot.The RAW264.7 cells were randomly divided into control group,induction group(1μg/ml LPS and 20 ng/ml IFN-γfor 24 h),saxagliptin group(after cell induction,the cells were cultured by saxagliptin culture medium for 24 hours),and AMPK inhibitor group(cells were pretreated by 10μmol/L AMPK inhibitor Compound C for 2 hours,then were induced and treated by saxagliptin).The cells were collected after corresponding treatment in each group,and the expression of macrophage polarization-related gene was detected by real-time fluorescence quantitative PCR.The expression changes in M1 macrophages and M2 macrophages were analyzed by immunofluorescence staining.Results Compared with control group,the serum levels of TG,TC and LDL-C in model group increased,while the HDL-C decreased(P<0.05).The aortic intima in model group was thicker than control group with obvious plaques(P<0.05).Compared with model group,the blood lipid levels and plaque formation of mice were significantly improved after saxagliptin treatment(P<0.05).Compared with control group,the expression of iNOS in aorta in model group increased,and the expression of Arg-1,p-AMPK and PPARαprotein decreased(P<0.05).Compared with model group,the expression of iNOS in aorta of saxagliptin group decreased,while the expression of Arg-1,p-AMPK and PPARαprotein increased(P<0.05).Compared with control group,the expression of PPAR-α,TGF-βand Arg-1 mRNA in RAW264.7 cells of induction group was down-regulated,while the expression of TNF-αand IL-6 mRNA was up-regulated(P<0.05);the positive ratio of M1-type markers iNOS increased(P<0.05),while the positive ratio of M2-type markers CD206 decreased(P<0.05).Compared with induction group,the expression of PPAR-α,TGF-βand Arg-1 mRNA in saxagliptin group was up-regulated,while the expression of TNF-αand IL-6 mRNA was down-regulated(P<0.05);the positive ratio of M1-type markers iNOS decreased(P<0.05),while the positive ratio of M2-type markers CD206 increased(P<0.05).Compared with saxagliptin group,the expression of PPAR-α,TGF-βand Arg-1 mRNA in AMPK inhibitor group was down-regulated,while the expression of TNF-αand IL-6 mRNA was up-regulated(P<0.05);the positive ratio of M1-type markers iNOS increased(P<0.05),while the positive ratio of M2-type markers CD206 decreased(P<0.05).Conclusion Saxagliptin can promote the polarization of macrophages to M2 to improve atherosclerosis,and its mechanism of action may be related to the activation of AMPK-PPARαpathway.