胰高血糖素样肽-1受体激动剂减轻高糖诱导的巨噬细胞炎性反应的机制

Glucagon-like Peptide-1 Receptor Agonist Alleviates High Glucose-induced Macrophage Inflammation

目的探究胰高血糖素样肽-1受体(glucagon-like peptide-1 receptor,GLP-1R)激动剂(exendin4)减轻高糖诱导的巨噬细胞炎性反应的机制。方法小鼠单核巨噬细胞白血病细胞(RAW264.7)分为3组分别做以下处理:对照组(RAW264.7细胞正常培养基培养)、高糖诱导组(RAW264.7细胞高葡萄糖培养基培养)、exendin-4组(RAW264.7细胞高葡萄糖培养基培养,添加exendin-4)。通过RT-qPCR检测不同处理组的GLP-1R mRNA水平,以及M1的促炎因子一氧化氮合酶(nitric oxide synthase,iNOS),白介素(interleukin,IL)-6、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和M2特异的基因IL-10,甘露糖受体1(mannose receptor 1,MRC-1),巨噬细胞半乳凝素1(macrophage galectin 1,MGL-1)、精氨酸酶(Arginase-1,ARG-1)的mRNA水平。通过免疫印迹(WB)检测VE-钙黏着蛋白和ZO-1,以及细胞间粘附分子-1(intercellular adhesion molecule-1,ICAM-1)和血管细胞粘附分子-1(vascular cell adhesion molecule-1,VCAM-1)的表达。通过蛋白质印迹分析p-STAT3和p-JNK蛋白表达。结果与对照组相比,高糖诱导组的GLP-1R mRNA水平降低(P<0.05),与高糖诱导组相比,exendin-4组的GLP-1R mRNA水平增加(P<0.05)。与对照组相比,高糖诱导组iNOS,IL-6和TNF-α的mRNA水平增加,IL-10、MRC-1、MGL-1和ARG-1的mRNA水平降低(P<0.05),与高糖诱导组相比,exendin-4组iNOS,IL-6和TNF-α的mRNA水平降低,IL-10、MRC-1、MGL-1和ARG-1的mRNA水平增加(P<0.05)。与对照组相比,高糖诱导组VE-钙黏着蛋白和ZO-1表达降低,ICAM-1和VCAM-1表达增加(P<0.05),与高糖诱导组相比,exendin-4组VE-钙黏着蛋白和ZO-1表达增加,ICAM-1和VCAM-1表达降低(P<0.05)。与对照组相比,高糖诱导组pSTAT3和p-JNK蛋白表达增加(P<0.05),与高糖诱导组相比,exendin-4组p-STAT3和p-JNK蛋白表达降低(P<0.05)。结论exendin-4抑制磷酸化STAT3和磷酸化JNK激活,促进M2极化和抑制M1极化,降低ICAM-1和VCAM-1表达,减轻高糖诱导的巨噬细胞炎性反应。

Objective To explore the mechanism by which the glucagon-like peptide-1 receptor(GLP-1R)agonist(exendin-4)reduces the inflammation and endothelial damage induced by high glucose in macrophages.Methods Mouse monocyte macrophage leukemia cells(RAW264.7)were divided into groups as follows:control group(RAW264.7 cells cultured in normal medium);high glucose induction group(RAW264.7 cells cultured in high glucose medium);exendin-4 group(RAW264.7 cells cultured in high glucose medium supplemented with exendin-4).The mRNA expression level of GLP-1R was detected by RT-qPCR.The mRNA expression levels of M1 pro-inflammatory factors nitric oxide synthase(iNOS),interleukin(IL)-6,tumor necrosis factor(TNF)-α(TNF-α)and M2 specific gene IL-10,mannose receptor 1(MRC-1),macrophage galectin 1(MGL-1),arginine-1(ARG-1)were also detected by RTqPCR.The expression levels of VE-cadherin,ZO-1,intercellular adhesion molecule-1(ICAM1)and vascular cell adhesion molecule-1(VCAM-1)were detected by Western blotting.The expression levels of p-STAT3 and p-JNK were analyzed by Western blotting.Results The expression level of GLP-1R mRNA was significantly decreased in the high glucose induced group compared with the control group(P<0.05).The expression level of GLP-1R mRNA was significantly increased(P<0.05)in the exendin-4 group compared with the high glucose induction group.In the high glucose induced group relative to the control group,the expression levels of iNOS,IL-6 and TNF-αmRNA were significantly increased,and the expression levels of IL-10,MRC-1,MGL-1 and ARG-1 mRNA were significantly decreased(P<0.05).These indicators were significantly improved in the exendin-4 group compared with the high glucose induced group(P<0.05).Compared with the control group,the expression levels of VE-cadherin and ZO-1 in the high glucose induced group were decreased,and the expression levels of ICAM-1 and VCAM-1 were increased(P<0.05),which were significantly reversed in the exendin-4 group(P<0.05).Compared with the control group,the protein expression levels of p-STAT3 and p-JNK in the high glucose induced group were increased(P<0.05).They were significantly decreased in the exendin-4 group when compared with the high glucose induced group(P<0.05).Conclusion Exendin-4 can inhibit the activation of the JNK-STAT3 pathway,promote M2 polarization and inhibit M1 polarization,reduce the expression of ICAM-1 and VCAM-1,and alleviate high glucose-induced macrophage inflammatory response.