胰岛素对高糖环境中巨噬细胞表型转换的影响

Effects of insulin on macrophage phenotype transformation under high glucose condition

目的·研究胰岛素对高糖培养的人单核细胞株THP-1细胞及糖尿病创面巨噬细胞表型转换的影响。方法·分别用正常糖(5.6 mmol/L)和高糖(25 mmol/L)培养THP-1细胞,PMA刺激贴壁后,LPS诱导分化为M1型巨噬细胞,胰岛素处理细胞6 h。使用RT-PCR、Western blotting检测M1型标记物诱导型一氧化氮合酶(iNOS)、肿瘤坏死因子α(TNF-α)、白介素-1β(IL-1β)以及M2型标记物精氨酸酶1(Arg1)、IL-10表达的变化。高脂饮食喂养联合小剂量链脲佐菌素(STZ)多次腹腔注射诱导2型糖尿病大鼠模型;血糖稳定5周后,在糖尿病大鼠背部制作2个直径1 cm的全层皮肤缺损创面,应用随机数字表法将创面随机分为胰岛素处理创面和生理盐水处理创面,分别滴加0.2 U胰岛素/20μL生理盐水或20μL生理盐水。于伤后第3、7、25日,观察创面巨噬细胞表型特征;以正常大鼠(n=3)作为正常对照。结果·经高糖培养后,由THP-1细胞诱导分化的巨噬细胞M1型标记物iNOS、TNF-αmRNA表达上调,而M2型标记物Arg1、IL-10 mRNA表达下调;胰岛素作用6 h后,iNOS、TNF-αmRNA表达下调,iNOS、IL-1β蛋白表达也下调,而Arg1、IL-10 mRNA及蛋白表达均上调。此外,高糖培养的巨噬细胞磷酸化NF-κB-p65水平明显升高,而胰岛素干预后磷酸化NF-κB-p65水平显著降低。与正常对照大鼠皮肤创面相比,糖尿病大鼠创面巨噬细胞iNOS表达明显升高;伤后第3、7日,生理盐水处理创面巨噬细胞iNOS高表达;伤后第25日,Arg1表达较低。伤后第7日起,胰岛素处理创面巨噬细胞iNOS表达开始下调;伤后第25日,Arg1的表达显著高于生理盐水处理创面。结论·胰岛素可诱导高糖环境中的巨噬细胞由M1表型向M2表型转换,其机制可能与NF-κB-p65的磷酸化有关。

Objective·To investigate the effects of insulin on high glucose-cultured human mononuclear cell line THP-1and macrophage phenotype transformation in diabetic wounds.Methods·THP-1cells were cultured with normal(5.6mmol/L)and high(25mmol/L)glucose,respectively,stimulated with PMA for differentiation,and induced to M1macrophages with LPS.After treated with insulin for6h,expression changes of M1type macrophage markers inducible nitric oxide synthase(iNOS),tumor necrosis factorα(TNF-α),and interleukin-1β(IL-1β),as well as M2type macrophage markers arginase1(Arg1)and IL-10were detected using real-time PCR and Western blotting.High fat diet feeding plus multiple intraperitoneal injections of low dose streptozotocin(STZ)were used to induce type II diabetes rat model.After blood glucose level has been stable for five weeks,two fullthickness skin wounds with the diameter of1cm were made on the back of DM rats.Wounds were randomly assigned to being treated with insulin(0.2U insulin/20μL saline)or saline(20μL saline)using the random number table.Characteristics of macrophage phenotypes were observed3,7,and25days after wounds were made.Normal rats(n=3)served as controls.Results·After being cultured with high glucose,the mRNA levels of M1markers iNOS and TNF-αwere up-regulated in LPS-induced THP-1cells,while the mRNA levels of M2markers Arg1and IL-10were down-regulated.After being treated with insulin for6h,mRNA levels of iNOS and TNF-αwere down-regulated,protein levels of iNOS,IL-1βwere down-regulated too,while mRNA and protein levels of Arg1and IL-10were up-regulated.In addition,the expression level of phosphorylated NF-κB-p65was significantly increased after high glucose culture and was significantly decreased after insulin intervention.Compared to normal rat skin wounds,the expression of iNOS in macrophages was significantly increased in wounds of diabetic rats.The expression of iNOS in macrophages was high in saline treated wounds3and7days after the wounds were made and the expression of Arg1was low25days after the wounds were made.In insulin treated wounds,the expression of iNOS started to decrease on day7after the wounds were made and the expression of Arg1was significantly higher than that in saline treated wounds on day25after the wounds were made.Conclusion·Insulin can induce macrophage phenotype transformation from M1to M2under high glucose condition and the mechanism may be associated with the phosphorylation of NF-κB-p65.