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/20 μL生理盐水或20 μL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-α mRNA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-α mRNA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-1 and macrophage phenotype transformation in diabetic wounds. Methods · THP-1 cells were cultured with normal (5.6 mmol/L) and high (25 mmol/L) glucose, respectively, stimulated with PMA for differentiation, and induced to M1 macrophages with LPS. After treated with insulin for 6 h, expression changes of M1 type macrophage markers inducible nitric oxide synthase (iNOS), tumor necrosis factor α (TNF-α), and interleukin-1β (IL-1β), as well as M2 type macrophage markers arginase1 (Arg1) and IL-10 were 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 full-thickness skin wounds with the diameter of 1cm were made on the back of DM rats. Wounds were randomly assigned to being treated with insulin (0.2 U insulin /20 μL saline) or saline (20 μL saline) using the random number table. Characteristics of macrophage phenotypes were observed 3, 7, and 25 days after wounds were made. Normal rats (n=3) served as controls. Results · After being cultured with high glucose, the mRNA levels of M1 markers iNOS and TNF-α were up-regulated in LPS-induced THP-1 cells, while the mRNA levels of M2 markers Arg1 and IL-10 were down-regulated. After being treated with insulin for 6 h, mRNA levels of iNOS and TNF-α were down-regulated, protein levels of iNOS, IL-1β were down-regulated too, while mRNA and protein levels of Arg1 and IL-10 were up-regulated. In addition, the expression level of phosphorylated NF-κB-p65 was significantly increased after