睾酮对巨噬细胞-脂肪细胞炎症因子生成及葡萄糖摄取的影响及机制

Effects and mechanism of testosterone on the production of inflammatory cytokines and glucose uptake in co-culture of RAW264. 7 macrophage and 3T3-L1 adipocytes

目的探讨睾酮对333-L1脂肪细胞-RAW264．7巨噬细胞间接共培养下，分别对脂肪细胞、巨噬细胞炎症因子白细胞介素（IL）-6、巨噬细胞趋化因子（MCP）-1生成的影响、脂肪细胞胰岛素敏感性、巨噬细胞表型的影响及分子机制。方法333-L1前脂肪细胞在双层细胞培养板（Transwell）细胞下室诱导成熟后，与细胞上室的RAW264．7巨噬细胞共培养，用10μmol／L睾酮处理24h，酶联免疫吸附测定（Elisa）检测培养液中IL-6、MCP-1的浓度，蛋白质印迹杂交（Western blot）检测核因子κB-p65（NF-κBp65）、细胞外调节蛋白激酶（ERK1／2）磷酸化的改变、CD16／32、CD206的表达。[3H]-2-脱氧葡萄糖掺入法测脂肪细胞的葡萄糖摄取率。结果睾酮能促进对333-L1-脂肪细胞与RAW264．7巨噬细胞间接共培养体系中炎症因子（IL-6，MCP-1）的生成，促进ERK1／2、NF-κBp65的活化，抑制脂肪细胞的葡萄糖摄取；睾酮能促进巨噬细胞向促炎性M1型巨噬细胞的分化。睾酮的上述作用可以完全被NF-κBp65的抑制剂吡咯烷二硫基甲酸盐（PDTC）逆转，可部分被ERK1／2的抑制剂PD98059逆转（70％～90％）。结论NF-κB、ERK1／2可能是睾酮促333-L1脂肪细胞．RAW264．7巨噬细胞间接共培养体系炎症因子生成、胰岛素抵抗、巨噬细胞向M，型分化的重要分子蛋白。

Objective To investigate the effects of testosterone （T） on inflammatory cytokines （IL- 6, MCP-1） production, insulin sensitivity of adipoeyte and changes of macrophage phenotypes in indirect coculture of RAW264. 7 macrophages and 3T3-L1 adipocytes. Methods 3T3-L1 preadipocytes were induced to mature in Transwell lower chamber, and then co-cultured with RAW264. 7 macrophages in the upper chambers for 72 hours. Testosterone 10 μmol/L was added into indirect co-culture for 24 h. ELISA was used to testing IL-6, MCP-1 concentrations in supernatant. Western blot was used to detecting the phosphorylation of NF kappa B, ERK1/2, and theexpression of CD16/32 and CD206. Glucose transport was assessed by [ 3H ] 2-deoxy glucose uptake in adipoeytes. Results Testosterone enhanced inflammatory cytokines （IL-6, MCP-1 ） production in indirect co-culture of 3T3-L1 adipocytes and RAW264. 7 macrophages, promoted the activation of ERK1/2 and nuclear factor kappa B p65, and inhibited glucose uptake in adipocytes. Testosterone facilitated the production of pro-inflammatory Ml macrophages. The above effects of testosterone can be completely reversed by PDTC, and can be partly reversed by PD98059 （70% -90% ）. Conclusion NF kappa B and ERK1/2 could be the key proteins for testosterone to promote the production of inflammatory factors, to lead to insulin resistance, and to make macrophages differentiate to pro-inflammatory phenotypes in co-culture of RAW264. 7 macrophages and 3T3-L1 adipocytes.