Toll样受体2(TLR2)基因敲除减轻小鼠胰岛素抵抗并促进巨噬细胞M2极化

Knockout of TLR2 gene attenuates insulin resistance and promotes M2 polarization of macrophages in mice

目的研究Toll样受体2(TLR2)基因缺失对小鼠胰岛素抵抗和巨噬细胞极化的影响。方法出生28 d雄性TLR2基因敲除(TLR2-/-)和野生型(WT)的C57BL/6小鼠各12只,采用PCR验证每只小鼠的基因型。基础饲养3个月后,进行葡萄糖耐量实验(GTT)和胰岛素抵抗实验(ITT);从外周血中分离单个核细胞,以粒细胞-巨噬细胞集落刺激因子(GM-CSF)/γ干扰素(IFN-γ)和M-CSF/白细胞介素4(IL-4)/IL-13分别诱导培养为M1型和M2型巨噬细胞,流式细胞术检测CD11b、F4/80、CD11c、CD206、早期生长反应蛋白2(EGR2),确定巨噬细胞表型。ELISA检测M1型巨噬细胞培养上清液中肿瘤坏死因子α(TNF-α)、IL-6的含量;M2型巨噬细胞培养上清液中IL-10的含量。结果PCR验证结果显示TLR2-/-和WT小鼠的基因型正确;行GTT后比较,TLR2-/-小鼠血糖调节能力比WT小鼠强,在30 min时最明显;行ITT后比较,WT小鼠比TLR2-/-小鼠调节血糖的能力和胰岛素敏感性均降低,在15 min差异明显;与WT小鼠相比,TLR2-/-小鼠M1型巨噬细胞的比率降低,M2型巨噬细胞比率增加;与WT小鼠相比,TLR2-/-小鼠M1型巨噬细胞培养上清液IL-6、TNF-α含量降低,TLR2-/-小鼠M2型巨噬细胞培养上清液IL-10含量增加。结论TLR2信号影响巨噬细胞的极化,使巨噬细胞倾向于M1表型,从而分泌促炎因子使机体处于低度炎症的状态,导致葡萄糖耐量和胰岛素敏感性的降低。

Objective To study the effect of deletion of Toll-like receptor 2(TLR2) gene on insulin resistance and polarization of macrophages in mice. Methods The wild-type(WT) and TLR2 knockout(TLR2-/-) C57BL/6 male mice, aged 28 days, were selected, with 12 mice in each group. The genotype of each mouse was identified by PCR. After mice were fed with basic diet for 3 months, the glucose tolerance test(GTT) and insulin resistance test(ITT) were performed. The mononuclear cells isolated from peripheral blood were stimulated with GM-CSF/IFN-γ and M-CSF/IL-4/IL-13, respectively, to induce differentiation to M1-like and M2-like macrophages. The CD11b, F4/80, CD11c, CD206 and early growth response 2(EGR2) were detected by flow cytometry to determine the phenotype of macrophages. The levels of TNF-α, IL-6 and IL-10 in the culture supernatant of macrophages were detected using ELISA. Results The result of PCR identification was consistent with the genotype of mice. Compared to WT mice, TLR2-/- mice exhibited the significantly improved glycemic control at 30 min during GTT and the significantly increased insulin sensitivity at 15 minutes during ITT. The flow cytometry showed that M1 markers decreased and M2 macrophages increased in the TLR2-/- mice. ELISA showed that the levels of IL-6 and TNF-α significantly decreased in the culture supernatant of M1 macrophages, while the level of IL-10 significantly increased in the culture supernatant of M2 macrophages in TLR2-/- mice compared with WT mice. Conclusion TLR2 signal has an effect on the polarization of macrophages and makes macrophages tend to switch to M1 phenotype. A higher number of pro-inflammatory factors secreted by M1 macrophages contribute to a low-grade inflammation state in the body, which leads to a decrease in glucose tolerance and insulin sensitivity.