Exendin-4对小鼠巨噬细胞一氧化氮生成和促炎/抗炎反应平衡的影响

Effects of exendin-4 on production of nitric oxide and promotion of IL-10/IL-12 balance in macrophages

目的:探讨新型糖尿病药物exendin-4(Ex-4)影响小鼠巨噬细胞一氧化氮(NO)生成和IL-10/IL-12分泌的量效-时效关系,以及是否经由PI3K/Akt/mTOR信号转导通路产生该作用。方法:体外培养小鼠RAW264.7巨噬细胞株,在有或者无细菌脂多糖(LPS)诱导下,给予不同浓度的Ex-4(1、10、50 nmol/L)刺激,并在培养4、8和24 h收集上清,分别采用Griess和ELISA方法检测上清NO和IL-10/IL-12的分泌水平。此外,分别给予mTOR抑制剂雷帕霉素或PI3K抑制剂LY294002预处理巨噬细胞1 h,再给予低、中浓度Ex-4(1、10 nmol/L)刺激,观察巨噬细胞NO的产生和IL-10/IL-12的分泌是否发生改变。结果:无论是否存在LPS,Ex-4均能显著促进小鼠RAW264.7细胞分泌IL-10,同时抑制IL-12和NO的分泌,并且星明显的时效-量效关系,中浓度Ex-4已达最大效应(与低浓度比较,P<0.05;与高浓度比较,P>0.05),该作用能被LY294002和雷帕霉素逆转(P<0.05或P<0.01),尤以雷帕霉素最为显著。结论:Ex-4可通过PI3K/Akt/mTOR信号通路调控单核细胞NO的分泌,并影响IL-10/IL-12的平衡,可能是其参与免疫调节的机制之一。

Objective： To investigate the dose-and time-dependent effects of exendin-4 on the production of nitric oxide （NO） and IL-10/IL-12 balance in maerophage, and to ascertain whether the phosphatidylinositol 3-kinase/ protein kinase B/mammalian target of rapamycin （PI3K/Akt/mTOR） pathway is involved. Methods： Murine macrophage RAW264.7 cell line was treated with different doses of exendin-4 （1,10,50 nmol/L） for 4, 8 or 24 hours with or without lipopolysaceharide （LPS）. In another set of experiment, RAW264.7 maerophage was cultured with low （1 nmol/L）or medium （10 nmol/L） dose of Ex-4 after 1-hour pretreatment with PI3K inhibitor （LY294002） or prototypic mTOR inhibitor （Rapamycin） for 4 hours. Cell-free supernatants were harvested for NO and IL-10/IL-12 assessment with Griess reagent and ELISA, respectively. Results： Compared with control group, exendin-4 could significantly increase the production of IL-10 while inhibit the secretion of NO and IL-12 with or without LPS in a dose-and time-dependent manner, and a medium dose of gx-4 exerted the most significant effect （P〈0.05 compared to low dose of Ex-4 but P〈0.05 compared to high-dose of Ex-4）. LY294002 and Rapamycin, the two PI3K/Akt/mTOR inhibitors, had the potential to reverse the effect of exendin-4 （P〈0. 05 or P〈0.01）, and between them, rapamycin was more effective. Conclusions：Exendin-4 might regulate, at least in part, the secretion of NO and IL-10/IL-12 balance in maerophage via PI3K/Akt/mTOR pathway, contributing to immune regulation.