摘要
目的:观察胰岛素对巨噬细胞破泡沫化过程中Toll样受体4(TLR4)表达及IL-6、TNF-α分泌的影响。方法:采用体外培养小鼠巨噬细胞系RAW264.7,氧化低密度脂蛋白(ox-LDL)诱导建立泡沫细胞模型,分为对照组、ox-LDL组、用胰岛素组、PI3K-AKT抑制剂组。油红O染色观察泡沫细胞模型的建立,取细胞上清用ELISA法检测白介素-6(IL-6)和肿瘤坏死因子-α(TNF-α)的水平;流式细胞术检测膜蛋白TLR4表达量,Western-blot检测TLR4、核因子-κB(NF-κB)的表达水平。结果:与对照组相比,ox-LDL处理过的巨噬细胞可向泡沫细胞转换,同时TLR4、NF-κB蛋白表达水平以及IL-6、TNF-α水平显著增加(P<0.05);而使用胰岛素干预后ox-LDL的作用显著减弱,TLR4、NF-κB蛋白表达水平以及IL-6、TNF-α水平显著降低(P<0.05 vs ox-LDLgroup);而使用PI3K-AKT抑制剂干预后,抑制剂显著降低胰岛素的作用,TLR4、NF-κB蛋白表达水平以及IL-6、TNF-α水平显著升高(P<0.05 vs ox-LDL+insulin)。结论:ox-LDL可诱导巨噬细胞向泡沫细胞转化,同时上调TLR4及NF-κB蛋白表达,增加炎性因子分泌,促进了AS进程,而胰岛素可使ox-LDL的作用显减弱,减少TLR4、NF-κB蛋白表达及炎性因子分泌,从而减轻AS进程,其机制可能与胰岛素通过PI3K-AKT抑制TLR4-NF-κB通路有关。
Objective： To explore the effect of insulin on the expression of toll-like receptor4 （TLR4） and IL-6 and TNF-α the formation ofmacrophage foam cells. Methods： Different treatments were given to macrophage by ox-LDL, insulin and wortmannin. After 24h, the intracellular lipid accumulation in macrophage was determined by Oil Red O staining. Levels of IL-6 and TNF-α were detected by ELISA kits. The expression of TLR4 was detected by flow cytometry （FCM） and western blot. The expression of NF-KB was analyzed by western blot. Results： Oil red O staining showed macrophages would transformed into foam cells in vitro if they were associated with the amount of ox-LDL, The protein level of TLR4 and its downstream NF-KB increased significantly detected by FCM and western blot respectively （P 〈 0.05, vs control）, and the level oflL-6 and TNF-α increased significantly detected by ELISA kits（P 〈 0.01 vs control）. Insulin significantly blunted the effects of ox-LDL, the protein level of TLR4 and NF-KB and the level of IL-6 and TNF-α decreased significantly （P 〈 0.05 vs ox-LDL group）. Moreover, wortmannin, which is the inhibitor of PI3K, could block the effects of insulin （P 〈 0.05 vs ox-LDL＋ insulin）. Conclusion： In vitro treatment by ox-LDL resulted in macrophages transforming into foam cell and the expression levels of TLR4 and NF-κB and the level of IL-6 and TNF-α were significantly increased, insulin significantly blunted the effects of ox-LDL, the expression levels of TLR4 and NF-κB and the level of IL-6 and TNF-α were significantly reduced, this may be inhibition of TLR4-NF-κB pathway in a PI3K-dependent way.
出处
《现代生物医学进展》
CAS
2013年第9期1605-1608,1604,共5页
Progress in Modern Biomedicine
基金
国家自然科学基金项目(81170184)
