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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。
AIM: To observe the effect of insulin on the expression of TLR4 and the secretion of IL-6 and TNF-α during the process of foam cell de-foaming in macrophages. Methods: Foam cell model was induced by macrophage cell line RAW264.7 and ox-LDL in vitro and divided into control group, ox-LDL group, insulin group, PI3K-AKT inhibitor group. Oil red O staining was used to observe the establishment of foam cell model. The cell supernatants were assayed for the levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) using ELISA. Flow cytometry was used to detect the expression of membrane protein TLR4 Western blot was used to detect the expression of TLR4 and NF-κB. Results: Compared with the control group, ox-LDL-treated macrophages could translocate to foam cells and the expression of TLR4 and NF-κB and the levels of IL-6 and TNF-α significantly increased (P <0.05) After intervention with insulin, the effect of ox-LDL was significantly weakened, and the expression of TLR4 and NF-κB and the levels of IL-6 and TNF-α were significantly decreased (P <0.05 vs ox-LDL group) (P <0.05 vs ox-LDL + insulin), the expression of TLR4 and NF-κB and the levels of IL-6 and TNF-α were significantly decreased. CONCLUSION: Ox-LDL can induce macrophages to foam cells, up-regulate the expression of TLR4 and NF-κB, increase the secretion of inflammatory cytokines and promote the progression of AS. Insulin can decrease the effect of ox-LDL and decrease the expression of TLR4 , NF-κB protein expression and inflammatory cytokine secretion, thereby reducing the AS process, the mechanism may be related to the inhibition of TLR4-NF-κB pathway by insulin through PI3K-AKT.