Toll样受体4在棕榈酸诱导RAW264.7巨噬细胞 炎症反应中的作用及机制

目的 探讨 Toll 样受体 4（TLR4）在棕榈酸诱导 RAW264.7 巨噬细胞炎症反应中的作用及可能机制。方 法 观察正常对照组（正常饮食）和高脂饲料组（高脂饮食诱导肥胖小鼠模型）C57BL/6J 小鼠血清自由脂肪酸（FFA） 水平及内脏脂肪组织炎症细胞因子肿瘤坏死因子-α（TNF-α）、白细胞介素-6（IL-6）和单核细胞趋化蛋白-1（MCP- 1）的表达；采用棕榈酸（150 μmol/L 组和 300 μmol/L 组）刺激小鼠 RAW264.7 巨噬细胞，观察这 2 组和空白对照 （Control）组、无 FFA 牛血清白蛋白（BSA）组的 TNF-α、IL-6 和 MCP-1 的表达和分泌，同时检测 TLR4 的蛋白表达和 核因子-κB（NF-κB）的激活情况；利用 siRNA 干扰实验抑制 TLR4，观察在 Control 组、BSA 组、棕榈酸（300 μmol/L） 组、棕榈酸（300 μmol/L）+Control siRNA 组、棕榈酸（300 μmol/L）+TLR4 siRNA 组中棕榈酸诱导巨噬细胞炎症因子的 表达和分泌。结果 高脂饲料组体质量和 Lee’s 指数高于正常对照组，血清中 FFA 水平升高，内脏脂肪组织中 TNF-α、IL-6 和 MCP-1 的表达明显增加（P＜0.05）。与 BSA 组比较，棕榈酸 150 μmol/L 组和棕榈酸 300 μmol/L 组 炎症因子 TNF-α、IL-6 和 MCP-1 的表达和分泌均明显增加，TLR4 和 NF-κB p65 磷酸化蛋白水平均增加（P＜0.05）。 与 BSA 组比较，棕榈酸 300 μmol/L 组 NF-κB p65 的核转运水平和细胞内水平明显升高（P＜0.05）。TLR4 被抑制 后，棕榈酸+TLR4 siRNA 组的 TLR4、TNF-α、IL-6 和 MCP-1 的 mRNA 表达和分泌水平明显低于棕榈酸组（P＜ 0.05）。结论 TLR4 可能参与棕榈酸诱导的 RAW264.7 巨噬细胞炎症反应，诱导炎症因子 TNF-α、IL-6 和 MCP-1 的释放，且其介导的炎症反应与 NF-κB 的激活有关。

Role of TLR4 in RAW264.7 macrophage inflammation induced by palmitic acid

objective To investigate the effect of toll-like receptor 4 (TLR4) in RAW264.7 macrophage inflammation induced by palmitic acid and its possible mechanisms thereof. Methods C57BL/6J male mice were fed normal (control group) or high-fat diet (high-fat group) for 18 weeks. The serum level of free fatty acids (FFA) and mRNA levels of inflammatory cytokines in visceral adipose (TNF-α, IL-6 and MCP-1) were detected. Then, palmitic acid (150 μmol/L and 300 μmol/L) was applied to stimulate RAW264.7 macrophage, the mRNA expression and the secretion level of cytokines were detected. Meanwhile, TLR4 protein level, the nuclear translocation and intracellular level of NF- κB p65 were also detected. After TLR4 inhibition by siRNA transfection, the mRNA expression and the secretion level of cytokines were detected. Results The body weight and Lee’s index were significantly higher in high-fat group than those of control group (P＜0.05). The serum level of FFA and mRNA levels of inflammatory cytokines (TNF - α, IL-6 and MCP-1) in visceral adipose were significantly higher in high-fat group than those of control group (P＜0.05). Compared with mice without FFA (BSA group), the mRNA expression and the levels of TNF-α, IL-6, and MCP-1 were significantly increased (P＜0.05), and the protein expression of TLR4 and NF - κB phosphorylated protein were up-regulated (P＜0.05) in mice treated with palmitic acid (150 μmol/L and 300 μmol/L). The nuclear translocation and intracellular level of NF-κB p65 were increased in mice treated with palmitic acid 300 μmol/L than mice in BSA group (P＜0.05). The mRNA levels of TNF-α, IL-6 and MCP-1 and their secretion level were significantly decreased after TLR4 siRNA transfection (P＜0.05). Conclusion These results suggest that TLR4 may mediate the inflammation in RAW264.7 macrophage stimulated by palmitic acid by inducing inflammatory cytokines (TNF-α, IL-6 and MCP-1), and which may be related with the activation of NF-κB.