地塞米松对脂多糖刺激THP-1释放炎性因子的影响及其机制

Effect of dexamethasone on release of pro-inflammatory cytokines from THP-1 induced by lipopolysaccharides

目的探讨地塞米松（Dexamethasone,DEX）对脂多糖（lipopolysaccharide,LPS）诱导的人外周血单核细胞（human peripheral mononuclear cell,THP-1）炎症因子释放的影响及其机制。方法体外培养人THP-1单核细胞,随机分为对照组（Control）、脂多糖组（LPS）和地塞米松组（DEX）。地塞米松组（1microg/ml）孵育地塞米松2 h后与脂多糖组（0.1μg/ml）均加入脂多糖刺激24 h。应用免疫蛋白印迹电泳法（Western blotting）检测各组细胞总蛋白、糖皮质激素受体（Glucocorticoid receptor ,GR）、磷酸化的糖皮质激素受体（p-GR）,IκB-α,磷酸化 IκB-α（p-IκB-α）,核因子κB （NF-κB）,磷酸化核因子κB （p-NF-ΚB）,巨噬细胞炎症趋化因子（MCP-1）的水平。用 ELISA 检测各组细胞培养上清中 MCP-1,肿瘤坏死因子-α（TNF-α）和白介素-6（IL-6）的表达水平,用实时定量 PCR（RT-PCR）检测各组细胞MCP-1,TNF-α和IL-6mRNA表达水平。结果 Westernblotting检测显示脂多糖组与对照组相比,脂多糖组 p-GR、p-NF-κB、p-IKB-α和 MCP-1蛋白表达水平明显升高（P〈0.05）,IκB-α表达明显下降（P〈0.05）,GR和 NF-KB表达水平无明显差异（P〉0.05）；RT-PCR 和 ELASA 检测显示MCP-1,TNF-α和IL-6分泌水平和mRNA 表达水平均明显增高（P〈0.05）。与脂多糖组相比,地塞米松组p-NF-κB,p-IKB-α和MCP-1蛋白水平表达明显下降,以及 MCP-1,TNF-α和 IL-6分泌和mRNA水平也均表达明显降低,但p-GR和IκB-α蛋白表达水平明显增加（P〈0.05）,但在 NF-κB和 GR表达水平依然无明显差异（P〉0.05）。结论地塞米松预处理可通过激活糖皮质激素受体而下调 NF-κB活化从而抑制脂多糖诱导单核细胞THP-1产生的炎症反应。

Objective To investigate the effects and potential mechanisms of dexamethasone （DEX）on pro-inflammatory cytokine release and expression from lipopolysaccharide （LPS）-induced THP-1 mononuclear cells.Methods The human THP-1 mononuclear cells were cultured and randomly divided into 3 groups：control group,LPS group,and DEX group.In DEX group,THP-1 cells were preincubated with DEX （1 mg/mL）for 2 h,followed by stimulation with DEX and LPS （0.1μg/mL）for 24 h.The expression of glucocorticoid receptor （GR）,p-GR,IκBα,p-IκBα,NF-κB,p-NF-κB and MCP-1 ,and the total protein in each group were measured by Western blotting.The levels of MCP-1 ,TNF-αand IL-6 in the supernatant of each group were measured by ELISA and RT-PCR. Results As compared with the control group,the expression levels of p-GR,p-NF-κB,p-IκBα,MCP-1,TNF-αand IL-6 in the LPS group were significantly increased,and IκBαdecreased （P〈0.05）,but there was no significant difference in the expression of IκBαand GR between the two groups （P〉0.05）.As compared with the LPS group,the expression levels of p-NF-κB,p-IκBα,MCP-1,TNF-αand IL-6 in the DEX group were significantly decreased,and IκB-αand p-GR increased （P〈0.05）. There was still no significant difference in the expression of NF-κB and GR between the two groups （P〉0.05）.Conclusions Dex pretreatment can inhibit inflammation that was induced by LPS-stimulating THP-1 mononuclear cells,and the mechanisms may be related to prevent NF-κB activation though GR activation.