烧伤血清对内皮细胞IκBα降解和NF-κB活化的影响

目的 了解烧伤血清对内皮细胞抑制性κB(IκBα)降解、核因子-KB(NF—KB)活化的影响，进一步探讨烧伤血清诱导内皮细胞活化分泌细胞因子的机制。方法 采用体外培养的内皮细胞，分别用正常人血清(对照组)、烧伤患者血清、烧伤患者血清 吡咯烷二硫代氨基甲酸盐(PDTC)刺激内皮细胞。采用Westem印迹法检测血清刺激30、60、90、120min后内皮细胞IκBα蛋白降解情况，电泳迁移率分析检测血清刺激30、60、120、240min后NF—κB活性的变化。结果烧伤血清刺激内皮细胞后30min，IκBα发生明显降解，刺激后60min达高峰，2h后表达逐渐升高；烧伤血清刺激内皮细胞后30min，NF-κB活性迅速升高，30～60min达高峰，2h后逐渐回复基础状态。PDTC能有效抑制烧伤血清作用条件下内皮细胞IκBα降解、NF-κB活化。结论 烧伤血清可诱导内皮细胞IκBα降解，活化NF—κB，从而在烧伤后内皮细胞分泌细胞因子过程中起重要作用。

Effects of burn patients' serum on IkappaBalpha degradation and NF-kappaB activation in endothelial cells in vitro]

OBJECTIVE: To investigate the effects of the serum from burned patients (burn serum) on inhibitor of kappaBalpha (IkappaBalpha) degradation and nuclear factor-kappaB (NF-kappaB) activation in the endothelial cells in order to explore the role of burn serum on endothelium activation. METHODS: Cultured endothelial cells (ECV-304) were stimulated by the serum from healthy volunteers, burn serum and burn serum with pyrrolidine dithiocarbamate (PDTC), respectively. Activation of endothelial NF-kappaB at 30, 60, 120, 240 min after stimulation was tested by electrophoretic mobility shift assay (EMSA) and the degradation of endothelial IkappaBalpha at these time points determined by Western blotting. RESULTS: Compared with that in the control group, cytosolic IkappaBalpha degradation in the endothelial cells occurred within 30 min after burn serum stimulation, and peaked at 60 min, followed by gradual recovery in the cytoplasm till reaching the pre-stimulation level at 2 h. Meanwhile, the activity of endothelial NF-kappaB was markedly increased, reaching the peak at 30 to 60 min with subsequent gradual decrease 2 h after stimulation. PDTC could effectively inhibit endothelial IkappaBalpha degradation and activation of NF-kappaB induced by the burn serum. CONCLUSION: Burn serum might induce the degradation of IkappaBalpha to activate NF-kappaB, which ultimately leads to cytokine release from the endothelium.