可溶型DC-SIGN对LPS诱导THP-1细胞炎性应答的调控作用

The regulatory effect of soluble DC-SIGN on LPS induced inflammatory response by THP-1 cells

目的探讨可溶型树突状细胞特异性的结合细胞间粘附分子的非整合素(sDC-SIGN)对脂多糖(LPS)刺激的单核细胞炎症反应的调节作用。方法以人急性单核细胞白血病细胞株(THP-1)为研究对象,分为空白组、200 ng/ml sDC-SIGN单独处理组、LPS单独刺激组、LPS刺激同时加100 ng/ml sDC-SIGN处理组和LPS刺激同时加200 ng/ml sDC-SIGN处理组,刺激后不同时间点收集上清和细胞。ELISA检测细胞上清中细胞因子含量、Q-PCR检测细胞内细胞因子的mRNA表达并利用Western blot方法检测细胞内细胞外调节蛋白激酶(ERK)和核转录因子-κB(NF-κB)信号通路的活化状况。结果不同浓度的sDC-SIGN均可抑制LPS刺激炎性细胞因子TNF-α、IL-6和IFN-α的mRNA表达和蛋白分泌,同时促进抑炎细胞因子IL-10的mRNA表达和蛋白分泌,且sDC-SIGN的抑制效果与浓度呈依赖关系;研究结果亦显示,sDC-SIGN减弱LPS刺激THP-1细胞诱导ERK和NF-κBP65的磷酸化水平。结论sDC-SIGN蛋白对LPS诱导的单核细胞炎症应答具有负向调控作用。

Objective To study the regulatory function of soluble dendritic cell-specific intercellular adhesion molecular 3-grabbing nonintegrin（sDC-SIGN）on lipopolysaccharides（LPS）-stimulated inflammatory responses in monocytes. Methods sDC-SIGN protein was prepared according to previous established methods with endotoxin removed. Human acute monocyticleukemia cell line（THP-1）were stimulated with 1 μg/ml LPS in the presence of different concentrations of sDC-SIGNprotein（0,100,and 200 ng/ml）. After 4 h stimulation,cells were collected for evaluating mRNA levels of TNF-alpha,IL-6,IFN-alpha and IL-10 by Q-PCR analysis. After 24 h stimulation,the culture supernatant was collected for detectingproduction of TNF-alpha,IL-6,IFN-alpha and IL-10 by ELISA. The phosphorylation levels of extrecellular regulatedprotein kinases（ERK）and NF-κB-P65 were examined at indicated time by western blot. Results sDC-SIGN proteininhibits the secretion of pro-inflammatory cytokines（TNF alpha,IL-6 and IFN-alpha）induced by LPS but promotes theproduction of anti-inflammatory cytokine IL-10. Accordingly,the trends of changes in mRNA expression levels of thecytokines were similar with those in the culture supernatant. It was worth noting that the inhibitory effect of sDC-SIGN was ina dose-dependent manner. Furthermore,it showed that sDC-SIGN protein could reduce the phosphorylation of ERK and NF-κB-P65 in THP-1 cells upon LPS stimulation. Conclusion sDC-SIGN protein has a negative regulatory role on theinflammatory response induced by LPS.