氯喹对脂多糖/内毒素诱导的RAW264.7细胞TLR4-MyD88非依赖信号转导途径的抑制作用  

Inhibition of chloroquine on TLR4-MyD88-independent signaling pathway in lipopolysaccharide/endotoxin-induced RAW264.7 cells

作　　者：王艳艳[1] 刘鑫[1] 曹红卫[1] 王宁[1] 鲁永玲[1] 郑江[1] 

机构地区：[1]第三军医大学西南医院综合实验研究中心,重庆400038

出　　处：《第三军医大学学报》2010年第9期869-873,共5页Journal of Third Military Medical University

基　　金：国家自然科学基金面上项目(30872681)~~

摘　　要：目的探讨氯喹对脂多糖/内毒素(lipopolysaccharide/endotoxin,LPS)诱导TLR4-MyD88非依赖途径的抑制作用。方法MTT法检测不同浓度氯喹(5、10、20、40、60μg/ml)对RAW264.7细胞活性的影响,ELISA检测培养体系中TNF-α、IL-6在2、6、12、18、24h的释放情况,RT-PCR检测TNF-α、IL-6、TLR4、TRAMmRNA的表达,EMSA检测NF-κB的活性,Westernblot检测IκBα磷酸化水平和IRF3的降解情况。结果氯喹浓度小于40μg/ml时对RAW264.7细胞活性无影响(P>0.05);应用氯喹预处理后LPS诱导的RAW264.7细胞TNF-α、IL-6在蛋白水平和核酸水平均受到明显抑制(P<0.05),TLR4和TRAM在核酸水平均受到显著抑制(P<0.01);EMSA结果显示NF-κB的活化受到抑制,而Western blot检测显示IκBα磷酸化及IRF3的降解也受到抑制。结论氯喹对LPS诱导的RAW264.7细胞TLR4-MyD88非依赖信号转导途径具有抑制作用。Objective To study the inhibitory effect of chloroquine on TLR4-MyD88-independent signaling pathway lipopolysaccharide/endotoxin （LPS）-induced RAW264.7 cells.Methods Effect of various chloroquine （5、10、20、40、60 μg/ml） concentrations on viability of RAW264.7 cells was detected by MTT test.TNF-α and IL-6 levels in culture supernatant were measured at 2,6,12,18,24 h by ELISA,and RT-PCR for the detection of expressions of TNF-α,IL-6,TLR4 mRNA and TRAM mRNA.EMSA was used to detect NF-κB activity.IκBα phosphorylation and IRF3 degradation were assayed by Western blotting.Results Chloroquine had no effect on the viability of RAW264.7 cells at a concentration of less than 40 μg/ml （P0.05）.The expression of TNF-α,IL-6,TLR4 and TRAM in LES-induced RAW264.7 cells at protein and mRNA level was significantly inhibited （P0.05,P0.01）.EMSA showed that LPS inhibited the activity of NF-κB and Western blotting showed that LPS could suppress the phosporylation of IκBα and the degradation of IRF3.Conclusion Chloroquine inhibits TLR4-MyD88-independent signaling pathway in LPS-induced RAW264.7 cells.

关 键 词：氯喹 内毒素 核因子κB 干扰素调节因子3 

分 类 号：R392.32[医药卫生—免疫学] R631.202[医药卫生—基础医学]