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.docVIP

PAGE PAGE 3 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 王艳艳，刘 鑫，曹红卫，王 宁，鲁永玲，郑 江 （400038 重庆，第三军医大学西南医院综合实验研究中心） [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法检测氯喹对RAW264.7细胞的活性影响；ELISA检测培养体系中TNF-α、IL-6的释放情况。RT-PCR检测TNF-α、IL-6、TLR4、TRAM mRNA的表达。EMSA检测NF-κB的活性，Western blot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时对细胞活性无影响（P0.05）；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。 [关键词] 氯喹；内毒素；核因子κB；干扰素调节因子3 [中图法分类号] [文献标志码] A Inhibition of chloroquine on TLR4/MyD88-independent signaling pathway within RAW264.7 cells induced by LPS Wang Yanyan,Lui Xin,Cao Hongwei,Wang Ning,Lu Yongling,Zheng Jiang (Medical Research Centre , Southwest Hospital , Third Military Medical University , Chongqing, 400038 , China) [Abstract] Objective To investigate the inhibitory effect of chloroquine on LPS-induced MyD88-independent TLR4 signaling pathway. Methods Influences of various concentrations of chloroquine on viability of RAW264.7 cells were measured by MTT test. Contents of TNF-α, IL-6 in culture supernatant were detected by ELISA, RT-PCR to detect TNF-α, IL-6，TLR4 mRNA and TRAM mRNA expression. EMSA to detect NF-κB activity.IκBα phosphorylation levels and the degradation of IRF3 detected by Western blotting. Results Chloroquine had no infulence on the viability of RAW264.7 when its concentration was under 40μg/ml（P 0.05）. Significant decrease of the release of TNF-α and IL-6 proteins and mRNAs induced by LPS was found(P 0.05).CQ reduced the expression of TRAM mRNA stimulated by LPS in RAW264.7 cells(P 0.01). The activation of NF-κB and the degradation of IRF3 were suppressed by LPS. Conclusion Chloroquine inhibits TLR4/MyD88-independent signaling pathway within RAW264.7 cells induced by LPS. [Key words] chloroquine; lipopolysaccharide; NF-κB;