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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 investigate the inhibitory effect of chloroquine on the TLR4-MyD88-independent pathway induced by lipopolysaccharide / endotoxin (LPS). Methods The effects of different concentrations of chloroquine (5, 10, 20, 40, 60μg / ml) on the activity of RAW264.7 cells were detected by MTT assay. The levels of TNF-α and IL- The release of TNF-α, IL-6, TLR4 and TRAM mRNA were detected by RT-PCR. The activity of NF-κB was detected by EMSA. The phosphorylation of IκBα and the degradation of IRF3 were detected by Western blot. Results Chloroquine pretreatment did not affect the viability of RAW264.7 cells when the concentration of chloroquine was less than 40 μg / ml (P> 0.05). After pretreatment with chloroquine, the levels of TNF-α and IL-6 in LPS-induced RAW264.7 cells were significantly increased at both protein and nucleic acid levels (P <0.01). The levels of TLR4 and TRAM were significantly inhibited at the nucleic acid level (P <0.01). The EMSA results showed that the activation of NF-κB was inhibited, and the phosphorylation of IκBα and the degradation of IRF3 were also inhibited by Western blot. Conclusion Chloroquine has an inhibitory effect on LPS-induced TLR4-MyD88 independent signal transduction pathway in RAW264.7 cells.