青蒿素对脂多糖诱导的小鼠巨噬细胞NF-κB信号通路激活和NO合成的影响

Effects of artemisinin on activation of NF-κB signaling pathway and NO synthesis in mouse macrophages induced by LPS

目的:研究青蒿素对脂多糖(LPS)诱导的小鼠巨噬细胞中核因子-κB(NF-κB)信号通路激活和NO合成的影响。方法:将小鼠巨噬细胞Raw264.7分成5组。LPS组细胞以含有1 mg/L LPS的细胞培养液培养;青蒿素20、40、80 mg/L组细胞分别以含有1 mg/L LPS和20、40、80 mg/L青蒿素的细胞培养液培养;空白对照组以不含LPS和青蒿素的细胞培养液培养。细胞培养24 h后,ELISA法检测培养上清中TNF-α、IL-1β含量,Griess试剂法检测培养上清中NO含量,Western blot法检测细胞中Toll样受体4(TLR4)、诱导型一氧化氮合酶(iNOS)、IκB-α蛋白,细胞质和细胞核中NF-κB p65蛋白的表达。结果:与空白对照组比较,LPS组细培养上清中TNF-α、IL-1β、NO含量升高(P<0.05),细胞中TLR4、iNOS蛋白表达水平增加,IκB-α蛋白表达水平降低,细胞质中NF-κB p65蛋白表达水平降低,细胞核中NF-κB p65蛋白表达水平升高(P<0.05)。与LPS组比较,各浓度青蒿素组TNF-α、IL-1β、NO含量降低,TLR4、iNOS蛋白表达水平降低,IκB-α蛋白表达水平增加,细胞质中NF-κB p65蛋白表达水平增加,细胞核中NF-κB p65蛋白表达水平降低(P<0.05);青蒿素80 mg/L组上述改变更显著(P<0.05)。结论:青蒿素可抑制LPS诱导的小鼠巨噬细胞NF-κB信号通路的激活和NO的合成。

Aim:To study the effects of artemisinin on NF-κB signaling pathway activation and NO synthesis of mouse macrophages induced by lipopolysaccharides(LPS).Methods:Mice macrophage Raw264.7 were allocated into 5 groups.LPS group was cultured with the medium containing 1 mg/L LPS;artemisinin 20,40 and 80 mg/L groups were cultured with the medium containing 1 mg/L LPS and 20,40 or 80 mg/L artemisinin,respectively;and the cells in the blank control group were cultured without LPS or artemisinin.After 24 hours culture,the contents of TNF-αand IL-1βin the supernatant were detected by ELISA method,the content of NO in the supernatant was detected by Griess reagent method.Western blot was used to detect the expressions of TLR4,iNOS and IκB-αin cells and the expressions of NF-κB p65 protein in cytoplasm and nucleus.Results:Compared with those of the blank control group,the contents of TNF-α,IL-1βand NO in the supernatant of LPS group increased(P<0.05);the expressions of TLR4 and iNOS proteins in cells increased,that of IκB-αprotein decreased(P<0.05);the expressions of NF-κB p65 protein in cytoplasm decreased,and that in the nucleus increased(P<0.05).Compared with those of LPS group,the contents of TNF-α,IL-1βand NO in the 3 artemisinin groups decreased(P<0.05);the expressions of TLR4 and iNOS proteins in cells decreased,IκB-αexpression increased(P<0.05);NF-κB p65 expression increased in cytoplasm,and decreased in the nucleus(P<0.05).The above changes were more significant in artemisinin 80 mg/L group(P<0.05).Conclusion:Artemisinin could inhibit the activation of NF-κB signaling pathway and NO synthesis in mouse macrophages induced by LPS.