白杨素通过JAK-STATs信号通路抑制内毒素诱导的巨噬细胞炎症反应

摘要

Objective To investigate the mechanism of chrysin in regulating lipopolysaccharide(LPS)-induced inflammation in RAW264.7 cells.Methods RAW264.7 cells were treated with different concentrations(0,5,10,20,40,60,80,100,150,and 200 μg/mL)of chrysin for 24 h,and the cell viability was measured using CCK-8.RAW264.7 cells were pre-treated with 10,30,or 60 μg/mL chrysin for 2 h before stimulation with LPS for different times.The levels of TNF-α,IL-6 and MCP-1 were detected by ELISA, and Western blotting was used to detect the phosphorylation of JAK-1, JAK-2, STAT-1 and STAT-3. The level of reactive oxygen species in RAW264.7 cells was detected by CM-H2DCFDA fluorescence probe.The effect of ROS on LPS-induced JAK-STATs signal and the inflammatory response of RAW264.7 cells was detected by ROS scavenger NAC. The transcription factors STAT-1 and STAT-3 nuclear translocation were observed by laser confocal microscopy. Results Chrysin below 60 μg/mL did not significantly affect the viability of RAW264.7 cells.At 10,30,and 60 μg/mL,chrysin dose-dependently inhibited the expression of iNOS induced by LPS. Chrysin treatment also inhibited LPS-induced phosphorylation of JAK-STATs, nuclear translocation of STAT1 and STAT3, release of TNF-α, IL-6 and MCP-1, and the production of ROS in RAW264.7 cells;ROS acted as an upstream signal to mediate the activation of JAK-STATs signaling pathway.Conclusion Chrysin blocks the activity of JAK-STATs mediated by ROS to inhibit LPS-induced inflammatory response in RAW264.7 cells.%目的 探讨白杨素对LPS诱导的RAW264.7细胞炎症的调控机制.方法 分别用不同浓度(0、5、10、20、40、60、80、100、150、200 μg/mL)白杨素作用RAW264.7细胞24 h后,采用CCK-8检测细胞活力值;分别用(10、30、60 μg/mL)白杨素预处理RAW264.7细胞2 h后,用LPS刺激RAW264.7细胞18 h后,采用ELISA法检测炎症因子TNF-α、IL-6和MCP-1的释放水平;分别用(10、30、60 μg/mL)白杨素预处理RAW264.7细胞2 h后,用LPS刺激RAW264.7细胞4 h后,运用Western blotting法检测JAK-1、JAK-2、STAT-1、STAT-3的磷酸化水平;分别用(10、30、60 μg/mL)白杨素预处理RAW264.7细胞2 h后,用LPS刺激RAW264.7细胞15 min后,运用CM-H2DCFDA荧光探针检测RAW264.7细胞内活性氧簇水平;运用ROS清除剂NAC处理RAW264.7细胞,检测ROS对LPS诱导RAW264.7细胞JAK-STATs信号和炎症反应的影响;运用激光共聚焦显微镜观察转录因子STAT-1和STAT-3核转位情况.结果 在白杨素浓度低于60 μg/mL时,对RAW264.7细胞活力没有显著影响,因此我们选择10、30、60 μg/mL白杨素作为抑制炎症作用的低、中、高剂量组;白杨素剂量依赖性地抑制LPS诱导的炎症蛋白iNOS的表达;白杨素剂量依赖性的下调LPS诱导的RAW264.7细胞中促炎因子TNF-α、IL-6和MCP-1的释放(P<0.01);白杨素抑制RAW264.7细胞中JAK-STATs信号活化并抑制STAT-1和STAT-3核转位;白杨素抑制RAW264.7细胞内ROS的产生;ROS作为上游信号介导JAK-STATs信号通路的活化.结论 白杨素能有效阻断ROS介导的JAK-STATs信号活化,从而抑制LPS诱导的RAW264.7细胞炎症反应.