南方医科大学学报

南方医科大学学报 ›› 2018, Vol. 38 ›› Issue (03): 243-.

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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

齐世美，李强，姜琦，戚之琳，章尧   

  • 出版日期:2018-03-20 发布日期:2018-03-20

Chrysin inhibits lipopolysaccharide-induced inflammatory responses of macrophages via JAK-STATs signaling pathway

  • Online:2018-03-20 Published:2018-03-20

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摘要： 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。

Abstract: 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 JAKSTATs signaling pathway. Conclusion Chrysin blocks the activity of JAK-STATs mediated by ROS to inhibit LPSinduced inflammatory response in RAW264.7 cells.