Qi Shi-Mei, Li Qiang, Jiang Qi, Qi Zhi-Lin, Zhang Yao
Department of Biochemistry, Wannan Medical College, Wuhu 241002, China. E-mail:
Nan Fang Yi Ke Da Xue Xue Bao. 2018 Mar 20;38(3):243-250. doi: 10.3969/j.issn.1673-4254.2018.03.02.
To investigate the mechanism of chrysin in regulating lipopolysaccharide (LPS)-induced inflammation in RAW264.7 cells.
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.
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.
Chrysin blocks the activity of JAK-STATs mediated by ROS to inhibit LPS-induced inflammatory response in RAW264.7 cells.
探讨白杨素调节脂多糖（LPS）诱导的RAW264.7细胞炎症反应的机制。
用不同浓度（0、5、10、20、40、60、80、100、150和200μg/mL）的白杨素处理RAW264.7细胞24小时，使用CCK-8检测细胞活力。RAW264.7细胞在用10、30或60μg/mL白杨素预处理2小时后，再用LPS刺激不同时间。通过ELISA检测TNF-α、IL-6和MCP-1的水平，并用蛋白质免疫印迹法检测JAK-1、JAK-2、STAT-1和STAT-3的磷酸化。用CM-H2DCFDA荧光探针检测RAW264.7细胞中的活性氧水平。用活性氧清除剂NAC检测活性氧对LPS诱导的JAK-STATs信号及RAW264.7细胞炎症反应的影响。通过激光共聚焦显微镜观察转录因子STAT-1和STAT-3的核转位。
60μg/mL以下的白杨素对RAW264.7细胞活力无明显影响。在10、30和60μg/mL时，白杨素剂量依赖性地抑制LPS诱导的iNOS表达。白杨素处理还抑制了LPS诱导的RAW264.7细胞中JAK-STATs的磷酸化、STAT1和STAT3的核转位、TNF-α、IL-6和MCP-1的释放以及活性氧的产生；活性氧作为上游信号介导JAK-STATs信号通路的激活。
白杨素通过阻断活性氧介导的JAK-STATs活性来抑制LPS诱导的RAW264.7细胞炎症反应。
