Objective: To investigate the effect of calenduloside E on lipopolysaccharide (LPS)-induced inflammatory response in RAW264.7 cells and explore the underlying molecular mechanism.
Methods: CCK-8 assay was used to examine the effect of different concentrations of calenduloside E (0-30 μg/mL) on the viability of RAW264.7 cells. The release of the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) in RAW264.7 cells in response to pretreatment with 6, 8, and 10 μg/mL calenduloside E for 2 h followed by stimulation with 100 ng/mL LPS was detected using enzyme-linked immunosorbent assay (ELISA). The expression levels of iNOS and COX-2 and the activation of JAK-stats, MAPKs and NF-кB signaling pathways in the treated cells were determined using Western blotting. A reactive oxygen species (ROS) detection kit was used to detect ROS production in the cells, and the nuclear translocation of the transcription factor stat3 was observed by laser confocal microscopy.
Results: Calenduloside E below 20 μg/mL did not significantly affect the viability of RAW264.7 cells. Calenduloside E dose-dependently decreased the expression levels of iNOS and COX-2 induced by LPS, inhibited LPS-induced release of TNF-α and IL-1β, and suppressed LPS-induced JAK1-stat3 signaling pathway activation and stat3 nuclear translocation. Calenduloside E also significantly reduced ROS production induced by LPS in RAW264.7 cells.
Conclusions: Calenduloside E inhibits LPS-induced inflammatory response by blocking ROS-mediated activation of JAK1-stat3 signaling pathway in RAW264.7 cells.
目的: 探讨金盏花苷E对脂多糖(LPS)诱导炎症反应的抑制作用及可能的分子机制。
方法: CCK-8实验检测不同浓度(0、2、4、6、8、10、20、25、30 μg/mL)的金盏花苷E对RAW264.7细胞活力的影响; 不同浓度的金盏花苷E(0、6、8、10 μg/mL)预处理RAW264.7细胞2 h, 然后用LPS(100 ng/mL)刺激细胞特定的时间, ELISA检测炎症因子TNF-α、IL-1β释放; Western blotting检测iNOS、COX-2的表达水平及JAK-stats、MAPKs及NF-кB信号途径的磷酸化; ROS检测试剂盒检测RAW264.7细胞内ROS含量; 激光共聚焦实验检测转录因子stat3的核转位。
结果: CCK-8结果显示, 金盏花苷E浓度在低于20 μg/mL时对RAW264.7细胞无明显毒性作用; 金盏花苷E浓度依赖性地下调LPS诱导的iNOS和COX-2的表达(P < 0.01 vs LPS组); 抑制LPS诱导的促炎细胞因子TNF-α及IL-1β的释放, 且1 0 μg/mL组抑制作用尤为显著(P < 0.01 vs LPS组); 抑制LPS诱导的JAK1-stat3信号途径激活及stat3的核转位; 降低LPS诱发的ROS产生(P < 0.01 vs LPS组)。
结论: 金盏花苷E通过抑制ROS介导的JAK1-stat3信号途径, 抑制LPS诱导的炎症反应。
Keywords: JAK1-stat3 signaling pathway; RAW264.7 cells; calenduloside E; inflammation; lipopolysaccharide.