Tang Tuo, Wang Shengnan, Cai Tianyu, Cheng Zhenyu, Qi Shimei, Qi Zhilin
Department of Biochemistry and Molecular Biology, Wannan Medical College, Wuhu 241002, China.
Anhui Provincial Key Laboratory of Active Biological Macro-molecules, Wannan Medical College, Wuhu 241002, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2019 Aug 30;39(8):904-910. doi: 10.12122/j.issn.1673-4254.2019.08.05.
To investigate the effect of calenduloside E on lipopolysaccharide (LPS)-induced inflammatory response in RAW264.7 cells and explore the underlying molecular mechanism.
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.
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.
Calenduloside E inhibits LPS-induced inflammatory response by blocking ROS-mediated activation of JAK1-stat3 signaling pathway in RAW264.7 cells.
研究金盏菊苷E对脂多糖（LPS）诱导的RAW264.7细胞炎症反应的影响，并探讨其潜在的分子机制。
采用CCK-8法检测不同浓度（0-30μg/mL）金盏菊苷E对RAW264.7细胞活力的影响。用酶联免疫吸附测定（ELISA）法检测RAW264.7细胞在用6、8和10μg/mL金盏菊苷E预处理2小时后再用100ng/mL LPS刺激时促炎细胞因子肿瘤坏死因子-α（TNF-α）和白细胞介素-1β（IL-1β）的释放。用蛋白质免疫印迹法测定处理后细胞中诱导型一氧化氮合酶（iNOS）和环氧化酶-2（COX-2）的表达水平以及JAK-信号转导和转录激活因子（JAK-stats）、丝裂原活化蛋白激酶（MAPKs）和核因子κB（NF-кB）信号通路的激活情况。用活性氧（ROS）检测试剂盒检测细胞内ROS的产生，并通过激光共聚焦显微镜观察转录因子信号转导和转录激活因子3（stat3）的核转位情况。
20μg/mL以下的金盏菊苷E对RAW264.7细胞活力无显著影响。金盏菊苷E剂量依赖性地降低LPS诱导的iNOS和COX-2的表达水平，抑制LPS诱导的TNF-α和IL-1β的释放，并抑制LPS诱导的JAK1-stat3信号通路激活和stat3核转位。金盏菊苷E还显著降低了LPS诱导的RAW264.7细胞内ROS的产生。
金盏菊苷E通过阻断RAW264.7细胞中ROS介导的JAK1-stat3信号通路激活来抑制LPS诱导的炎症反应。
