Liu Jing, Liu Ying, Li Bin, Zhou Ya
Department of Pathogenic Biology and Immunology, Ningxia Medical University, Yinchuan 750004; Department of Clinical Laboratory, Xianning Center Hospital of Hubei Province, Xianning 437100, China.
Department of Pathogenic Biology and Immunology, Ningxia Medical University, Yinchuan 750004, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2015 May;31(5):585-9, 595.
To observe the effects of sophoridine on lipopolysaccharide (LPS)-induced secretion of tumor necrosis factor α (TNF-α) and interleukin 1β (IL-1β) as well as the expressions of Toll-like receptor 4 (TLR4) and c-Jun in RAW264.7 cells and explore the molecular mechanism of anti-LPS of sophoridine.
RAW264.7 cells were cultured and divided into four groups: macrophage control group (using serum-free DMEM to incubate cells), sophoridine control group (using 31.25 mg/L sophoridine-added DMEM to incubate cells), LPS group and sophoridine intervention group (using 100 μg/L LPS DMEM to incubate cells for 60 minutes, then throwing away LPS and adding serum-free DMEM or 31.25 mg/L sophoridine DMEM to incubate cells). Cells and culture medium were collected respectively at 5, 30, 60 and 120 minutes after the above treatment. The expression levels of TLR4 and c-Jun mRNA were determined by reverse transcription PCR (RT-PCR), and the expression of c-Jun protein in RAW264.7 cells was measured by immunocytochemistry and Western blotting; The levels of TNF-α and IL-1β in cell culture medium were analyzed by ELISA.
Compared with macrophage control group, sophoridine control group had no statistical difference in each index. Compared with macrophage control group, the expressions of TLR4 mRNA, c-Jun mRNA and protein as well as the secretion of TNF-α and IL-1β significantly increased at each time point in LPS group, and maintained the level to 120 minutes. Sophoridine suppressed the expressions of TLR4 mRNA, c-Jun mRNA and protein, and reduced the secretion of TNF-α and IL-1β in LPS-stimulated RAW264.7 cells in sophoridine intervention group.
Sophoridine down-regulated the secretion of TNF-α and IL-1β in LPS-induced RAW264.7 cells via inhibiting the expressions of TLR4 and c-Jun.
观察槐定碱对脂多糖（LPS）诱导的肿瘤坏死因子α（TNF-α）和白细胞介素1β（IL-1β）分泌以及RAW264.7细胞中Toll样受体4（TLR4）和c-Jun表达的影响，探讨槐定碱抗LPS的分子机制。
培养RAW264.7细胞并分为四组：巨噬细胞对照组（用无血清DMEM培养细胞）、槐定碱对照组（用含31.25 mg/L槐定碱的DMEM培养细胞）、LPS组和槐定碱干预组（用100 μg/L LPS的DMEM培养细胞60分钟，然后弃去LPS并加入无血清DMEM或31.25 mg/L槐定碱的DMEM培养细胞）。上述处理后分别于5、30、60和120分钟收集细胞和培养基。采用逆转录聚合酶链反应（RT-PCR）检测TLR4和c-Jun mRNA的表达水平，用免疫细胞化学和蛋白质印迹法检测RAW264.7细胞中c-Jun蛋白的表达；采用酶联免疫吸附测定（ELISA）分析细胞培养基中TNF-α和IL-1β的水平。
与巨噬细胞对照组相比，槐定碱对照组各指标无统计学差异。与巨噬细胞对照组相比，LPS组各时间点TLR4 mRNA、c-Jun mRNA和蛋白的表达以及TNF-α和IL-1β的分泌均显著增加，并维持至120分钟。在槐定碱干预组中，槐定碱抑制LPS刺激的RAW264.7细胞中TLR4 mRNA、c-Jun mRNA和蛋白的表达，并减少TNF-α和IL-1β的分泌。
槐定碱通过抑制TLR4和c-Jun的表达下调LPS诱导的RAW264.7细胞中TNF-α和IL-1β的分泌。
