槐定碱抑制脂多糖诱导的RAW264.7细胞炎症因子分泌及机制  

Sophoridine suppresses inflammatory cytokine secretion by lipopolysaccharide-induced RAW264.7 cells and its mechanism

作　　者：刘静[1,2] 刘瑛[1] 李宾 周娅[1] 

机构地区：[1]宁夏医科大学病原生物学与免疫学系,宁夏银川750004 [2]湖北省咸宁市中心医院检验科,湖北咸宁437100 [3]湖北省咸宁市中心医院心血管内科,湖北咸宁437100

出　　处：《细胞与分子免疫学杂志》2015年第5期585-589,595,共6页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(30660227);宁夏自然科学基金(NZ0783);银川市科技计划项目基金(银财发[2009]308)

摘　　要：目的观察槐定碱对脂多糖(LPS)诱导RAW264.7细胞分泌肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)时,对Toll样受体4(TLR4)、c-Jun表达的影响,探讨槐定碱抗LPS分子机制。方法培养RAW264.7细胞,实验分为4组:巨噬细胞对照组,以无血清DMEM孵育细胞;槐定碱处理组,以含31.25 mg/L槐定碱DMEM孵育细胞;LPS组及LPS刺激后槐定碱处理组,分别以浓度为100μg/L LPS DMEM孵育细胞60 min后弃去LPS,而后分别加入无血清DMEM或31.25 mg/L槐定碱DMEM继续培养。以上4组分别于处理完毕后5、30、60、120 min收集细胞及培养液。采用反转录PCR检测RAW264.7细胞TLR4、c-Jun mRNA的表达,Western blot法及免疫细胞化学技术检测RAW264.7细胞c-Jun蛋白的表达;ELISA检测培养液中TNF-α、IL-1β分泌量。结果槐定碱处理组与巨噬细胞对照组各指标间未见显著性差异;LPS组各时间点RAW264.7细胞的TLR4、c-Jun mRNA及c-Jun蛋白表达,培养液中TNF-α、IL-1β分泌量均显著高于巨噬细胞对照组,并维持至120 min未见下降;LPS刺激后槐定碱处理组显著抑制LPS刺激的RAW264.7细胞TLR4、c-Jun mRNA及c-Jun蛋白表达,并减少TNF-α、IL-1β分泌。结论槐定碱通过抑制TLR4及c-Jun表达,下调LPS诱导的RAW264.7细胞TNF-α、IL-1β的分泌。Objective To observe the effects of sophoridine on lipopolysaccharide （LPS）-induced secretion of tumor necrosis factor a （TNF-a） and interleukin 113 （IL-I~） as well as the expressions of Toll-like receptor 4 （TLR4） and c-Jun in RAW264. ？ cells and explore the molecular mechanism of anti-LPS of sophoridine. Methods RAW264.7 cells were cultured and divided into four groups： macrophage control group （ using serum-free DMEM to incubate cells）, sophondine 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. Results 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. Conclusion 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.

关 键 词：槐定碱 脂多糖 RAW264.7细胞 TLR4 C-JUN TNF-α IL-1β 

分 类 号：R285.5[医药卫生—中药学] R392.5[医药卫生—中医学]