天山雪莲总酚酸对脂多糖刺激的RAW264.7细胞炎症因子释放的影响及其机制研究  

Study on Effects of Total Phenolic Acids from Saussureae Involucratae Herba on Release of Inflammatory Mediators in Lipopolysaccharide-stimulated RAW264.7 Macrophages and Its Mechanism

作　　者：颜丽珊 邱新宇 康建英 顾春宇 张硕峰[1] 张超[1] 王亦巍 孔静 罗赣[1] 张翼[1] YAN Li-shan;QIU Xin-yua;KANG Jian-ying;GU Chun-yu;ZHANG Shuo-feng;ZHANG Chao;WANG Yi-wei;KONG Jing;LUO Gan;ZHANG Yi(School of Chinese Materia Medica,Beijing University of Chinese Medicine,Beijing 102488,China;Beijing Johamu Pharmaceutical Co.,Ltd.,Beijing 102433,China)

机构地区：[1]北京中医药大学中药学院,北京102488 [2]北京远大九和药业有限公司,北京102433

出　　处：《中国现代中药》2023年第10期2109-2118,共10页Modern Chinese Medicine

基　　金：国家自然科学基金青年科学基金项目(82003957)。

摘　　要：目的:研究天山雪莲总酚酸(PSI)抑制脂多糖(LPS)刺激的RAW264.7细胞炎症因子释放的作用及其作用机制。方法:采用大孔吸附树脂分离PSI,利用超高效液相色谱法(UPLC)对其进行成分分析。建立LPS诱导的RAW264.7细胞炎症模型。采用噻唑蓝法检测细胞活力;Griess法检测一氧化氮(NO)的生成;酶联免疫吸附法(ELISA)检测炎症因子释放水平;蛋白免疫印迹(Western blot)技术检测Toll样受体4(TLR4)信号通路关键蛋白的表达水平;免疫荧光技术检测核转录因子(NF)-κB亚基p65、激活子蛋白1(AP-1)亚基c-Jun和干扰素调节因子3(IRF3)的入核情况。结果:PSI主要含有绿原酸(23.60%),质量浓度为12.50~100.00μg·mL^(-1)时能显著抑制LPS刺激下炎症因子NO、前列腺素E2(PGE2)、肿瘤坏死因子(TNF)-α、白细胞介素(IL)-10、IL6、趋化因子单核细胞趋化蛋白(MCP)-1、巨噬细胞炎症蛋白(MIP)-1α和调节激活正常T细胞表达分泌因子(Rantes)的释放,并能剂量依赖性地降低TLR4信号通路关键蛋白κB抑制因子(IκB)α、IκB激酶(IKK)α/β、p65、TANK结合激酶1(TBK1)、IRF3、p38、胞外信号调节激酶(ERK)1/2及c-Jun的磷酸化水平,同时抑制LPS诱导的p65、c-Jun和IRF3的入核。结论:PSI能抑制LPS刺激下RAW264.7细胞炎症因子的产生,其作用机制可能与抑制TLR4信号通路有关。Objective:To investigate the effects of total phenolic acid from Saussureae Involucratae Herba(PSI)on the secretion of inflammatory mediators in LPS-stimulated RAW264.7 cells and explore the underlying mechanisms.Methods:Enrichment of phenolic acid of Saussureae Involucratae Herba was performed by macroporous adsorbent resin column.PSI was characterized by ultra-performance liquid chromatography(UPLC).The lipopolysaccharide(LPS)-induced RAW264.7 macrophages were used as the inflammatory cell model.Cell viability was determined by MTT assay.The production of nitric oxide(NO)was measured by Griess assay.The release of inflammatory cytokines and chemokines was detected by enzyme-linked immunosorbent assay(ELISA).The expression levels of the key components involved in the Tolllike receptor 4(TLR4)signaling pathway were determined by Western blot.The nuclear translocation of NF-κB/p65,AP-1/c-Jun and IRF3 was detected by immunofluorescence.Results:The enriched PSI mainly contains chlorogenic acid(23.60%).The releasement of inflammatory mediators,including NO,PGE2,TNF-α,IL-10,IL-6,MCP-1,MIP-1αand Rantes was significantly decreased in PSI-treated(12.50~100.00μg·mL^(-1))RAW264.7 cells after LPS exposure.PSI treatment concentration-dependently downregulated the phosphorylation levels of IKKα/β,IκBα,p65,TBK1,IRF3,p38,ERK1/2 and c-Jun.Furthermore,PSI treatment remarkably suppressed the nuclear translocation of p65,c-Jun and IRF3.Conclusion:PSI mitigated the LPS-induced release of inflammatory mediators in RAW264.7 cells,which may be associated with the inhibitory effects of PSI on the TLR4 signaling pathway.

关 键 词：天山雪莲总酚酸 脂多糖 炎症 TLR4信号通路 

分 类 号：R285.3[医药卫生—中药学]