当归苯酞riligustilide抑制LPS诱导的RAW 264.7细胞炎症反应及机制研究  

Inhibitory Effect and Mechanism of Riligustilide from Danggui(Angelica sinensis)on LPS-induced Inflammatory Response in RAW 264.7 Cells

作　　者：刘员 王欢欢 吕洁丽[1] 张来宾[1] LIU Yuan;WANG Huanhuan;LYU Jieli;ZHANG Laibin(School of Pharmacy,Xinxiang Medical University,Xinxiang 453003,Henna,China)

机构地区：[1]新乡医学院药学院,河南新乡453003

出　　处：《中华中医药学刊》2024年第7期140-146,I0026,共8页Chinese Archives of Traditional Chinese Medicine

基　　金：国家自然科学基金项目(81773898);河南省优秀青年科学基金项目(212300410066)。

摘　　要：目的研究当归苯酞二聚体riligustilide(DG2)对脂多糖(lipopolysaccharides, LPS)诱导的RAW 264.7巨噬细胞炎症反应的抑制作用,并探讨其作用机制。方法通过LPS诱导建立RAW 264.7细胞炎症模型,采用噻唑蓝(methyl thiazolyl tetrazolium, MTT)法考察DG2对RAW 264.7细胞存活率的影响,Griess法考察DG2对LPS诱导的RAW 264.7细胞释放炎症介质一氧化氮(nitric oxide, NO)的影响,酶联免疫吸附测定法(enzyme-linked immunosorbent assay, ELISA)考察DG2对LPS诱导的RAW 264.7细胞分泌炎症因子[肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)和白细胞介素-6(interleukin-6,IL-6)]的影响,Western blotting法考察DG2对LPS诱导的RAW 264.7细胞诱导型一氧化氮合酶(inducible nitric oxide synthase, iNOS)和环氧化酶-2(cyclooxygenase-2,COX-2),以及丝裂原活化蛋白激酶(mitogen-activated protein kinase, MAPK)、核转录因子κB(nuclear factor-κB,NF-κB)和信号传导及转录激活蛋白(signal transducer and activator of transcription, STAT)信号通路的影响,免疫荧光法考察DG2对LPS诱导的RAW 264.7细胞STAT3核转位的影响。结果DG2浓度在64μmol/L下对RAW 264.7细胞存活率无影响,DG2能显著降低LPS诱导的RAW 264.7细胞释放NO的水平(P<0.001)[IC_(50)=(26.13±5.75)μmol/L],与阳性对照槲皮素的作用相当[IC_(50)=(26.06±2.28)μmol/L];还能够显著抑制炎症因子IL-6和TNF-α的生成(P<0.01,P<0.001)。DG2能够显著抑制LPS诱导的RAW 264.7细胞iNOS和COX-2蛋白的表达(P<0.01,P<0.001),显著抑制p-STAT3、磷酸化蛋白激酶B(p-AKT)和p-p38的蛋白表达(P<0.05,P<0.01),同时抑制STAT3的核转位。结论DG2可抑制LPS诱导的RAW 264.7细胞炎症反应,其机制可能与下调STAT、NF-κB和MAPK信号通路有关。Objective To investigate the inhibitory effect and mechanism of riligustilide(DG2),a phthalide dimer from Danggui(Angelica sinensis)on lipopolysaccharides(LPS)-induced inflammatory response in RAW 264.7 macrophages.Methods The inflammation model of RAW 264.7 cells was established by LPS induction.The effect of DG2 on the survival rate of RAW 264.7 cells was detected by MTT method,and the effect of DG2 on the levels of inflammatory mediator nitric oxide(NO)in LPS-induced RAW 264.7 cells was determined by Griess method.Enzyme-linked immunosorbent assay(ELISA)was used to investigate the effect of DG2 on the secretion of tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)in LPS-induced RAW 264.7 cells.Western blotting was used to detect the effects of DG2 on inducible nitric oxide synthase(iNOS)and cyclooxygenase-2(COX-2)in RAW 264.7 cells induced by LPS,as well as mitogen-activated protein kinase(MAPK),nuclear factor-κB(NF-κB)and signal transducer and activator of transcription(STAT)signaling pathways.Immunofluorescence was used to observe the effect of DG2 on the nuclear translocation of STAT3 in RAW 264.7 cells induced by LPS.Results DG2 at the concentration of 64μmol/L had no effect on the survival rate of RAW 264.7 cells.DG2 significantly reduced the level of NO released by LPS-induced RAW 264.7 cells(P<0.001)[IC_(50)=(26.13±5.75)μmol/L],which was similar to that of positive control quercetin[IC_(50)=(26.06±2.28)μmol/L].It also significantly inhibited the production of inflammatory factors IL-6 and TNF-α(P<0.01,P<0.001).In addition,DG2 significantly inhibited the LPS-induced expressions of iNOS and COX-2 proteins(P<0.01,P<0.001),as well as phosphorylation STAT3(p-STAT3),phosphorylation protein kinase B(p-AKT)and p-p38 proteins(P<0.05,P<0.01).Moreover,it inhibited the nuclear translocation of STAT3 in LPS-induced RAW 264.7 cells.Conclusion DG2 could inhibit LPS-induced inflammatory response in RAW 264.7 cells,and the mechanism may be related to the down-regulation of STAT,NF-κB and MAPK signaling pat

关 键 词：当归 苯酞二聚体 抗炎 RAW 264.7细胞 作用机制 

分 类 号：R284[医药卫生—中药学]