摘要
该研究以LPS诱导的RAW264. 7细胞炎症模型为载体,观察荆防药对正丁醇提取部位分离物A组分的体外抗炎作用,及其调控NF-κB,PI3K/AKT信号通路的抗炎作用机制。建立LPS诱导的RAW264. 7细胞炎症模型,荆防药对正丁醇提取部位分离物A组分预处理3 h,取细胞上清ELISA法测定炎症因子IL-6,IL-1β,TNF-α含量; Griess法测定NO含量; RT-PCR法测定RAW264. 7炎症细胞中IL-6,IL-1β,TNF-α,IFN-γ,i NOS,PI3K,AKT,CHUK,NF-κB1,Rela mRNA表达; Western blot法测定细胞中PI3K,AKT,NF-κB p50,p65,p105总蛋白及磷酸化蛋白p-PI3K,p-AKT,p-p65的表达水平。并采用LC-MS及数据库对比鉴定分离物A中可能的化学成分。结果显示,荆防药对正丁醇提取部位分离物A组分显著抑制LPS致RAW264. 7细胞炎症模型上清中NO,IL-6,IL-1β,TNF-α炎症因子的释放(P<0. 05或P<0. 01),降低IL-6,IL-1β,TNF-α,IFN-γmRNA表达水平,显著下调炎症模型细胞中PI3K,AKT mRNA表达及蛋白磷酸化表达水平(P<0. 05或P<0. 01);显著降低炎症模型细胞NF-κB p50,p-p65,i NOS蛋白水平,以及NF-κB1,Rela mRNA表达水平,上调CHUK基因表达。成分分析共鉴定出分离物A组分含化合物196种,其中isoobtusilactone,5-O-甲基维斯阿米醇苷,蒽贝素(embelin),升麻素苷含量较高。综上,荆防药对正丁醇提取部位分离物A组分体外对LPS诱导的RAW264. 7细胞炎症模型具有较好抗炎效应,作用发挥与其抑制PI3K/AKT信号通路活化及NF-κB信号通路活化有关,蒽贝素可能为其抗炎作用发挥的主要有效成分之一。
The LPS-induced RAW264. 7 cells inflammation model was used as a carrier to investigate the in vitro anti-inflammation effects of Jingfang n-butanol extraction(JFNE) isolated fraction A and explore its preliminary anti-inflammation mechanism by observing the regulatory effect on PI3 K/AKT signaling pathway and NF-κB pathway. The RAW264. 7 cells inflammation model was established by stimulating with LPS for 12 h. After 3 h pre-treatment with fraction A,the contents of interleukin-6(IL-6),interleukin-1β(IL-1β) and tumor necrosis factor(TNF-α) in the supernatant of RAW264. 7 cells inflammation model were determined by ELISA and the contents of NO in supernatant were assayed by Griess. Reverse transcription-polymerase chain reaction(RT-PCR) method was used to determine the expression of IL-6,IL-1β,TNF-α,IFN-γ,i NOS,PI3 K,AKT,CHUK,NF-κB1 and Rela mRNA in RAW264. 7 inflammatory cells,and the expression levels of phosphorylated and total PI3 K/AKT protein,NF-κB p50,p65,p-p65,p105 protein in cells were determined via Western blot. In addition,LC-MS and database were used to identify the possible chemical constituents in fraction A. The results showed that fraction A could significantly reduce the release levels of NO,IL-6,IL-1β and TNF-α in the supernatant and the expression of IL-6,IL-1β,TNF-α,IFN-γ,i NOS,PI3 K,AKT,CHUK,NF-κB1 and Rela mRNA in RAW264. 7 inflammation model cells(P<0. 05 or P<0. 01) and significantly inhibit the phosphorylation expression levels of PI3 K and AKT protein and mRNA expressions(P<0. 05 or P<0. 01). Moreover,fraction A could significantly reduce the levels of NF-κB p50,p-p65 and i NOS protein,as well as NF-κB1,Rela mRNA expressions in RAW264. 7 cells,and increase the expression of CHUK gene.A total of 196 compounds were identified from fraction A in the composition analysis,and isoobtusilactone,5-O-methyl-vismitol,emebel(embelin) and prim-O-glucosylcimifugin showed high contents. The results all above showed that fraction A had a certain antiinflammatory effect in LPS-induced
作者
饶志粒
曹海娟
石博宇
罗杰
刘小波
曾南
RAO Zhi-li;CAO Hai-juan;SHI Bo-yu;LUO Jie;LIU Xiao-bo;ZENG Nan(Pharmacy College of Chengdu University of Traditional Chinese Medicine,Chengdu 611137,China)
出处
《中国中药杂志》
CAS
CSCD
北大核心
2019年第5期1026-1033,共8页
China Journal of Chinese Materia Medica
基金
国家自然科学基金项目(81473399)
成都市科技局项目(2015-HM01-00187-SF)
