基于NF-κB/MAPK信号通路探讨天山堇菜七叶内酯对脂多糖诱导RAW 264.7细胞的保护作用及机制  

The anti-inflammation effects of Aesculetin from Viola tianshanica Maxim via NF-κB and MAPK signaling pathways in RAW 264.7 cells stimulated by LPS and its mechanism

作　　者：王雪 刘燕 史玉柱 WANG Xue;LIU Yan;SHI Yu-zhu(Xinjiang Institute of Materia Medica,Urumqi 830011,China)

机构地区：[1]新疆维吾尔自治区药物研究所,新疆乌鲁木齐830011

出　　处：《中国药理学通报》2022年第9期1340-1349,共10页Chinese Pharmacological Bulletin

基　　金：新疆维吾尔自治区天山青年计划优秀青年科技人才培养项目(No 2018Q052)。

摘　　要：目的考察天山堇菜七叶内酯(aesculetin,AESN)对脂多糖(LPS)诱导小鼠单核巨噬细胞白血病细胞(RAW 264.7)炎症模型的保护作用及机制。方法实验设置空白对照组、LPS模型组、AESN不同浓度(0.16、0.8、4、20μmol·L^(-1))给药组以及对照药吲哚美辛(INDO,10μmol·L^(-1))组,建立LPS诱导RAW 264.7炎症模型。MTS法检测AESN对RAW 264.7细胞毒;Griess法检测一氧化氮(NO)分泌水平;ELISA法检测细胞上清TNF-α、IL-6、IL-1β的含量;qRT-PCR方法检测TNF-α、IL-6、IL-1β,一氧化氮合酶(iNOS)mRNA表达水平;细胞免疫荧光的方法检测iNOS、p-NF-κB p65、IκBα、p-p38、p-ERK1/2蛋白表达;检测AESN对环氧酶1/2(COX 1/2)的抑制活性。结果AESN在0.16、0.8、4、20μmol·L^(-1)可明显抑制LPS诱导RAW 264.7分泌炎症介质NO,抑制iNOS mRNA及蛋白表达水平;降低细胞上清中炎症因子TNF-α、IL-6、IL-1β的含量及mRNA表达水平。AESN还能明显抑制p-NF-κB p65蛋白表达,抑制IκBα降解,从而抑制p-NF-κB p65发生核转位;还能明显抑制pp38、p-ERK1/2核转位。AESN对COX-1、COX-2具有明显的抑制活性,IC分别为:28.1、2.3μmol·L^(-1)。结论AESN具有较好的抗炎作用,其作用机制与抑制NF-κB/MAPK信号通路有关。Aim To investigate the anti-inflammatory effects of Aesculetin from Viola tianshanica Maxim in LPS-stimulated RAW 264.7 cells and the underlying mechanism.Methods RAW 264.7 cells were divided into control group,model group(LPS),0.16,0.8,4,20μmol·L^(-1) AESN groups(different concentrations of AESN+LPS)and positive control group(10μmol·L^(-1) Indomethacin+LPS).LPS(1 mg·L^(-1)) was used to stimulate RAW 264.7 cells for 24 h to establish inflammatory model.MTS assay was used to detemine cytotoxicity of Aesculetin in RAW 264.7 cells.Griess method was used to detect NO secretion in LPS-stimulated RAW 264.7 cells.ELISA was applied to determine the contents of TNF-α,IL-6 and IL-1βin cell culture supernatant.qRT-PCR was employed to detect the mRNA expression levels of TNF-α,IL-6,IL-1βand iNOS.Immunofluorescence assay was used to evaluated the protein expressions of iNOS,p-NF-κB p65,IκBα,p-p38 and p-ERK1/2.Enzyme assay was used to detect the inhibition activity of Aesculetin on cyclooxygenase 1/2(COX 1/2).Results Aesculetin significantly inhibited the secretion of inflammatory mediator NO,mRNA and protein expression of iNOS in LPS-induced RAW 264.7 at 0.16,0.8,4 and 20μmol·L^(-1).The contents of TNF-α,IL-6 and IL-1βin supernatant significantly decreased,and the mRNA expression levels of TNF-α,IL-6 and IL-1βwere also reduced by Aesculetin.Aesculetin also obviously inhibited the protein degradation of IκBαand inhibited the nuclear translocation of p-NF-κB p65,p-p38,p-ERK1/2.In addition,Aesculetin had significant inhibitory activities on COX-1 and COX-2,and the ICwas 28.1μmol·L,2.3μmol·L^(-1),respectively.Conclusions AESN has good anti-inflammatory effect,and its mechanism is closely related to the inhibition of NF-κB and MAPK signaling pathways.

关 键 词：天山堇菜 七叶内酯 信号通路 RAW 264.7 抗炎 作用机制 

分 类 号：R-332[医药卫生] R284.1R345.57R364.5R971.1