艾纳香油对NF-κB及Nrf2/HO-1信号通路的作用研究

Effect of Blumea balsamifera(L.)DC Oil on NF-κB and Nrf2/HO-1 Signal Pathway

旨在通过研究NF-κB、Nrf2/HO-1通路来揭示艾纳香油(Blumea balsamifera(L.) DC Oil, BBO)发挥抗炎效果的作用机制。本研究利用LPS诱导的RAW264.7巨噬细胞炎症模型,试验分为空白组、LPS模型组、BBO组(低、中、高剂量)、BBO阴性对照组,每组3个重复,采用Hoechst 33342和PI双染检测BBO对细胞凋亡的影响;采用ELISA和分光光度法检测BBO对LPS诱导巨噬细胞后分泌IL-1β、TNF-α、PGE_(2)、LTB_(4)、NO含量及iNOS活力的影响;采用RT-PCR检测BBO对TNF-α、IL-1β的mRNA表达水平的影响;采用Western blotting检测BBO对NF-κB及Nrf2/HO-1信号通路关键蛋白水平的影响。结果显示,BBO在80μg·mL^(-1)剂量范围内可以扭转LPS导致的巨噬细胞形态变化及细胞凋亡发生;与LPS模型组相比,BBO 60~80μg·mL^(-1)剂量下可以极显著抑制LPS诱导的细胞炎性因子和炎症介质IL-1β、TNF-α、PGE_(2)、LTB_(4)、NO的分泌及iNOS活力(P<0.01),并极显著抑制LPS导致的细胞IL-1β、TNF-α mRNA表达(P<0.01);同时,BBO 60~80μg·mL^(-1)剂量下极显著下调LPS导致的COX-2、5-LOX、p-IKKα、p-P65、p-IκB-α、胞质Nrf2蛋白表达(P<0.01),极显著促进HO-1、核Nrf2蛋白表达(P<0.01),并呈现剂量依赖性关系。结果提示,艾纳香油有良好的抑制细胞炎性和抗凋亡效果,其可能通过抑制NF-κB通路中关键蛋白磷酸化和炎症因子的产生从而促进Nrf2/HO-1通路中主要抗炎基因表达最终发挥抗炎作用。

The purpose of this study was to reveal the mechanism of anti-inflammatory effect of BBO by studying NF-κB and Nrf2/HO-1 pathway.RAW264.7 macrophage inflammation model induced by LPS was used.The control group,LPS model group,BBO group(low,middle and high dose)and BBO negative control group were set up in this experiment,with 3 repeats in each group.The effect of BBO on apoptosis was detected by Hoechst 33342 and PI double staining.The contents of IL-1β,TNF-α,PGE 2,LTB 4,NO and the activity of iNOS in LPS-induced macro-phage were detected by ELISA and spectrophotometry.The effect of BBO on the mRNA expression of TNF-αand IL-1βwas detected by RT-PCR.The effect of BBO on the key proteins expression in NF-κB and Nrf2/HO-1 signal pathway was detected by Western blotting.The results showed that BBO could reverse the morphological changes and apoptosis of macrophages induced by LPS within the dose range of 80μg·mL^(-1).Compared with LPS model group,BBO of 60-80μg·mL-1 could extremely significantly inhibit the secretion of LPS-induced cellular inflammatory factors and inflammatory mediators IL-1β,TNF-α,PGE 2,LTB 4,NO and iNOS activity(P<0.01);BBO could also extremely significantly inhibit the expression of IL-1βand TNF-αmRNA(P<0.01)induced by LPS.At the same time,BBO of 60-80μg·mL^(-1) could extremely significantly down-regulate the protein expression of COX-2,5-LOX,p-IKKα,p-P65,p-IκB-αand cytoplasmic Nrf2 induced by LPS(P<0.01),and extremely significantly promote the protein expression of HO-1 and nuclear Nrf2 in a dose-dependent manner(P<0.01).The results suggest that BBO has good effects on anti-inflammatory and anti-apoptotic.It may play an anti-inflammatory role by inhibiting the phosphorylation of key proteins and the production of inflammatory factors in NF-κB pathway and promote the expression of major anti-inflammatory genes in Nrf2/HO-1 pathway.