黄杞苷干预NF-κB信号通路抑制巨噬细胞炎症反应及氧化应激

Engeletin suppresses inflammatory response and oxidative stress of macrophage through interfering the NF-κB signaling pathway

目的探讨黄杞苷对脂多糖(LPS)诱导的小鼠RAW264.7巨噬细胞增殖、炎症反应及氧化应激的干预效果及分子机制。方法RAW264.7细胞随机分为对照组、LPS组和不同浓度黄杞苷干预组,于不同培养时间用四甲基偶氮唑蓝(MTT)法检测细胞的增殖;ELISA检测上清液中肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)和白细胞介素-1β(IL-1β)浓度;试剂盒检测细胞活性氧(ROS)、超氧化物(O_(2)·^(-))和一氧化氮(NO)的释放量;Western blot检测NF-κB信号通路中IκBα、p65、p-p65蛋白的表达。结果LPS组较对照组细胞增殖增加,而100、200、400、600、800μmol/L黄杞苷均能抑制LPS诱导的细胞增殖,其中400μmol/L黄杞苷干预效果最明显。LPS组细胞产生的TNF-α、IL-6、IL-1β、ROS、O_(2)·^(-)和NO较对照组升高,400μmol/L黄杞苷可降低LPS诱导TNF-α、IL-6、IL-1β、ROS、O_(2)·^(-)和NO的产生。LPS组细胞IκBα蛋白水平下调,p65蛋白、p-p65蛋白水平上调,而黄杞苷可抑制LPS诱导的IκBα蛋白水平下调,p65蛋白、p-p65蛋白水平上调。结论黄杞苷可能通过干预NF-κB信号通路,抑制LPS诱导RAW264.7细胞增殖、炎症因子和ROS/活性氮簇(RNS)产生,显示了重要的抗炎和抗氧化功效,为其治疗炎症性疾病的深入研究提供一定的依据和借鉴。

Objective To explore the intervention effect and molecular mechanism of engeletin on the proliferation,inflammatory response,and oxidative stress in mouse RAW264.7 macrophages induced by lipopolysaccharide(LPS).Methods RAW264.7 cells were randomly divided into the control group,the LPS group and the intervention groups with different engeletin concentrations.Cell proliferation was detected by methyl thiazolyl tetrazolium(MTT)assay at different culture times.The concentrations of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6)and interleukin-1β(IL-1β)in supernatant were detected by enzyme-linked immunosorbent assay(ELISA).The release of cell reactive oxygen species(ROS),superoxide(O_(2)·^(-))and nitric oxide(NO)was detected by corresponding kits.Expression of IκBα,p65 and p-p65 in NF-κB signaling pathway was analyzed by Western blot.Results Compared with the control group,cell proliferation in the LPS group was increased,100,200,400,600 and 800μmol/L engeletin could inhibit LPS-induced cell proliferation,and 400μmol/L engeletin intervention was most effective.TNF-α,IL-6,IL-1β,ROS,O_(2)·^(-)and NO produced by cells in the LPS group were higher than those in the control group.400μmol/L engeletin could decrease the production of LPS-induced TNF-α,IL-6,IL-1β,ROS,O_(2)·^(-)and NO.In the LPS group,protein level of IκBαwas down-regulated,and p65 and p-p65 were up-regulated;engeletin could rescue these phenotypes.Conclusion Engeletin may inhibit cell proliferation and the generation of inflammatory factors,ROS/reactive nitrogen species(RNS)in RAW264.7 cells under LPS induction through interfering NF-κB signal pathway.It shows an important anti-inflammatory and antioxidant effect,and provides reference for research of engeletin in the treatment of inflammatory diseases.