墓头回提取液通过抑制NF-κB信号通路调节LPS体外诱导的巨噬细胞活化及体内诱导的炎症反应  

Modulation of LPS-induced macrophage activation and inflammatory response by Patrinia heterophylla extracts via inhibition of the NF-κB signaling pathway

作　　者：胡新新 赵丽[2] 谢英华 韩曦瑶 刘熠晗 王秋云 周佳楠 闻梦静 HU Xinxin;ZHAO Li;XIE Yinghua;HAN Xiyao;LIU Yihan;WANG Qiuyun;ZHOU Jianan;WEN Mengjing(Department of Hematology,Shanghai Fifth People's Hospital,Fudan University,Shanghai 200240,China;Clinical Molecular Cytogenetics and Immunology Laboratory,The First Hospital of Lanzhou University,Lanzhou 730000,China)

机构地区：[1]复旦大学附属上海市第五人民医院血液科,200240 [2]兰州大学第一医院临床细胞分子遗传与免疫检验室,730000

出　　处：《免疫学杂志》2024年第12期861-869,共9页Immunological Journal

基　　金：上海市闵行区自然科学研究课题(2023MHZ098)。

摘　　要：目的探究药用植物墓头回(MTH)提取物是否通过抑制核因子κB(NF-κB)信号通路调节脂多糖(LPS)体外诱导的巨噬细胞活化及体内诱导的炎症反应。方法细胞水平研究:将RAW264.7细胞分为6组,即对照组、LPS组、LPS+地塞米松(DEX)组、LPS+MTH(低、中、高剂量)组,其中LPS组、LPS+DEX组、LPS+MTH组细胞加入终质量浓度100 ng/ml LPS诱导,LPS+DEX组额外加入DEX(终质量浓度5.0 ng/ml),LPS+MTH组额外加入MTH提取物(终质量浓度0.1、0.2和0.4 mg/ml)。用CCK-8法检测细胞活性,用Transwell检测细胞侵袭,用流式细胞术检测细胞凋亡,用实时荧光定量PCR及酶联免疫吸附实验分别检测白细胞介素-6(IL-6)、白细胞介素-17(IL-17)和肿瘤坏死因子-α(TNF-α)的表达及分泌水平,用Griess法检测细胞一氧化氮(NO)的水平,用流式细胞染色实验检测总活性氧(ROS)水平,用蛋白质免疫印迹实验检测p65和核因子κB抑制蛋白(IκB)磷酸化水平,用荧光素酶报告基因实验检测NF-κB转录活性。动物水平研究:将大鼠50只随机分5组,10只/组,即对照组、LPS组、LPS+DEX组、LPS+MTH低剂量组(6 g/kg)、LPS+MTH高剂量组(24 g/kg)。将LPS注射至大鼠整个肺部,于建模前36 h、24 h、12 h和建模后12 h、24 h各组分别灌胃给药。末次给药12 h,取肺泡灌洗液,检测肺泡灌洗液的IL-6、IL-17、TNF-α及NF-κB信号通路相关指标。结果细胞水平研究:与对照组比,LPS组细胞活性、侵袭、凋亡、IL-6、IL-17和TNF-αmRNA及蛋白、NO、ROS、p65蛋白S536位点和IκB蛋白S32位点磷酸化水平、NF-κB转录活性均明显增高(均P<0.05),与LPS组比,3个LPS+MTH组的细胞活性、侵袭、IL-6、IL-17和TNF-αmRNA及蛋白、NO、ROS、p65蛋白S536位点和IκB蛋白S32位点磷酸化水平、NF-κB转录活性均明显降低(均P<0.05),凋亡明显增高(均P<0.05),以上指标均呈MTH剂量依赖性趋势。动物水平研究:与对照组比,LPS组IL-6、IL-17、TNF-α、p65蛋白S536位点和IκB�Objective To investigate whether the extracts of Patrinia heterophylla(MTH)inhibits the activation of macrophages induced by lipopolysaccharide(LPS)in vitro and the inflammatory response induced in vivo by suppressing the nuclear factor kappa B(NF-κB)signaling pathway.Methods Cellular studies:RAW264.7 cells were divided into 6 groups:control group,LPS group,LPS+Dexamethasone(DEX)group and LPS+MTH(low,medium,high dose)groups.The LPS group,LPS+DEX and LPS+MTH group were induced with a final concentration of 100 ng/ml LPS.While the LPS+DEX group was additionally treated with 5.0 ng/ml DEX,the LPS+MTH group was additionally treated with MTH(final concentrations of 0.1,0.2 and 0.4 mg/ml).The cell activity was detected using CCK-8 assay,cell invasion was detected using Transwell assay,cell apoptosis was detected using flow cytometry,and interleukin-6(IL-6),interleukin-17(IL-17)and tumor necrosis factor-α(TNF-α)were detected using real-time fluorescence quantitative PCR and enzyme-linked immunosorbent assay,respectively.The expression and secretion levels of nitric oxide(NO)in cells were detected by Griess assay,total reactive oxygen species(ROS)levels were detected by flow cytometry.The phosphorylation level of p65 and inhibitor ofκB(IκB)were detected by protein immunoblotting assay.The transcriptional activity was detection by luciferase reporter gene assay.Animal studies:50 rats were randomly divided into 5 groups,10 per group,namely the control group,LPS group,LPS+DEX group,LPS+MTH low-dose group(6 g/kg)and LPS+MTH high-dose group(24 g/kg).LPS was injected into the lungs of rats,and the groups were orally administered at 36 h,24 h,12 h before modeling,and 12 h,24 h after modeling.12 h after the last administration,bronchoalveolar lavage fluid was collected,and the IL-6,IL-17,TNF-αand NF-κB signaling pathway-related indicators in the bronchoalveolar lavage fluid were measured.Results Cellular studies:Compared with the control group,the cell activity,invasion,apoptosis,IL-6,IL-17 and TNF-αmRNA and protein,NO,R

关 键 词：墓头回 巨噬细胞 脂多糖 炎症反应 核因子ΚB 

分 类 号：R285[医药卫生—中药学]