基于NF-κB信号通路探讨苓桂术甘汤对脂多糖诱导的小鼠急性肺损伤的抗炎保护作用  

Anti-inflammatory and Protective Effect of Linggui Zhugantang on LPS-induced Acute Lung Injury in Mice via NF-κB Signaling Pathway

作　　者：丁薇 汪文来[2] 何湛湛 陈香云 刘珍洪[3,4] 陶旭光 赵培彰[5] 杨桢 赵红霞 DING Wei;WANG Wenlai;HE Zhanzhan;CHEN Xiangyun;LIU Zhenhong;TAO Xuguang;ZHAO Peizhang;YANG Zhen;ZHAO Hongxia(School of Traditional Chinese Medicine(TCM),Beijing University of Chinese Medicine,Beijing 102488,China;Institute of Basic Theory for Chinese Medicine,China Academy of Chinese Medical Sciences,Beijing 100700,China;Encephalopathy Research Institute,Beijing University of Chinese Medicine,Beijing 100700,China;Key Laboratory of TCM Syndrome Treatment of Encephalopathy,National Administration of TCM,Beijing 100700,China;West China Clinical Medical College,Sichuan University,Chengdu 610041,China)

机构地区：[1]北京中医药大学中医学院,北京102488 [2]中国中医科学院中医基础理论研究所,北京100700 [3]北京中医药大学中医脑病研究院,北京100700 [4]国家中医药管理局脑病中医证治重点研究室,北京100700 [5]四川大学华西临床医学院,成都610041

出　　处：《中国实验方剂学杂志》2023年第15期14-21,共8页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(82104822);中国博士后科学基金项目(2021M700520);中国中医科学院科技创新工程黑地黄丸重大攻关项目(CI2021A00606);中国中医科学院自主选题(YZX202237,YZX202241,YZ2020042,YZ2020019);北京市中医药科技发展基金项目(JJ2018-99)。

摘　　要：目的:观察苓桂术甘汤对脂多糖(LPS)诱导急性肺损伤(ALI)模型小鼠的疗效及相关作用机制。方法:将72只7周龄SPF级C57BL/6小鼠按体质量随机分为正常组、模型组、地塞米松组(5 mg·kg^(-1))、苓桂术甘汤高、中、低剂量组(9.36、4.68、2.34 g·kg^(-1)),每组12只。除正常组外,其余各组采用鼻滴法予LPS(50μg/只)构建小鼠ALI模型。给药组分别在造模前连续灌胃给药7 d。造模后12 h取小鼠肺组织,测定双肺湿/干质量比(W/D);苏木素-伊红(HE)染色观察肺组织病理形态学变化;支气管肺泡灌洗液(BALF)采用细胞计数仪计算总细胞数,瑞氏吉姆萨染色检测炎性细胞的分类(中性粒细胞、巨噬细胞)与数量;酶联免疫吸附测定法(ELISA)检测BALF中炎性细胞因子肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)的表达水平;蛋白免疫印迹法(Western blot)检测核转录因子-κB(NF-κB)炎症通路中NF-κB抑制蛋白α(IκBα)、NF-κB p65及二者磷酸化蛋白的表达,并计算磷酸化蛋白/总蛋白比值。结果:与正常组比较,模型组小鼠肺组织严重损伤,肺泡完整结构被破坏,肺泡壁增厚,大量炎性细胞与红细胞浸润,肺水肿显著加重(P<0.01)。BALF中总细胞与炎性细胞数量,IL-6、TNF-α及肺组织NF-κB p65、IκBα磷酸化蛋白表达显著上调(P<0.01);与模型组比较,苓桂术甘汤高、中、低剂量组与地塞米松组小鼠肺损伤明显缓解,肺水肿显著减轻(P<0.01)。BALF中总细胞与中性粒细胞数量,IL-6、TNF-α及肺组织NF-κB p65、IκBα磷酸化蛋白表达显著下调(P<0.01),巨噬细胞数量明显减少(P<0.05)。结论:苓桂术甘汤对LPS-ALI小鼠具有抗炎保护作用,能有效减轻炎症、利水消肿,其机制可能与抑制NF-κB通路活化有关。Objective:To observe the therapeutic effect and underlying mechanism of Linggui Zhugantang on lipopolysaccharide(LPS)-induced acute lung injury(ALI)in mice.Method:Seventy-two 7-week-old C57BL/6 mice of SPF grade were randomly divided into a normal group,a model group,a dexamethasone group(5 mg·kg^(-1)),and high-,medium-,and low-dose Linggui Zhugantang groups(9.36,4.68,2.34 g·kg^(-1)),with 12 mice in each group.Except for the normal group,the remaining groups underwent intranasal instillation of LPS(50μg per mouse)for the induction of the ALI model.The treatment groups received oral administration for 7 days prior to modeling.After 12 hours of modeling,mouse lung tissues were taken to measure the wet/dry weight ratio(W/D).Hematoxylin-eosin(HE)staining was performed to observe the pathological morphological changes in lung tissues.Bronchoalveolar lavage fluid(BALF)was collected for total cell count using a cell counter,and Wright-Giemsa staining was conducted to classify and quantify inflammatory cells(neutrophils and macrophages).Enzyme-linked immunosorbent assay(ELISA)was used to determine the expression levels of inflammatory cytokines tumor necrosis factor-α(TNF-α)and interleukin-6(IL-6)in BALF.Western blot analysis was performed to detect the expression of nuclear factor-κB(NF-κB)inhibitory proteinα(IκBα),NF-κB p65,and their phosphorylated proteins,and the ratio of phosphorylated protein/total protein was calculated.Result:Compared with the normal group,the model group exhibited severe lung tissue damage,disrupted alveolar structure,thickened alveolar walls,infiltration of extensive inflammatory cells and red blood cells,and significantly aggravated lung edema(P<0.01).The total cell count,inflammatory cell count,expression levels of IL-6,and TNF-αin BALF,as well as NF-κB p65 and phosphorylated IκBαin lung tissues,were significantly upregulated in the model group(P<0.01).Compared with the model group,high-,medium-,and low-dose Linggui Zhugantang groups,as well as the dexamethasone group,showed im

关 键 词：苓桂术甘汤 脂多糖 急性肺损伤 炎症 核转录因子-κB(NF-κB)信号通路 

分 类 号：R2-0[医药卫生—中医学] R22R285.5R289R33