达原饮调控TLR/MAPK/NF-κB通路防治H1N1感染诱发急性肺损伤的作用机制  

Dayuanyin Regulates TLR/MAPK/NF-κB Pathway for Preventing and Treating Acute Lung Injury Induced by H1N1 Infection

作　　者：李承泽 褚福浩[2] 李园 刘运泽 郑浩呈 王思慈 顾益枭 朱万鸿 张若诗 宋行健 盖聪 丁霞 LI Chengze;CHU Fuhao;LI Yuan;LIU Yunze;ZHENG Haocheng;WANG Sici;GU Yixiao;ZHU Wanhong;ZHANG Ruoshi;SONG Xingjian;GAI Cong;DING Xia(School of Traditional Chinese Medicine(TCM),Beijing University of Chinese Medicine,Beijing 100102,China;School of Chinese Materia Medica,Beijing University of Chinese Medicine,Beijing 100102,China;National Institute of TCM Constitution and Prevention of Disease,Beijing University of Chinese Medicine,Beijing 100102,China;Dongzhimen Hospital,Beijing University of Chinese Medicine,Beijing 101121,China)

机构地区：[1]北京中医药大学中医学院,北京100102 [2]北京中医药大学中药学院,北京100102 [3]北京中医药大学国家中医体质与治未病研究院,北京100102 [4]北京中医药大学东直门医院,北京101121

出　　处：《中国实验方剂学杂志》2024年第18期52-60,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家中医药管理局中医药创新团队及人才支持计划项目(ZYYCXTD-C-202006);合肥微尺度物质科学国家研究中心开放课题项目(KF2021104)。

摘　　要：目的:探究达原饮对H1N1感染诱发的急性肺损伤的药效及其潜在机制。方法:使用超高效液相色谱-四级杆静电场轨道阱质谱仪对达原饮水煎液进行药物成分分析。将48只雌性BALB/c小鼠随机分为正常组、模型组、奥司他韦组(19.5 mg·kg^(-1))、达原饮低、中、高剂量组(2.73、5.46、10.92 g·kg^(-1))。正常组和模型组给予去离子水灌胃,其他各组给予对应药物灌胃。给药第3天,正常组给予生理盐水滴鼻,其他各组在鼻内接种A/RP/8/34(H1N1)构建流感病毒感染的动物模型。连续给药7 d,每日称体质量。末次给药12 h后进行取材,肺组织称重,计算肺指数;苏木素-伊红(HE)染色观察肺组织及支气管病理形态学变化;微球免疫分析系统(CBA)检测血清中的γ干扰素(IFN-γ)、趋化因子(C-X-C基序)配体1(CXCL1)、肿瘤坏死因子-α(TNF-α)、趋化因子配体2(CCL2)、白细胞介素-12p70(IL-12p70)、趋化因子配体5(CCL5)、白细胞介素-1β(IL-1β)、趋化因子(C-X-C基序)配体10(CXCL10)、粒细胞-巨噬细胞集落刺激因子(GM-CSF)、白细胞介素-10(IL-10)、干扰素-β(IFN-β)、干扰素-α(IFN-α)、白细胞介素-6(IL-6)。基于质谱结果联合网络药理学,分析达原饮防治甲流病毒引起的急性肺损伤的作用机制。通过蛋白免疫印迹法(Western bolt)检测肺组织中的细胞外信号调节激酶1/2(ERK1/2)、p38丝裂原活化蛋白激酶(p38 MAPK)、核转录因子-κB(NF-κB),及其磷酸化蛋白的表达;实时荧光定量聚合酶链式反应(Real-time PCR)检测肺组织中的髓样分化因子88(MyD88)、Toll样受体3(TLR3)、Toll样受体7(TLR7)、Toll样受体8(TLR8)的mRNA表达。结果:质谱分析确定达原饮水煎液中含有芍药苷、黄芩素等57种化合物。与正常组比较,模型组小鼠体质量下降(P<0.01),肺部水肿和出血,肺指数升高(P<0.01),IFN-γ、IL-12p70、CCL5、IL-1β、CXCL10、GM-CSF、IFN-β、IL-6显著升高(P<0.01)。与模型组比较,达原饮�Objective To investigate the therapeutic effect of Dayuanyin on acute lung injury induced by H1N1 infection and decipher the potential mechanism.Method The constituents in Dayuanyin were analyzed by ultra-high performance liquid chromatography-quadrupole-exactive orbitrap mass spectrometry(UHPLC-Q-Exactive Orbitrap MS).Forty-eight female BALB/c mice were randomized into normal,model,oseltamivir(19.5 mg·kg^(-1)),and low-,medium-,and high-dose(2.73,5.46,10.92 g·kg^(-1))Dayuanyin groups.The normal and model groups were administrated with deionized water by gavage,and the other groups were administrated with the corresponding drugs by gavage.On day 3 of drug administration,the normal group received nasal inhalation of normal saline,and the other groups were inoculated intranasally with A/RP/8/34(H1N1)for the modeling of influenza virus infection.Mice were administrated with drugs continuously for 7 days and weighed daily.Sampling was performed 12 h after the last administration,and the lung tissue was weighed to calculate the lung index.Hematoxylin-eosin staining was performed to observe the pathological and morphological changes of the lung tissue and bronchi.The cytometric bead array(CBA)was used to measure the serum levels of interferon-gamma(IFN-γ),C-X-C motif ligand 1(CXCL1),tumor necrosis factor-alpha(TNF-α),chemokine ligand 2(CCL2),interleukin-12p70(IL-12p70),chemokine ligand 5(CCL5),interleukin-1β(IL-1β),chemokine(C-X-C motif)ligand 10(CXCL10),granulocyte-macrophage colony-stimulating factor(GM-CSF),interleukin-10(IL-10),interferon-beta(IFN-β),interferon-alpha(IFN-α),and interleukin-6(IL-6).According to the results of mass spectrometry and network pharmacology,we analyzed the mechanism of Dayuanyin in treating acute lung injury caused by H1N1.The protein levels of extracellular signal-regulated kinase 1/2(ERK1/2),p38 mitogen-activated protein kinase(p38 MAPK),nuclear factor-kappa B(NF-κB),and their phosphorylated forms were determined by Western blot.The mRNA levels of myeloid differentiation factor

关 键 词：达原饮 流感病毒 急性肺损伤 炎症 Toll样受体(TLR)/丝裂原活化蛋白激酶(MAPK)/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