摘要
目的基于网络药理学和实验验证探讨苏黄止咳胶囊治疗变应性咳嗽(AC)的作用机制。方法(1)根据药物相似度(DL≥0.18)和口服生物利用度(OB≥30%)从TCMSP数据库中筛选苏黄止咳胶囊的有效活性成分,通过SwissADME数据库预测有效成分的重要靶点。利用GeneCards数据库收集与变应性咳嗽相关的疾病靶点,并通过Venny数据库获得苏黄止咳胶囊和变应性咳嗽的交集基因。利用STRING数据库和Cytoscape 3.9.0软件构建苏黄止咳胶囊治疗变应性咳嗽的蛋白质互作(PPI)网络,根据拓扑参数筛选出核心靶点,并通过“药物-有效成分-靶点”网络筛选出苏黄止咳胶囊的有效活性成分。借助DAVID数据库对苏黄止咳胶囊治疗变应性咳嗽的交集基因进行GO功能和KEGG通路富集分析,并使用AutoDockTool4进行分子对接验证。(2)将36只豚鼠分为正常组、模型组及苏黄止咳胶囊低、中、高(0.15、1.5、0.31 g·kg^(-1))剂量组和阳性药氯雷他定组(0.8 mg·kg^(-1))各6只,通过腹腔注射环磷酰胺(30 mg·kg^(-1))、卵蛋白(2 mg)和氢氧化铝(100 mg)诱导变应性咳嗽模型;模型复制第1天开始灌胃给药,每天1次,连续给药3周,每周对豚鼠体质量进行记录。将人支气管上皮细胞(HBEC)分为正常组(10%空白血清)、模型组(10%空白血清)、苏黄止咳胶囊高剂量组(10%苏黄止咳胶囊高剂量含药血清)和氯雷他定组(10%氯雷他定含药血清),使用神经生长因子(NGF,100 ng·mL^(-1))进行模型复制。采用大动物全身体积描记系统(WBP)检测豚鼠的咳嗽敏感性;收集支气管肺泡灌洗液(BALF),用于炎症细胞的分类计数和后续炎症因子检测;苏木精-伊红(HE)染色检测豚鼠肺组织病理变化;ELISA法检测豚鼠血清和BALF中白细胞介素1β(IL-1β)、肿瘤坏死因子α(TNF-α)水平,检测豚鼠血清和肺组织匀浆中P物质(SP)、神经激肽A(NKA)、神经激肽B(NKB)和降钙素基因相关肽(CGRP)水平;RT-PCR法检测豚鼠肺组织中SP mRNA和神经激肽1受体(NK-1R)mRNA表达水平;Western Blot法检测豚鼠和HBEC细胞中磷酸化大鼠肉瘤(p-Ras)、大鼠肉瘤(Ras)、磷酸化丝裂原活化蛋白激酶3(p-ERK)、丝裂原活化蛋白激酶3(ERK)、磷酸化丝裂原活化蛋白激酶1(p-MEK)和丝裂原活化蛋白激酶1(MEK)蛋白表达水平。结果(1)共筛选出苏黄止咳胶囊95种化合物和703个相关靶点,其中柳叶鱼黄素、白花前胡素E和白花前胡甲素是其主要成分;共发现与变应性咳嗽相关的靶点1494个,将苏黄止咳胶囊和变应性咳嗽所有的靶点进行交集分析,共发现263个交集基因,其中丝裂原活化蛋白激酶3(MAPK3,别称ERK1)和丝裂原活化蛋白激酶激酶1(MAP2K1,别称MEK)是苏黄止咳胶囊治疗变应性咳嗽的关键靶点。GO富集和KEGG通路富集分析结果显示,苏黄止咳胶囊治疗变应性咳嗽的机制主要涉及炎症反应、ERK1和ERK2级联的正向调节、PI3K-Akt信号通路、MAPK信号通路、cAMP信号通路和Ras信号通路等信号通路。分子对接结果表明,柳叶鱼黄素、白花前胡素E和白花前胡甲素成分与ERK1和MAP2K1靶点之间具有良好的结合能力。(2)实验结果显示,在动物实验中,苏黄止咳胶囊能增加变应性咳嗽模型豚鼠的体质量(P<0.05),明显降低咳嗽次数(P<0.001),延长咳嗽潜伏期(P<0.001),改善肺组织气道病理上皮细胞损伤及肺泡损伤情况,降低BALF中细胞总数、中性粒细胞、嗜酸性粒细胞比例(P<0.05,P<0.001)及IL-1β(P<0.001)、TNF-α(P<0.05)分泌水平,升高巨噬细胞比例(P<0.01),降低血清中IL-1β(P<0.05)、TNF-α(P<0.01)、SP(P<0.001)、NKA(P<0.001)、NKB(P<0.05)和CGRP(P<0.001)分泌水平,降低肺组织中的SP、NKA、NKB的分泌水平(P<0.001)及SP mRNA和神经激肽1受体(NK-1R)mRNA表达水平(P<0.001),降低肺组织中p-Ras/Ras、p-MEK/MEK和p-ERK/ERK水平(P<0.05),降低背根神经节中p-Ras/Ras、p-MEK/MEK和p-ERK/ERK水平(P<0.05);在细胞实验中,苏黄止咳胶囊能明显降低NFG细胞模型中p-Ras/Ras、p-MEK/MEK和p-ERK/ERK的水平(P<0.05)。结论苏黄止咳胶囊能有效改善变应性咳嗽豚鼠咳嗽敏感性、肺部炎症和神经源性炎症,其作用机制可能与抑制MAPK/ERK信号通路有关。
Objective To investigate the therapeutic mechanisms of Suhuang Zhike Capsules(SHZKC)in treating atopic cough(AC)through network pharmacology and experimental validation.Methods(1)Active ingredients of Suhuang Zhike Capsules(SHZKC)were screened from the TCMSP database based on drug-likeness(DL≥0.18)and oral bioavailability(OB≥30%),with target prediction performed using SwissADME;disease targets for atopic cough(AC)were retrieved from GeneCards,and overlapping targets with SHZKC were identified via Venny;the proteinprotein interaction(PPI)network for SHZKC against AC was constructed using STRING and Cytoscape 3.9.0,with core targets screened by topological parameters and bioactive compounds identified through the"herb-active ingredienttarget"network;GO functional and KEGG pathway enrichment analyses of overlapping targets were conducted using DAVID,followed by molecular docking validation with AutoDockTools.(2)Thirty-six guinea pigs were divided into six groups(n=6 each):normal control,AC model,SHZKC low-/medium-/high-dose(0.15,0.31,1.5 g·kg^(-1)),and positive control(Loratadine,0.8 mg·kg^(-1));AC was induced by intraperitoneal Cyclophosphamide(30 mg·kg^(-1)),Ovalbumin(2 mg),and Aluminum Hydroxide(100 mg),with treatments administered via daily gavage for 3 weeks starting from modeling day 1;body mass was recorded weekly.Human bronchial epithelial cells(HBECs)were grouped into:normal(10%blank serum),model(10% blank serum),SHZKC high-dose(10%SHZKC medicated serum),and Loratadine(10% Loratadine medicated serum),with NGF(100 ng·mL^(-1))for modeling.Evaluations included:whole-body plethysmography(WBP)for cough sensitivity;bronchoalveolar lavage fluid(BALF)collection for inflammatory cell counts and cytokine assays;hematoxylin-eosin(HE)staining for lung pathology;ELISA for IL-1β and TNF-α in serum/BALF,and SP/NKA/NKB/CGRP in serum/lung homogenate;RT-PCR for SP mRNA and NK-1R mRNA in lung tissue;Western Blot for p-Ras,Ras,p-ERK,ERK,p-MEK,and MEK expression in lung tissue/HBECs.Results(1)A total of 95 compounds and 703 potential targets of SHZKC were identified,with myricetin,imperatorin E,and oxypeucedanin being its primary active components.Additionally,1494 targets associated with allergic cough were screened.Intersection analysis between SHZKC and allergic cough targets revealed 263 shared genes,among which mitogen-activated protein kinase 3(MAPK3,also known as ERK1)and mitogen-activated protein kinase kinase 1(MAP2K1,also known as MEK)emerged as key therapeutic targets of SHZKC in treating allergic cough.Gene Ontology(GO)enrichment and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analyses demonstrated that the therapeutic mechanisms of SHZKC in allergic cough primarily involve inflammatory responses,positive regulation of ERK1/2 cascade,PI3K-Akt signaling pathway,MAPK signaling pathway,cAMP signaling pathway,and Ras signaling pathway.Molecular docking results indicated that pectolinarigenin,praeruptorin E,and dl-praeraptorin a exhibited strong binding affinity with ERK1 and MAP2K1.(2)Experimental results demonstrated that in animal models,SHZKC significantly increased body mass in guinea pigs with allergic cough(P<0.05),markedly reduced cough frequency(P<0.001),and prolonged cough latency(P<0.001).It also ameliorated pathological epithelial cell damage and alveolar injury in lung tissues,while decreasing total cell counts,neutrophil,and eosinophil proportions in BALF(P<0.05,P<0.001),as well as IL-1β(P<0.001)and TNF-α(P<0.05)secretion levels.SHZKC further elevated macrophage proportions(P<0.01)and reduced serum levels of IL-1β(P<0.05),TNF-α(P<0.01),substance P(SP)(P<0.001),neurokinin A(NKA)(P<0.001),neurokinin B(NKB)(P<0.05),and calcitonin gene-related peptide(CGRP)(P<0.001).Additionally,it lowered SP,NKA,and NKB secretion levels in lung tissues(P<0.001)and downregulated mRNA expression of SP and neurokinin-1 receptor(NK-1R)(P<0.001).Moreover,SHZKC reduced the phosphorylation ratios of p-Ras/Ras,p-MEK/MEK,and p-ERK/ERK in lung tissues(P<0.05)and dorsal root ganglia(P<0.05).In cell experiments,SHZKC significantly decreased the levels of p-Ras/Ras,p-MEK/MEK,and p-ERK/ERK in the NGF-induced cell model(P<0.05).Conclusion SUHZKC alleviates cough sensitivity,pulmonary inflammation,and neurogenic inflammation in AC by inhibiting the MAPK/ERK signaling pathway.
作者
张莲英
唐明文
叶小丹
黄智兰
李亚清
余燕
谢纬
李敏芳
ZHANG Lianying;TANG Mingwen;YE Xiaodan;HUANG Zhilan;LI Yaqing;YU Yan;XIE Wei;LI Minfang(Artemisia Research Center,Guangzhou University of Chinese Medicine,Guangzhou 510405 Guangdong,China;The Fourth School of Clinical Medicine,Guangzhou University of Chinese Medicine,Shenzhen 518033 Guangdong,China;Shenzhen Hospital of Traditional Chinese Medicine,Shenzhen 518033 Guangdong,China;Dept.of Community Health,Shenzhen Hospital of Traditional Chinese Medicine,Shenzhen 518033 Guangdong,China;The Second School of Clinical Medicine,Guangzhou University of Chinese Medicine,Guangzhou 510120 Guangdong,China)
出处
《中药新药与临床药理》
北大核心
2025年第10期1720-1736,共17页
Traditional Chinese Drug Research and Clinical Pharmacology
基金
广东省中医药局科研项目(20241262)
深圳市“医疗三名工程”项目(SZZYSM202311001)
“丹龙”青年医师创新发展项目(HXQNJJ-2023-16)
深圳市中医药传承创新人才培养512工程(深府办函〔2020〕1号)。
