基于蛋白质组学和网络药理学探讨小儿化痰止咳颗粒治疗过敏性咳嗽的作用机制  

Proteomics and Network Pharmacology Reveal Mechanism of Xiaoer Huatan Zhike Granules in Treating Allergic Cough

作　　者：杜友启 徐旖旎[1] 廖佳佳 龙朝文 邰诗蝶 杜友文 李松 甘诗泉 沈祥春[1] 陶玲[1] 杨熟英 付凌云[1] DU Youqi;XU Yini;LIAO Jiajia;LONG Chaowen;TAI Shidie;DU Youwen;LI Song;GAN Shiquan;SHEN Xiangchun;TAO Ling;YANG Shuying;FU Lingyun(Guizhou Medical University,Anshun 561113,China;Guizhou Kehui Pharmaceutical Co Ltd.,Guiyang 550014,China)

机构地区：[1]贵州医科大学,贵州安顺561113 [2]贵州省科晖制药有限公司,贵阳550014

出　　处：《中国实验方剂学杂志》2025年第3期69-79,共11页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：贵阳市科技计划项目(筑科合同[2022]5-11号);贵州省科技创新基地(黔科合中引地[2023]003);贵州省高层次创新型人才十层次人才项目(黔科合平台人才-GCC[2023]048);贵州省科技厅科技成果转化项目(黔科合成果[2022]一般010)。

摘　　要：目的:基于蛋白质组学、网络药理学共同探讨小儿化痰止咳颗粒(XEHT)在治疗过敏性咳嗽(AC)可能涉及的药理作用机制。方法:通过腹腔注射含卵蛋白(OVA)2 mg和氢氧化铝100 mg的混悬液1 mL致敏后,以1%OVA雾化构建豚鼠AC模型,随机分为模型组、XEHT低、中、高剂量组(1、5、20 g·kg^(-1))与孟鲁斯特钠组(1 mg·kg^(-1))每组6只,正常组6只。给药组按剂量灌胃给予相应药液,正常组和模型组灌胃给予同体积生理盐水,1次/d,连续给药10 d,以1%OVA雾化刺激豚鼠,观察咳嗽次数;苏木素-伊红(HE)染色观察肺组织病理变化;酶联免疫吸附测定法(ELISA)检测肺泡灌洗液(BALF)中C反应蛋白(CRP)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)、超氧化物歧化酶(SOD)、丙二醛(MDA)及血清中免疫球蛋白G(IgG)、免疫球蛋白A(IgA)的含量;免疫组织化学法(IHC)观察肺部组织中IL-6、TNF-α的蛋白表达;透射电镜观察肺泡Ⅱ型上皮细胞超微结构;实时荧光定量聚合酶链式反应(Real-time PCR)测定肺组织中IL-6、白细胞介素-1β(IL-1β)、TNF-αmRNA水平。采用非标记蛋白质组学技术(Label Free)检测各干预组间差异蛋白;网络药理学预测XEHT治疗AC的作用靶点,将蛋白组学及网络药理学的结果进行京都基因与基因组百科全书(KEGG)富集分析并寻找相同的作用通路。结果:与正常组比较,模型组咳嗽次数显著增加(P<0.01),BALF中CRP、TNF-α含量显著升高、SOD显著降低(P<0.01),IL-6、MDA升高(P<0.05),血清中IgA、IgG含量升高(P<0.05,P<0.01),肺组织充血及炎症细胞浸润明显,IL-6和TNF-α蛋白表达水平升高(P<0.01),Ⅱ型上皮细胞可见大范围低电子密度水肿区,细胞器明显肿胀、空泡化,胞核固缩或染色质稀疏、溶解,IL-6、IL-1β、TNF-α的mRNA表达水平显著升高(P<0.01);与模型组比较,各给药组咳嗽次数减少(P<0.01)、BALF中CRP、TNF-α、IL-6、MDA含量减少(P<0.05、P<0.01),SOD活性增加(P<0.05Objective:To explore the pharmacological mechanism involved in the treatment of allergic cough(AC)by Xiaoer Huatan Zhike granules(XEHT)based on proteomics and network pharmacology.Methods:After sensitization by intraperitoneal injection of 1 mL suspension containing 2 mg ovalbumin(OVA)and 100 mg aluminum hydroxide,a guinea pig model of allergic cough was constructed by nebulization with 1%OVA.The modeled guinea pigs were randomized into the model,low-,medium-and high-dose(1,5,20 g·kg^(-1),respectively)XEHT,and sodium montelukast(1 mg·kg^(-1))groups(n=6),and another 6 guinea pigs were selected as the blank group.The guinea pigs in drug administration groups were administrated with the corresponding drugs by gavage,and those in the blank and model groups received the same volume of normal saline by gavage,1 time·d^(-1).After 10 consecutive days of drug administration,the guinea pigs were stimulated by 1%OVA nebulization,and the coughs were observed.The pathological changes in the lung tissue were observed by hematoxylin-eosin staining.The enzyme-linked immunosorbent assay was performed to measure the levels of C-reactive protein(CRP),interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),superoxide dismutase(SOD),and malondialdehyde(MDA)in the bronchoalveolar lavage fluid(BALF)and immunoglobulin G(IgG)and immunoglobulin A(IgA)in the serum.Immunohistochemistry(IHC)was employed to observe the expression of IL-6 and TNF-αin the lung tissue.Transmission electron microscopy was employed observe the alveolar typeⅡepithelial cell ultrastructure.Real-time PCR was employed to determine the mRNA levels of IL-6,interleukin-1β(IL-1β),and TNF-αin the lung tissue.Label-free proteomics was used to detect the differential proteins among groups.Network pharmacology was used to predict the targets of XEHT in treating AC.The Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis was performed to search for the same pathways from the results of proteomics and network pharmacology.Results:Compared with the blank group,

关 键 词：过敏性咳嗽 小儿化痰止咳颗粒 蛋白质组学 网络药理学 作用机制 

分 类 号：R256[医药卫生—中医内科学] R441[医药卫生—中医学] R285