目的 探究槟榔的成分及其促进口腔黏膜下纤维化的机制.方法 采用Thermo QE plus液相色谱串联高分辨质谱仪和Compound discover 3.2数据处理软件进行槟榔中药化学成分分析,将检测到的化合物以质谱响应值排序,分析排名前20化合物的活性.化合物活性来源根据《中国药典(2015版)》汇总,数据查询基于PubChem、Chemical book以及Scifinder数据库.借助网络药理学方法分析槟榔影响口腔黏膜下纤维化(OSF)的潜在活性成分、核心靶点及主要影响的生物功能、信号通路.通过整合人类基因数据库(Genecards)、基因组百科全书数据库(KEGG)等数据库获取OSF的作用靶点.以OB≥30%为条件,在中药系统药理学技术平台(TCMSP)中筛选出槟榔可作用于靶点的化合物,并构建靶点-化合物、化合物-中药、靶点-化合物-中药网络.运用MOE软件的分子对接技术,分析成分-靶点结合的可能性,再利用Pymol软件进行分子对接可视化.最后通过免疫组化在临床样本中验证槟榔是否影响PI3K-Akt、MAPK两条通路涉及的主要蛋白,初步验证槟榔诱导OSF的潜在作用机制.结果 基于网络药理学和槟榔粗提物中筛选出前10的化合物与OSF核心靶点中核心交集基因,确定了槟榔可能通过调控PI3K-Akt与MAPK通路.通过免疫组化在临床样本中验证,细胞膜上的PI3K蛋白表达量在OSF患者组中表达下降(P=0.0002),p-PI3K(P=0.0002)表达量上升,进一步激活下游的AKT1蛋白表达增加(P=0.0006),加剧磷酸化蛋白p-AKT蛋白的表达及堆积(P=0.0013);而在MAPK通路中,OSF患者组对比正常组,通过上调JNK蛋白(P=0.0130),诱导下游AP-1复合蛋白c-jun及c-fos转录因子的活性增加,促使其向细胞核聚集;且OSF患者组较正常组血浆的IL-6(P<0.0001)与IL-8(P=0.0095)含量均上升.结论 槟榔碱、槟榔次碱、异去甲槟榔碱等槟榔中的主要活性成分可能通过刺激PI3K-Akt与MAPK信号通路,促进炎症介质IL-6及IL-8的表达,诱导胶原增生,导致口腔黏膜下纤维化病变.
Objective To explore the pharmacologically active components in areca nut that induce oral submucosal fibrosis(OSF)and the possible mechanism.Methods The chemical components in areca nut were analyzed using Thermo QE plus liquid chromatography tandem high-resolution mass spectrometer and Compound discover 3.2 data processing software.The chemical activity of the top 20 compounds was analyzed based on Chinese Pharmacopoeia(2015),PubChem,Chemical book,and SciFinder databases.The potential active components,core targets,biological functions and signaling pathways affecting OSF were analyzed by network pharmacology.The targets of OSF were obtained by integrating Genecards and KEGG databases.The compounds acting on the targets were selected from the Systematic Pharmacology Technology Platform of Traditional Chinese Medicine(TCMSP),and the target-compound,compound-TCM,target-compound-TCM network was constructed.Molecular docking was used to analyze the component-target binding.Immunohistochemistry was used to examine the expressions of key proteins in the PI3K-Akt and MAPK pathways in clinical samples of OSF.Results The core intersection target genes between the top 10 active ingredients in areca nut extract and OSF involved mainly the PI3K-Akt and MAPK pathways.In the clinical samples,the expressions of PI3K protein decreased and the expressions p-PI3K,AKT1 and P-Akt all increased significantly in OSF tissue,where increased JNK protein expression and enhanced activity of c-Jun and c-Fos transcriptional factors were also detected.The OSF patients had significantly elevated plasma levels of IL-6 and IL-8 compared with healthy individuals.Conclusion The main active ingredients including arecoline,arecaine,and guvacine are capable of activating the PI3K-Akt and MAPK pathways to promote the expressions of inflammatory mediators IL-6 and IL-8 and induce collagen hyperplasia,thus leading to the occurrence of oral submucosal fibrosis.
李睿镈;高歌;谢曦;罗海彬
海南大学热带生物资源教育部重点实验室//海南大学药学院,海南 海口 570228
槟榔;口腔黏膜下纤维化性变;网络药理学;分子对接
areca nut;oral submucosal fibrosis;network pharmacology;molecular docking
《南方医科大学学报》 2024 (005)
930-940 / 11
国家自然科学基金(82360731);海南省自然科学基金(821MS027,823RC467,KJRC2023B10) Supported by National Natural Science Foundation of China(82360731).
10.12122/j.issn.1673-4254.2024.05.15
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