基于网络药理学和分子对接技术探讨沉香治疗胃溃疡的作用机制

Exploring the Mechanism of Aquilariae Lignum Resinatum in the Treatment of Gastric Ulcer Based on Network Pharmacology and Molecular Docking Techniques

目的 基于网络药理学方法结合动物体内实验探讨沉香治疗胃溃疡的作用机制。方法 (1)通过TCMSP数据库检索并筛选沉香的活性成分及其潜在作用靶点;通过OMIM数据库、GeneCard数据库检索胃溃疡的疾病相关靶点;通过Venny平台对沉香的活性成分作用靶点与胃溃疡的重要疾病靶点取交集,获得沉香治疗胃溃疡的关键靶点(共同靶点);采用Cytoscape 3.7.1软件构建沉香治疗胃溃疡的“药物-活性成分-关键靶点”网络,分析关键活性成分;将共同靶点导入STRING数据库,构建蛋白互作(PPI)网络,筛选核心靶点;将共同靶点导入DAVID数据库进行GO功能及KEGG通路富集分析;通过AutoDock软件对关键活性成分与核心靶点进行分子对接。(2)将SD大鼠随机分为正常组、模型组、阳性药组(奥美拉唑,0.2 g·kg^(-1))及沉香提取物低、中、高剂量组(0.1、0.3、0.9 g·kg^(-1)),每组7只;灌胃给药(10 mL·kg^(-1)),每天1次,连续7 d;末次给药1 h后,大鼠灌胃给予无水乙醇(5 mL·kg^(-1))复制急性胃黏膜损伤模型。计算大鼠溃疡指数及溃疡抑制率;采用HE染色法观察大鼠胃组织病理学变化;Western Blot法检测胃组织相关蛋白的表达;免疫组化法检测胃组织中Bcl-2、Bax、Caspase3蛋白表达。结果 (1)筛选出沉香活性成分9个,预测得到活性成分作用靶点173个,胃溃疡疾病相关靶点1 297个;得到交集靶点(共同靶点)110个,即沉香治疗胃溃疡的关键靶点。关键活性成分包括槲皮素、β谷甾醇、6,7-二甲氧基-2-(2-苯乙基)色酮、6,7-二甲氧基-2-[2-(4-甲氧基苯基)乙基]色原酮、波尔定碱等;AKT1、TP53、IL6、VEGFA、JUN、TNF、CASP3、MYC、PTGS2、MAPK1等可能是沉香治疗胃溃疡的核心靶点;GO功能富集分析得到580个生物学过程条目、51个细胞组分条目、101个分子功能条目;KEGG富集共得到110条信号通路,主要涉及癌症通路、TNF信号通路、乙型肝炎通路。6,7-二甲氧基-2-(2-苯乙基)色酮与AKT1、TP53、IL6有较强的结合性,β谷甾醇与AKT1、VEGFA有较强的结合性。(2)与模型组比较,沉香提取物各剂量组大鼠胃黏膜组织的出血点明显减少,溃疡和糜烂程度降低,溃疡指数均显著降低(P<0.01);沉香提取物低、中、高剂量组的溃疡抑制率分别为19.12%、40.43%、64.48%;沉香提取物各剂量组大鼠胃组织的p-PI3K/PI3K、p-Akt/Akt、p-IκBα/IκBα、p-P65/P65蛋白表达均显著下调(P<0.01),抗凋亡蛋白Bcl-2表达显著上调(P<0.01),Bax、Caspase3等凋亡蛋白表达显著下调(P<0.01)。结论 沉香可能通过多成分、多靶点、多通路发挥抗炎、抗凋亡作用来治疗胃溃疡。

Objective To explore the mechanism of action of Aquilariae Lignum Resinatum in the treatment of gastric ulcer based on network pharmacology approach combined with in vivo animal experiments. Methods(1)Searching and screening the active components of Aquilariae Lignum Resinatum and their potential targets through TCMSP database;searching the disease related targets of gastric ulcer through OMIM database and GeneCard database;intersecting the active component targets of Aquilariae Lignum Resinatum with the important disease targets of gastric ulcer through Venny platform to obtain the key targets(common targets) of Aquilariae Lignum Resinatum for the treatment of gastric ulcer;Cytoscape 3.7.1 software was used to construct a "drugsactive components-key targets" network for the treatment of gastric ulcer with Aquilariae Lignum Resinatum and to analyze the key active components;the common targets were imported into the STRING database to construct a protein-protein interaction(PPI) network to screen the core targets;the common targets were imported into the DAVID database GO function and KEGG pathway enrichment analysis were performed;molecular docking was performed key active components and core targets via AutoDock software.(2)SD rats were randomly divided into normal group, model group, positive drug group(Omeprazole, 0.2 g · kg-1) and Aquilariae Lignum Resinatum extract low-,medium-and high-dose(0.1,0.3,0.9 g·kg^(-1))groups,with 7 rats in each group;the drug was administered by gavage(10 mL·kg^(-1))once a day for consecutive 7 days;one hour after the last dose,the rats were gavaged absolute ethyl alcohol(5 mL·kg^(-1))to replicate the acute gastric mucosal injury model. The ulcer index and ulcer inhibition rate of rats were calculated;the histopathological changes of gastric tissue were observed by HE staining;the expression of gastric tissue-related proteins was detected by Western Blot;the protein expressions of Bcl-2, Bax and Caspase3 in gastric tissue was detected by immunohistochemistry. Results(1) Screening of9 active components of Aquilariae Lignum Resinatum, 173 active component action targets were predicted and1 297 targets related to gastric ulcer disease;110 intersecting targets(common targets) were obtained, i.e. key targets of Aquilariae Lignum Resinatum for the treatment of gastric ulcer. The key active components included quercetin,β-sitosterol,6,7-dimethoxy-2-(2-phenylethyl)chromone,6,7-dimethoxy-2-[2-(4-methoxyphenyl)ethyl] chromogenic ketone, boldine, etc.;AKT1, TP53, IL6, VEGFA, JUN, TNF, CASP3, MYC, PTGS2and MAPK1 may be the core targets of Aquilariae Lignum Resinatum in the treatment of gastric ulcer;GO functional enrichment analysis yielded 580 biological process entries,51 cellular component entries and 101 molecular functional entries;KEGG enrichment yielded a total of 110 signaling pathways,mainly involving cancer pathway,TNF signaling pathway and hepatitis B pathway. 6,7-Dimethoxy-2-(2-phenylethyl)chromone showed strong binding to AKT1,TP53 and IL6,and β-sitosterol showed strong binding to AKT1 and VEGFA.(2)Compared with the model group,the bleeding points of gastric mucosa tissue of rats in all dose groups of Aquilariae Lignum Resinatum extract were significantly reduced, the degree of ulceration and erosion was decreased, and the ulcer index was significantly reduced in all groups(P<0.01);the ulcer inhibition rates of low-,medium-and high-dose groups of Aquilariae Lignum Resinatum extract were 19.12%,40.43% and 64.48%,respectively;the protein expressions of p-PI3K/PI3K,p-Akt/Akt,p-IκBα/IκBα and p-P65/P65 were significantly down-regulated in the gastric tissue of rats in all dose groups of Aquilariae Lignum Resinatum extract(P<0.01),the expression of anti-apoptotic protein Bcl-2 was significantly up-regulated(P<0.01), and the apoptotic expressions of Bax and Caspase3 were significantly down-regulated(P<0.01). Conclusion Aquilariae Lignum Resinatum may exert anti-inflammatory and anti-apoptotic effects through multi-components,multi-targets and multi-pathways to treat gastric ulcers.