三黄泻心汤保护大鼠应激性胃溃疡的作用机制

Mechanism of Sanhuang Xiexintang in Protecting Stress Gastric Ulcer in Rats

目的:通过网络药理学、分子对接及动物实验探讨探讨三黄泻心汤(SHXXT)保护大鼠应激性胃溃疡(SGU)的分子作用机制。方法:从中药系统药理学数据库与分析平台(TCMSP)、中医药综合数据库(TCMID)、在线中医药生物信息学分析工具(BATMAN-TCM)及Swiss Target Prediction数据库搜集和筛选SHXXT中活性成分及相应靶点;从在线人类孟德尔遗传数据库(OMIM)、药物靶标数据库(TTD)、GeneCards数据库及PharmGKB数据库中筛选SGU相关靶点;通过Cytoscape 3.9.1软件构建中药-活性成分-靶点(H-C-T)网络;通过蛋白质相互作用平台(STRING)数据库对药物和疾病交集靶点进行蛋白质-蛋白质相互作用(PPI)分析;通过生物学信息注释数据库(DAVID)进行基因本体(GO)富集分析和京都基因与基因组百科全书(KEGG)通路分析;采用AutodockVina 1.2.2分子对接软件对活性成分与关键靶点进行验证,并通过动物实验进一步验证实验结果。结果:筛选获得55种活性成分,预测得到SHXXT保护SGU的潜在靶基因255个,PPI分析表明蛋白激酶B(Akt)、第10号染色体上缺失与张力蛋白同源的磷酸酶(PTEN)、肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、环氧合酶-2(COX-2)为SHXXT保护SGU的核心靶点,GO和KEGG分析显示SHXXT可能通过调节磷脂酰肌醇3-激酶(PI3K)/Akt信号通路和炎症进程等多种生物过程影响SGU过程。分子对接结果显示,活性成分和关键靶点均有良好的结合能力。动物实验表明,与空白组比较,模型组大鼠溃疡指数(UI)显著升高(P<0.01),血清中TNF-α、IL-1β水平显著升高(P<0.01),胃黏膜组织中PTEN的磷酸化水平显著下调(P<0.01),PI3K、核转录因子-κB(NF-κB)、Akt的磷酸化水平显著上调(P<0.01)。与模型组比较,给药组大鼠UI显著降低(P<0.01),血清中TNF-α、IL-1β水平显著降低(P<0.01),胃黏膜组织中PTEN的磷酸化水平显著上调(P<0.05),PI3K、Akt、NF-κB的磷酸化水平明显下调(P<0.05)。结论:应用网络药理学预测、分子对接模拟及动物实验验证等方法证实SHXXT调控PI3K/Akt/NF-κB信号通路调节大鼠炎症反应,进而保护SGU大鼠胃黏膜。

Objective:To explore the molecular mechanism of Sanhuang Xiexintang(SHXXT)in protecting stress gastric ulcer(SGU)in rats through network pharmacology,molecular docking,and animal experiments.Method:The active ingredients and corresponding targets in SHXXT were collected and screened from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP),Traditional Chinese Medicine Information Database(TCMID),Bioinformation Analysis Tool for Molecular Mechanism of Traditional Chinese Medicine(BATMAN-TCM),and Swiss Target Prediction database.SGU-related targets were screened from the Online Mendelian Inheritance in Man(OMIM),Therapeutic Target Database(TTD),GeneCards database,and PharmGKB database.Herbal-ingredient-target(H-C-T)network was constructed by using Cytoscape 3.9.1 software.Protein-protein interaction(PPI)of drug and disease intersection targets was analyzed by using the Protein Interaction Platform(STRING)database.Gene ontology(GO)enrichment analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis were conducted through the Database for Annotation Visualization and Integrated Discovery(DAVID).The active ingredients and key targets were validated using AutodockVina 1.2.2 molecular docking software,and the experimental results were further validated through animal experiments.Result:The 55 active ingredients were screened,and 255 potential target genes for SHXXT treatment of SGU were predicted.The PPI analysis showed that protein kinase B(Akt),phosphatase and tensin homolog deleted on chromosome ten(PTEN),tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),and cyclooxygenase-2(COX-2)are the core targets of SHXXT for protecting SGU.GO and KEGG analyses showed that SHXXT may affect the development of SGU by regulating various biological processes such as the phosphoinositide 3-kinase(PI3K)/Akt signaling pathway and inflammatory processes.The molecular docking results showed that both the active ingredients and key targets had good binding ability.Animal experiments showed that compared with the blank group,the ulcer index(UI)of the model group was significantly increased(P<0.01),and the serum levels of TNF-α and IL-1β significantly increased(P<0.01).The phosphorylation level of PTEN in gastric mucosal tissue was significantly down-regulated(P<0.05).The phosphorylation levels of PI3K,Akt,and nuclear factor kappa-B(NF-κB)were significantly up-regulated(P<0.05).Compared with the model group,the UI of the treatment group was significantly reduced(P<0.01),and the serum levels of TNF-αand IL-1β were significantly reduced(P<0.01).The phosphorylation level of PTEN in gastric mucosal tissue was significantly up-regulated(P<0.01),and the phosphorylation levels of PI3K,Akt,and NF-κB were significantly downregulated(P<0.01).Conclusion:The application of network pharmacology prediction,molecular docking simulation,and animal experimental validation confirms that SHXXT regulates the PI3K/Akt/NF-κB signaling pathway to regulate the inflammatory response of rats and thus protects the gastric mucosa of SGU rats.