基于网络药理学和实验验证探讨二氢杨梅素治疗炎症性肠病的作用机制

Mechanism of dihydromyricetin in the treatment of inflammatory bowel disease based on network pharmacology and experimental verification

[目的]通过网络药理学和体外实验预测并验证二氢杨梅素(DHM)治疗炎症性肠病(IBD)的潜在靶点及作用机制。[方法]使用Pubchem数据库获取DHM作用靶点,通过GeneCards、OMIM、TTD、PharmGKB、Drugbank数据库获取IBD疾病靶点,获取药物与疾病的交集靶点。利用STRING数据库构建蛋白互作(PPI)网络并使用Cytoscape可视化筛选核心靶点。运用R语言进行基因本体(GO)功能分析和京都基因和基因组百科全书(KEGG)通路富集分析。体外实验验证DHM的抗炎作用及可能机制。[结果]DHM作用靶点与IBD疾病靶点相交共得到42个交集靶点,并筛选出5个核心靶点凝血因子Ⅱ(F2)、内皮型一氧化氮合酶3(NOS3)、丝氨酸羟甲基转移酶1(SHMT1)、造血细胞激酶(HCK)、二氢叶酸还原酶(DHFR)。GO功能分析和KEGG富集分析显示DHM可能通过黏附连接、趋化因子、Ras信号通路等发挥作用。实时荧光定量聚合酶链反应(RT-PCR)显示DHM能抑制脂多糖(LPS)诱导的HT-29细胞炎症因子肿瘤坏死因子-α(TNF-α)、白细胞介素-1β(IL-1β)、白细胞介素-18(IL-18)mRNA水平升高。RT-PCR提示在LPS诱导后靶点NOS3、HCK mRNA表达水平升高,而DHM干预后其表达水平下降。蛋白免疫印迹法(Western blot)检测显示DHM能有效恢复LPS诱导的HT-29细胞连接蛋白的下调。[结论]研究揭示了DHM通过多通路、多靶点治疗IBD,并可能通过恢复细胞间连接改善屏障功能受损有效治疗结肠炎。

[Objective]To predict and verify the potential target and mechanism of dihydromyricetin(DHM)in the treatment of inflammatory bowel disease(IBD)through network pharmacology and in vitro experiments.[Methods]The targets of dihydromyricetin were obtained from Pubchem database,and the targets of inflammatory bowel disease were obtained from GeneCards,OMIM,TTD,PharmGKB and Drugbank database,and the intersection targets of drug and disease were screened.Build PPI networks with STRING databases and visually screen core targets using Cytoscape.GO function analysis and KEGG path enrichment analysis were performed in R language.The anti-inflammatory effect of dihydromyricetin and its possible mechanism were verified in vitro.[Results]A total of 42 intersecting targets of dihydromyricetin and inflammatory bowel disease were obtained,and 5 core targets F2,NOS3,SHMT1,HCK and DHFR were selected.GO functional analysis and KEGG enrichment analysis showed that dihydromyricetin may play its role through adhesion linkage,chemokines,Ras signaling pathway and so on.RT-PCR showed that dihydromyricetin could inhibit the increase of TNF-α,IL-1βand IL-18 mRNA levels which were induced by LPS in HT-29 cells.RT-PCR indicated that the expression levels of NOS3 and HCK mRNA increased after LPS induction,but decreased after DHM intervention.Western Blot analysis showed that dihydromyricetin could effectively restore the LPS-induced downregulation of HT-29 cell connexin.[Conclusion]This study reveals that dihydromyricetin can treat inflammatory bowel disease through multi-pathway and multi-target,and may be effective in the treatment of colitis by restoring intercellular connectivity to improve impaired barrier function.