基于网络药理学及实验验证探讨清化祛瘀健脾方治疗溃疡性结肠炎的作用机制

Study on the mechanism of Qinghua Quyu Jianpi Decoction in treating ulcerative colitis based on network pharmacology and experimental verification

关键词:  清化祛瘀健脾方  溃疡性结肠炎  网络药理学  分子对接  动物实验

Key words:  Qinghua Quyu Jianpi Decoction  ulcerative colitis  network pharmacology  molecular docking  animal experiment

摘要:

[目的] 旨在从网络药理学和实验验证的角度探讨清化祛瘀健脾方治疗溃疡性结肠炎的潜在药理机制。[方法] 通过网络数据库筛选清化祛瘀健脾方与溃疡性结肠炎相关基因，进一步遴选出交集基因，对交集基因进行GO功能和京都基因和基因组百科全书（KEGG）富集分析，确定清化祛瘀健脾方对溃疡性结肠炎的药理作用；同时建立蛋白-蛋白互作网络，并进行功能模块分析。筛选核心基因，通过分子对接，筛选结合最稳定的分子进行实验验证。最后，采用苏木精-伊红染色、免疫组织化学染色及蛋白质免疫印迹方法验证清化祛瘀健脾方发挥疗效的分子机制。[结果] 从清化祛瘀健脾方中筛选出86个活性成分和224个潜在靶点，与疾病靶点取交集获得254个共同靶点。这些共同靶点主要参与炎症反应、DNA转录、蛋白激酶活性、细胞凋亡、细胞增殖调控等过程，表明清化祛瘀健脾方通过影响这些过程来减轻炎症反应。进一步对蛋白-蛋白互作网络进行了核心基因分析及分子对接验证，发现低氧诱导因子-1（HIF1A）、信号转导及转录激活蛋白3（STAT3）、MYC和丝裂原活化蛋白激酶3（MAPK3）可能是清化祛瘀健脾方主要作用靶点。最后，对分子对接预测结合能最稳定的靶标MAPK3进行动物实验验证，发现清化祛瘀健脾方可以明显减轻溃疡性结肠炎模型小鼠结肠炎症反应，促进MAPK3蛋白的表达。[结论] 网络药理学及分子对接预测了清化祛瘀健脾方治疗溃疡性结肠炎的功能成分及作用靶点，并通过动物实验证实了该中药复方可能通过激活MAPK信号通路，促进肠黏膜愈合而发挥抗炎作用，为清化祛瘀健脾方等中药复方的深入研究提供了新思路。

Abstract:

[Objective] To investigate the potential pharmacological mechanism of Qinghua Quyu Jianpi Decoction in the treatment of ulcerative colitis from the perspective of network pharmacology and experimental validation. [Methods] Genes related to ulcerative colitis with Qinghua Quyu Jianpi Decoction were screened through the network database，further selected the shared genes，and performed GO function and KEGG enrichment analysis on the shared genes to determine the pharmacological effects of Qinghua Quyu Jianpi Decoction on ulcerative colitis. Meanwhile，a protein-protein interaction network and performed functional module analysis were established. The shared core genes were screened and the most stable binding molecules were selected for experimental validation by molecular docking. Finally，hematoxylin-eosin staining，immunohistochemical staining and Western blot assays were used to further confirm the molecular mechanism of the efficacy of the Qinghua Quyu Jianpi Decoction. [Results] The 86 active ingredients and 224 potential targets were screened from the formula，and 254 common targets were obtained by intersecting with the disease targets. These common targets were mainly involved in inflammatory response，DNA transcription，protein kinase activity，apoptosis，cell proliferation regulation and other processes，suggesting that Qinghua Quyu Jianpi Decoction could improve the inflammatory response by affecting these processes. Further，core gene analysis and molecular docking validation of the protein-protein interaction network were performed，and HIF1A，STAT3，MYC and MAPK3 were screened as possible main action targets of Qinghua Quyu Jianpi Decoction. Finally，MAPK3，the target with the most stable binding capacity predicted by molecular docking，was validated in animal experiments，and it was found that Qinghua Quyu Jianpi Decoction could significantly reduce the colonic inflammation response and promote MAPK3 protein expression in mice with ulcerative colitis. [Conclusion] The network pharmacology and molecular docking predicted the functional components and targets of Qinghua Quyu Jianpi Decoction for the treatment of ulcerative colitis，and animal experiments confirmed that the compound might activate MAPK signaling pathway and promote intestinal mucosal healing and play anti-inflammatory effect，which provides a new idea for the in-depth research of Qinghua Quyu Jianpi Decoction and other herbal compounds.