基于网络药理学和体外实验探讨椿皮散治疗溃疡性结肠炎和肛窦炎的作用机制

Study on the mechanism of Chunpi San in the treatment of Ulcerative colitis and anal sinusitis based on network pharmacology and in vitro experiments

摘要:

目的 用网络药理学分析椿皮散治疗溃疡性结肠炎（UC）和肛窦炎的活性成分和潜在分子通路，并使 用分子对接技术和体外实验加以验证。方法 根据文献资料确定椿皮散的活性成分，使用在线数据库得到活性成 分与 UC、肛窦炎相关靶点，使用 Venny2.1软件将活性成分靶点与疾病靶点取交集后上传至 STRING分析平台，在 Cytoscape v3.10.1 软件绘制蛋白互作网络（PPI）图并筛选核心靶点，在微生信在线分析平台对交集靶点进行 GO、 KEGG 富集分析，然后采用 AutoDock Vina 1.2.3 软件进行分子对接分析，最后用 Jurkat T 细胞进行体外实验验证。 细胞用1 μmol/L离子霉素（P）和50 μg/L佛波酯（I）激活后，分别加入0.25和0.50 g/L 椿皮散浸膏处理，使用CCK8法 检测细胞存活率，ELISA试剂盒检测上清液中炎症因子IL-2水平，Western blot 检测细胞内MAPK通路相关蛋白相 对含量。结果 筛选到椿皮散活性成分靶点 638 个，UC和肛窦炎相关靶点1 609个，交集靶点162个，核心靶点24个。 KEGG 富集分析显示主要包括MAPK、PI3K-AKT等信号通路，GO功能分析主要集中在细胞氧化应激和信号传导 等功能。分子对接结果验证了排名前10的活性成分与排名前10的某些核心靶点具有良好的结合特性。体外实验 结果也表明，P/I 能够激活 Jurkat T 细胞炎症因子 IL-2 的释放，并上调 MAPK 通路相关蛋白 p-RAF-1、p-ERK1/2、 STAT3和p-STAT3，而椿皮散浸膏能够显著降低上述蛋白水平。结论 椿皮散可能通过多成分、多靶点和多途径对 UC和肛窦炎发挥治疗作用。

Abstract:

Abstract: Objective To analyze the active ingredients and potential molecular pathways of Chunpi San (CPS) in the treatment of ulcerative colitis (UC) and anal sinusitis (AS) through network pharmacology, with experimental validation via molecular docking and in vitro functional assays. Methods Based on literature review, the active ingredients of CPS were determined. Online databases were used to obtain the active ingredients and UC/AS related targets. Venny 2.1 software was used to intersect the active ingredient targets with disease targets and upload them to the STRING analysis platform. A protein-protein interaction network (PPI) map was constructed in Cytoscape v3.10.1 software and core targets were screened. GO and KEGG enrichment analyses were performed on the intersecting targets on the WeChat online analysis platform. AutoDock Vina 1.2.3 software was used for molecular docking analysis, and Jurkat T cells were used for in vitro experimental validation. After activation with 1 μmol/L ionomycin (I) and 50 μg/L phorbol ester (P), cells were treated with 0.25 g/L and 0.50 g/L CPS extract, respectively. Cell survival rate was assessed using CCK-8 assays, the inflammatory cytokine IL-2 level in the supernatant was measured using an ELISA kit, and the relative content of MAPK pathway related proteins in cells was detected by Western blot. Results A total of 638 CPS active ingredient targets, 1 609 UC and AS related targets, 162 intersecting targets, and 24 core targets were screened. KEGG enrichment analysis showed that these mainly involve signaling pathways such as MAPK and PI3K-AKT, while GO functional analysis primarily focuses on cellular oxidative stress and signal transduction functions. The molecular docking results confirmed that the top ten active ingredients have good binding affinities with certain core targets among these. In vitro experimental results also showed that P/I can activate the release of the inflammatory cytokine IL-2 in Jurkat T cells and upregulate MAPK pathway related proteins, including p-RAF-1, p-ERK1/2, STAT3, and p-STAT3, whereas CPS extract can significantly reduce the levels of these proteins. Conclusion CPS may exert therapeutic effects on UC and AS through multiple components, targets, and pathways.