桂枝加桂汤治疗免疫性肝损伤的作用机制研究

Study on Therapeutic Mechanism of Guizhi Jiagui Decoction against Immune Liver Injury

摘要: 目的：基于网络药理学、分子对接和细胞体外实验探究桂枝加桂汤治疗免疫性肝损伤(immune liver injury, ILI)的作用机制。方法：通过ETCM检索桂枝加桂汤中的活性成分，利用Pubchem、Swiss Target数据库预测成分的作用靶点；采用Genecards、OMIM数据库获取ILI疾病相关靶点；绘制韦恩图并获取交集靶点；使用String平台构建“药物–疾病”共同靶点的蛋白互作网络，分析网络并筛选出关键靶点；利用Metascape平台对交集靶点开展GO功能注释与KEGG通路富集分析；采用Cytoscape3.9.0软件构建“成分–靶点–通路”网络，并筛选出关键活性成分；采用分子对接进行验证。用脂多糖(lipopolysaccharide, LPS)诱导小鼠腹腔巨噬细胞构造炎症模型，设置空白对照组、LPS模型组(1 μg·ml⁻¹)、LPS + 10-姜酚组(15.625 μmol·l⁻¹)探究重要活性成分10-姜酚的抗炎效果。采用酶联免疫吸附试验(ELISA)检测巨噬细胞上清中炎性因子IL-6、IL-1β及TNF-α的水平。结果：获得200个活性成分和69个交集靶点，GO分析得到1908个条目；通路富集结果显示，54条显著富集的通路主要与PPAR、AMPK、JAK-STAT等信号传导途径相关；获得关键活性成分有10-姜酚、6-姜酚、6-姜烯酚等，重要靶点基因有AKT1、TNF、ESR1等；分子对接显示重要活性成分与重要靶点基因结合活性较好。体外实验显示，与LPS组相比，10-姜酚组细胞上清中IL-6、IL-1β、TNF-α含量显著减少。结论：桂枝加桂汤可能通过10-姜酚、6-姜酚、6-姜烯酚等化学成分作用于AKT1、TNF、ESR1等靶点基因，进而调控PPAR信号通路、AMPK信号通路、JAK-STAT信号通路等通路，发挥治疗ILI的作用。

Abstract: Objective: To investigate the mechanism of Guizhi Jiagui Decoction in the treatment of immune liver injury (ILI) using network pharmacology, molecular docking, and in vitro cell experiments. Methods: Active components screened from the ETCM database for Guizhi Jiagui Decoction had their targets mapped using PubChem and Swiss Target Prediction, while disease targets for ILI were collated from GeneCards and OMIM. A Venn diagram was constructed to identify common targets. A protein–protein interaction (PPI) network of the common targets was built using the STRING platform and analyzed to identify key targets. GO and KEGG enrichment analyses of the overlapping targets were performed via the Metascape platform. To visualize and prioritize important active ingredients, a “component–target–pathway” interaction network was visualized with Cytoscape 3.9.0. To further assess the predicted interactions, the binding stability of key components to core targets was examined via molecular docking. For in vitro validation, lipopolysaccharide (LPS) was used to induce an inflammatory state in mouse peritoneal macrophages, establishing the experimental model. The experimental groups included a blank control group, an LPS model group (1 μg·ml⁻¹), and an LPS + 10-gingerol group (15.625 μmol·l⁻¹). ELISA was employed to detect the levels of IL-6, IL-1β, and TNF-α in the macrophage culture supernatant. Results: A total of 200 active ingredients and 69 common targets were identified. GO enrichment analysis yielded 1908 entries, and KEGG analysis revealed 54 significantly enriched pathways, primarily involving PPAR, AMPK, JAK-STAT, and related signaling pathways. Key active components included 10-gingerol, 6-gingerol, and 6-shogaol, while important target genes included AKT1, TNF, and ESR1. The docking simulations demonstrated favorable binding interactions between the primary active constituents and the central targets. In vitro experiments demonstrated that, compared with the LPS group, the 10-gingerol treatment group exhibited significantly reduced levels of IL-6, IL-1β, and TNF-α in the cell supernatant. Conclusion: Guizhi Jiagui Decoction may exert therapeutic effects against ILI by targeting genes such as AKT1, TNF, and ESR1 via active components including 10-gingerol, 6-gingerol, and 6-shogaol, thereby modulating PPAR, AMPK, JAK-STAT, and other related pathways.