丹参-郁金药对缓解肝癌作用机制研究

Study on the mechanism of Danshen-Yujin herbal drug pair treating hepatocellular carcinoma

关键词:  肝癌  丹参  郁金  网络药理学  生物信息学  细胞自噬  免疫浸润

Key words:  hepatocellular carcinoma  Danshen  Yujin  network pharmacology  bioinformatics  cellular autophagy  immune infiltration

摘要:

[目的]基于网络药理学及生物信息学研究丹参-郁金药对治疗肝癌的物质基础及作用机制。[方法]首先，依据中医药系统药理学数据库与分析平台（TCMSP），对药物的活性成分作进一步筛选，并找出与之相关的靶点；其次，根据GeneCards/OMIM数据库筛选出肝癌相关靶点，随机生存森林算法进一步筛选出与生存有差异的靶点；然后，将药物靶点和疾病靶点进行交集分析。通过基因本体论（GO）和京都基因与基因组百科全书（KEGG）等生物信息学软件对共有靶点进行功能注释和富集分析，利用Cytoscape软件进行网络拓扑计算和模块分析，预测其活性成分与靶点的结合特性；同时进行免疫浸润分析，从免疫的角度解释关键基因与免疫的关系；通过基因集富集分析（GSEA）探索共有靶点参与的通路，通过基因集差异分析（GSVA）进一步分析高低表达组之间的信号通路差异情况。最后，通过构建多因素回归模型，根据模型中各个影响因素对结局变量的贡献程度，给每个影响因素的每个取值水平进行赋分，然后再将各个评分相加得到总评分，从而计算出预测值。[结果]从丹参-郁金药对中筛选得到80个活性成分，与肝癌的交集靶点共31个。主要成分为黄酮类化合物、二萜类化合物、酚酸类化合物、甾醇类化合物、内酯类化合物等。GO与KEGG富集分析结果显示，共有靶点所涉及的生物过程主要包括小分子代谢过程的调控（regulation of small molecule metabolic process）、对雌二醇的响应（response to estradiol）、DNA结合转录因子活性的调控（regulation of DNA-nding transcription factor activity）等，信号通路主要与FoxO信号通路（FoxO signaling pathway）、HIF-1信号通路（HIF-1 signaling pathway）、p53信号通路（p53 signaling pathway）等相关。随机生存森林算法分析发现CCNB1、VEGFA、MAPK3在生存中的差异具有统计学意义，可能是丹参-郁金药对治疗肝癌的关键靶点。免疫细胞浸润分析显示关键基因和多种免疫细胞相关。同时，GSEA通路富集分析提示关键基因与多条信号通路相关。最后，通过关键基因表达量将他们回归分析的结果通过列线图的形式展现，Nomogram模型具有较好的预测效能。[结论]丹参-郁金药对可能通过调控P53信号通路、FoxO信号通路、HIF-1信号通路等，靶向调控肝癌的自噬、氧化应激、免疫调节、凋亡等病理过程，同时CCNB1、VEGFA、MAPK3等可能是丹参-郁金药对治疗肝癌的关键靶基因。该研究初步揭示了丹参-郁金治疗肝癌具有多靶点、多通路的潜在作用机制，为进一步研究提供了理论依据，为中医药现代化和国际化作出贡献。

Abstract:

[Objective] To investigate the material basis and mechanism of action of the Danshen（Salvia miltiorrhiza Bunge）-Yujin （Curcuma aromatica Salisb）in treating hepatocellular Carcinoma using network pharmacology and bioinformatics. [Methods] Firstly， the active ingredients of the herbs were screened and their related targets identified using the TCMSP database. Secondly， hepatocellular Carcinoma-related targets were screened from the GeneCards and OMIM databases，followed by further prioritization of survival-significant targets using the random survival forest algorithm. Subsequently，intersection analysis was performed to identify common targets shared between the herb and disease targets. These common targets underwent functional annotation and enrichment analysis（GO and KEGG）. Cytoscape software was used for network topology calculations and module analysis to predict the binding characteristics between active components and targets. Immune infiltration analysis was conducted to elucidate the relationship between key genes and the immune system from an immunological perspective. Gene set enrichment analysis（GSEA）explored pathways involving the common targets，and gene set variation analysis（GSVA）further analyzed signaling pathway differences between highand lowexpression groups. Finally，a multifactor regression model was constructed；scores were assigned to each level of the influencing factors based on their contribution to the outcome variable within the model. These individual scores were summed to obtain a total score，which was used to calculate the predicted value. [Results] A total of 80 compounds were screened from DanshenYujin，31 of which had overlapping targets with HCC. The major components were flavonoids，diterpenoids，phenolic acids，sterols， lactones，etc. GO enrichment results showed significant enrichment of biological processes，such as regulation of small molecule metabolism，response to estradiol，regulation of DNA-binding transcription factor activity. KEGG enrichment showed that genes were particularly enriched in FoxO pathway，HIF-1 pathway，p53 pathway，and other pathways. Randomized survival forest algorithm analysis revealed statistically significant differences in survival for CCNB1，VEGFA，and MAPK3，which may be the key target genes of Danshen-Yujin in the treatment of HCC；immune infiltration analysis showed that the three key genes were associated with a variety of immune cells，and involved in autophagy-related signaling pathways. Meanwhile，GSEA pathway enrichment analysis suggested that the key genes were associated with multiple signaling pathways. Finally，the results of the regression analyses were presented in the form of nomogram by key gene expression，and the Nomogram model had good predictive efficacy. [Conclusion] The Danshen-Yujin herb pair may modulate pathological processes in hepatocellular carcinoma，such as autophagy，oxidative stress，immune regulation， and apoptosis，by targeting signaling pathways including P53，FoxO，and HIF-1. Furthermore，CCNB1，VEGFA，and MAPK3 may serve as key target genes of Danshen-Yujin in treating hepatocellular carcinoma. This study preliminarily reveals the potential multi-target and multi-pathway mechanism of Danshen-Yujin in the treatment of hepatocellular Carcinoma，providing a theoretical basis for further research and contributing to the modernization and internationalization of traditional Chinese medicine.