基于动态转录组学和网络药理学整合的中药复方治疗肝癌腹水作用机制的研究

Ascites is a common complication of primary liver cancer. It greatly affects the quality of life and confers poor prognosis of patients with liver cancer. Currently, traditional Chinese medicine (TCM) has been widely used as an adjuvant therapy against this disease. Recent studies have demonstrated that TCM using for patients with liver cancer ascites may take obvious advantages in the control of disease advancement, the improvement of symptoms and the quality of patients' life. However, it is necessary but very difficult to investigate the anti-hepatoma mechanisms of TCM with multi-herbs, multi-ingredients, multi-targets and multi-pathways. In this study, Gansui Banxia Tang, a classic TCM formula from Chinese medical sage Zhang Zhongjing, will be selected as an example. At first, the dynamic gene expression regulation network of liver cancer concomitant with ascites will be constructed and analyzed; Then, the candidate effector molecules and the corresponding compositive compounds of Gansui Banxia Tang will be screened by structural similarity comparison of chemical compounds and computer aided drug virtual screening technology; After that, following networks: effective compound-candidate target network, effective compound-pathway network, candidate target-known anti-cancer target network and candidate target-disease network will be constructed using network pharmacological methods and comparied with the dynamic gene expression regulation network of liver cancer mentioned above, in order to clarify the anti-hepatoma mechanisms of Gansui Banxia Tang and predict its candidate therapeutic targets; Finally, the key pathways which Gansui Banxia Tang may be involved in and candidate therapeutic targets will be validated by experiments using H22 hepatocellular carcinoma implanted mouse models. This study may explain the mechanisms of Gansui Banxia Tang for the treatment of liver cancer ascites, and meanwhile, may predict the potential targets of this fomula, which will facilitate to elucidate the compatible mechanism of the complex prescription and provide basis for an alternative approach to investigate novel TCM formula on the network pharmacology level.

中药复方治疗肝癌腹水独具优势，如何诠释其科学内涵是中医药现代化亟待解决的关键问题之一。本课题以来自《金匮要略》、临床治疗肝癌腹水及晚期肝癌效果确切的泻水名方甘遂半夏汤为范例，整合动态转录组学和网络药理学方法，分别构建"不同阶段肝癌的动态基因表达调控网络"和"甘遂半夏汤化学成分→候选效应分子→通路→疾病的多级网络"，并将二者进行比对和关联分析，从而挖掘该方对不同阶段肝癌的效应机制和潜在作用靶标。最后，采用小鼠H22肝癌腹水移植瘤模型，对关键通路和靶标进行实验验证。本课题按照"数据收集和整合→疾病和药物网络构建→网络比对和关联分析→药物作用机制挖掘→药物网络靶标预测→实验验证"的研究模式，全面揭示甘遂半夏汤对肝癌发生发展动态过程的作用机制，阐明其矫正不同阶段肝癌失衡网络的分子机理，破解其治疗肝癌腹水的科学内涵，为同类中医复方的研究提供新的思路和模式。

甘遂半夏汤是东汉张仲景《金匮要略》的经方之一，其治疗肝癌腹水及晚期肝癌的疗效确切。然而，该方中的“甘遂-甘草”为“十八反”配伍禁忌范畴，大大限制了其临床应用。为探明甘遂半夏汤对抗肝癌的干预特点及作用机制，本课题建立不同进展阶段肝癌组织中差异表达基因的相互作用网络，根据网络节点的拓扑特征和功能属性，发现肝癌早期分子网络是以调节细胞免疫、细胞增殖和细胞周期的功能模块为主，肝癌中、晚期分子网络是以调节细胞迁移和血管浸润的功能模块为主；其次，基于药物结构相似性原理，预测甘遂半夏汤各化学组分的候选靶标，并通过网络分析发现其与肝癌早期的细胞免疫调节和肝癌晚期的细胞迁移功能模块紧密关联；再次，基于肝癌移植瘤裸鼠模型，进一步验证甘遂半夏汤对肝癌移植瘤生长的显著抑制作用，及其对JAK-STAT、G蛋白偶联、脂肪酸代谢等通路中关键靶点的调节作用。为进一步拓展研究该方中“甘草-甘遂”反药组合配伍禁忌的网络调节机制，本课题采用均匀设计与药效学实验相结合的分析方法，发现甘草在不同的剂量和配比条件下，既可以拮抗也可以协同甘遂抗小鼠肝癌腹水的药效作用；同时，发现“甘草-甘遂”反药组合在对抗肝癌腹水中的配伍机制可能与调节抗利尿激素分泌调控网络的失衡相关，进而找到这种配伍关系的网络调节机制，即当甘草增强甘遂的抗腹水作用时，二者分别下调其候选靶标PIK3CG和ADRB1，从而减少已知抗腹水靶标精氨酸抗利尿激素受体（AVPR2）的表达，使其与抗利尿激素的相互作用减少，并抑制抗利尿激素的分泌，实现促进腹水排空的效果；当甘草拮抗甘遂的抗腹水作用时，甘草抑制了甘遂对其候选靶标ADRB1表达的调节，从而增加AVPR2的表达，使其与抗利尿激素的相互作用增强，导致水的再吸收增加。本研究一方面全面揭示甘遂半夏汤对肝癌发生发展动态过程的作用机制，并阐明其矫正不同阶段肝癌失衡网络的分子机理；另一方面以药物的主要功效和临床治疗作用为依据，观察“十八反”代表性反药配伍组合“甘遂-甘草”对多个抗腹水药效指标的干预影响，并探索其可能的影响因素，还首次将网络药理学分析方法引入到中药“十八反”配伍机制的研究中，为阐释中药“十八反”配伍禁忌的新涵义奠定方法学基础。相关研究结果已发表SCI论文7篇，其中3篇影响因子大于5，1篇为封面论文，并先后获得中国中西医结合学会科技奖和中国中医科学院科技奖二等奖。