基于代谢调控网络干预的重楼总皂苷组分配伍机制研究

Chinese medicine (CM), whose therapeutic characteristics are regarded as multi-component, multi-target and multi-pathway integrally regulation, can effectively inhibit malignant tumor involved in multi-gene, multiple signaling pathways. However, it's difficult to understand the complex CM's material base and mechanism of action. To clarify the component compatibility mechanisms of CM, one has to start with the investigation of the metabolic regulatory network alterations in response to the CM intervention. This method can be used to evaluate the effects of CM from a holistic view, discover the law of the CM prescription-symptom relationship, and guide safe medication and rational administration. Therefore, it is significant for the promotion of the innovation and development of modern CM. Rhizoma Paridis, as a common CM, is used as one of the main materials in the anti-tumor CM prescription. With the support of the Drug Creation Project from Science and Technology in China, we found that Rhizoma Paridis Saponins (RPS), which are the effective part of Rhizoma Paridis, showed strong anti-lung cancer and antihepatocarcinoma activities. Meanwhile, the anti-tumor effect and the toxicity of the total RPS were better than those of the separate saponins. To reveal the component compatibility mechanisms of RPS, we will adopt chemomics, metabonomics and network biology approaches to reconstruct the metabolic regulatory network in response to the different parts of the RPS intervention. This metabolic regulatory network maight help us elucidate the pathways involved in different parts of RPS intervening tumor formation and progression, and approaches of metabolic disturbance consequences of the minimum toxic doses of them inducing toxic reaction. All in all, this project will lay the groundwork for the interpretation of Rhizoma Paridis Saponins' material base and mechanism of action. Furthermore, it may serve as an indication for the safe medication and rational administration of RPS. At last, similar strategies would be good for the study on the component compatibility mechanisms of other single Chinese herb.

中药因多成分、多靶点、多途径整合调节的治疗特色，对涉及多基因、多信号通路的恶性肿瘤有较好的治疗作用。但由于中药成分的复杂性，导致其物质基础不明确，作用机制研究较为困难。从药物干预的代谢调控网络入手，研究中药组分配伍机制，可以整体评价中药的效应，研究中医方证相应规律，指导中药的安全、合理用药，对现代中药创制具有推动意义。中药重楼是抗肿瘤方剂中常用药味。课题组在国家"重大新药创制"基金的支持下，发现其有效部位重楼总皂苷对肺癌、肝癌等具有较强的抗肿瘤活性，且总皂苷抗肿瘤活性及安全性优于不同极性皂苷组分及单体。为揭示总皂苷中各化学物质系统内在关系，本项目拟采用化学物质组学、代谢组学、网络生物学等方法，构建不同组分在抗肿瘤及毒性剂量下对机体的代谢调控网络影响。这些为阐明重楼总皂苷的物质基础及作用机制奠定基础，对其安全、合理用药有指导意义，同时为以现代药理学方法研究单味中药组分配伍机制发挥示范作用。

重楼皂苷（RPS）作为滇重楼中主要活性成分，前期报道其具有较好的抗肿瘤、抑制肺转移的作用。为进一步明确其抗肿瘤机制，本项目首先建立了DEN诱导的肝癌及肺转移模型、H22肝癌模型及Lewis肺腺癌模型。给药一阶段后，通过组织病理切片分析、血液生化分析、免疫组化、蛋白电泳、PCR、基因芯片、蛋白芯片、代谢组学等方法，最终发现RPS有效的缓解肝损伤，抑制肺腺癌的形成。基因芯片表明RPS调节了多种差异基因包括转移、细胞周期、免疫、粘附、迁移、血管生成、凋亡等的表达。蛋白芯片也表明RPS通过抑制VEGFD、VEGFR3、RAGE、IL6R、IL17BR、CXCL16等细胞因子或受体的表达，进而抑制NF-κB、干预PI3K/Akt和MAPK磷酸化等通路，调控核因子NF-κB、HIF-1A、STAT3和Jun等，最终抑制肿瘤细胞血管生成、淋巴血管生成、粘附、炎症、侵袭酶等表达。此外，RPS可通过抑制脂肪酸的氧化、糖异生途径阻断机体的能量供应，同时抑制了甘氨酸、丙氨酸的产生，进而阻断肿瘤的生长。从相关代谢酶角度分析，RPS可调控p53/mTOR-c-Myc-HIF-1α通路，进而降低GLUT1、HK2、PKM2、LDHA相关基因，干预糖代谢。上调的p53也可提高GLS，降低ATP的基因表达，进而抑制能量供给。同时，RPS降低了FASN的mRNA水平，进而抑制脂质合成，干预肿瘤生长。总之，RPS显示了多靶点的抗肿瘤特性。.尽管RPS显示了较好的抗肿瘤活性，其毒性表征仍值得研究。在本项目中，通过90天喂养试验，RPS显示了一定的肝、肺损伤。为了增加RPS药效，降低其毒副作用，通过药物筛选，发现姜黄提取物与RPS配伍可改善肿瘤小鼠生活质量，增加RPS抗肿瘤活性。45天动物喂养试验表明，姜黄素可通过缓解RPS引发的肝功能指标异常（ALT、AST、AKP、γ-GT），氧化应激指标异常（ROS、MDA、8-OHdG、Trx、TXNIP），炎症相关因子（COX-2、IL-1beta、NF-kappaB）的释放，以及代谢紊乱（RPS抑制了脂肪酸的氧化、糖分解及TCA循环途径，紊乱了甘氨酸、丝氨酸、苏氨酸等代谢通路）等，降低RPS引发的肝、肺损伤。.总之，本研究从药效及安全性两方面研究RPS的特点，同时配伍姜黄素可更好的提高RPS药效，发挥中药多靶点抗肿瘤作用，为抗肿瘤药物的研发提供研究思路与方法。