紫红獐牙菜抗HBV物质群对代谢网络整体调节机制研究

Hepatitis B is a serious worldwide disease caused by hepatitis B virus (HBV) infection, Swertia punicea Hemsl. is widely used as a folk medicine to treat viral hepatitis. However, its active components and mechanism of action are still not clear. Some ioslated compounds showed significant anti-HBV actives. The applicant proposed a hypothesis that anti-HBV active substance groups of S. punicea treat hepatitis B by adjusting the metabolic network of the whole.Therefore, we plan to combine "Phytochemommics" and "Metabolommics" to explore the anti-HBV active components group in the S. punicea, and further research their action mechanism via the metabolic network integrated regulation. Using the Phytochemommics method to find and enrich the active components and their secondary metabolites, then determine their anti-HBV active by in vitro/ in vivo experiments to establish the active components in S. punicea. Based on the Metabolomics methods and techniques to compare the whole metabolic network differences among healthy model, HBV duck model and the model treated with active components. The pattern recognition and canonical correlation analysis will be combined to establish the dynamic interactions between them, using the physiological determine indexes as linkage to scale the relationship between "active components and metabolites network " and coupling with biological pathways analysis to explore their synergy therapeutic mechanism from metabolites network integrated regulation. The study will play a good role in the study of the active substances of traditional Chinese medicine.

乙肝是严重危害人类健康的世界性疾病。紫红獐芽菜具有清肝利胆，清热解毒功效，民间广泛用于治疗病毒性肝炎，但其药效物质基础和作用机制尚不清晰。前期从紫红獐芽菜分离得到的化合物显示了确切的抗HBV活性。申请人提出紫红獐牙菜抗HBV活性物质群通过对代谢网络整体调节发挥其治疗乙肝的假说。拟用“植物化学组学和代谢组学” 相结合的模式，寻找富集各类活性次生代谢产物，通过体内外实验测定其抗HBV活性，建立活性物质群库；融合代谢组学研究方法和技术，系统研究健康模型、雏鸭乙肝模型和经活性物质群治疗后内源性代谢物整体变化；利用模式识别和典型相关性分析，以生理检测指标为连结点，标量化“活性物质群-内源性代谢物”间相关性；建立活性物质群与机体内源性代谢物的相互作用动态关系，结合生物代谢通路分析，从代谢网络整体调节的角度探寻活性物质群抗HBV协同作用机制。这对中药的物质基础研究将起到很好的范例作用.

民族药紫红獐牙菜 (Swertia punicea Hemsl) 系龙胆科獐牙菜属植物，俗称“肝炎草”，性寒味苦，能清肝利胆，清热解毒，民间广泛用于肿瘤、肝炎、黄疸等疾病的治疗。现代研究表明，紫红獐牙菜具有保肝利胆、强心、抗真菌、抗肿瘤等药理活性。.本项目从紫红獐牙菜中分离并鉴定46个化合物，包括2个新化合物及1个新的天然产物，活性研究表明部分化合物具有良好的抗癌和抗氧化活性， stigmasterol对BGC-823、HeLa和HepG-2细胞株具有较好细胞毒活性，IC50值分别为：6.74、6.36和2.5μM， oleanolic acid和swerchirin对Hep G-2细胞具有中等强度抑制活性，IC50值分别为：12.03和13.58μM。mangiferin的抗氧化活性（48.2μM）明显强于Vc（49.6μM）；bellidifodin、norbellidifolin、 swerpunilactone B的IC50值略低于Vc，分别为68.9μM、50.8μM和152.4μM。.对獐牙菜中分离的化合物进行快速筛选，围绕药效成分、GSTs 活性及荧光信号三者间的响应关系，快速发现swerchirin(C7)具有很强的体外 GSTs抑制活性，其可靠性已通过分子对接、细胞成像、GSTs 表达检测及肝肿瘤顺铂体外用药的敏感性考察等多角度初步验证。Swerchirin处理 HepG2 细胞能显著抑制胞内 GSTs 活性，下调HepG2 细胞中 GSTs 的表达与阳性对照依他尼酸 EA（500 μM）相当。Swerchirin抗癌活性不显著，但可能通过抑制GSTs活性而有效地增强化疗药物或其他抗癌药物的抗癌效果，实验结果表明顺铂对细胞生长的抑制作用明显优于C7，但顺铂联合C7用药后，细胞存活率明显低于顺铂单独用药。C7的IC50 值最高（23.97±0.023 μM），顺铂联合C7用药的IC50 值最低，且显著低于顺铂单独用药（3.54±0.018 μM vs 9.24±0.009 μM），两药合用效果远大于单独用药，细胞凋亡实验结果也表明联合用药后，顺铂诱导的细胞凋亡数由58.22 %升高为80.89 %，而C7无显著诱导凋亡的能力。Swerchirin作为GSTs酶的有效抑制剂，具有顺铂对HepG2细胞的增敏作用，有望成为临床抗癌药物的有效药物伴侣。