基于入血活性成分追踪和代谢组学研究走马胎抗肝癌的体内药效物质基础及其代谢机制

Chinese medicines have perfect effects on hepatocarcinoma, but their active compounds are complicated and the mechanisms are not known, which are limiting their application in anti-hepatocarcinoma. Ardisia gigantifolia Stapf is widely applied in Guangxi. Our study and other reports have found Ardisia gigantifolia Stapf had strong activity for anti-hepatocarcinoma, but its efficacy material base in vivo and mechanisms remain unknown. In the present study, a bioactivity-guided fractionation screening platform, including fractionation by preparative high performance liquid chromatography (preparative HPLC), cytotoxity screening by HepG2, Sk-hep1 and Huh7 cell lines, and identification by liquid chromatography coupled to mass spectrometer (LC/MS), was developed for fast screening and identifying potential anti-liver cancer constituents of Ardisia gigantifolia Stapf. Further, we used a nuclear magnetic resonance based metabolomic approach to gain potential the biomarkers in serum and urine of liver cancer in Sk-hep1 xenograft mice in vivo, and in cell and cell medium of HepG2, Sk-hep1 and Huh7 in vitro . We also used Ardisia gigantifolia Stapf treatments in the same model to test its potential therapeutics and explore its anti-tumor mechanisms in vivo and in vitro. Our research will demonstrate the pharmacological mechanism of “multiple-components, multiple-targets and system therapy” of Ardisia gigantifolia Stapf for anti-hepatocarcinoma and provide a new approach for the research of Chinese medicine in anti-hepatocarcinoma.

中药抗肝癌有较好的疗效，但因其成分复杂，作用机制不明，限制其在肝癌的应用。走马胎为广西民间应用较多的中药，我们前期研究及文献报道，走马胎具有较强的抗肝癌活性，但其体内抗肝癌直接作用物质及作用机制尚未阐明。本研究通过制备型HPLC将走马胎大鼠含药血清分段收集，以抗肝癌细胞活性（MTT）为导向，快速筛选走马胎抗肝癌的体内活性成分，再通过LC/MS技术对其进行结构鉴定，锁定走马胎抗肝癌体内药效物质基础。以HepG2、Sk-hep1和Huh7为体外肝癌模型，以Sk-hep1异源移植小鼠为体内肝癌模型，进一步利用NMR技术检测走马胎对体内和体外肝癌模型代谢网络的影响，从代谢水平整体和全面地研究走马胎抗肝癌作用机制。走马胎抗肝癌体内直接作用物质和代谢机制的阐明，有利于深入揭示走马胎“多组分、多靶点和系统治疗”的作用特点，为走马胎在肝癌防治方面的应用和推广提供依据，并为中药抗肝癌提供新的研究思路和方法。

走马胎为广西民间应用较多的中药，我们前期研究及文献报道，走马胎具有较强的抗肝癌活性，但其抗肝癌直接作用物质及作用机制尚未阐明。课题组以抗肝癌活性为导向，分离得到走马胎抗肝癌活性单体百两金皂苷B，发现其对肝癌HepG2、Sk-hep1和Huh-7细胞增殖均有较强的抑制作用，并能促进其凋亡。通过HPLC-ELSD指纹图谱发现走马胎药材含有较高含量的百两金皂苷B（0.013%-0.198%）。通过血清药理学发现灌胃给予10g/kg/d走马胎醇提物和水提物（连续3d）并不能明显抑制肝癌细胞增殖，而灌胃给予0.3g/kg/d走马胎醇提物（连续15d）则可明显抑制肝癌细胞增殖。LC-MS全谱测定发现走马胎含药血清中百两金皂苷B入血困难。制备型HPLC将走马胎大鼠含药血清分段收集，发现其极性较高的富集段可能具有抗肝癌作用。体外转录组学和代谢组学发现走马胎醇提物主要通过影响细胞周期、p53和MAPK信号通路，降低肝癌细胞中多种氨基酸含量、增加不饱和脂肪酸合成、促进谷胱甘肽的氧化等而具有抗肝癌作用。体内灌胃给予走马胎醇提物（0.3g/kg/d，连续15d）和腹腔注射百两金皂苷B（4mg/kg/w）均有一定抑制裸鼠成瘤作用。代谢组学发现走马胎醇提物可能通过升高血清去氧紫草素、丁子香酚、肉桂醛、牛磺酸和前列腺素J2，降低血清L-犬尿氨酸水平而具有抗肝癌作用；百两金皂苷B则可能升高瘤组织内鞘氨醇、维生素B6、苯甲醛和贝母分碱，降低瘤组织内肌酐等含量，升高成瘤裸鼠血清中西奥骨化醇、丁子香酚、红景天苷、黄腐酚、蛇床子素、去铁酮、尿囊素和吲哚-3-乙酸，降低成瘤裸鼠血清中油酸等而具有抗肝癌作用。本研究的完成将推动走马胎在抗肝癌方面的推广和应用，并为中药和民族药的药效物质基础和作用机制研究提供新的思路和方法。共发表论文5篇，培养研究生1名。