壮药“国虾薄”热处理产物抗NSCLC有效成分研究

Blocking the apoptotic pathway is the principal cause of the occurrence, development and drug resistance of tumor. Recently it is found that apoptosis induced via death receptor signal transduction pathways and mitochondrial pathways are the main mechanism of anti-tumor treatment. "Gocaekmbaw", the Zhuang-medicine, possesses the effects of through the three channels and two ways, and showed heat clearing and detoxicating activities. In the preliminary studies, we found that the ethanol extract of the heat-processed Gocaekmbaw by high temperature and pressure conditions showed stronger non-small cell lung carcinoma (NSCLC) A549 cells inhibitory activity compared to the crude plant and possessed apoptosis induction effect. However, the active constituents of Gocaekmbaw and their mechanism are not clear yet. In this study, the active constituents are isolated and identified from the heat-processed Gocaekmbaw using the chromatography and spectra data. Using MTT assay, cell morphology assay, flow cytometry analysis, enzyme activity assay, and immunoblotting analysis, the active constituents are demonstrated effects in A549 cell proliferation, apoptosis, cell cycle, expression of apoptosis-related factor of caspase-8, caspase-9 and caspase-3, mitochondrial membrane potential (Δψm) and expression of apoptosis related proteins Bax, Bcl-2 of cytochrome c. It is verified the effect of anti lung cancer of the heat-processed Gocaekmbaw and its active fractions from the animal level using T/C and inhibition rate measured of A549 nude mice. They will be provided a reliable scientific data for the development of new anti-lung cancer drugs.

细胞凋亡通路受阻是导致肿瘤的发生、发展及产生耐药的主要原因，通过死亡受体信号转导途径和线粒体途径诱导细胞凋亡，是近年来发现的抗肿瘤治疗的主要机制。壮药"国虾薄"通三道两路，具有清热解毒的作用。前期研究发现，国虾薄热处理产物与原药材相比具有很强的抑制非小细胞肺癌（NSCLC）A549细胞增殖活性，并诱导细胞凋亡。但其有效成分尚不明确，作用机制尚不清楚。本研究拟运用色谱及波谱等方法，分离、鉴定国虾薄有效成分；运用MTT法、流式细胞术、Western blot技术等，检测有效成分对A549细胞增殖、细胞凋亡、细胞周期、细胞凋亡相关的因子Caspase-8、Caspase-9及Caspase-3的表达、线粒体膜电位及与细胞凋亡相关蛋白Bax、Bcl-2、Cytochrome c的表达；并通过A549裸小鼠移植瘤模型，从动物水平验证国虾薄热处理产物抗肺癌的作用，为开发新型抗肿瘤药物奠定基础。

壮药“国虾薄”热处理产物具有较高的抑制A549细胞增殖作用，其主要活性成分为极性偏小的皂苷类化合物。随着温度的升高，这些成分的含量也逐渐增高；国虾薄提取物通过HP20大孔树脂吸附，20%、50%、70%、95%乙醇洗脱，富集95%乙醇洗脱部位，得到有效部位；通过硅胶柱色谱、反相柱色谱等方法，分离得到11个皂苷类化合物，其中达木林C、达木林D、gypenoside Jh1为新化合物。用CCK-8法检测发现这11个化合物均具有浓度依赖性抑制A549细胞增殖作用，尤是达木林B具有最强的抑制活性。此外，发现达木林B对A549细胞及人正常胚肺成纤维细胞MRC-5具有选择性抑制A549细胞作用，尤其以20ug/mL的选择性最强，24h时就出现明显的选择性。达木林B浓度为20ug/mL 时，A549细胞克隆率低于3%。用流式细胞术及Western Blotting从细胞周期及细胞凋亡两大方面探究达木林B对A549细胞的作用机制，发现Damulin B能有效阻滞A549细胞于G0/G1期；能通过线粒体信号介导的内源性途径诱导A549细胞凋亡。通过构建Lewis肺癌荷瘤C57BL/6J小鼠模型，发现与模型组相比，经灌胃国虾薄总皂苷的小鼠肿瘤体积明显降低，脾指数和胸腺指数明显增加，肿瘤组织内微血管的形成及肿瘤的侵袭转移被明显抑制。划痕及Transwell小室实验发现国虾薄总皂苷含药血清具有较明显的抑制A549细胞的迁移和侵袭能力，促使小鼠机体的免疫细胞由Th2向Th1型逆转而发挥抗肿瘤作用，LCMS-IT-TOF检测其发挥作用的主要成分为gypenoside L、gypenoside LI、达木林B及达木林A等。国虾薄皂苷单体gypenoside LVI灌胃，其代谢产物与国虾薄总皂苷代谢产物相吻合。因此，gypenoside L、gypenoside LI、达木林B及达木林A可以作为先导化合物为进一步设计合成新型的抗NSCLC提供参考。