基于抑制硫氧还蛋白还原酶活性的甘肃道地药材抗癌成分筛选

The homeostasis of redox is a crucial foundation for the cells to undertake the normal physiological activites. It has been demonstrated that the small protein system, which is composed of the thioredoxin(Trx), the thioredoxin reductase (TrxR) and the reductive coenzymes II(NADPH) in cells of human and mammalian, plays a cardinal role in maintaining the redox balance. The massive evidence showed that over-expression of Trx and TrxR have been observed in tumor cells and it has emerged as a target of the drug design. It has been reported that the Gansu geoherbs such as Radix Astragali and Radix Angelicae Sinensis displayed the remarkable effectiveness to the anti-cancer assay. However, the mechanism underlying the anti-cancer is currently still elusive. In this study, the anticancer screening will be performed in a variety of tumor cell lines and tumor-bearing mice via determination of the inhibition of TrxR by the various components isolated from Gansu genuine herbs using the biochemistry, molecular biology and immunohistochemical etc. strategies. This research aims to obtain the anti-tumor effective ingredients from Gansu geoherbs and investigate the expression and activity of TrxR in vitro and in vivo within cells as well as whole organism treated with the active ingredients. The results of this study will elucidate the anticancer mechanism of Gansu genuine herbs and be the potentiality for promoting the development of local herbs medicines. Moreover, it possesses the significance for accelerating the Chinese medicinal herbs into the international market.

氧化还原平衡是细胞完成正常生理活动的重要基础。由硫氧还蛋白（Thioredoxin，Trx）、硫氧还蛋白还原酶（Thioredoxin reductase，TrxR）和还原性辅酶Ⅱ（NADPH）构成的小分子蛋白系统在人和哺乳类细胞内的氧化还原平衡维持中起非常重要的作用。大量研究表明Trx和TrxR在肿瘤细胞中过表达，因而是目前肿瘤药物设计的靶标蛋白。甘肃多种道地药材如黄芪、当归等报导具有明显的抗癌药效，但抗癌机理不明。本研究拟采用生物化学、分子生物学及免疫组化等方法，以多种肿瘤细胞系及荷瘤小鼠为材料，以是否抑制TrxR活性为选择标准，对分离纯化得到的药材成分逐一进行抑癌活性筛选，以期得到甘肃道地药材的抗肿瘤有效成分，并揭示这些有效成分在细胞和整体水平、体内和体外环境抑制TrxR表达和活性的规律。研究结果对阐明甘肃道地药材抗癌机理、针对性开发地方药材品种和加快中药材走向国际市场具有重要意义。

大量资料表明TrxR在肿瘤细胞中过表达，是目前肿瘤药物设计的靶标。甘肃道地药材黄芪、红芪等报导有明显的抗癌活性，但抗癌机理不明。本研究采用生物化学、细胞生物学及分子生物学等方法，在建立工艺提取分离并纯化黄芪多糖、黄芪黄酮、红芪多糖与红芪黄酮的基础上，以四种肿瘤细胞系及荷瘤小鼠进行抑癌活性筛选。以黄芪多糖、黄芪总皂苷、槲皮素、DLF、黄芪黄酮、白藜芦醇、青蒿素在细胞水平分别对四种肿瘤细胞生长增殖影响及对TrxR2 表达进行体外研究， MTT法检测活性成分对四种肿瘤细胞的半数抑制浓度（IC50）。以黄芪多糖、大黄素、DLF对荷H22小鼠肝癌细胞株皮下移植瘤昆明小鼠进行体内研究。FQ-PCR检测黄芪多糖、大黄素、DLF对小鼠肿瘤组织TrxR2、ASK1、Bcl-2及caspase-3表达影响。建立了适宜于提取红芪与黄芪多糖的回收机组法工艺流程。响应面分析法在单因素试验基础上优化提取工艺，获得红芪总黄酮与黄芪总黄酮提取最佳工艺条件，经HPD722和聚酰胺大孔吸附树脂联用纯化红芪总黄酮与黄芪总黄酮，获得较高含量红芪总黄酮与黄芪总黄酮，为更好的开展药理活性研究奠定基础。体外试验研究结果显示活性成分对四种肿瘤细胞抑制生长增殖以及抑制TrxR2 表达。黄芪多糖作用Hela细胞（IC50 61.19μg/mL）与HepG2细胞（IC50 43.54μg/mL）24h后，黄芪多糖显著抑制TrxR2表达(p＜0.05)。体内试验研究结果表明黄芪多糖、大黄素、DLF对荷H22小鼠肝癌细胞株皮下移植瘤昆明小鼠具有抑瘤效应，并抑制musTrxR2基因表达。结果表明在细胞或整体水平黄芪多糖、黄芪总皂苷、大黄素、槲皮素、DLF、黄芪黄酮、白藜芦醇、青蒿素有抗肿瘤活性，抑制TrxR2基因表达。研究结果对阐明甘肃道地药材抗癌机理、针对性开发地方药材品种和加快中药国际化具有重要意义。