基于PKM2靶点的青砖茶调控糖酵解药效物质及作用机制研究

Cancer is a kind of metabolic disease. Glycolysis is the main cause of infinite proliferation of cancer cells. M2-type pyruvate kinase (PKM2) is a central point of regulation in the process of glycolysis which is overexpressed in cancer cells. PKM2 is associated with cancer proliferation, glycolysis and metastasis. In recent years, how to obtain new activators which made a lowly active PKM2 become into a highly active PKM2 has become one of research hotspots in colorectal cancer. Qingzhuan dark tea is a unique post-fermented tea. It is not only an indispensable beverage but also a traditional Chinese medicine for the people in the northwest of China. Our pre-study found that the secondary metabolites of Qingzhuan dark tea and young leaves were significantly different. The ethanolic extracts of Qingzhuan dark tea showed inhibitory effects on the proliferation and glycolysis of colorectal cancer cells. It is expected to find new natural PKM2 activators from the active components of Qingzhuan dark tea which has therapeutic potential in colorectal cancer. In our future study, firstly, the purification and isolation of active components of Qingzhuan dark will be guided by HPLC. Secondly, the inhibitory activities of colorectal cancer proliferation and glycolysis of isolated compounds will be evaluated on cells SW480 and SW620. Finally, the mechanisms of active compounds of Qingzhuan dark tea will be studied by biology techniques such as western blotting analysis and quantitative real-time polymerase chain reaction (qPCR). This project is expected to find new lead compounds with regulating glycolysis based on PKM2 activation, which provide pharmacy and pharmacology basis for developing novel candidate drugs targeted on the treatment of colorectal cancer.

M2型丙酮酸激酶（M2 type pyruvate kinase, PKM2）是肿瘤糖酵解的关键酶，在肿瘤细胞内过表达且与肿瘤增殖、糖酵解和转移密切相关。寻求将低活性的PKM2转变为高活性的PKM2激动剂已成为结肠癌新药研究的热点。青砖茶是一种独特的真菌后发酵茶，具有多种药理作用。我们组前期对比研究中首次发现青砖茶与其发酵前原料的次生代谢物成分有明显差别，且青砖茶提取物能明显抑制结肠癌细胞增殖和糖酵解，有望从中发现新颖的天然PKM2激动剂。本课题将以青砖茶的差别成分和抑制糖酵解活性为依据，采用谱学跟踪，定向分离纯化青砖茶的活性组分，同时以结肠癌细胞SW480和SW620为模型，筛选抑制结肠癌细胞增殖和糖酵解作用的化合物，并通过蛋白印迹法、qPCR及免疫共沉淀等手段来初步探讨活性化合物在细胞内作用的机制。期望发现有效的先导化合物，为发现新颖的治疗结肠癌活性物质提供药学和药理学依据。

青砖茶是以老青茶为原料，经长时间独特真菌发酵后压制而成。饮用青砖茶，不仅具有预防和治疗高血脂、糖尿病等疾病的功效，还具有显著减肥、抗菌以及抗癌等功效。而老青茶功效不如青砖茶。青砖茶的功效成分系统地分离纯化研究并不多，且青砖茶的各种功效仅限于人们的认知，有针对性的研究尚且不足。因此，阐明青砖茶的药效物质基础和作用机制有利于青砖茶这一宝贵地方传统资源的利用。.本项目以老青茶和青砖茶的次生代谢物和活性为依据，从而获得高活性的抑制糖酵解分子并研究其分子作用机制，进而为治疗结肠癌提供天然的药物先导小分子。具体研究内容及重要结果如下：.1、完成了青砖茶差别成分的提取、粗分离和粗组分结肠癌细胞生物功能测试，利用生物活性和现代谱学追踪对活性组分进行了有效地分离纯化，最终鉴定了33个差别化合物，化合物类型主要是三萜类和多酚类；.2、对单体化合物进行了抑制结肠癌细胞SW480、SW620和DLD-1的生物活性筛选，发现了化合物8和17在浓度为50μmol/L时有较好的抑制活性，其它化合物在浓度100μmol/L下没有显示出明显的活性，从而为进一步的作用机制研究提供了基础。.3、研究了不同浓度的化合物8和17对结肠癌细胞SW480和DLD-1活性的影响、迁移能力的影响以及葡萄糖消耗量、乳酸生成量的影响。发现了化合物8和17能够抑制 DLD-1和SW480细胞的活性，并且这一抑制作用随着药物浓度的升高、作用时间的延长而增强，呈现浓度、时间依赖性。50μmol/L单体化合物8和17作用于24h后迁移的SW480和DLD-1细胞的细胞数目百分比明显减少，但是这两个化合物对结肠癌细胞葡萄糖消耗量、乳酸生成量，结果无显著差异。关于其分子机制有待进一步研究。.4、发现了其他具有抑制糖酵解的化合物。共发表论文1篇和授权发明专利3项。.上述结果为地方特色产业青砖茶的大力开发和应用提供理论支持和数据参考，为开发新一代天然的高效低毒的治疗结肠癌的药物小分子奠定了基础。