紫草素干预白血病细胞生长的巯基修饰机制研究

Previous prescriptions contained extracts from Zicao "Lithospermum erythrorhizon Sieb. et Zucc" in TCM had been proved their chemotherapeutic effects on leukemia, which were closely related to "Blood cooling and Circulation invigorating" in TCM. Originally, the drug-induced differentiation and apoptosis were seemed as two mechanisms differing in leukemia chemotherapy. However, currently, those two mechanisms were found related to intracellular redox homeostasis at various levels. Recently, our group and others' found the leukemia cells were sensitive to Shikonin among all tumor cell lines. The compound was further proved its stronger thoil-modified activities. For example, Shikonin could induced leukemia cell differentiation and apoptosis. The mechanism(s) involved into this cytotoxicities become an urgent theoretical problem. Thus current project aim to determine the role of cellular redox homeostasis and the involvement of thiols modification in association with Shikonin treatments in previous setup leukemia differentiation and/or apoptosis modes. Any breakthrough in the project will not only help to understand the reason of the Shikonin special cytotoxicity on leukemia cells, but also gives additional evidences and supports to "The redox regulation theory".

一些含紫草方剂具有治疗白血病及防止复发的作用，这种作用被认为与紫草"凉血活血"的功效密切相关。一般认为白血病细胞的诱导分化与凋亡是药物化疗的两种机制，但后来研究发现二者在不同层次与细胞内氧化还原态变化有关。近年来，我们和其它研究者陆续发现白血病细胞对紫草素敏感性远高于其它肿瘤细胞株，且紫草素具有明显的巯基反应特性和氧化还原通路的调控特性，其既诱导白血病细胞分化又能引发凋亡。紫草素通过怎样的机制来发挥这些作用成为急需探索的一个重要理论问题。本项目拟利用前期工作建立的白血病细胞分化/凋亡评价模型，在细胞氧化还原态变化及巯基修饰性上对紫草素抑制白血病细胞作用的可能机制进行系统研究。本项目的突破不但有助于深入理解白血病细胞对紫草素极其敏感的化学实质，而且是对"氧化还原调控理论"的丰富和补充。

急性白血病细胞对源自紫草的小分子迈克尔反应受体化合物譬如紫草素、阿卡宁、乙酰基紫草素具有高度的敏感，这些分子被认为白血病的化疗中潜在的候选药物。本项目旨在细胞氧化还原态变化及巯基靶向性上对紫草素抑制白血病细胞作用的可能机制进行系统研究。结果显示紫草素浓度依赖性抑制HL-60、U937细胞增殖，诱导细胞从分化到凋亡，其IC50比其它两个紫草萘醌更低。紫草素处理组观察到显著地谷胱甘肽消耗及Nrf2-ARE途径激活。进一步通过氧化还原稳态切换及巯醇蛋白结合实验发现紫草素对巯基分子有直接地化学修饰作用。Nrf2下游基因譬如HMOX1, GCLC, GCLM, NQO1的上调证实了紫草素的潜在Nrf2激动作用。Nrf2激动剂对紫草素引起的HL-60及U937细胞分化有正协同作用，通过Nrf2的shRNA沉默或加入抑制剂后发现负协同作用。通过网络药理学发现紫草素不仅对Nrf2途径有直接作用，而且间接通过其它蛋白相互作用来影响该通路，总共23个差异基因及45个癌基因在其中扮演重要角色。结果揭示了鸟苷酸单磷酸合成酶（GMPS）是调控白血病细胞从分化到死亡的关键分子。紫草素能浓度依赖性的层级化开启内质网应激（ER-stress）途径。GSK2656157抑制内质网应激的同时增加了紫草素的诱导分化作用，MG132增加了内质网应激的同时增加了紫草素的诱导凋亡作用。所有这些证据表明萘醌尤其是紫草素抑制AML细胞增殖作用源于其化学方面消耗巯醇类分子。这产生了对细胞氧化还原稳态的扰动，进而开启了Nrf2/ARE途径以及内质网应激途径，使得细胞从分化切换到凋亡。这些发现不但有助于理解紫草素对白血病细胞特殊的细胞毒性，更提供了氧化还原调控理论的证据，这有助于理解中药凉血的辩证机理。