基于TLRs/NF-κB通路的藏药镰形棘豆抗肿瘤作用机制研究

Researchers suggest that tumor may originate from chronic inflammation as early as 19th century. Recent evidence from xenograft models and human trials has also demonstrated that inflammation plays an important role in tumorgenesis and progression. Oxytropis falcata Bunge (O. falcata) is honored as "the king of herbal medicine" in Tibetan medicine for its prominent activities. Previous studies revealed that this herb possesses analgesic and anti-inflammatory effects. In addition, O. falcata exerts good antitumor activities in vitro. However, its antitumor mechanism and the relationship between antitumor and anti-inflammation remain unclear. In this project, we aim to study its antitumor mechanism from a novel viewpoint. Our study will focus on the Toll-like receptors that are the beginning of cytokine expression. Additionally, TLRs/NF-κB signaling will be the target pathway, which is closely associated with inflammation and tumor. In these experiments, several specific ligands and inhibits of this pathway will be used as control. The studies will be carried out to explore the effects of O. falcata on regulating of upstream receptors, key linkers and downstream factors on TLRs/NF-κB pathway in vitro. The antitumor activity of O. falcata will also be confirmed using mouse xenograft models. In general, we strive to reveal the molecular mechanism of antitumor effect of O. falcate by exploring the potential target and the possible intervention on TLRs/NF-κB signaling pathway, and to seek the interior relations between its anti-inflammatory and antitumor activity. This study may provide the further medicinal value and scientific connotation of Tibetan medicine O. falcata. It is also expected to provide a new approach for national drug innovation and modernization.

早在19世纪学者就提出肿瘤可能源自慢性炎症。现代研究表明炎症在肿瘤的发生发展中起着重要作用，近年来炎症与肿瘤的关系引起了人们的重视。藏药镰形棘豆因疗效突出享有草药之王的美誉。前期研究表明镰形棘豆除传统的镇痛抗炎功效外，还具有良好的抗肿瘤作用，但抗肿瘤机制如何，与抗炎之间有何关联尚不明晰。本项目从细胞因子表达的源头TLRs入手，选择与炎症和肿瘤密切相关的TLRs/NF-κB信号通路为靶点，以通路特异性配体和阻断剂为对照，采用现代分子生物学方法，研究镰形棘豆对肿瘤细胞TLRs/NF-κB通路的上游受体、中间关键环节和下游因子表达的影响，比较体内外对此通路影响的异同，探讨镰形棘豆的作用靶点和可能的干预环节，寻找其抗炎和抗肿瘤作用间的内在联系，以期从分子水平揭示镰形棘豆抗肿瘤的作用机制。开展本项研究可以进一步开发镰形棘豆的药用价值，深入挖掘民族药的科学内涵，为民族药的创新研发和现代化开辟新的途径。

本研究对藏药镰形棘豆国内外相关文献资料进行了综述，并基于TLRs/NF-κB通路对镰形棘豆抗肿瘤作用机制进行了研究。首先提取、分离得到镰形棘豆生物碱、黄酮和挥发油提取部位，并对三个部位进行了定性和定量分析。体外实验证明，生物碱、黄酮和挥发油对A549肺癌细胞均有显著的抑制作用；其中，以挥发油抑瘤效果最好。进一步的研究表明，挥发油抑制A549细胞生长的机制可能与阻断TLRs /NF-κB通路有关；另外，还与其下调炎症因子IL-1β、IL-6、TNFα、VEGF、MMP-2的表达有关。通过对镰形棘豆黄酮提取物进一步分离，得到了5个黄酮类单体成分；其中7-羟基二氢黄酮（7-HF）和2', 4'-二羟基二氢查尔酮（2', 4'-DDC）对A549肺癌细胞均有较好的抑制作用；且能抑制LPS诱导的A549细胞生长，其抑制肿瘤细胞生长的机制可能与下调TLR4、MyD88、TRAF-6、NF-κB蛋白水平有关；同时，7-HF和2', 4'-DDC还可通过下调LPS诱导的炎症因子IL-1β、IL-6、TNFα、VEGF、MMP-2的分泌实现抗肿瘤作用。镰形棘豆生物碱（OFA）和黄酮（OFF）含药血清均能促进A549细胞凋亡，呈现剂量依赖性；OFA和OFF的含药血清均阻滞A549的生长在G0/G1期。另外，镰形棘豆醇提物、生物碱和黄酮提取物对Lewis肺癌荷瘤小鼠均有显著的抑瘤作用，其中生物碱的抑瘤作用最为显著，高剂量组的抑瘤率达40.2%，与阳性对照药CTX作用相当；均可诱导小鼠体内Lewis肺癌细胞凋亡，其中生物碱组诱导凋亡比例较高，高剂量组达到55.3%。生物碱、黄酮活性部位均将细胞生长阻滞在S期。生物碱对小鼠Lewis肺癌细胞抑制作用可能与阻断其TLRs/NF-κB通路相关。综上结果，镰形棘豆抗癌作用可能与其干预和逆转炎癌转化机制有关。