丹参酚酸/丹参酮类对血小板介导肿瘤血行转移的整合效应研究

As a Chinese Medicine which promoting blood circulation and removing blood stasis, Salvia has an identified antithrombotic effect and was widely used in cardiovascular disease. Its application of frequency in TCM anticancer prescription is also high. Our team had confirmed that salvia can inhibit tumor angiogenesis and metastasis in previous experiments, in addition the hydrophilic phenolic acids and the lipophilic tanshinones have different targets. The review literature shows that the key step of tumor hematogenous metastasis is that platelet functions are hijacked by malignant tumors, which mediate emboli formation and tumor angiogenesis. The mechanism of tumor emboli formation is similar to thrombus formation. Both of them are mediated by abnormal activated platelet. So we give a hypothesis that the active ingredient groups of salvia have an integration effect on tumor hematogenous metastasis. The active ingredient of salvia including natural ratio (A)、phenolic acids (B) and tanshinones (C) will be extracted. (A-B) or (A-C) will be got by the strategy of selective component knockout. The modern techniques such as factorial design combined with ELISA, Western Blot, Realtime PCR, plasmid stability expression analysis technology will be used to confirm how Salvianolic acid and tanshinone have an effect on the key step of tumor hematogenous metastasis mediated by platelet (platelet activity and aggregation, tumor cells adhesion and migration, tumor angiogenesis). Methods of mathematical pharmacology will be applied to analyze whether the synergistic effect of combining two parts components is bigger than simple sum of them. This project provides a support for clinical application of salvia from a new perspective.

活血化瘀药丹参抗血栓作用明确，在心血管疾病中广泛应用，抗癌方中应用频度也很高，课题组前期实验证实丹参对肿瘤血管生成和转移有抑制作用，且脂溶性丹参酮类和水溶性丹酚酸类作用靶点不同。纵观文献可知，肿瘤发生血行转移关键环节是血小板功能被恶性肿瘤劫持，从而介导癌栓形成和肿瘤血管生成。而癌栓形成机制与血栓相似，均由异常活化血小板介导，因此课题组提出丹参通过两组分组合对血行转移产生整合效应的假说。拟提取丹参活性成分天然配比A、丹酚酸类B、丹参酮类C，利用选择性成分敲除策略获得A-B和A-C，采用析因设计结合酶联免疫、Western Blot、Realtime PCR、质粒稳转表达分析等技术，确认丹参酚酸类和丹参酮类对血小板介导肿瘤血行转移中关键环节（血小板活化、聚集，肿瘤细胞黏附、迁移、肿瘤血管生成）的影响，应用数学药理学的方法分析两组分组合是否产生大于加和的协同作用，从新的视角为临床应用丹参提供支撑

活血化瘀药广泛运用于肿瘤治疗。肿瘤转移过程复杂而活血化瘀类药物种类繁多。将丹参作为代表性活血化瘀中药，按照脂溶性、水溶性成分分类，选择各自代表成分丹参素、丹酚酸B、隐丹参酮以及丹参酮IIA等主要活性成分进行研究，探究两类不同活性成分对血小板聚集、活化和粘附的效应，以及对血小板介导的肿瘤细胞相关粘附、整合素β1、β3、mmp2/9、TIMP和CXCR4 mRNA水平的影响；建立小鼠黑色素瘤、肝癌、乳腺癌等体内外模型。初步发现丹参水溶性成分如丹参素对血小板与肿瘤细胞的粘附作用明显，其机制主要是通过下调Integrinβ3，MMP2/9和CXCR4表达发挥作用，丹酚酸B通过调节AT1R蛋白表达和VEGFA产生从而抑制肝癌血管生成；而丹参脂溶性溶性成分如丹参酮IIA、隐丹参酮对血小板活化的作用要明显，隐丹参酮主要是通过调控NF-κB和STAT3信号而调节TNF-α的表达，此外还通过促进血管内皮细胞内源性线粒体凋亡信号通路最终诱导内皮细胞的凋亡。.基于前期研究和文献调研，还对丹参水溶性酚酸类成分丹参素的加强肿瘤免疫功能进行了探索。将人乳腺癌ZR-75-1细胞和NK细胞共孵育，并外源性给予TGF-β1来排除其它血小板释放物的干扰，探讨NK细胞对ZR-75-1细胞杀伤效应，研究血小板释放因子TGF-β1对NK细胞肿瘤免疫监视的干预作用。结果显示，丹参素能有效抑制血小板释放的TGF-β1含量，逆转TGF-β1对NKG2DL-NKG2D信号轴的抑制作用，恢复NK细胞的抗肿瘤活性，抑制肿瘤的发生发展。.由此可见，丹参不同类成分能够干预肿瘤迁移、侵袭，尤其是血管生成的各个环节，通过不同靶点发挥血管生成抑制效应从而抑制肿瘤转移。其中丹参酚酸类成分作用更倾向于抑制血小板与肿瘤细胞的粘附，丹参酮类成分作用更倾向于抑制血小板活化，两类成分对血小板与肿瘤细胞是作用于不同环节但相互协同，而在抑制血管生成方面是通过不同途径发挥相同作用，最终在血小板介导的肿瘤转移过程中发挥整合效应。