“大气下陷”视角下升陷汤干预肺癌播散肿瘤细胞定植血管周围小生境的生物力学行为研究

Disseminated tumor cells (DCC) colonize the perivascular niches was recognized to the beginning of the lung cancer metastatic colonization. " pectoral qi collapse " was proposed to better understand the biomechanical property changes in the thoracic cavity, which correlates with the metastatic behavior of lung DCC’ shedding, circulatory extravasation, and colonization around the perivascular niche. As the representative prescription for " Invigorating qi and ascending qi collapse " treatment, the Shengxian decoction has been demonstrated notable therapeutic effect on lung cancer metastasis, and previous study has shown that the Shengxian decoction affect the biomechanical of the A549 cell. Recent studies have found that overexpression of L1 cell adhesion molecules (L1CAM) can enhance the potential of metastatic colonization through activating the mechanical transduction effectors of YAP/MRTF and resulting in the lung DCC replacing the pericytes. Therefore, a reasonable hypothesis was proposed that the Shengxian decoction could intervene the lung metastasis by affecting the DCC colonization around the perivascular niche, where the cascade reaction of ILK-YAP/MRTF was blocked due to the inhibition of the L1CAM expression. To further examine this hypothesis, we designed a series of experiments to investigate the lung cancer metastasis model in vivo and lung microvascular niche model in vitro. By monitoring the biomechanical behavior of A549-LM and the location and expressions of L1CAM-ILK-YAP/MRTF pathway factors around the perivascular niches, the scientific connotation of " invigorating qi and ascending qi collapse " can be revealed.

播散肿瘤细胞（DCC）定植血管周围小生境是肺癌转移性定植的启动事件。“大气下陷”是胸腔生物力学特性变化的中医认识，与肺癌DCC脱落、外渗、定植血管周围小生境的转移行为表征关联。“益气升陷”代表方升陷汤干预肺癌转移疗效确切，且前期研究发现其与细胞的生物力学有关。最新研究表明L1细胞黏附分子（L1CAM）过表达可激活YAP/MRTF机械转导效应器，导致肺癌DCC竞争性取代周细胞，促进转移性定植发生。据此提出研究假说：升陷汤通过抑制L1CAM表达，调控ILK-YAP/MRTF级联反应，干预肺癌DCC定植血管周围小生境的生物力学行为，阻延转移的发生。本项目拟构建肺转移小鼠模型及肺微血管小生境体外模型，通过监测A549-LM在血管周围小生境中生物力学行为以及L1CAM-ILK-YAP/MRTF通路因子定位表达，揭示升陷汤干预肺癌DCC定植血管周围小生境的调控机制，阐释“益气升陷”治法的科学内涵。

升陷汤是一种由黄芪、知母、柴胡、桔梗、升麻组成的中药方剂，具有益气升阳之功效。然而，升陷汤的抗癌作用却鲜有报道。在此，我们基于网络药理学及实验验证，探讨了升陷汤治疗肺腺癌的治疗潜力与作用机制。项目组首先使用高效液相色谱（HPLC）鉴定了升陷汤水煎液的化学指纹图谱，明确了升陷汤标准煎液的物质基础。然后，基于网络药理学对升陷汤在肺腺癌治疗中的关键靶点、信号传导路径等进行了研究。即通过运用PharmMapper查询活性成分对应靶点。通过肿瘤基因组图谱（TCGA）得到肺腺癌（LUAD）差异表达基因，并补充5个在线平台的靶基因数据。而后取两者靶点交集，利用STRING数据库实现与二者共同靶点的蛋白相互作用的PPI网络构建，并通过网络可视化软件Cytoscape 3.9.0绘制关联网络图。最后，通过体外验证升陷汤及其含药血清对人肺腺癌细胞的体外增殖抑制作用，并通过裸鼠的肿瘤形成实验证实了升陷汤对肺腺癌的抑制活性及其作用机制。HPLC分析结果表明，升陷汤中存在包含新芒果苷、芒果苷、毛蕊异黄酮葡萄糖苷、异阿魏酸、木犀草素、芒柄花素和柴胡皂苷A在内的多种抗癌活性成分，提示其具有一定的抗癌潜能；网络药理学研究表明升陷汤治疗肺腺癌的潜在靶点基因有197个，PPI网络关键靶点主要为ALB、AKT1、EGFR、SRC、HSP90AA1、CASP3和MMP9等，且7种活性成分与关键靶点均具有较高的结合活性，主要通过抑制细胞增殖、调节凋亡与免疫来达到治疗肺腺癌的目的；体外实验结果表明，升陷汤对人肺腺癌细胞A549具有增殖抑制作用，同时血清药理评价显示升陷汤含药血清对多种人肺腺癌细胞（A549、SK-LU-1和NCl-H1975）具有细胞毒性作用。同时，升陷汤的肿瘤抑制活性也通过裸鼠体内的肿瘤形成试验得到证实。结果表明，升陷汤治疗可诱导肿瘤坏死，并下调血清中缺氧诱导因子HIF-1α。升陷汤的体内生物安全性评估显示小鼠模型的毒性水平较低。我们的研究首次提供了升陷汤有效抑制肺腺癌细胞生长的科学证据，因此可为肺腺癌治疗开发新型抗癌药物。