益气活血药—槐耳清膏“超常”活化MEK/ERK信号通路介导气滞血瘀证肺癌凋亡的机制研究

Clinical researches show that invigorating qi and promoting blood circulation drug “Huaier extract” could repress cell cycle and induce apoptosis in lung cancer with syndrome of qi stagnation and blood stasis, but the mechanism remains unclear. Recent studies have shown that excessive activation of MEK/ERK signaling pathway caused tumor cells apoptosis. Our previous study expounds that that Huaier extract could induce apoptosis of lung cancer cells, and cause the cellular nucleus overactivation of p-ERK1/2 protein, a core factor in MEK/ERK signaling pathway, accompanied with activation of apoptotic factors like p-P53, p-P38, Caspase-3 and so on. Based on the previous studies, it is hypothesized that the invigorating qi and promoting blood circulation drug “Huaier extract” induce tumor apoptosis in lung cancer with syndrome of qi stagnation and blood stasis through excessive activation of MEK/ERK signaling pathway. In this study, we will establish tumor-bearing nude mice with the syndrome of qi stagnation and blood stasis, and adopt confocal laser scanning microscopy, mass-spectrometric technique and flow cytometry in vitro, in vivo and clinical trial to explore the apoptosis mechanism of excessive activation of MEK/ERK signaling pathway from the point of morphology, function and molecule regulation in lung cancer with syndrome of qi stagnation and blood stasis after using invigorating qi and promoting blood circulation drug “Huaier extract”. Therefore, this project will provide theoretical basis and clues on clinical application of invigorating qi and promoting blood circulation drug “Huaier extract” in lung cancer with syndrome of qi stagnation and blood stasis.

临床研究显示益气活血药-槐耳清膏能够阻遏气滞血瘀证肺癌细胞周期进程，诱导肿瘤细胞凋亡，但其详细作用机制不详。最新研究表明MEK/ERK信号通路的“超常”活化可促使肿瘤细胞发生凋亡。申请人前期研究结果显示：槐耳清膏可诱导肺癌细胞凋亡，同时导致p-ERK1/2蛋白（MEK/ERK信号通路核心因子）的过度激活和向细胞核内的迁移，并伴随p-P53、p-P38及Caspase-3等凋亡因子的活化。据此提出假说：益气活血药-槐耳清膏通过“超常”活化MEK/ERK信号通路介导气滞血瘀证肺癌细胞的凋亡。本研究拟通过构建气滞血瘀证肺癌裸鼠荷瘤模型，在体外实验、动物实验及临床试验中，利用激光共聚焦扫描显微技术、质谱分析技术及流式细胞术等，从形态、功能及分子调控层面，探索益气活血药-槐耳清膏干预MEK/ERK信号通路诱导气滞血瘀证肺癌凋亡的分子机制，以期为槐耳清膏临床治疗气滞血瘀证肺癌提供理论依据和线索。

气滞血瘀证是肺癌重要临床证型之一，临床研究显示益气活血药-槐耳清膏能够阻遏气滞血瘀证肺癌细胞周期进程，诱导肿瘤细胞凋亡，但其详细作用机制不详。最新研究表明MEK/ERK信号通路的“超常”活化可促使肿瘤细胞发生凋亡。本研究通过构建气滞血瘀证肺癌裸鼠荷瘤模型，在体外实验、动物实验及临床试验中，利用激光共聚焦扫描显微技术、RNAseq分析技术及流式细胞术等，从形态、功能及分子调控层面，探索槐耳清膏干预MEK/ERK信号通路诱导气滞血瘀证肺癌凋亡的分子机制。研究结果显示：体外实验中，槐耳清膏通过影响气滞血虚型肺癌细胞内质网膜稳定性，可促使其释放Ca2+进入胞浆，一方面介导MEK/ERK信号通路的“超常”活化，导致其下游P53/P38的激活，另一方面使得肺癌细胞氧化应激调控失衡，两者共同促进肺癌细胞的凋亡；体内实验中，槐耳清膏可以有效抑制肺癌细胞的生长，表现为成瘤体积和重量的下降，同时肿瘤内同样出现MEK/ERK信号通路异常活化，另外早期气滞血瘀证型肺癌患者术中标本中提取的CAFs细胞可介导EGFR核心盐藻糖基化增加肿瘤细胞的转移活性，而术后口服槐耳颗粒可减少肿瘤转移机率。本研究结果提示：气滞血瘀证肺癌发生发展过程中涉及多条恶性生物学行为相关信号通路的激活，益气活血药槐耳颗粒通过干预MEK/ERK信号通路有效控制肺癌恶性生物学行为并可改善患者预后。本研究率先以槐耳清膏为研究基础，以MEK/ERK信号通路超常活化为主线，探讨益气活法对气滞血瘀证肺癌介导的生物学恶性行为及信号调控网络的干预机制。为立足基因水平认知益气活血法治疗气滞血瘀证肺癌的分子机制提供线索，并为槐耳清膏临床治疗气滞血瘀证肺癌提供理论依据和线索。