基于MAPK/ERK和PI3K/AKT信号通路交互串话研究小檗碱联合芒柄花黄素协同抑制鼻咽癌的分子机制

Berberine and formononetin have broad spectrum antitumor effects, and there is no report on the synergistic antitumor effect of the combination of two monomers. Nasopharyngeal carcinoma is the most common malignant tumor in the head and neck in China. Berberine can improve the sensitivity of radiotherapy for nasopharyngeal carcinoma and inhibit the proliferation, tumor formation and migration of nasopharyngeal carcinoma cells. Our new results showed that berberine and formononetin have synergistic effect on inhibiting proliferation and inducing apoptosis of nasopharyngeal carcinoma cells, and significantly reduced the expression of p-MEK, p-ERK1/2, PI3K and p-AKT protein. In this study, by NPC cells cultured in vitro and nasopharyngeal carcinoma cell nude mice model, the effects of berberine combined with formononetin on the proliferation, cell cycle, apoptosis, migration and invasion of nasopharyngeal carcinoma cells will be investigated using real-time cellular analysis, flow cytometry, overexpression of gene and RNAi, CoIP, laser scanning confocal microscope, electron microscope and Western blot, and the optimum compatibility of berberine and formononetin concentrations on anti nasopharyngeal carcinoma will be screened. By setting the activator group and inhibitor group, key gene knockout group and high expression group, the molecular mechanism underlying the combined action of berberine and formononetin will be elucidated partly through the crostalk between MAPK/ERK and PI3K/AKT signaling pathways. The study will provide experiment basis for the clinical application of berberine combination with formononetin in the treatment of nasopharyngeal carcinoma, and provide a new model for the research on the application of Chinese medicine monomer.

小檗碱和芒柄花黄素联合应用是否具有协同抗肿瘤效应尚无报道。鼻咽癌是我国最常见头颈部恶性肿瘤，小檗碱能提高鼻咽癌放疗敏感性，抑制鼻咽癌细胞增殖、成瘤和迁移。课题组发现小檗碱联合芒柄花黄素具有协同抑制鼻咽癌细胞增殖和诱导凋亡的效应，同时显著降低p-MEK、p-ERK1/2、PI3K和p-AKT蛋白表达。本研究拟通过体外培养鼻咽癌细胞和移植瘤裸鼠模型，采用实时无标记细胞功能分析技术、流式细胞术、基因过表达和RNAi技术、CoIP、激光共聚焦、电镜和Western blot等探讨小檗碱和芒柄花黄素联合应用对鼻咽癌细胞增殖、凋亡、迁移和侵袭的影响，筛选出协同抗鼻咽癌的合适配伍浓度；通过抑制剂、激活剂、AKT和ERK双基因沉默和过表达，从MAPK/ERK和PI3K/AKT信号通路交互串话阐明药物联合作用部分机制，为小檗碱联合芒柄花黄素治疗鼻咽癌的临床应用提供实验依据，为中药单体联合应用研究提供新模式。

鼻咽癌高发于中国华南地区，严重威胁我国人民生命健康。早期鼻咽癌治疗效果好，然而70%的患者就诊时已为中晚期，治疗费用高且效果差。寻找安全有效的治疗药物是鼻咽癌领域研究的重要问题。小檗碱联合芒柄花黄素是否具有抗鼻咽癌的协同效应，其作用机制是什么。项目采用了细胞培养、HE染色、Western-blot、免疫荧光和流式细胞术等技术分八部分进行了研究，主要结果如下：（1）明确了小檗碱联合芒柄花黄素能够抑制鼻咽癌细胞增殖、迁移、侵袭和诱导凋亡的协同效应，协同抑制鼻咽癌细胞移植瘤生长；（2）小檗碱联合芒柄花黄素能够通过线粒体途径和内质网途径协同诱导鼻咽癌细胞凋亡；（3）小檗碱联合芒柄花黄素通过调控MAPK/ERK1/2信号通路介导的自噬抑制鼻咽癌细胞增殖；（4）小檗碱联合芒柄花黄素能够通过调控MAPK/ERK1/2、TGFβ1/ERK1/2、和VEGFC/VEGFR3信号通路，下调鼻咽癌细胞迁移与侵袭相关蛋白和下调移植瘤组织血管形成和淋巴管形成相关蛋白表达，抑制鼻咽癌细胞迁移与侵袭；（5）小檗碱联合芒柄花黄素对PI3K/AKT信号通路的影响不显著；（6）小檗碱联合芒柄花黄素能够通过下调LINC00319抑制鼻咽癌细胞增殖、迁移与侵袭和诱导凋亡。提示小檗碱与芒柄花黄素的联合应用可作为治疗鼻咽癌的方案，其作用机制与调控MAPK/ERK1/2、TGFβ1/ERK1/2和VEGFC/VEGFR3信号通路和LINC00319密切相关。本项目已发表期刊论文17篇，其中SCI论文6篇，1篇论文获得奖励；参加国内会议19人次，省内会议10人次；会议报告2次，参加国际会议1次；申请专利2项，获批专利1项；培养毕业硕士研究生4名，今年拟毕业博士研究生1名。