土家药鸡血七抗乳腺癌药效物质基础及 CyclinD1-CDK4/6-Rb通路介导的作用机制研究

Breast cancer is one of the most common malignant tumors in women all over the world. In recent years, the development of CDK4/6 inhibitors have made significant progress in the treatment of breast cancer. Jixue Qi is the classic “Qi” and “Blood” drug of Tujia ethnomedicine and it has unique effects on the mammary cancer caused by qi stagnation and blood stasis. Our group have isolated some flavones with unique new skeleton and their precursors. In vitro anti-breast cancer cells proliferation experimental results revealed that the effect of flavone in Jixue Qi are comparable to the effects of taxol, and pre experiment results showed the flavones in Jixue Qi down-regulate some proteins involved in Cyclin D1-CDK4/6-Rb signaling pathway in breast cancer cell lines, but its pharmacodynamic material basis and mechanism are not clear yet. This study intends to Jixue Qi as the research object, anti proliferation model of breast cancer cell lines guided isolation and rapidly identify the target compounds by UPLC-Q-TOF-MS and NMR were used to discover the active compounds, and advanced isolation methods were used to obtain the active compounds. In vitro and in vivo models of anti-cancer were used to confirm the anti breast cancer effect of Jixue Qi, analyze the expression of genes and proteins involved in D1-CDK4/6-Rb signaling pathway to discover its potential mechanisms of anti-breast cancer effects. this research will contribute to the application of Jixue Qi for anti-breast cancer and lay the material and theoretical foundation for the clinical application of Jixue Qi.

乳腺癌是全球女性最常见的恶性肿瘤，近年CDK4/6抑制剂在乳腺癌治疗上取得重大进展。鸡血七是土家族经典“七药”和“血药”，能舒筋活血、行气镇痛，对气滞血瘀所致的“乳岩”疗效独特。课题组前期从鸡血七中分离到具有独特新骨架的黄酮及其前体，体外抗乳腺癌细胞增殖显示效果与紫杉醇相当；且其对乳腺癌细胞株CyclinD1-CDK4/6-Rb信号通路相关蛋白有下调作用，但其物质基础和作用机理不明。本研究拟以鸡血七为研究对象，通过乳腺癌细胞株体外增殖模型为活性导向，利用UPLC-Q-TOF-MS和NMR快速识别目标化合物，运用现代分离手段获取鸡血七中活性成分，以抗乳腺癌体内外模型研究鸡血七黄酮的抗癌效果，应用现代生物化学技术对CyclinD1-CDK4/6-Rb信号通路相关蛋白和基因的影响进行检测，揭示其抗乳腺癌作用的物质基础，阐明其抗乳腺癌分子作用机制，为该民族药的临床应用奠定物质理论基础。

土家药鸡血七 (Pronephrium penangianum (Hook.)Holtt.) 系金星蕨科披针新月蕨属植物披针新月蕨的干燥根茎，又名“鸡血莲”、“土当归”等，性凉味苦，能活血调经、散瘀止痛、除湿，民间广泛用于月经不调、跌打损伤、风湿痹痛等疾病的治疗。现代研究表明，鸡血七具有抗炎、抗氧化、抗肿瘤等药理活性。.本项目从鸡血七中分离并鉴定50个化合物，其中有10个新化合物，包括2个特殊结构的红色化合物及一系列鸡血七中特有的黄烷-4-醇类化合物。活性研究表明部分化合物具有良好的抗癌活性，四种已知黄烷4-醇糖苷abacopterins A和C、eruberin B和triphyllin A对多种细胞具有中等强度的抗增殖活性。abacopterins A和C、eruberin B和triphyllin A对HepG-2细胞系的IC50值分别为20.02, 28.29, 21.93和31.77 μM；对HCT-116细胞系的IC50值分别为15.77, 15.50, 19.61和25.17 μM； 对BGC-823细胞系的IC50值分别为18.43, 20.77, 24.13和21.66 μM；abacopterins A和C 对MCF-7细胞系有抑制作用，其IC50值分别为38.74和54.13 μM。.运用一种新型荧光增强DNA模板Ag簇纳米材料tween 80自组装作为检测Cyt c的探针，检测了Dox和鸡血七中分离的单体化合物(2S,4S)-6,8-dimethyl-7-hydroxy-4′-methoxy-4, 2″ -oxidoflavan-5-O-α-D-glucopyranoside（J5）治疗引起的凋亡肿瘤细胞释放Cyt c的能力。结果发现在HCT-116细胞系中J5诱导的Cyt c释放仅次于阳性对照药，同时采用MTT法检测J5对HCT-116细胞系的影响，结果显示J5对HCT-116细胞系表现出较高的细胞毒性，IC50为2.5 μM，这一结果暗示了J5作为一种有前途的抗结肠癌候选药物的可能性。.开发了一种新的药物传递系统，利用从鸡血七中分离的化合物(J5)治疗宫颈癌。以普鲁士蓝纳米粒子为载体，以红细胞膜为伪装，以叶酸为表面修饰剂，建立给药体系，并将其运用于宫颈癌体内外模型的化疗和光热联合治疗，实验结果显示这种新的药物传递系统增强了单体化合物J5的