基于肿瘤炎性微环境的消癌解毒方的效应物质基础研究

Xiao’ai Jiedu Fang was prescribed under the guidance of "cancer pathogen" pathogenic theory put forward by Prof. Zhou Zhong-ying, one of the nation-level Chinese medicine masters. Clinical and experiment results all showed that it may have certain anti-tumor effect and intervene tumor inflammatory microenvironment. As the pathogenesis of "cancer pathogen" is similar and closely related to inflammatory microenvironment, this project is planned to perform in vitro efficacy interventional experiments based on literature research and the previous experiments, combined with "plant chemical separation" and "activity tracking". With the application of modern chromatography spectrum analysis technology, Xiao’ai Jiedu Fang solution, its composition and monomer components are dynamically intervened, screened and verified respectively. Combining techniques concerning molecular biology, cytobiology, and epigenetics, the experiments are aimed to observe and analyze the interventional efficacy of inflammatory microenvironment of tumors such as tumor cells, immune cells and related cytokines as well as to track the separated part and monomer. With the help of computer molecular simulation technology, this study is to reveal the pharmacodynamic material basis and molecular mechanism of intervened effect of compound prescriptions on inflammatory microenvironment and explore the compatibility mechanism to supplement cancer pathogenesis theory, for providing foundation for clinical diagnosis of cancer and further development and utilization of Xiao’ai Jiedu Fang.

消癌解毒方是在国医大师周仲瑛教授的“癌毒”病机理论指导下配伍形成的。临床诊疗及药效实验结果均表明其具有确切的抗肿瘤效果和干预肿瘤炎性微环境作用，基于“癌毒”病机与炎性微环境的相似相通、关系密切，本项目结合文献研究及前期实验基础，拟通过体外药效干预实验，“植化分离”与“活性追踪”相结合，应用现代色谱波谱等分析技术，结合分子生物学、细胞生物学、表观遗传学等手段，分别采用消癌解毒方、复方组份及其部位、相关单体成分进行动态干预、筛选和验证，观察分析对以肿瘤细胞、免疫细胞及相关细胞因子为代表的肿瘤炎性微环境干预效应，追踪分离活性部位及单体，辅以计算机分子模拟技术，研究揭示复方干预炎性微环境的药效物质基础及分子机制，探讨复方配伍机理、完善癌毒病机理论，为肿瘤的临床诊疗以及复方的进一步开发利用提供依据。

本课题系围绕消癌解毒方药效物质基础及其对干预肿瘤炎性微环境的分子机制进行研究。通过对癌毒病机理论的系统分析和总结，以更好地选择实验模型和方法。采用液质联用、核磁共振、柱层析等技术，对消癌解毒方中化学成分进行系统分析，并研究其在不同组织中的分布情况，共鉴定出消癌解毒方中48个化学成分，其中芦丁和半枝莲碱B含量分别为0.41μg.g-1、0.60 μg.g-1；从白花蛇舌草鉴定出12个成分；从天葵子鉴定出21个成分，得到3个化合物，其中小檗碱为首次从天葵子中分离得到；小鼠灌胃后组织分布实验表明，芦丁、野黄芩苷和半枝莲碱B在组织中分布广泛，野黄芩苷在组织内含量相对较低，芦丁主要分布于肝脏，野黄芩苷只在肺中检测到，半枝莲碱B主要分布在肝脏和肾脏中。结肠癌荷瘤小鼠模型药效实验表明，消癌解毒方通过抑制调节性T细胞的分化，降低肿瘤组织中Foxp3蛋白表达，进而发挥抗肿瘤功效。通过DEN诱导大鼠肝癌模型，探讨复方对大鼠肝癌模型肿瘤微环境的影响，结果表明其能够降低肝癌大鼠血清和肝组织中IL-1β、IL-6表达，降低α-SMA蛋白、TGF-β1蛋白、SMAD7蛋白、SMAD4蛋白表达，通过影响TLR4/MYD88/NF-κB信号通路、Bcl-xl/Caspase-3信号通路影响肿瘤生长，并降低CD68、CD206、Arg-1、Dectin-1的表达。通过复方含药血清干预M2型巨噬细胞条件下的SMCC-7721肝癌细胞，结果表明含药血清对2型巨噬细胞培养条件下SMCC-7721细胞的增殖具有时间、剂量依赖性的抑制作用。为探讨对4T1荷瘤小鼠肿瘤生长的影响及其作用机制，复方与顺铂联用结果表明其通过调控组蛋白去甲基化酶，对乳腺癌细胞4T1荷瘤小鼠的肿瘤产生抑制效果。采用MDA-MB-231细胞探讨复方通过调控PI3K/AKT通路，抑制乳腺癌细胞增殖的作用及其机制，结果表明消癌解毒方可以降低PI3K/AKT的磷酸化水平，上调p53和p21的表达。此外肿瘤细胞迁移、侵袭机制实验表明，4’-羟基汉黄芩素能够抑制SW620小迁移、侵袭，其可能是通过影响VEGF、AKT、p-AKT、PI3K、p-PI3K蛋白的表达发挥作用。在该项目资助下已发表了1篇SCI论文，17篇国内核心期刊论文，另有多篇在投。