基于“肠道菌群-肠黏膜免疫”共调节作用探讨黄芪-莪术药对抗结肠癌的作用机制

Intestinal microenvironmental imbalance caused by intestinal microecological imbalance which via co-regulation of intestinal flora disorder and immune escape is an important cause of colon cancer. The chemokine pathway SDF-1/CXCR4 is an important connection between the intestinal flora and the intestinal mucosa immunity. Our previous studies showed that the drug pair of “Astragalus membranaceus-Curcuma wenyujin” has a clear regulatory effect on colon cancer, chemokines and immune function. Based on this, the applicant follows the TCM's overall concept and puts forward the hypothesis that“The drug pair of Astragalus membranaceus-Curcuma wenyujin exerts anti-colon cancer effect by regulating intestinal microflora-intestinal mucosal immune function through SDF-1/CXCR4, and this regulation effect is closely related to the ratio of drug pair ratio and dose-effect relationship.” The project is planned to work from the following aspects: ① To construct CT26.WT orthotopic transplantation tumor model to optimize the optimal ratio and dose of Astragalus membranaceus-Curcuma wenyujin; ② To study the regulatory effect of Astragalus membranaceus-Curcuma wenyujin on imbalanced intestinal microecological of colon cancer in vivo; ③ The immunomagnetic beads technology was used to isolate CD4+ T cells, and the co-cultured with CT26.WT-GFP to explore the mechanism of Astragalus membranaceus-Curcuma wenyujin on regulating "intestinal flora-intestinal mucosal immunity" in vitro. We aims to reveal the scientific connotation of Astragalus membranaceus-Curcuma wenyujin, the classic drug pair, and to provide scientific basis for the development of clinical drugs and innovative drugs which against colon cancer.

肠道菌群紊乱及免疫逃逸共同作用导致的肠道微生态失衡是引发结肠癌的重要病因，趋化因子传导通路SDF-1/CXCR4是连接肠道菌群与肠黏膜免疫的重要桥梁。本团队前期研究发现黄芪-莪术对结肠癌肠道菌群、趋化因子和免疫功能均有显著调节作用。基于此，申请者提出“黄芪-莪术药对可能是通过SDF-1/CXCR4调控肠道菌群-肠黏膜免疫功能继而发挥抗结肠癌作用，且该调控作用与药对配比和量-效紧密关联”的假说。本项目拟从以下几方面展开：①构建CT26.WT原位移植瘤模型筛选黄芪-莪术最佳配比和最优剂量；②整体动物模型研究黄芪-莪术调控结肠癌失衡的肠道微生态作用；③免疫磁珠技术分离CD4+T细胞与CT26.WT-GFP共培养模型及结肠癌细胞株模型，以探讨黄芪-莪术抗结肠癌的“肠道菌群-肠黏膜免疫”作用机制。旨在揭示黄芪-莪术经典药对相使配伍的科学内涵，为临床用药和抗结肠癌创新药物的研发提供科学依据。

肠道菌群紊乱及免疫逃逸共同作用导致的肠道微生态紊乱是引发结肠癌的重要原因。本研究①先以结肠癌原位移植瘤小鼠模型研究黄芪-莪术不同配伍比例下对结肠癌的干预效应，采用主成分分析和聚类分析得到黄芪-莪术不同配比抗结肠癌效应最适配比为2:1。后应用UPLC/MS技术方法测得黄芪-莪术不同配比下主要活性成分含量，发现黄芪-莪术配比组中有明确抗肿瘤活性的黄芪甲苷、β-榄香烯、莪术二酮、姜黄素成分含量合煎比单煎成分含量高，能够解释黄芪-莪术在配伍使用后抗肿瘤效应更优。②黄芪-莪术对结肠癌小鼠损伤的肠道机械屏障有修复作用，能够扭转肠道微生物数量的改变，代谢产物SCFAs的含量也得到升高。因此，黄芪-莪术药对抗肿瘤作用可能是通过改善肠道菌紊乱而发挥作用。③通过将黄芪、莪术中主要活性成分与作用靶点蛋白相结合，通过分子对接预测其结合能。结果显示，黄芪、莪术中主要活性成分能够与SDF-1/CXCR4/NF-κB信号通路蛋白形成稳定结合，发生相互作用。而后我们运用WB和Q-PCR方法在体验证分子对接结果。结果表明，黄芪-莪术给药组可显著回调以上蛋白和mRNA表达水平。表明黄芪-莪术在体内抑制结肠癌生长的作用机制，可通过SDF-1/CXCR4/NF-κB通路来关联肠道菌群紊乱及肠道免疫的调控。④黄芪-莪术可能通过抑制巨噬细胞中Sp1表达导致ZFAS1转录表达被抑制，促进miR-153-3p靶向结合CCR5并降低CCR5表达，从而导致巨噬细胞M2极化被抑制，最终阻止结直肠癌的生长和转移。黄芪-莪术药对抗肿瘤作用可能是通过调控肠道免疫而发挥作用。⑤ 黄芪-莪术抑制结肠癌肿瘤生长和转移的潜在机制可能部分修复了结肠癌紊乱的代谢状态。这为探索结肠癌的诊断生物标志物提供了科学依据，也为黄芪-莪术药对作为一种创新的临床抗结肠癌药物开发提供依据。