基于Toll样受体家族调控肠道菌群与宿主免疫平衡调节的中医扶正祛邪理论对晚期结直肠癌疗效机制研究

Colorectal cancer is one of the malignant tumor with highest morbidity. TCM (Traditional Chinese Medicine) plays a great role in treating advanced colorectal cancer. Our previous research has revealed that TCM ‘Strengthening the Body Resistance to Eliminate Pathogenic Factors’ theory can moderate Th1/Th2 immune balance by secreting various of cell factors so that it can treat advanced colorectal cancer. However, the specific mechanism of the treating effectiveness remains unclear. These years’ researches have revealed that the secreting of cell factors like IL-6 and IFN can be controlled by Toll-like receptors (TLRs). TLRs widely locate on intestinal epithelial cells. The gut microbiome is the central role between TLRs and colorectal cancer development. They are also an important reacting place and metabolic pathway for most oral-taken TCM herb medicines and have a intimate connection with TCM ‘Strengthening the Body Resistance to Eliminate Pathogenic Factors’ theory. In this study, we will use three different CT-26 colorectal cancer model with normal, sterile and TLRs gene knock-out mice to compare the differences and relationships of TLRs expression on intestinal and tumor tissues, gut microbiome distribution and treating effectiveness under these three conditions. Thus we can analysis the effect of TCM ‘Strengthening the Body Resistance to Eliminate Pathogenic Factors’ theory on TLRs controlling gut microbiome and host immune balance and its possible signal pathway. We will also use small sample randomized clinical research to analysis TCM advantage people’s characters on TLRs spectrum, gut microbiome distribution and immune functions.

结直肠癌是目前我国发病率较高的恶性肿瘤，中医药在治疗晚期结直肠癌中起到重要的作用。我课题组在前期基础研究证实中医扶正祛邪法能够通过分泌多种细胞因子，调节机体Th1/Th2免疫平衡，进而治疗晚期结直肠癌，然而其具体疗效机制仍未明确。近年研究显示以IL-6、IFN为主的细胞因子分泌与Toll样受体（TLRs）调节相关。TLRs广泛存在肠上皮细胞中，肠道菌群是其与肠癌发展过程中的重要环节，同时也是口服中草药主要作用场所与代谢途径，与中医扶正祛邪理论有着密切联系。因此本课题拟通过建立无菌、正常、TLRs基因敲除三种CT26肠癌小鼠模型，对比不同条件下肠道组织TLRs、肠道菌群分布与疗效之间的差异性与相关性，分析中医扶正祛邪法对TLRs调控肠道菌群与宿主免疫平衡的作用，阐释其可能信号通路；通过小样本随机对照临床研究，分析中医优势人群TLRs谱、肠道菌群分布及免疫学特征。

前期研究基础表明，以祛邪胶囊为代表的中医扶正祛邪法能够延长晚期结直肠癌患者生存期、提高生活质量，基础研究提示其疗效与促进机体免疫平衡调节有关。本研究通过临床研究与基础实验研究，进一步从肠道菌群及免疫平衡机制对中医扶正祛邪法治疗晚期结直肠癌的作用机制进行了探讨。其中临床研究部分通过随机对照双盲安慰剂对照临床试验，共纳入40例晚期结直肠癌维持治疗期患者，按1：1比例随机分为中医扶正加祛邪组（常规扶正中医治疗基础上联合祛邪胶囊）及单纯扶正组（常规扶正中医治疗基础上联合安慰剂胶囊），进行1个月干预，治疗前后收集患者粪便标本进行16sRNA多样性分析，并采集外周血TB淋巴细胞亚群分析。结果显示，中医扶正+祛邪组相比单纯扶正组能够显著改善晚期结直肠癌患者治疗前后外周血T细胞水平，且治疗后扶正+祛邪组CD4+T细胞绝对值明显高于单纯扶正组（p=0.024）。此外，中医扶正+祛邪（祛邪胶囊）组与单纯扶正组均能改善肠道菌群中有益菌与有害菌的比例，但扶正+祛邪组更为显著，患者肠道菌群拟杆菌门及志贺菌门比例下降，厚壁菌、普雷沃氏菌门比例上升，F/B比值上升，扶正+祛邪干预能够显著提高疣微菌、双歧杆菌等有益菌比例，下调有害菌如变形菌门、蓝细菌等物种比例。通过环境因子分析发现扶正+祛邪组对肠道菌群的改变与外周血CD4+T以及CD8+T细胞水平有一定相关性。基础研究通过祛邪胶囊干预BALB/c小鼠移植瘤模型及无菌条件下、菌群移植后小鼠模型发现：肠道菌群是祛邪胶囊发挥疗效的介质，祛邪胶囊干预后引起了Firmicutes丰度的升高和Bacteriodetes的丰度的下降。此外，肠道菌群代谢产物－丁酸介导myosin11调控的直接抗肿瘤作用，myosin11蛋白表达与祛邪胶囊干预存在相关性，并且与受到肠道菌群的调节。综上，以祛邪胶囊为代表的中医扶正+祛邪法能够显著提高晚期结直肠癌患者T细胞免疫，明显改善结直肠癌肠道菌群中有益菌与有害菌分布比例，并且其抗肿瘤机制与菌群调节有关，免疫调节、短链脂肪酸分泌及相关蛋白通路可能是这一机制的潜在靶点。