基于肠道菌群甘草多糖促进肠上皮γδT细胞分化的抗肿瘤作用及其机制研究

Traditional Chinese medicine has unique advantages in the prevention and treatment of malignant tumor. The previous study confirmed that Glycyrrhiza Uralensis Polysaccharides(GCP) from licorice, one of the commonly used tonic herb, has good antitumor effect. However, the bioavailability of oral GCP is low, and it is difficult to fully explain the way and mechanism of how GCP exert its effect. In view of the regulation role of intestinal microflora on tumor immunotherapy, we propose that based on TLR4 pathway, GCP can promote the differentiation of intestinal intraepithelial γδT cells (γδ+ IELs) and play the anti-tumor effect by adjusting the intestinal flora. In order to clarify the role of GCP in promoting the differentiation of γδ+ IELs, we take tumor bearing mice as the research object, through intestinal microflora clearance and intestinal microflora reconstruction, using DNA sequencing, PCR, WB and flow cytometry to clarify the effect of GCP on the differentiation of γδ+ IELs, based on the regulation of intestinal microflora. TLR4△IEC tumor bearing mice are used and co-culture of siRNATLR4 silencing or overexpression of intestinal epithelial cells and γδT cells are used to clarify the mechanism of the anti-tumor effect of GCP on the differentiation of γδ+ IELs based on TLR4 pathway. This study provides modern scientific support for the theory of “Fuzheng peiben” of traditional Chinese medicine, also it provides the theoretical basis for anti tumor polysaccharides of traditional Chinese medicine.

中医药在防治恶性肿瘤方面具有独特的优势。前期研究证实，补益类中药甘草，其多糖具有良好的抗肿瘤功效。然而口服甘草多糖生物利用度低，难以阐释其发挥药效的作用途径和机制。鉴于肠道菌群对肿瘤免疫治疗的调控作用，我们提出基于TLR4通路，甘草多糖通过调节肠道菌群，促进肠上皮γδT细胞分化，发挥抗肿瘤作用的假说。本课题以荷瘤鼠为研究对象，通过肠道菌群清除及菌群重建，采用DNA测序、PCR、流式细胞术等方法，明确基于肠道菌群甘草多糖促进肠上皮γδT细胞分化的作用；以TLR4△IEC荷瘤鼠为研究对象，考察特异性敲除肠上皮细胞TLR4对γδT细胞分化及抗肿瘤作用的影响；通过沉默和过表达TLR4的肠上皮细胞与γδT细胞共培养，明确肠上皮TLR4对γδT细胞分化及抗肿瘤作用的影响；揭示基于肠上皮TLR4通路甘草多糖调节肠道菌群促进肠上皮γδT细胞分化的抗肿瘤作用及机制。课题将为“扶正培本”理论提供丰富的科学内涵

手术和放化疗是恶性肿瘤主要治疗手段，受治疗时间窗和严重毒副作用的限制，威胁肿瘤患者的生存质量。中医“扶正培本”指导下的补益类多糖显示出良好的抗肿瘤作用，本研究探讨口服生物利用度低的甘草多糖（GCP）抑瘤作用的潜在机制。结果显示，GCP基于调节肠道菌群丰度和多样性，尤其显著提高Enterorhabdus、Odoribacter、Ruminococcaceae_UCG_014、 Enterococcus 、 Ruminiclostridium_5等属的丰度，促进肠上皮γδT细胞表达NKG2D和CD27、分泌IFN-γ，同时血清促炎细胞因子增高、移植瘤浸润CD27+γδT细胞增多，且具显著的抑瘤作用。分子生物学实验结果提示，基于调节肠道菌群，GCP通过肠粘膜TLR4/Myd88非依赖通路影响肠上皮γδ分化，利用Tlr4f/f cre T基因鼠确证了GCP影响肠上皮γδ分化的分子机制。体外实验结果显示，GCP粪便上清基于肠上皮细胞TLR4/Myd88非依赖通路促进γδT细胞分化发挥抗肿瘤的分子机制。综上，研究阐明了基于肠上皮TLR4通路口服GCP调节肠道菌群促进肠上皮γδT细胞分化的抗肿瘤作用机制，为GCP防治肿瘤临床应用提供了科学依据。