产丁酸菌通过影响肠道菌群调控Wnt通路防治肠腺瘤恶变的机制

Alterations of gut microbiota and bile acid metabolism play vital roles in intestinal carcinogenesis. Our preliminary data have shown deoxycholate (DCA) promoted the development of colorectal cancer (CRC) in Apcmin/+ mice, which can only spontaneously develop intestinal adenomas. By transplanting feces from DCA-induced canceraous Apcmin/+ mice could also induced intestinal carcinogenesis in homologous mice. Furthermore, we found that the abundance of butyrate-producing bacteria and butyrate level were decreased, a low-grade inflammation state of intestine was induced and also Wnt/β-catenin signaling pathway was activated during the tumor development. Meanwhile，it has been shown that the abundance of butyrate-producing bacteria reduced in CRC patients. Based on these previous studies, we suppose that manipulation of intestinal micrbiota by butyrate-producing bacteria can alleviate the low-grade inflammation of intestine and inhibit the process of DCA-induced intestinal carcinogenesis through Wnt signaling pathway. Our proposal is firstly to evaluate the effects of butyrate-producing bacteria on the intestinal cancer development, changes of gut microbiota and intestinal barrier functions during intestinal cancer development in Apcmin/+ mice, which will be analyzed by pathological evaluation and pyrosequencing analysis. Secondly, we will investigate butyrate level in feces, proinflammatory cytokines secretion and Wnt signaling pathway activation using the techniques of gas chromatography, qPCR and Western blot. In vitro, we will evaluate the cell growth and manipulation of Wnt signaling pathway by using intestinal preneoplastic cell lines and CRC cells stimulated by butyrate-producing bacteria, and further screen the differential functional components of the bacteria. This project will lay a foundation for the butyrate-producing bacteria supplement in the prevention and treatment of CRC.

肠道菌群失衡和胆汁酸代谢紊乱在肠腺瘤恶变中起重要作用。我们发现脱氧胆酸（DCA）可诱导Apcmin/+小鼠肠腺瘤恶变，且植入该恶变鼠的粪菌液亦能诱导同系鼠腺瘤恶变；并发现：该过程中肠道产丁酸菌及其代谢物丁酸减少，肠粘膜呈低度炎症状态以及Wnt/β-catenin信号通路活化。文献也证实肠癌患者产丁酸菌丰度减少，故我们推测“补充产丁酸菌可能通过纠正肠道菌群失衡，抑制肠粘膜低度炎症，调控Wnt通路进而拮抗腺瘤恶变”。本研究首先给予DCA诱导腺瘤恶变的Apcmin/+小鼠产丁酸菌，采用病理学及焦磷酸测序等方法评价肠道肿瘤、菌群结构及肠粘膜屏障的变化；进而应用气相色谱、qPCR及Western blot分析该过程中肠道丁酸、促炎因子及Wnt通路的改变；最后探讨不同产丁酸菌对结肠肿瘤前细胞和结肠癌细胞生长及Wnt通路关键分子的调控，并筛选出不同功能组分。本项目将为产丁酸菌临床干预肠腺瘤恶变奠定基础。

近年来结直肠癌(Colorectal cancer, CRC)的发病率和死亡率在全球范围显著增加。除了遗传背景，饮食因素也参与了结直肠癌的发生与发展。肠道菌群失调与肠道癌变密切相关。我们发现，在Apcmin/+小鼠肠腺瘤恶变过程中，肠道产丁酸菌及其代谢物丁酸减少，肠粘膜呈低度炎症状态以及Wnt/β-catenin信号通路活化。此外，在结直肠癌患者中观察到产丁酸菌显著减少。然而，产丁酸菌对肠道肿瘤发展的潜在益处及其机制仍需进一步阐明。我们的科学假说是：补充产丁酸菌可能通过纠正肠道菌群失衡，抑制肠粘膜低度炎症，调控Wnt通路进而拮抗腺瘤恶变。本研究首先采用病理学及16S rRNA测序等方法探索了补充丁酸梭菌在高脂饮食（HFD）诱导Apcmin/+小鼠肠腺瘤恶性转化过程中的作用；进而采用qPCR、Western blot、siRNA转染等方法探讨了丁酸梭菌对结肠癌细胞增殖、Wnt/β-catenin通路及GPRs的调控机制；最后以人正常结肠组织、腺瘤及腺癌为研究对象，采用免疫组化方法探讨了肠腺瘤恶性转化过程中GPR43和GPR109A的变化。我们主要发现：丁酸梭菌可抑制HFD诱导Apcmin/+小鼠腺瘤恶变；丁酸梭菌改善HFD诱导的小鼠肠道菌群失调，并影响Apcmin/+小鼠胆汁酸和SCFAs代谢水平；丁酸梭菌通过抑制Wnt/β-catenin信号通路、激活G蛋白偶联受体拮抗HFD诱导的肠腺瘤恶性转化。本项目从肠癌高发的重要环境因素及肠道菌群失衡入手，采用了多种研究方法，从不同角度探索了产丁酸菌调控Wnt/β-catenin信号通路拮抗HFD诱导肠腺瘤恶性转化过程中的作用及机制，为揭示产丁酸菌拮抗肠腺瘤恶变的分子机理提供新的科学依据，并为产丁酸菌临床干预肠腺瘤恶变奠定了基础。成果以论文的形式提交，已在SCI期刊发表论文14篇，核心期刊3篇，使7名博硕士研究生获得系统的科研技能训练。