内源性大麻素系统在肠道菌群紊乱致IBS发病机制中的研究

Intestinal flora disturbance has been confirmed to participate in the pathogenesis of irritable bowel syndrome (IBS). Oral probiotics could colonize in the intestinal mucosa, and improve IBS symptoms by regulating the intestinal microbiota. Recent studies show that the endocannabinoid system (ECS) in intestinal mucosa can be involved in the regulation of intestinal physiological function. ECS is closely related with the intestinal flora, especially the probiotics, but the underlying mechanism is still unknown. Our group aims to explore the roles of ECS in microbiota-epithelum communication, and its downstream effects on mucosal barrier function, immune function and neurological regulation. Based on the results of our latest research, we would construct ECS knockdown intestinal epithelial cell lines in bacteria-cell co-culture system, IBS rat model, endocannabinoid receptor knockout rats as experimental groups. By using ECS agonist and antagonist, probiotics and IBS patients’ bacterial supernatant as variable factors, we could observe the reaction of the intestinal epithelial cells using cytological and biological tools. How the intestinal bacteria participate in pathogenesis of IBS, and whether ECS play an important role in probiotics treatment is what we care about. This study will provide probiotics an important basis for the microbiota targeted treatment in IBS.

肠道菌群失调已证实在肠易激综合征(irritable bowel syndrome, IBS)发病中发挥作用，口服益生菌可定植于肠道上皮，通过调节肠道微生态，改善IBS症状。新近发现肠道黏膜上皮内源性大麻素系统(endocannabinoid system, ECS)的激活可调节肠道屏障、炎症水平以及内脏疼痛等功能，且与益生菌的诱导密切相关，但具体机制不清。本课题组旨在从肠道菌群-肠上皮细胞相互作用方面探讨ECS参与肠道屏障、免疫和神经功能调节的机制。在前期研究基础上，我们设计构建ECS正常/低活性肠上皮细胞-肠道菌株共培养体系和IBS模型、敲基因大鼠模型，以ECS激动/抑制剂、益生菌和患者肠菌群上清为刺激因素，运用多种功能学和分子生物学技术，从细胞和动物水平观察ECS如何参与肠道菌群对上皮的相互作用，并探讨其内在作用机制。本研究结果将为针对性利用益生菌治疗IBS提供重要理论基础。

本课题的学术理论依据是肠道菌群紊乱可能通过影响肠上皮细胞内源性大麻素系统（Endocannabinoid system, ECS）活性改变而参与导致肠易激综合征（Irritable bowel syndrome, IBS）发病。益生菌经口服定植后，可能调节肠上皮细胞ECS及下游信号通路而表达屏障修复蛋白、免疫调节因子和相关神经因子，纠正IBS患者肠道屏障、感觉和动力异常，恢复肠道功能。.本研究通过建立大鼠母婴分离模拟IBS、CB受体敲基因鼠模型，以及体外肠上皮细胞和肠道菌株共培养体系，以内源性大麻素配体及受体拮抗剂、益生菌培养液及菌体和IBS病人菌群上清液等为刺激因素，综合运用免疫组化、Western blot、RT-PCR、细胞流式等多种分子生物学技术，从分子、细胞及动物水平多方面观察和验证益生菌干预肠道菌群是否通过ECS发挥IBS治疗作用，探讨益生菌在参与治疗IBS特征性症状的病理生理机制，从而为选择性利用益生菌治疗IBS提供理论基础。.本项目执行期间，各项实验按照研究计划认真执行，并按实际研究进展实时调整实验方案，最终获得预期成果，其中发现的重要结果有：IBS患者中肠道屏障功能产生异常；IBS患者肠道黏膜上皮CB受体表达上调；IBS动物模型肠道黏膜上皮CB受体表达上调，并与益生菌相关；乳酸杆菌与肠胶质细胞共培养，可诱导其活性和SP的表达改变。.所获得的研究成果具有以下创新之处：CB I和CB II在结肠黏膜细胞广泛表达，主要表达部位在结肠腺体上皮细胞。CB I和CB II的表达在IBS患者显著高于健康对照者，提示此受体蛋白参与IBS相关肠道症状的发生过程。肠道菌群的异常改变是和ECS密切相关的，且此机制参与IBS的发病过程。益生菌干预可通过调节肠上皮细胞ECS，进而发挥改善IBS症状的治疗效果。