维生素A辅助治疗持续性腹泻病改善肠道菌群失调的作用及机制研究

In the recent years, there is a significantly increased trend in the incidence of persistent diarrhea. The persistent diarrhea of infants is 19.0% in the diarrhea, which have been the fifth disease in the department of digestion of Chongqing Children's Hospital. Some studies have demonstrated that vitamin A (VA) is an independent risk factor to the incidence of persistent diarrhea. Our previous study found that VA deficiency was 81.9% and fecal microbial dysbiosis was 80.8% in the children of persistent diarrhea. Moreover, the children numbers of moderate and severe dysbiosis in the fecal microbial were both significantly increased in the VA deficiency group compared with the VA normal group. And VA adjuvant therapy could obviously enhance the cure rate for the persistent diarrhea. However, what's the regulatory molecular mechanism during the VA adjuvant treatment? Whether can VA regulate intestinal microbial to improve intestine barrier function? There are still unknown. On the basis of the last funding of NSFC, we will take VA adjuvant therapy as a key point. In the current project will determine the effects of VA nutritional level on the intestinal microbial diversity through the analysis of clinical investigation. The targets of VA deficiency leading to intestinal microbial disorder will be discovered by the animal model of germfree mice with VA deficiency, and the mechanism of VA supplementation will be investigate in the diarrhea rat model with intestinal microbial dysbiosis. Finally, the Caco-2 cells will be used to reveal the specific signaling pathway of VA improving intestinal barrier function. The design of the present project displayed a better research method of finding a scientific question from clinical survey, investigating possible mechanism using animal models and then revealing specific signaling pathways in vitro. The aim is to expound the mechanism of VA adjuvant therapy to persistent diarrhea of children. The project can establish reliable experimental basis for the new clinic treatment strategy from improving nutritional statue of children.

近年来持续性腹泻病发病率呈显著上升趋势，婴幼儿持续性腹泻约占腹泻病的19.0%，属于我院消化科前五位病种。研究表明维生素A（VA）是持续性腹泻的一个独立危险因素，本项目前期研究发现持续性腹泻患儿中81.9%存在VA缺乏，菌群失调者80.8%，VA缺乏组中重度菌群失调比例显著高于VA正常组，VA辅助治疗可显著提高患儿治愈率。然而VA辅助治疗的机理是什么？VA是否调节肠道菌群促进肠道屏障功能？目前尚不清楚。本项目拟在前一个NSFC项目基础上，以VA辅助治疗为切入点，通过临床调查分析明确VA水平对肠道菌群的影响，利用无菌鼠模型寻找VA缺乏导致肠道菌群失调的作用靶点，在肠道菌群失调腹泻动物中探讨VA补充机理，在Caco-2细胞中揭示VA促进肠道屏障功能的信号通路。从临床调查发现问题，到动物模型探讨可能机制，再到体外揭示信号通路，旨在阐明VA辅助治疗作用机理，为临床提供新的治疗策略奠定实验基础。

近年来随着人们生活水平的不断提高，儿童持续性腹泻病（PD）发病率呈显著上升趋势，婴幼儿PD约占腹泻病的19%。我们的研究发现VA联合Zn的补充治疗，不仅可以缓解PD患儿的VA缺乏营养状态，而且可以改善PD患儿的贫血，显著提高治疗效果，为临床治疗婴幼儿PD提供了一种新的策略。进一步根据PD儿童血清中的VA水平分析，发现VAD的PD儿童粪便菌群的多样性下降，Enterococcus是VAD的PD儿童的优势菌属，而Escherichia coli和Clostridium butyricum是VAN的PD儿童粪便中的优势菌属，表明不同VA营养水平的PD患儿粪便中的菌群存在差异。随后我们构建了不同VA营养水平的持续性腹泻大鼠模型，发现VAD降低大鼠体重、增加大鼠腹泻程度；血清视黄醇水平、紧密连接中的claudin、Occludin蛋白水平及大鼠的抓力均受到腹泻和VA水平交互作用的影响；而腹泻是降低大鼠体重、增加血清DAO含量、损伤肠绒毛结构、降低ZO-1表达水平的独立影响因素，VAD显著增加血清中zonulin的水平。表明VA缺乏可能会加重腹泻病的严重性，引起小肠粘膜损伤；而VA补充可调节上皮间紧密连接蛋白表达水平，改善肠道损伤缓解腹泻。进一步的机制研究揭示了VA可通过RAR上调TLR4表达水平，促进TLR4与ZO-2的相互作用，改善肠上皮屏障功能。基于上述机制研究，该项目又在TLR4-/-KO小鼠上构建了VAD或VAN模型，进一步阐明了TLR4在VA调控肠粘膜菌群，维持肠道微生态中发挥了重要的作用。.目前VA联合Zn辅助治疗PD儿童已在我院消化科得到推广应用，该项目已发表SCI论文6篇，CSCD核心论文2篇；参加国际国内学术会议10余人次，其中中华医学会全国儿科学学术会议分组报告3人次，壁报及书面交流4人次；培养毕业研究生5名，其中博士研究生1名，硕士研究生4名。