派伊尔结内生Lactobacillus reuteri L3对肠粘膜免疫的调节及在抑郁症中的作用

Depression is one major causes of morbidity and mortality and is associated with impaired health-related quality of life. The microbiota–gut–brain axis is associated with alterations in behaviour and cognition. Peyer’s patches (PP) is entry site for bacteria. PP contain immunocompetent cells and is thought to be the key inductive sites for mucosal immune responses. Our previous study observed that the changes in the composition of indigenous opportunistic bacteria in the PP were associated with obesity. However, the role of PP indigenous opportunistic bacteria in neuronal immune system and depression has received little attention to date. In this study, we will use an depression animal model induced by chronic social defeat stress (CSDS) to prove the hypothesis that the changes in the composition of indigenous opportunistic bacteria in the PP are associated with depression. Lactobacillus reuteri L3 isolated from the PP is used to modulate mucosal immune responses. Illumina high-throughput sequencing is used to describe gut flora structures, while Flow cytometry is used to analysis the composition of T lymphocyte in PP. RT-PCR and Western blot are also used to estimate the expression of gene and protein, respectively. The data predict novel pathways that might underlie microbiota-mediated effects on brain and behavior, thus presenting targets for investigations into mechanisms and potential therapy.

抑郁症作为一种神经心理疾病，具有高发病率和高死亡率，肠腔菌群-肠道-大脑轴与行为、认知密切相关。研究发现派伊尔结是菌群与免疫细胞发生接触，并实现肠粘膜免疫应答的关键部位；我们前期发现抑郁样肥胖小鼠派伊尔结内菌群结构紊乱。然而，派伊尔结内生菌群能否通过调节肠粘膜免疫而影响抑郁尚缺乏全局性和机理性的认识。本项目拟以慢性社会挫败应激导致的抑郁样小鼠为模型，通过靶向派伊尔结免疫应答的抗炎性内生Lactobacillus reuteri L3对其干预，借助Illumina高通量检测、流式细胞术、代谢组学、RT-PCR、免疫印迹等技术系统阐明派伊尔结内生菌群通过诱导粘膜免疫应答，导致肠通透性改变，引起神经炎症，继而通过吲哚胺-2,3-双加氧酶等表达影响神经递质的合成，以调节抑郁行为。研究结果将为高效微生态制剂的研究提供新思路，并对临床合理开展抑郁症的预防及治疗提供重要理论依据。

本项目研究抑郁下PP内生菌群结构组成并对菌群与肠粘膜免疫、脑炎症和精神行为开展研究。. CSDS建抑郁样模型，Illumina高通量对PP内生菌群测序分析，结果发现抑郁敏感组PP内生菌群显著区别于正常及抑郁抵抗组。表现为正常及抵抗组以Proteobacteria为主，敏感组以Firmicutes为主。敏感组Candidatus Arthromitus显著升高，Oxalobacteraceae, Burkholderia-Paraburkholderia, Rhodococcus, Sphingobium、Pseudomonas及Lactobacillus显著降低。PP内生菌群与肠腔菌群存在显著差异，分属Candidatus Arthromitus和Bacteroides两种肠型。.流式细胞术对PP内淋巴细胞亚群进行分析；HE及AB-PAS染色、RT-PCR等对肠粘膜免疫进行分析，发现敏感组回肠炎细胞浸润增加、黏液层变薄。PP和脾脏CD3+CD4+ /CD3+CD8+ 显著升高，Foxp3+ 调节性T cell显著下降。回肠LPS、 IgA、TNF-α和IL-6显著升高，IL-10显著降低，TNF-α、TGF-β、IFN-γ、IL-23及IL-6 mRNA表达显著高于正常组。. ELISA测定血液LPS、细胞因子及神经递质； RT-PCR测定脑中炎症基因表达，结果发现敏感组血液BDNF、5-HT和DA显著降低，LPS显著升高。前额皮质IL-10显著降低，IL-6显著升高。BDNF基因表达显著下调，IDO、TNF-α、IL-6及NF-κb基因表达显著上调（P< 0.05）。. 用一株靶向PP免疫应答的Lactobacillus reuteri L3对抑郁样小鼠进行干预，行为学实验显示Lactobacillus reuteri L3能改善抑郁样行为。干预后能显著调节PP内生菌群结构，表现为Lactobacillus和Pseudomonas显著增加，Cupriavidus显著减少等。Lactobacillus reuteri L3灌胃后，血液中FITC-葡萄聚糖及回肠TNF-α、IL-6、IFN-γ都显著降低，肠及前额皮质、海马炎性基因表达显著下调，Iba-1阳性小胶质细胞显著减少。. 本项目对临床抑郁症预防及治疗有重要理论意义。.