高尿酸血症肠免疫应答机制研究

Hyperuricemia is associated with several metabolic syndrome, while the effect of hyperuricemia on intestinal immunity remains unclear. Although the involvement of gut microbiota in hyperuricemia has been evidenced, but it is still unknown whether the changes in microbiota result in alterations of gut pathology. Our previous studies found that gut microbiota of hyperuricemia mouse was disturbance and were enriched in microbiota that is associated with intestinal inflammation. Consequently, intestinal permeability was significantly increased with age in hyperuricemia mouse. Furthermore, uric acid level was positively correlated with intestine permeability. KEGG pathway classification and functional enrichment showed Toll-like receptor signaling pathway and TNF-signaling pathway involved in intestinal immune. Molecular biology experiment further evidenced the expression of TLR2, TLR5 and TNF-α were abnormal. Inflammatory cells infiltration were observed in hyperuricemia mouse. These indicates a disorder of the intestinal immune system in hyperuricemia mice. Based on the previous study, we further investigate the changes of intestinal immune system and the influence of intestinal flora on intestinal immune system in hyperuricemia mouse: (1) the change of intestinal immune system in hyperuricemia mouse; 2) changes of gut microbial composition and metabolites in hyperuricemia mouse; (3) effects of intestinal microbiome on intestinal immune system and intestinal barrier. The purpose of this study is to evidenced the changes of intestine immune in hyperuricemia mice and provided target treatment from new perspective.

高尿酸血症与多种代谢性疾病的发生密切相关，但高尿酸血症对肠免疫的影响并不清楚。虽然高尿酸血症伴随肠菌群紊乱，但肠道菌群的改变是否会导致肠免疫应答变化仍不知。我们前期研究表明高尿酸血症小鼠肠稳态失调，与炎症相关的菌群增多，肠渗透性升高，同时发现尿酸水平与肠渗透性增加呈正相关性。RNA-Sep分析表明与微生物模式识别相关的Toll-样受体和与炎症相关的TNF信号通路被显著富集。肠黏膜TLR2、TLR5表达异常，TNF-α表达上调，炎性细胞浸润，这表明高尿酸血症小鼠肠免疫应答异常。在前期研究基础上，我们进一步展开研究：（1）探索高尿酸血症小鼠肠免疫应答变化；（2)明确引起高尿酸血症小鼠肠免疫应答的肠道微生物及代谢产物；（3）研究肠道微生物对肠免疫应答的作用机制。通过研究，明确高尿酸血症小鼠肠免疫应答及机制，为高尿酸血症的治疗提供新的靶标和理论依据。

高尿酸血症与多种代谢性疾病的发生密切相关，但高尿酸血症对肠免疫的影响并不清楚。虽然高尿酸血症伴随肠菌群紊乱，但肠道菌群的改变是否会导致肠免疫应答变化及肠道菌群是否是高尿酸血症肠屏障破坏的主要因素仍不知。在前期研究的基础上，我们进一步深入研究高尿酸血症肠免疫应答机制，以及肠道菌群对肠免疫的调节作用，明确高尿酸血症肠免疫应答机制及肠屏障损伤因素。我们发现高尿酸血症小鼠肠渗透性显著高于正常小鼠，且与尿酸水平具有显著的正相关性（r=0.79，p<0.01）。空肠和回肠中occludin 的表达显著降低，结肠组织中紧密连接蛋白无明显变化，但紧密连接蛋白ZO-1,occludin的位置发生变化。与正常小鼠相比，虽然回肠、空肠、结肠组织结构没有明显变化，但是高尿酸血症小鼠回肠、空肠、结肠有明显的淋巴细胞（CD3+）浸润现象。通过RNA-seq测序及分子实验验证发现结肠组织中Toll受体、TNF和NF- kappa B 信号通路被显著富集， TLR2、TLR4和 TLR5及炎症因子IL-1β、TNF-α表达量升高，且血清中LPS和TNF-α水平显著高于正常小鼠，表明高尿酸血症小鼠肠免疫应答异常。菌群测序发现高尿酸血症小鼠肠道菌群发生紊乱，有益菌群减少，促炎症相关的菌群增多。同样地，采用别嘌呤醇和嗪氧酸钾构建的高尿酸血症模型小鼠与基因敲除小鼠相似，肠渗透性升高，菌群显著不同。此外，我们发现降低尿酸显著降低肠渗透性，但菌群也随之发生了改变。而干扰高尿酸血症小鼠菌群后，与高尿酸血症小鼠相比，虽然尿酸没有明显变化，但是肠渗透性显著降低，说明菌群改变肠屏障可以不依赖尿酸。进一步在无菌小鼠基础上构建高尿酸血症小鼠，发现虽然尿酸水平显著高于正常小鼠，但肠渗透性没有明显变化，说明菌群是调控高尿酸血症小鼠肠屏障的关键因素。