高尿酸血症对肠道ABCG2/PDZK1表达与调控的分子机制研究

Hyperuricemia is the key pathophysiological basis of gout. The most common reason generating hyperuricemia is verified the obstacle of urate excretion by current researches. The intestine is known the most important organ involved the excretion of uric acid besides the kidney. After a lot of contemporary studies in genetics have been completed, many genes related with the polymorphism of hyperuricemia are testified, according to results from these studies. The disfunction of ABCG2 can mediate decreased intestine urate excretion, which is a common cause of hyperuricemia. PDZK1 is a kind of an important structural protein regulated ABCG2 function, by potential molecular interaction with ABCG2. And also it is directly regulated by inflammatory signals in several cells. The inflammation stimulated by monosodium urate is the centre of gout through the activation of TLR2/4/NLRP3/ASC/Caspase-1 pathway, as the current direction and hotpot in studies about gout. According to these backgrounds, we want to focus on the animal model of hyperuricemia and cultured epthelium cells from human intestine, in vitro, and to determinate the expression and regulation of ABCG2/PDZK1 in the intestine by hyperuricemia in this study. With this research we will be done, the key pathway and molecules involved in the regulation hyperuricemia with ABCG2/PDZK1 expression and function will be elaborated systemically. Using these results, we can find and select potential targets to treat gout patients with kidney failure or saturated urate clearation in the further.

高尿酸血症是痛风的关键病理生理基础，尿酸排泌障碍是临床最常见的高尿酸血症的诱因。肠道是除肾脏以外最主要的尿酸排泌器官。通过人群大样本遗传学研究已发现众多高尿酸血症易感候选基因，其中ABCG2和SLC2A9是已知与亚裔人群罹患高尿酸血症关系最为密切的位点。ABCG2功能下调可通过肠道排泌尿酸减少介导高尿酸血症的发生。PDZK1是潜在能与ABCG2互作并调控功能的重要结构蛋白，其本身也受到炎症信号调控。由尿酸刺激TLR2/4/NLRP3/ASC/Caspase-1活化介导的炎症是痛风发病的中心环节，也是当前本病研究的新方向和热点。本项目立足上述前沿背景，试图通过小鼠高尿酸血症模型及肠道上皮细胞的体外研究平台系统地阐述高尿酸血症本身对ABCG2/PDZK1的可能调控作用，初步揭示参与调控的主要信号分子和途径，由此进一步为肾功能不全和尿酸清除率已饱和的痛风患者筛选治疗靶点的研究奠定基础。

目的.尿酸排泌障碍是临床最常见的高尿酸血症的诱因。肠道是除肾脏以外最主要的尿酸排泌器官。ABCG2功能下调可通过肠道排泌尿酸减少介导高尿酸血症的发生。PDZK1是潜在能与ABCG2互相作用及调控功能的重要结构蛋白。本项目立足上述背景，通过小鼠高尿酸血症模型及肠道上皮细胞的体外研究平台系统地阐述高尿酸血症本身对ABCG2/PDZK1的可能调控作用，初步揭示参与调控的主要信号分子和途径。由此进一步为肾功能不全和尿酸清除率已饱和的痛风患者筛选治疗靶点的研究奠定基础。.方法.加入可溶性尿酸盐刺激人肠道上皮细胞系Caco-2和HT-29，RT-qPCR和Westernblot分别检测PDZK1/ABCG2 mRNA和蛋白的表达，免疫荧光检测PDZK1/ABCG2分子的细胞定位和相对表达强度的变化，流式细胞仪检测ABCG2功能变化。分别用siRNA敲低ABCG2或PDZK1，RT-qPCR和Westernblot交互检测尿酸干预下的ABCG2/PDZK1之间的上下游关系，流式细胞仪检测ABCG2功能变化。.酵母膏联合氧嗪酸钾处理建立高尿酸血症小鼠动物模型，检测高尿酸血症小鼠肠道尿酸排泌的情况及与肠道内ABCG2/PDZK1表达的相关性；ELISA检测高尿酸血症小鼠外周血炎症因子的表达。..结果.（1）可溶性尿酸盐能够促进人肠道上皮细胞HT-29、Caco-2细胞中PDZK1/ABCG2的表达，这一过程受NLRP3/ASC/Caspase-1和P13K/Akt信号通路调控。.（2）ABCG2定位于胞浆和胞膜，尿酸刺激后ABCG2由胞浆向胞膜转移，.（3）PDZK1能够调节ABCG2在HT-29、Caco-2细胞中的表达。.（4）高尿酸血症小鼠空肠、结肠ABCG2显著增加。. (5) 高尿酸血症小鼠外周血促炎因子（IL-1β、TNF-α、MCP-1）升高，抑炎因子（IL-10、TGF-β）减少。