Background and aim

Intestinal epithelial dysfunction is the foundation of various intestinal and extra-intestinal diseases, while the effects and mechanism of uric acid on the intestinal barrier are little known. TSPO has been shown to be related to the generation of ROS and is involved in regulating inflammation, whether uric acid drives intestinal epithelial dysfunction through TSPO-mediated NLRP3 inflammasome activation is unknown.

Methods

UOX gene knockout mouse (UOX-/-) were used for models of hyperuricemia. Fluorescein isothiocyanate (FITC)-labeled dextran was used to assess in vivo intestinal permeability. Serum lipopolysaccharide (LPS) and culture supernatants IL-1β were measured using ELISA Kit. IEC-6 exposed to different concentrations of uric acid was used for in vitro experiment. Protein content and mRNA were assessed using Western blotting and Q-PCR, respectively. Intracellular ROS was determined using flow cytometry and fluorescence microscope. Mitochondrial membrane potential was detected on an immunofluorescence. Small interfering RNA transfection was used to assess the interaction between translocator protein (TSPO) and NLRP3 inflammasome. N-acetyl-L-cysteine (NAC) was used as ROS scavenger.

Results

Our results showed that hyperuricemia mice were characteristic by increased intestinal permeability. Hyperuricemia upregulated TSPO, increased production of ROS and activated NLRP3 inflammasome, which resulted in lower expression of occludin and claudin-1. In vitro, we showed that soluble uric acid alone increased the expression of TSPO, depolarized mitochondrial membrane potential, increased ROS release and activated NLRP3 inflammasome, which further reduced the expression of occludin and claudin-1. Silencing TSPO suppressed NLRP3 inflammasome activation and increased expression of claudin-1 and occludin, which was accompanied by lower levels of ROS. Scavenging ROS also significantly inhibited NLRP3 inflammasome activation without change of TSPO, indicating that TSPO-mediated NLRP3 inflammasome activation was dependent on ROS.

Conclusions

In conclusion, uric acid drives intestinal barrier dysfunction through TSPO-mediated NLRP3 inflammasome.

中文翻译：

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尿酸通过 TSPO 介导的 NLRP3 炎性体激活驱动肠道屏障功能障碍

 背景和目的

肠上皮功能障碍是多种肠道及肠外疾病的基础，而尿酸对肠道屏障的影响和机制却鲜为人知。 TSPO已被证明与ROS的产生有关并参与调节炎症，但尿酸是否通过TSPO介导的NLRP3炎症小体激活驱动肠上皮功能障碍尚不清楚。

 方法

UOX基因敲除小鼠（UOX-/-）用于高尿酸血症模型。异硫氰酸荧光素 (FITC) 标记的葡聚糖用于评估体内肠道通透性。使用 ELISA 试剂盒测定血清脂多糖 (LPS) 和培养上清液 IL-1β。采用暴露于不同浓度尿酸的IEC-6进行体外实验。分别使用蛋白质印迹和 Q-PCR 评估蛋白质含量和 mRNA。使用流式细胞术和荧光显微镜测定细胞内ROS。通过免疫荧光检测线粒体膜电位。小干扰 RNA 转染用于评估易位蛋白 (TSPO) 和 NLRP3 炎性体之间的相互作用。 N-乙酰基-L-半胱氨酸（NAC）用作ROS清除剂。

 结果

我们的结果表明，高尿酸血症小鼠的特征是肠道通透性增加。高尿酸血症上调 TSPO，增加 ROS 的产生并激活 NLRP3 炎症小体，从而导致 occludin 和 claudin-1 的表达降低。在体外，我们发现单独的可溶性尿酸会增加 TSPO 的表达，使线粒体膜电位去极化，增加 ROS 释放并激活 NLRP3 炎症小体，从而进一步降低 occludin 和 claudin-1 的表达。沉默 TSPO 可以抑制 NLRP3 炎症小体的激活，并增加 claudin-1 和 occludin 的表达，同时降低 ROS 水平。清除ROS也显着抑制NLRP3炎症小体激活，且不改变TSPO，表明TSPO介导的NLRP3炎症小体激活依赖于ROS。

 结论

总之，尿酸通过 TSPO 介导的 NLRP3 炎性体驱动肠道屏障功能障碍。