基于HNF-1α/4α对尿酸转运蛋白OAT1/3的转录调节研究矢志方减轻肾小管炎症降尿酸的机制

Hyperuricemia induces renal tubular epithelial cells inflammatory injure, leading to uric acid excretion dysfunction. Previous research of our research group found that Chinese medicine compound Shizhifang can inhibit IL-1β release, improve tubular inflammation and up-regulating OAT1/OAT3 expression, reduce serum uric acid level. However, the mechanism of Shizhifang inhibiting inflammation to regulate the expression of OAT1/OAT3 is still unclear. Literature reports that HNF-1α and HNF-4α are the major transcriptional regulators of uric acid transporters OAT1 and OAT3, and IL-1β inhibits the expression of transcription factors HNF-1α and HNF-4α. Based on this, we hypothesized that Shizhifang inhibits IL-1β release, promotes HNF-1α/HNF-4α to regulate OAT1/OAT3 transcriptional expression and increase uric acid excretion. In this project, the high uric acid cells model of HK-2 was induced by uric acid in vitro, and the hyperuricemia mouse model was established in vivo with potassium oxonate to study the regulation mechanism of IL-1β-HNF-1α/4α-OAT1/3 by Shizhifang. This study provides further experimental evidence for the reduction of renal tubular inflammation to reduce blood uric acid by Shizhifang which has the role of dispelling phlegm and eliminating dampness and promoting blood circulation to remove blood stasis.

高尿酸血症引起肾小管炎症，导致尿酸排泄减少。课题组前期研究发现，中药复方矢志方能够抑制IL-1β释放，减轻肾小管炎症，并显著上调肾小管尿酸转运蛋白OAT1/OAT3表达，降低血尿酸水平。然而，矢志方如何抑制炎症调节OAT1/OAT3表达，具体机制仍不清楚。文献报道，HNF-1α和HNF-4α是尿酸转运蛋白OAT1、OAT3的主要转录调节因子，IL-1β抑制HNF-1α/4α表达。据此，我们提出假说：矢志方通过抑制IL-1β释放，促进HNF-1α/HNF-4α调节OAT1/OAT3转录表达，增加尿酸排泄。本项目在体外以尿酸诱导HK-2高尿酸细胞模型，在体内以氧嗪酸钾建立高尿酸血症小鼠模型，研究矢志方对IL-1β- HNF-1α/4α -OAT1/3的调节作用机制。本研究为矢志方“化痰祛湿、活血化瘀”减轻肾小管炎症从而降低血尿酸提供进一步实验依据。

项目背景：高尿酸血症引起肾小管炎症，导致尿酸排泄减少。 .主要研究内容：体外培养HK-2细胞，分别以尿酸、IL-1β inhibitor、IL-1β、尿酸+IL-1β inhibitor、尿酸+IL-1β干预24h，另培养HK-2细胞，分正常组、尿酸模型组、矢志方组（300ug/mL）、尿酸+矢志方组干预24h，分别检测炎症因子IL-1β，IL-18、IL-6、TNF-α水平及HNF-1α、HNF-4α、OAT1、OAT3蛋白的表达。体内实验采用氧嗪酸钾（250mg/kg）和腺嘌呤（50mg/kg）灌胃建立尿酸性肾病小鼠模型，分正常组、模型组、非布司他组和矢志方组，每组10只。2周后，检测各组小鼠血尿酸、肾功能指标，HE、Masson染色观察肾组织普通病理，Western blot、免疫荧光检测肾组织炎症因子IL-1β及HNF-1α、HNF-4α、OAT1、OAT3的表达。.重要结果：体外实验发现，与0ug/mL尿酸比较，200、400、600ug/mL尿酸干预HK-2细胞24h，炎症因子IL-1β，IL-18、IL-6、TNF-α水平升高，HNF-1α、HNF-4α、OAT1、OAT3表达减少；以IL-1β干预HK-2，炎症因子及HNF-1α/HNF-4α、OAT1/OAT3表达水平与200ug/mL尿酸干预效果相当；抑制IL-1β或以矢志方组干预尿酸细胞模型，可见IL-1β表达减少、尿酸转运相关蛋白表达改善。体内实验发现，模型组小鼠血尿酸水平升高、肾功能降低，病理可见肾小管管壁变薄、管腔扩张、纤维组织增生，Western blot、免疫荧光检测显示肾组织IL-1β表达增加、HNF-1α、HNF-4α、OAT1、OAT3表达减少；而非布司他组和矢志方组小鼠血尿酸水平下降、肾功能改善、肾损伤减轻，肾组织IL-1β表达减少、HNF-1α、HNF-4α、OAT1、OAT3表达增加。.关键数据：尿酸诱导炎症因子抑制HNF-1α/HNF-4α和OAT1/OAT3导致肾小管炎症；矢志方可降低肾组织炎症因子IL-1β水平促进HNF-1α/HNF-4α和OAT1/OAT3表达，减轻肾小管炎症和肾损伤，降低血尿酸。.科学意义：本研究为运用矢志方“化痰祛湿、活血化瘀”减轻高尿酸血症肾小管损伤降低血尿酸提供了进一步的实验依据。