从ROS-Caspase-1—GSDMD经典炎性通路研究矢志方减轻尿酸诱导肾小管上皮细胞焦亡的机制与作用靶点

Pyroptosis is a cell programmed death found in recent years triggered by activated caspase-1 cleaving GSDMD. Studies confirm that pyroptosis of tubular epithelical cells plays an important role in tubular injury. Shizhifang is an experiential formula that used clinically in the treatment of hyperuricemia and uric acid nephropathy. Based on the previous studies, we found that Shizhifang can effectively inhibit ROS release and regulate NLRP3-ASC-Caspase-1 axis activation in hyperuricemic rats to reduce tubular epithelical cells pyroptosis and improve kidney inflammation damage. However, it is still necessary to explore how the pathway of ROS-Caspase-1-GSDMD affect pyroptosis and which molecular maybe the target of Shizhifang regulate pyroptosis pathway. Based on this, we propose a hypothesis that Shizhifang's protective effect on the kidney of hyperuricemic model mice may be related to the regulation of some key protein targets in the ROS-Caspase-1-GSDMD inflammatory pathway and the reduction of uric acid-induced renal tubular epithelial cell pyroptosis. This project will establish in vitro uric acid-induced renal tubular epithelial cell injury model, and inhibit molecules of ROS、NLRP3 and Caspase-1 to explore the mechanisms and precise targets of Shizhifang suppressing pyroptosis. Further, we will establish the oxonic acid potassium-induced hyperuricemia mouse model to verify the mechanism and targets of the Shizhifang playing a role in anti-pyroptosis and provide a scientific basis for further research and application.

细胞焦亡是由活化的Caspase-1裂解GSDMD触发的细胞程序性死亡，肾小管上皮细胞焦亡在肾小管炎症损伤中具有重要的作用。前期发现治疗尿酸性肾病的临床经验方矢志方通过抑制尿酸诱导的ROS释放和NLRP3-ASC-Caspase-1轴活化，减轻肾小管上皮细胞焦亡、改善肾脏炎症。但矢志方如何调控ROS-Caspase-1-GSDMD通路影响细胞焦亡且作用于哪一靶点仍需研究。据此我们提出假说:矢志方对高尿酸血症模型小鼠的肾脏保护作用可能与调控ROS-Caspase-1-GSDMD炎性通路中某关键靶点，减轻尿酸诱导的肾小管上皮细胞焦亡有关。本项目在体外建立尿酸诱导的肾小管上皮细胞损伤模型，分别抑制ROS、NLRP3、Caspase-1，研究矢志方抑制细胞焦亡的机制和靶点，进一步建立氧嗪酸钾诱导的高尿酸血症小鼠模型，验证矢志方发挥抗焦亡作用的机制和精确靶点，为进一步研发和推广应用提供科学依据。

本项目着眼于高尿酸血症肾小管上皮细胞炎性损伤的病理学基础，定位在尿酸诱导的ROS释放、NLRP3炎性体活化导致肾脏炎症的机制。结合新进发现Caspase-1—GSDMD经典炎性通路在细胞焦亡中的关键和特异性作用，以及中医药对肾脏炎症的调控作用，思考具有“化痰祛湿、活血化瘀”作用的矢志方对细胞焦亡的作用机制，探索矢志方发挥抗焦亡与炎症损伤的关键靶点，我们提出科学假说：矢志方对高尿酸血症小鼠的肾脏保护作用可能与调控ROS-Caspase-1—GSDMD炎性通路中某关键蛋白靶点，减轻尿酸诱导的肾小管上皮细胞焦亡有关。本项目在体外建立尿酸诱导的肾小管上皮细胞损伤模型，分别抑制ROS、NLRP3、Caspase-1，研究矢志方抑制细胞焦亡的机制和靶点。进一步建立氧嗪酸钾诱导的高尿酸血症小鼠模型，验证矢志方发挥抗焦亡作用的机制和精确靶点，为进一步研发和推广应用提供科学依据。本项目的研究将为矢志方“化痰祛湿、活血化瘀”防治高尿酸血症肾小管上皮细胞炎性损伤提供进一步实验依据。.本项目执行四年，采用生化分析、光镜、透射电镜、免疫组化、免疫荧光、Western blot、RT-PCR和Elisa等手段，通过体内外实验完成对科学假说的验证。体内实验结果显示，矢志方能有效降低高尿酸血症小鼠血尿酸，保护肾功能，减轻小管损伤，抑制肾间质胶原纤维沉积；矢志方能有效抑制高尿酸血症小鼠肾组织ROS产生，抑制NLRP3和Caspase-1、GSDMD、N-GSDMD等焦亡相关蛋白的表达，减轻肾脏炎性损伤。体外实验结果显示，ROS抑制剂Mito-Tempo可以抑制尿酸诱导的NRK-52E细胞ROS水平升高，矢志方干预后与Mito-Tempo具有相似效果；NLRP3炎性体抑制剂MCC950可以通过抑制尿酸诱导NRK-52E细胞和高尿酸血症小鼠肾组织中NLRP3炎性体的活化及组装，抑制细胞焦亡，减轻焦亡相关蛋白和基因的表达，同时减轻炎性因子IL-1β、IL-18水平；矢志方干预细胞后与MCC950具有相似的效果。另外通过UPLC-Q-TOF/MS检测矢志方给药血清的主要原型产物和代谢产物。以上结果表明，矢志方可能通过抑制肾组织ROS产生、抑制尿酸诱导NLRP3炎性体的激活，从而减轻尿酸诱导肾小管上皮细胞焦亡的发生。