基于P-selectin／PSGL-1介导的MAPK信号通路研究清肾颗粒对肾纤维化血管内皮损伤的干预

Vascular endothelial cell injury plays an important role in renal fibrosis, so studying the mechanism is important in postponing the process of Chronic Kidney Disease（CKD）. Damp-heat syndrome is closely related to inflammatory state of the kidney, while Clearing away Heat, Resolving Dampness and Removing Blood Stasis（CRR）Therapy and Qingshen Granule（a Traditional Chinese Medicine having the effect of CRR）can protect vascular endothelial cells and improve renal function through suppressing serum P-selectin and improved NO/ET ratio imbalance in CKD patients, but the signal pathway of P-selectin mediated and its regulation mechanism is not clear. The data have shown that P-selectin and its ligand PSGL-1 could mediated vascular endothelial inflammatory injury and renal fibrosis via the regulation mechanism of MAPK signal pathway. The renal fibrosis model rats of 5/6 nephrectomy and human umbilical vein endothelial cell（HUVEC）injury model induced by Lipopolysaccharides（LPS）are established in this project to study the role of 4 signaling pathways in MAPK family mediated by P-selectin/PSGL-1 on the process of vascular endothelial inflammatory injury and epithelial–mesenchymal transitions（EndMT）, in order to elucidate the mechanism of renal fibrosis from the point of view of vascular endothelial cell injury in, and further observe the the interventional effect of Qingshen Granule on the above signaling transduction and vascular endothelial cell injury, revealing the mechanism of anti-renal fibrosis.

血管内皮细胞损伤在肾纤维化发生及进展中起重要作用，研究其发病机制对延缓CKD进展有重要意义。湿热证与肾脏炎症反应密切相关，具有清热化湿祛瘀功效的中药清肾颗粒通过降低CKD患者P-selectin水平，改善NO/ET失衡，达到保护血管内皮、改善肾功能作用，但对于P-selectin介导的相关信号通路及其调控机制尚不清楚。研究表明P-selectin与其配体PSGL-1结合后通过对MAPK信号通路调控机制介导了血管内皮炎性损伤和肾纤维化过程。本研究以5/6肾切除大鼠肾纤维化模型和LPS诱导人脐静脉内皮细胞（HUVEC）损伤模型为研究对象，探讨P-selectin/PSGL-1对MAPK家族中4条信号通路的调控机制及其在内皮细胞炎性损伤和内皮细胞-间充质转化（EndMT）中的作用，从内皮细胞损伤角度阐明肾纤维化机制；并观察清肾颗粒对上述信号通路转导和内皮损伤的干预作用，揭示其抗肾纤维化机理。

血管内皮细胞损伤在肾纤维化发生及进展中起重要作用，研究其发病机制对延缓慢性肾脏病的进展有重要意义。本研究系统探讨了P-selectin/PSGL-1通路对MAPK家族4条信号通路（ERK1/2、JNK、p38、BMK1/ERK5）的调控机制及其在肾纤维化中作用；并探讨清肾颗粒抗肾纤维化的作用机理；研究方法涉及Western blot、RT-PCR、免疫组化、免疫荧光等。.（1）在体实验：5/6肾切除肾纤维化大鼠肾脏中P-selectin/PSGL-1通路及MAPK家族4条信号通路（ERK1/2、JNK、p38、BMK1/ERK5）均被激活，导致内皮细胞炎性损伤和内皮细胞-间充质转化（EndMT）；中药清肾颗粒通过抑制P-selectin/PSGL-1及MAPK通路活化，减轻内皮炎性损伤，延缓EndMT和肾纤维化进程。.（2）体外实验：在LPS刺激下（24h，50ng/ml），HUVEC细胞中P-selectin/PSGL-1通路及MAPK家族上述4条信号通路均被激活。分别应用上述通路抑制剂后，HUVEC细胞中炎症因子表达下降，内皮损伤减轻，EndMT进程延缓。进一步发现P-selectin/PSGL-1通路主要对MAPK家族中p38和JNK通路发挥调控作用；而JNK和ERK1/2通路对P-selectin/PSGL-1通路似乎还有反向调节作用。清肾颗粒通过抑制HUVEC细胞中P-selectin/PSGL-1介导的MAPK信号通路活化，进而减轻HUVEC细胞的炎性损伤，延缓EndMT进展。.该项目从整体、细胞和基因蛋白表达层面深入研究P-selectin/PSGL-1通路对MAPK家族4条信号通路的调控作用，从内皮细胞炎性损伤角度阐明肾纤维化机制；并基于上述机制阐明中药清肾颗粒抗肾纤维化的作用机理，为其临床应用提供更充分的理论依据。该项目已验证了科学假说，完成了预期计划。