基于瘦素介导的JAK/STAT信号通路调控炎症效应机制研究清肾颗粒对肾间质纤维化干预作用

Renal tubular epithelial-mesenchymal transition（EMT）is the key step to renal interstitial fibrosis, so studying the mechanism could play an important role in postponing the CRF process. CRF 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）Therapy can retard renal fibrosis and improve renal function through suppressing serum Leptin and the inflammation of the kidney, but Leptin Signaling transduction pathway and its regulation of inflammatory signaling mechanism is not clear. The data have shown that Leptin plays an important role in the process of renal fibrosis, JAK/STAT pathway is the main signal transduction pathway of Leptin; As the major regulation and initiation factors in inflammatory, NF-κB might result in renal tubular EMT and renal interstitial fibrosis; It has been established that the cross-talk mechanism exists between JAK/STAT and NF-κB signaling pathway. The rats model of unilateral ureter obstruction（UUO）and renal tubular epithelial cells（HK-2）are established in this project to study the role of JAK/STAT signaling pathway mediated by Leptin on NF-κB pathway and inflammatory effect; and further observe the the interventional effect of Qingshen Granule on Leptin signaling transduction, revealing the mechanism of anti-renal fibrosis.

肾小管上皮细胞转分化（EMT）是肾间质纤维化发生的关键，研究其发病机制对延缓肾衰竭进展有重要意义。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复方清肾颗粒可以通过抑制血清瘦素的生成及肾脏炎症反应，发挥抗肾间质纤维化作用，但对于瘦素信号转导通路及其调控的相关炎症信号机制尚不清楚。研究表明，瘦素在肾脏纤维化过程中发挥重要作用，而JAK/STAT通路是瘦素的主要信号转导通路；NF-κB作为炎症效应的主要调控和启动因子，导致肾小管EMT及肾间质纤维化进展；现已证实，JAK/STAT和NF-κB两条信号通路之间存在串话机制。本研究以单侧输尿管梗阻（UUO）肾间质纤维化大鼠和人肾小管上皮细胞（HK-2）为研究对象，探讨瘦素介导的JAK/STAT信号通路对NF-κB通路及炎症效应的调控作用；在此基础上观察清肾颗粒对瘦素信号通路转导的干预作用，揭示其抗肾脏纤维化的作用机制。

本研究系统探讨了瘦素介导的JAK/STAT信号通路对NF-κB通路及炎症效应的调控作用，及其在肾小管上皮细胞转分化（EMT）及肾间质纤维化的机制，并深入阐述了清肾颗粒抗肾间质纤维化的作用机理；研究方法涉及了ELISA、EMSA、Western blot、RT-PCR、免疫组织及细胞化学等。主要研究内容与结果如下： .（1）在体实验：瘦素通过介导JAK/STAT信号通路活化，进而激活NF-κB诱导的炎症反应，导致UUO大鼠的肾小管EMT和肾间质纤维化进程；中药清肾颗粒能够通过抑制瘦素生成及其介导的JAK/STAT通路活性，进而降低NF-κB活性及其诱导的炎症效应，从而抑制肾小管EMT进程，延缓肾间质纤维化进展。.（2）体外实验：根据实验结果选择24小时为药物作用于HK-2细胞的适宜时间，瘦素刺激浓度为100ng/ml。给予瘦素刺激后，HK-2细胞中JAK/STAT信号通路活性增加、NF-κB活性及炎症因子表达、细胞转分化进程均增强；而应用JAK抑制剂AG490后，HK-2细胞的NF-κB活性及炎症效应因子表达、细胞转分化进程均减弱；应用NF-κB抑制剂PDTC后，HK-2细胞的炎症反应及转分化进程亦能够得到减弱；清肾颗粒含药血清能够通过抑制瘦素生成及其介导的JAK/STAT通路活性，进而抑制NF-κB活性及其诱导的炎症效应，抑制HK-2细胞转分化进程。.该项目从整体、细胞和基因蛋白表达层面深入研究瘦素介导的JAK/STAT信号通路对NF-κB通路及炎症效应的调控作用，从而明确瘦素抑制肾小管EMT、抗肾间质纤维化的作用机制；并阐明中药清肾颗粒对对瘦素信号通路调控机制的干预作用，及其对肾小管EMT、肾间质纤维化的影响，从而发现清肾颗粒抗肾脏纤维化作用的新靶点，为其临床应用提供更充分的理论依据。该项目已验证了预定设想，完成了预期计划。