基于氧化应激介导NF-κB信号活化研究清肾颗粒对肾小管上皮细胞转分化的影响

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 and high expression of inflammatory factors in a previous studies, while Clearing away Heat, Resolving Dampness and Removing Blood Stasis（CRR）and Qingshen Granule（a Traditional Chinese Medicine having the effect of CRR）Therapy can retard renal fibrosis and improve renal function through suppressing the inflammation of the kidney, but signaling pathway of anti-inflammatory effects is not clear.The data show that activated NF-κB is the key factor of initiating inflammation and it directly involve in the progress of renal tubular EMT and renal interstitial fibrosis. Oxidative stress play the important role in renal tubular EMT and it can induce the NF-κB activity. The rats model of unilateral ureter obstruction（UUO）and the cell model of renal tubular EMT induced by high glucose are established in this project to study the signaling mechanisms between oxidative stress and NF-κB, the effects on renal tubular EMT and renal interstitial fibrosis，and further observe the mechanism of oxidative stress activating NF-κB adjusted by Qingshen Granule. As a result, a new target for anti-inflammation and anti-fibrosis effect of Qingshen Granule can be revealed.

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慢性肾衰进展有重要意义。湿热证与肾脏炎症状态及高表达炎症因子密切相关，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肾脏炎症反应发挥抗肾脏纤维化、改善肾功能作用，但其抗炎作用的信号转导机制尚不清楚。研究表明，NF-κB信号活化是启动炎症反应的重要因素，直接参与肾小管EMT、肾间质纤维化的发生发展；而氧化应激在肾小管EMT中起重要作用，能够诱导NF-κB信号活化。本研究以单侧输尿管梗阻（UUO）的肾间质纤维化大鼠和高糖诱导的人肾小管上皮细胞（HK-2）转分化的细胞模型为研究对象，从整体、细胞和基因蛋白表达层面深入研究氧化应激对NF-κB信号通路的调控机制及其该机制在肾小管EMT、肾间质纤维化中的作用；并进一步探讨清肾颗粒对氧化应激介导NF-κB信号活化的影响，揭示其发挥抗炎、抗肾脏纤维化的作用机制。

本研究系统探讨了氧化应激介导的NF-κB信号通路活化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：氧化应激通过介导NF-κB信号通路活化，进而激活炎症反应，导致单侧输尿管梗阻（UUO）大鼠的肾小管EMT和肾间质纤维化进程；应用NF-κB特异性抑制剂PDTC阻断NF-κB活性后，大鼠肾小管EMT和肾间质纤维化进程减弱；中药清肾颗粒能够通过抗氧化应激作用，抑制UUO模型大鼠肾脏组织中NF-κB介导的炎症反应，进而抑制肾小管EMT进程，从而延缓肾间质纤维化进展。.（2）体外实验：根据实验结果选择48小时为药物作用适宜时间，PDTC组药物浓度选用5μmol/L较为适宜。给予高糖刺激后，HK-2细胞中氧化应激反应、NF-κB活性及炎症因子表达、细胞转分化进程均增强；而应用NF-κB抑制剂PDTC后，HK-2细胞的炎症因子表达、细胞转分化进程均减弱；清肾颗粒含药血清能够通过抗氧化应激作用，抑制高糖诱导的HK-2细胞中NF-κB介导的炎症反应，进而抑制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探讨了氧化应激介导的炎症反应在肾间质纤维化进程中的作用；并阐明中药清肾颗粒对肾间质纤维化的作用机制，明确其抗炎、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。