抗肾间质纤维化新药——氟非尼酮抑制肾小管上皮细胞凋亡的研究

Renal interstitial fibrosis is the common pathway and pathological basis of chronic kidney disease progress to end-stage renal disease. The efficacy of the currently available treatment is disappointing and new anti-fibrotic medicine with multiple targets and pathways is urgently needed. Fluorofenidone(AKF-PD) is a new compound drug designed by Central South University. Its pre-clinical study was already completed and the application for Phase I clinical trials of new drugs has been submitted. In our previous study, we found AKF-PD could inhibit apoptosis of tubular epithelial cells in vitro and in vivo, which plays an important role in tubular atrophy and renal interstitial fibrosis.And its effect on apoptosis may be related to the inhibition of signaling pathways such as Fas pathway, mitochondrial pathway and endoplasmic reticulum stress. In our present experiments, we will use cells, animals and various molecular biology techniques to test if AKF-PD can affect the key points of the three apoptosis pathways This project will help us to understand the pharmacological mechanisms of AKF-PD , improve its use in clinic, and establish the theoretical foundation for the development of other antifibrotic drugs.

肾间质纤维化是所有慢性肾脏病进展到终末期肾病的共同途径和病理基础，临床上缺乏有效治疗药物，开发新型多通道多靶点抗纤维化药物迫在眉睫。氟非尼酮（AKF-PD）是中南大学自主设计的新型化合物，已完成了抗肾间质纤维化的临床前研究，并进行了国家I类新药的I期临床试验申报。肾小管上皮细胞凋亡是导致肾小管萎缩及肾间质纤维化的重要原因，课题组前期研究发现，AKF-PD在体外细胞实验及体内动物实验中均具有抑制肾小管上皮细胞凋亡的作用， 并且其作用可能与影响Fas通路、线粒体通路及内质网应激等信号通路有关。本课题拟进一步通过细胞和动物实验，综合应用多种分子生物学技术，明确AKF-PD是否可分别作用于这3条细胞凋亡通路的关键环节，从而多靶点抑制肾小管上皮细胞凋亡，发挥抗肾间质纤维化作用。本项目将有助于加深对AKF-PD药理作用机制的认识，为AKF-PD在临床上的应用及其他抗纤维化新药的研发奠定理论基础。

肾间质纤维化是所有慢性肾脏病进展到终末期肾病的共同途径和病理基础，是决定患者预后的关键因素，但临床上缺乏有效治疗药物，开发新型多通道多靶点抗纤维化药物迫在眉睫。氟非尼酮（AKF-PD）是中南大学自主设计的新型抗纤维化化合物，已进入国家I类新药的I期临床试验阶段。肾小管上皮细胞凋亡是导致肾小管萎缩及肾间质纤维化的重要原因，课题组前期研究发现，AKF-PD具有抑制肾小管上皮细胞凋亡的作用，提示AKF-PD可能通过抑制肾小管上皮细胞凋亡而发挥抗肾间质纤维化的作用。本课题在此基础上，进一步明确AKF-PD抑制肾小管上皮细胞凋亡的分子机制。.在本研究中，我们首先构建血管紧张素Ⅱ(AngⅡ)诱导肾小管上皮细胞（NRK-52E）凋亡的细胞模型，通过荧光染色、TUNEL染色以及流式细胞仪检测法证实AKF-PD可明显抑制AngⅡ诱导的NRK-52E细胞凋亡；进一步通过Real-time PCR、Western blot等方法证实，AKF-PD能影响AngⅡ刺激NRK-52E细胞后Fas通路、线粒体通路及内质网通路中多个重要因子的表达。在体内动物实验中，我们通过HE染色、Masson染色及Ⅰ型胶原（COLⅠ）Real-time PCR及免疫组化证实AKF-PD可明显抑制单侧输尿管梗阻（UUO）大鼠肾间质纤维化；通过TUNEL染色及PCNA免疫组化证实AKF-PD可明显抑制UUO大鼠肾小管上皮细胞凋亡，而对肾小管上皮细胞增殖无明显影响；通过Real-time PCR、Western blot及免疫组化的方法证实AKF-PD可影响UUO大鼠肾组织中Fas通路、线粒体通路及内质网通路中多个重要因子的表达。.本研究通过体外细胞实验及体内动物实验，证实AKF-PD可分别作用于Fas通路、线粒体通路及内质网通路的关键环节，从而多靶点抑制肾小管上皮细胞凋亡，发挥抗肾间质纤维化作用。本研究将有助于加深对AKF-PD药理作用机制的认识，为AKF-PD在临床上的应用及其他抗纤维化新药的研发奠定理论基础。