肾康丸通过原癌基因AEG-1调控AEG-1/PTEN泛素化降解通路抑制糖尿病肾病足细胞骨架重构的机制研究

The podocyte cytoskeletal remodeling is the molecular basis of podocyte injury which plays a pivotal role in the development of diabetic nephropathy (DN), but the underlying mechanism is still unclear. Under the support of the National Natural Science Foundation of China, a series of studies of us show that astrocyte elevated gene-1(AEG-1), a oncogene is overexpressed in podocyte of DN and may induce podocyte cytoskeletal remodeling by promoting ubiquitin degradation of the tumor suppressor phosphatase and tensin homology deleted on chromosome ten (PTEN). There still no relevant data have been found so far. This effect of AEG-1 may inhibited by the traditional Chinese medicine Shenkang pill, which developed for the treatment of DN by us and has obtained the certificate of national invention patent. We hypothesize that AEG-1 play a critical role in the pathogenesis of DN by promoting podocyte cytoskeletal remodeling via AEG-1/PTEN ubiquitination degradation pathway. And that Shenkang pill exerts DN renal protective effect by inhibiting AEG-1 and regulating AEG-1/PTEN ubiquitination degradation pathway to inhibit podocyte cytoskeletal remodeling. There we view AEG-1 as an entry point of this project and study the effects of Shenkang pill on the AEG-1 induced podocyte cytoskeletal remodeling via the AEG-1/PTEN ubiquitination degradation pathway, using co-immunoprecipitation, ultrasound-mediated gene transfer, magnetic bead perfusion for glomerular isolation, both in vivo and in vitro. Our results are expected to elucidate a new mechanism of Shenkang pill for the treatment of DN, establish a new target for DN treatment, and reveal a new pathway of podocyte injury in DN.

足细胞骨架重构是糖尿病肾病（DN）的关键致病原因足细胞损伤的分子基础，但发生机制未明。在国家自然科学基金资助下，项目组的系列研究发现DN足细胞中高表达的原癌基因AEG-1可能通过促进抑癌因子PTEN泛素化降解诱导足细胞骨架重构而参与DN发生发展，迄今未见相关报道；自主组方治疗DN的中药肾康丸（已获国家发明专利）可能抑制AEG-1的这一作用。推测：AEG-1通过促进PTEN泛素化降解诱导DN足细胞骨架重构导致足细胞损伤而致病；肾康丸通过抑制AEG-1调控AEG-1/PTEN泛素化降解通路抑制足细胞骨架重构而发挥DN肾保护作用。本项目拟以AEG-1为切入点，采用超声微泡基因转移、磁珠灌注法分离肾小球等方法，从体内外两方面研究肾康丸对AEG-1促进PTEN泛素化降解诱导DN足细胞骨架重构的影响。研究结果有望阐明一种肾康丸治疗DN的新机制，确立一个DN治疗的新靶点，揭示一条DN足细胞损伤的新通路。

糖尿病肾病（diabetic nephropathy，DN）发病率日益增高、发病机制复杂、治疗棘手、预后差、危害大。在国家自然科学基金资助下（81072848、81673792），本项目组的前期系列研究发现DN足细胞中高表达的原癌基因 astrocyte elevated gene-1（AEG-1）可能通过促进抑癌因子phosphatase and tensin homology deleted on chromosome ten（PTEN）泛素化降解诱导足细胞骨架重构而参与DN发生发展，自主组方治疗DN的中药肾康丸可能抑制AEG-1的这一作用。. 本项目以2型DN自发性模型db/db小鼠、STZ小鼠为动物模型，以足细胞、肾小管上皮细胞等肾脏固有细胞为细胞模型，采用超声微泡基因转移、磁珠灌注法分离肾小球、免疫印迹、免疫组化、PCR、Pull down等方法，在体内外研究：（1）肾康丸及其组方中重要有效成分山玉兰的根皮提取物衍生物MCL（自主合成）对 AEG-1 调控 AEG-1/PTEN 泛素化降解通路抑制 DN 足细胞骨架重构的影响；（2）circRNA-010383在DN肾脏纤维化中的调控作用；（3）FOXO3a在DN环境下氧化应激介导的足细胞损伤中的作用；（4）Brahma-related gene-1(BRG-1)在介导肾脏纤维化中的作用。.本项目的研究成果：（1）明确了 AEG-1 通过促进 PTEN 泛素化降解诱导 DN 足细胞骨架重构，揭示了 DN 新的发病机制和干预靶点；（2）阐明了肾康丸以及MCL通过AEG-1调控AEG-1/PTEN泛素化降解通路抑制DN足细胞骨架重构进而发挥其 DN 肾脏保护作用的分子机制，为今后进一步研发拥有自主知识产权的 DN 候选新药肾康丸及MCL提供了扎实的实验基础和理论依据；（3）发现了circRNA-010383在DN肾脏纤维化中的重要负性调控作用以及FOXO3a在DN环境下氧化应激介导的足细胞损伤中的重要作用，丰富了DN新的发病机制研究，为进一步深化肾康丸及MCL的DN调控机制研究提供了新的线索；（4）拓展研究发现BRG-1是介导肾脏纤维化的关键蛋白之一，为进一步探索肾脏纤维化的发病机制提供了新的研究线索和干预靶点。