基于氧化苦参碱上调Id2表达抑制糖尿病肾小管上皮细胞转分化的作用及机制研究

Renal tubular epithelial-to-mesenchymal transition (EMT) are the main pathological changes of diabetic nephropathy (DN), which is a common cause of end-stage renal disease and treatment difficulty. Previous studies have suggested that the expression of Id2 decreased as a key role in development of EMT. And Id2 must combine with basic helix-loop-helix(bHLH) transcription factors to adjust the role of downstream target genes. Twist is a kind of the structure of the bHLH transcription factor and Id2 also can combine with Snail in the tumor diseases, both are the important regulatory factor for epithelial-to-mesenchymal transition. However, the regulating mechanisms have not been clearly clarified. BMP-7 can up-regulate the expression of Id2 against the transforming growth factor-β/ Smad2/3 signal caused by high glucose-induced EMT, but its mechanisms of regulation on the renal tubular EMT in DN are unknown. In our previous investigation, Oxymatrine may significant ameliorate renal tubular interstitial fibrosis and inhibit renal tubular EMT through Id2, but the underlying mechanisms have not clearly clarified. Based on this, this study was performed to the multiple means and around the regulation pathway of upstream and downstream of Id2 and to clarify the protection mechanisms of Oxymatrine and its targets. In further research, the focus will be on the sophora resources reasonable use and increase "diabetic nephropathy" attending to provide new experimental basis function. Furthermore, Oxymatrine could promote the research of Chinese medicine treatment application and has important social significance.

肾小管上皮细胞向间充质细胞转分化(EMT)是DN的主要病理特征，病程进展可引起终末期肾衰竭，治疗困难。研究表明，Id2表达减少参与EMT过程，而Id2又必须与bHLH转录因子结合才能发挥调节下游靶基因的作用，Twist是一种bHLH结构的转录因子，在肿瘤中Id2也可通过结合Snail发挥作用，二者均为EMT的重要调节因子，但DN中Id2对Twist、Snail的作用机制并不清楚；BMP-7可通过上调Id2水平而对抗TGF-β1/Smad2/3信号导致的EMT，但BMP-7如何调控Id2表达的机制也不清楚。前期研究发现氧化苦参碱可能通过Id2改善DN肾纤维化，但具体机制仍不清楚。基于此，本项目采用多手段进行干预，围绕Id2的上下游调控通路系统分析氧化苦参碱的保护机制，阐明其作用靶点，为苦参资源的合理利用和增加“糖尿病肾病”功能主治提供新的实验依据，促进中医药研究的治疗应用具有重要的社会意义。

糖尿病肾病(DN)是糖尿病(DM)最常见和最严重的慢性微血管并发症之一。长久以来，有关DN的发病机制，国内外学者进行了大量广泛的研究，但是迄今为止确切的机制尚未充分阐明，亦无有效的治疗药物。本项目建立两种动物模型：DM大鼠动物模型，SD大鼠随机分为NC组、DM模型组、过表达BMP-7组及氧化苦参碱干预(OMT)组和db/db小鼠模型，分为DM组和OMT组。生化、病理形态变化、Western blot、Real-time PCR法等进行相应指标的检测。结合单独敲低或共敲低Smad1/5/8及针对MAPKs系统主要三个信号分子特异性阻断剂，外源性给予rhBMP-7或OMT加高糖培养，观察Id2、Smads、MAPKs及EMT和ECM相关指标的变化；Co-IP来验证Id2与Twist蛋白之间的相互作用关系。研究结果显示，高糖状态下BMP-7可通过BMP-7/Smad1/5/8或抑制MAPKs通路磷酸化来上调Id2的表达以达到抗纤维化作用，Twist可促进EMT过程及使ECM的沉积增多，Id2可能是通过与Twist相互结合，从而抑制Twist对下游靶基因的转录调控作用；而Id2不影响Snail本身的表达，仅通过与Snail结合影响其对下游靶蛋白的转录调控，从而发挥抑制EMT作用；OMT可阻断高糖对Id2的抑制作用或可影响BMP-7/MAPKs通路，促进Id2的恢复，进一步促使Id2与Twist相互结合，从而影响Twist对下游靶基因的调控，抑制EMT的进程，从而改善DN肾小管-间质纤维化。本项目明确了Id2对Twist、Snail的作用及影响下游靶基因的作用机制及BMP-7如何调控Id2表达的机制；明确了氧化苦参碱防治糖尿病肾纤维化的作用及其可能机制，为苦参资源的合理利用、阐明其作用靶点和增加“糖尿病肾病”功能主治提供了新的实验依据，也为延缓糖尿病肾纤维化的进展及治疗带来新的希望。