细胞焦亡在糖尿病肾病足细胞损伤中的机制研究

Pyroptosis is a new form of programmed cell death with inflammatory response. Recent studies have demonstrated that the pyroptosis of podocytes plays an important role in diabetic nephropathy (DN), however, the underlying molecular mechanism is not yet clear. Our previous studies have shown that DN is closely related to oxidative stress, meanwhile, the expression of MALAT1, a long noncoding RNA, and the nuclear factor erythroid -2p45 related factor 2 (NRF2) play important roles in both oxidative stress and pyroptosis；moreover, miR-200c can be involved in oxidative stress by targeting NRF2, whereas MALAT1 is the endogenous competitive RNA (ceRNA) of miR-200c. Therefore, the project aimed to investigate the relationship between oxidative stress and podocyte pyroptosis in diabetes via establishing glomerular podocytes cell model and DN animal model exposed to high glucose, thereby clarifying whether the mechanism is related to MALAT1 / miR-200c / NRF2 regulation. The contents including: 1) Studying the relationship between oxidative stress and pyroptosis of podocytes exposed to high glucose; 2) Investigating the potential molecular mechanism on the relationship between the oxidative stress-associated podocyte pyroptosis and the expression of NRF2, and the regulatory relationship between the expression of MALAT1 and miR-200c exposed to high glucose; 3) Investigating the effect and its underlying molecular mechanism of atorvastatin on podocyte pyroptosis exposed to high glucose. Our study will provide new ideas for prevention and treatment of DN.

细胞焦亡是近几年新发现的一种细胞程序性死亡方式。研究发现，足细胞焦亡与糖尿病肾病（DN）密切相关，具体机制尚不明确。课题组前期研究表明DN与氧化应激关系密切，而长链非编码RNA MALAT1与NRF2在氧化应激和细胞焦亡中均起重要作用。此外，miR-200c可通过靶向NRF2参与氧化应激，且MALAT1是miR-200c的内源竞争RNA（ceRNA）。因此，本项目通过建立高糖环境下的足细胞培养和DN动物模型，研究糖尿病中氧化应激和足细胞焦亡的关系，及其可能机制是否与MALAT1/miR-200c/NRF2调控相关。内容包括：1.高糖环境下氧化应激与足细胞焦亡的相互关系；2. 高糖环境下，氧化应激相关的足细胞焦亡是否与NRF2相关，同时MALAT1及miR-200c在其中所起的作用及具体机制；3.高糖环境下阿托伐他汀对足细胞焦亡的影响及其可能机制。通过以上研究，以期为DN的防治提供新的思路。

随着全球老龄化加速，糖尿病发病率显著上升，伴随着糖尿病主要并发病-糖尿病肾病（diabetic nephropathy, DN ）的发病率也明显增加。蛋白尿是DN的主要临床特征，而肾小球滤过屏障(glomerular filtration barrier，GFB)是阻止白蛋白尿发生的关键因素。足细胞是肾小球的特异性细胞，位于GFB结构的最外层，足细胞的完整对于维持GFB的正常功能不可或缺。近年来，足细胞损伤的相关研究如焦亡、氧化应激等已成为研究DN 发病机制的热点。本课题负责人在前期的课题研究中发现，足细胞在高糖培养环境下，感官上发现足细胞的伸展性发生了明显改变，同时体内实验发现，DN 大鼠白蛋白尿增多，电镜下足细胞形态改变明显，足突大量融合，其具体机制目前尚未明确。所以本课题负责人查阅大量文献及通过生信分析预测，在高糖环境下阿托伐他汀可能通过MALAT1/miR-200c/NRF2调控足细胞氧化应激，从而调控足细胞焦亡，目前国内外还没有相关文献报道。为了验证这一猜想，课题负责人从细胞和动物层面分别验证。在细胞层面，通过WB、qRT-PCR、流式细胞术、细胞丙二醛(MDA)、谷胱甘肽（GSH）、超氧化物歧化酶（SOD）检测氧化应激及焦亡水平。结果表明，阿托伐他汀可通过MALAT1/miR-200C/NRF2信号通路保护足细胞免受高糖诱导的焦亡和氧化应激的影响。这一研究结果为 DN 的临床诊断及治疗提供方向及基础。