基于内质网应激的miRNAs调控研究糖尿病肾病足细胞"毒损肾络" 病理机制及滋阴益气活血解毒法的干预作用

Injured podocytes has become increasingly prominent importance during the early stage of diabetic nephropathy(DN) , but its exact mechanism has not been clarified. It is widely thinked that podocytes apoptosis is closely related. Endoplasmic reticulum stress(ERS) is the key mechanism during development and progression of DN, and it is related with podocytes apoptosis, and it is regulated by miRNAs in the glioma cell C6. So far there is no reported at home and abroad that miRNAs regulate ERS during the podocytes apoptosis in DN. Our preliminary study confirmed that the mRNA expression of GRP78 was increased , otherwise the protein expression of GRP78 was decreased in the injured podocytes. It is probably a new mechanism about podocytes apoptosis that the protein expression of GRP78 is regulated by miRNAs before the transcription. To provide that hypothesis, we will observe that miRNAs is differentially expressed, and how miRNAs regulate ERS of podocytes apoptosis in the models of DN (genetically modified MKR mouse) and vitro injured podocytes using molecular biology, laser capture microscopic separation techniques and so on. At the same time, all will be verified by the traditional Chinese Medical prescription based the method of tonifying yin and qi, promoting blood flow and detoxificating. Our aim is to enrich the scientific connotation of the pathogenesis of " kidney damage by poison " in DN, and provide scientific clinical diagnosis and look forward to new therapeutic targets for DN.

足细胞损伤在糖尿病肾病(DN)早期病变阶段凸显重要，但其确切机制尚未阐明，多认为与足细胞凋亡密切相关。内质网应激（ERS）是DN发生发展的关键机制，在足细胞凋亡中具有重要地位，胶质瘤C6细胞中ERS受控于miRNAs。但有关miRNAs调控ERS，介导DN足细胞凋亡的研究，迄今国内外尚无报道。我们预实验中发现，受损足细胞内GRP78 mRNA 表达增高，而GRP78 蛋白表达降低。我们推测GRP78蛋白表达存在转录前miRNAs调控，这可能是DN足细胞凋亡的新机制。为证实这一假说，我们以DN小鼠（转基因MKR鼠）和体外诱导受损足细胞为模型，采用分子生物学、激光捕获显微分离等技术，观察miRNAs差异表达，miRNAs对ERS的调控及其与足细胞凋亡的相关性，并以滋阴益气活血解毒法中药复方干预。旨在丰富DN"因毒致虚，毒损肾络"病机的科学内涵，为2型糖尿病肾病寻找新的治疗靶点和治疗药物。

糖尿病肾病（DN）是严重威胁糖尿病患者生命健康的常见并发症之一，其中足细胞是糖尿病肾病发生、发展的关键靶细胞，足细胞损伤在糖尿病肾病(DN)早期病变阶段凸显重要，足细胞的丢失是蛋白尿形成的启动机制之一，但其确切机制尚未阐明，多认为与足细胞凋亡密切相关。. 已有研究证实，内质网应激（ERS）相关凋亡信号途径的激活在过度 ERS致足细胞凋亡中具有重要作用，提示过度ERS在糖尿病肾损伤进程中扮演重要角色。miRNAs是小分子、非编码调控RNA，对足细胞的生长发育和功能都具有重要作用。miRNAs异常表达与肾脏疾病发生密切相关，miRNAs参与内质网应激标志分子GRP78的转录后调控，因此调控miRNAs表达可成为治疗足细胞病变的重要靶标。而miRNAs是否调控ERS介导DN足细胞的凋亡是本课题的研究重点。我们推测miRNAs参与内质网应激标志分子GRP78的转录后调控，从而影响糖尿病肾病足细胞凋亡，在糖尿病肾病的“因毒致损，毒损肾络”病机转化中具有重要作用。. 本课题结合前期研究工作，以MKR鼠2型糖尿病肾病转基因动物模型 、以及体外高糖诱导的肾小球足细胞为研究对象，分别从在体、体外实验两方面，旨在探讨miRNAs调控内质网应激介导糖尿病肾病足细胞凋亡的分子机制及滋阴益气活血解毒立法的左归降糖益肾方的干预作用与干预机制。研究结果表明：左归降糖益肾方可减少MKR转基因2型糖尿病肾病鼠尿微量白蛋白，可能与维持足细胞裂孔膜nephrin、podocin的表达，保护足细胞结构功能有关。预测的miRNAs与GRP78 mRNA及蛋白的表达水平密切相关。证实了高糖状态下肾组织及足细胞内存在内质网应激及miRNAs的表达异常。而以滋阴益气、活血解毒立法组方的左归降糖益肾方可以通过调控miRNAs(miRNA-181a，miRNA-30d)的表达水平，进而影响内质网应激标志分子GRP78的表达水平；同时，通过降低内质网应激凋亡信号分子AKT1、Caspase-12、Bcl-2、Bax的表达水平，从而保护肾小球足细胞。