E2F1/EZH2/Beclin-1通路在糖尿病肾病肾间质纤维化中的作用及白藜芦醇干预研究

Diabetic nephropathy (DN) is one of the most serious complications of diabetes. The pathogenesis of DN has not yet been well elucidated and it is lack of effective measures to treat DN. Research indicates that epithelial-mesenchymal transition in renal tubular epithelial cells is one of important pathogenesis of renal fibrosis associated with DN. Moreover, autophagy plays an important role in the occurrence of EMT, but the exact mechanism is unclear. Recent studies have shown that improve the level of autophagy could promote the degradation of Snail1, which is well known as a master regulators of EMT, and thus suppress the occurrence of EMT. Previous experiment results of the applicant and our research group revealed that E2F1/EZH2/Beclin-1 pathway was close related to the occurrence and development of DN, speculating a mediator role of this pathway on high glucose-induced autophagy suppression and EMT. At present research, renal tubular epithelial cells, diabetic mice and E2F1 gene knockout mice will be served as study subjects to further investigate whether E2F1/EZH2/Beclin-1 pathway mediates high glucose-induced autophagy suppression and EMT in vivo and in vitro and to explore whether this pathway and its biological effect mediates anti-fibrosis effect of resveratrol. This research may be helpful to further clarify the pathogenesis of DN and to provide experimental basis in looking for effective drug molecular targets.

糖尿病肾病（DN）是糖尿病最严重的并发症之一，其发病机制尚未充分阐明并缺乏有效治疗措施。研究表明肾小管上皮细胞的上皮-间充质转化（EMT）是DN肾纤维化的重要发病机制之一，而自噬在EMT的发生中起重要作用，但机制不甚清楚。最新研究显示，提高细胞自噬水平能促进EMT始动因子Snail1的降解，进而抑制EMT的发生。申请者及课题组前期结果表明E2F1/EZH2/Beclin-1通路可能与DN的发生发展密切相关，推测该通路可能介导了高糖对自噬的抑制以及对EMT的诱导作用。本项目将以肾小管上皮细胞、糖尿病小鼠及E2F1基因敲除小鼠为研究对象，在体内和体外深入研究高糖是否通过E2F1/EZH2/Beclin-1通路抑制自噬，促进EMT，并探讨白藜芦醇是否可通过调控E2F1/EZH2/Beclin-1通路的效应发挥抗纤维化作用，为进一步阐明DN发病机制、寻找有效药物分子靶标提供实验依据。

糖尿病肾病（DN）是糖尿病严重的并发症，肾小管上皮细胞-间充质转化（EMT）是DN肾纤维化的重要发病机制之一，Snail1通过自噬降解减少可能是EMT 发生的重要因素，而E2F1/EZH2/Beclin-1通路与自噬关系密切。本项目以肾小管上皮细胞和糖尿病小鼠研究对象，利用过表达和RNA干扰技术在体内外深入研究E2F1/EZH2/Beclin-1通路是否通过抑制自噬减少Snail1降解而促进EMT发生，并探讨白藜芦醇是否可通过调控该通路发挥抗纤维化作用。结果表明：糖尿病大鼠肾组织和高糖培养的肾小管上皮细胞中LC3Ⅱ/Ⅰ、Beclin-1蛋白表达减少，p62表达增多，自噬受到抑制，发生EMT，ECM沉积增多。促进自噬（自噬激动剂雷帕霉素）可部分逆转高糖诱导的肾小管上皮细胞 EMT并减少ECM沉积；而抑制自噬（自噬抑制剂氯喹）可促进高糖诱导的肾小管上皮细胞 EMT并增加ECM沉积。高糖状态下自噬受抑是导致Snail 1降解减少的主要原因，进而导致大鼠肾小管上皮细胞发生EMT和ECM沉积增多，促进肾脏纤维化的发生。我们还发现与正常对照组比较，在DM小鼠肾组织及高糖培养的肾小管上皮细胞中E2F1、EZH2表达增加，E2F7表达减少。分别敲低内源性E2F1和EZH2或过表达E2F7后可以部分恢复细胞自噬水平，抑制EMT的发生，减少ECM沉积；分别过表达E2F1和EZH2或敲低E2F7后则可抑制细胞自噬水平，促进EMT的发生，增加ECM沉积。且E2F1可以通过调控EZH2的表达调控肾小管上皮细胞自噬水平而影响EMT的发生。白藜芦醇能通过抑制E2F1的表达和上调E2F7的表达，促进大鼠肾小管上皮细胞自噬水平并抑制EMT发生。通过本研究为进一步阐明DN发病机制、寻找有效药物分子靶标防治DN的发生发展提供了实验依据。