DPP-4/CD26及其对NRF2负性调控诱导糖尿病溃疡延迟愈合作用及机制研究

Diabetic uclers (DUs) healing is a chronic process, and its pathogenesis has not been well elucidated. The dysfuction of skin keratinocytes and fibroblasts greatly contributes to the delay of diabetic wound healing. Our previous research found that dipeptidyl peptidase 4（DPP-4, also called CD26）inhibitors accelerate the healing process of DUs , which partly owing to the well-orchestrated integration of skin keratinocytes and fibroblasts induced by increased substrates of this enzyme, such as chemokines. Moreover, in several kinds of cells, DPP-4 inhibition actives endogenous antioxidative nuclear factor E2 related factor 2 (NRF2) pathway, which has been well established to play a positive role in diabetic wound healing by ameliorating re-epithelialization and extracellular matrix remodeling, meanwhile improving the frustrated functions of keratinocytes and fibroblasts under high glucose condition, such as migration, proliferation etc. Therefore, we supposed that DPP-4/CD26 may delay the diabetic wound healing by postponing the critical pathophysiological processes, as well as deteriorating repair functions of keratinocytes and fibroblasts, which through the negative regulation on the natural substrates of this enzyme and NRF2 pathway. In this project, animal models (DPP-4/CD26 conditional knockout mice) and skin keratinocytes/fibroblasts (including NRF2 -/- cell line) combined with advanced experimental techniques, for example single cell tracking, will be used to reveal the role of DPP-4/CD26 on diabetic wound healing, and explore the effects of DPP-4/CD26 on cell functions. The potential molecular mechanisms and the role of NRF2 in this process also will be analyzed, aiming to further understand the mechanism of DUs, and provide new strategies and evidence for the treatment of DUs.

糖尿病溃疡（DUs）迁延不愈，其发病机制尚未完全阐明。皮肤表皮和成纤维细胞功能紊乱是导致DUs延迟愈合的重要原因，课题组前期发现二肽基肽酶4（DPP-4，即CD26）抑制剂可明显促进DUs愈合，机制可能部分源于该酶底物（如趋化因子）水平升高优化创缘表皮和成纤维细胞协同作用。此外，抑制DPP-4可使得抗氧化核因子E2相关因子2（NRF2）激活，后者可通过改善高糖条件下受损细胞功能，如迁移、增殖，促进创口再上皮化和胞外基质重塑，加速DUs愈合。推测DPP-4/CD26通过对其天然底物及NRF2负性调控恶化表皮和成纤维细胞修复作用，延缓创口愈合重要进程，诱导DUs延迟愈合。拟以条件敲除小鼠DUs 模型及表皮/成纤维细胞（NRF2-/-）为研究对象，揭示DPP-4/CD26对细胞功能的影响，明确在DUs中的作用；寻找下游可能途径，探索NRF2在其扮演的角色；为深入理解DUs形成机理提供新的依据。

糖尿病溃疡（DUs）迁延不愈，其发病机制尚未完全阐明。皮肤表皮和成纤维细胞功能紊乱是导致DUs延迟愈合的重要原因，课题组前期发现二肽基肽酶4（DPP-4，即CD26）抑制剂可明显促进DUs愈合，机制可能部分源于该酶底物（如趋化因子）水平升高优化创缘表皮和成纤维细胞协同作用。此外，抑制DPP-4可使得抗氧化核因子E2相关因子2（NRF2）激活，后者可通过改善高糖条件下受损细胞功能，如迁移、增殖，促进创口再上皮化和胞外基质重塑，加速DUs愈合。研究内容包括: 第一建立DM常规动物模型和高糖条件下皮肤细胞（表皮细胞Hacat、成纤维细胞HSF）培养，观察DPP-4/CD26在DUs过程中的变化；通过对DPP-4/CD26蛋白表达及活性的调控，明确DPP-4/CD26在DUs愈合中的重要作用；检测NRF2通路、趋化因子及其受体等相关指标，寻找DPP-4/CD26在促进DUs愈合下游可能途径。第二采用基因敲除小鼠及细胞株，结合过表达 DPP-4/CD26 LV-shRNA 慢病毒、质粒/siRNA及抑制DPP-4/CD26活性，揭示NRF2通路、趋化因子及其受体在DPP-4/CD26改善DUs愈合中的重要地位。得出重要结果包括：DPP-4i在体外和STZ诱导的小鼠中加速糖尿病创口愈合、DPP-4i促进SDF-1a产生成纤维细胞间接驱动角质形成细胞的EMT、DPP-4i在糖尿病小鼠中诱导SDF-1a表达和EMT、DPP-4i减轻小鼠糖尿病创口的瘢痕形成、抑制mGPDH上调NRF2信号通路促进高糖条件下表皮细胞群体迁移、mGPDH缺失通过NRF2调控黑色素瘤的发生和转移促进小鼠创口愈合、P62通过调控垂体促性腺激素合成分泌影响雌性生殖、离体敲除GPD2促进高糖培养的成纤维细胞分泌功能从而促进创口愈合、离体敲除GPD2促进高糖培养的成纤维细胞分泌功能从而促进创口愈合、高糖条件下成纤维细胞敲除GPD2促进TGF-β/Smad信号通路加快DUs愈合。遗憾的是NRF2不影响DPP-4i对糖尿病创面愈合的促进作用，这和我们最初的预想不一致，但是我们发现了DPP-4i促进SDF-1a产生成纤维细胞间接驱动角质形成细胞的EMT从而加速糖尿病创口愈合。本课题研究明确了DPP-4/CD26在DUs愈合中的重要作用，探讨其成为DUs有效干预靶点的可能性，为深入理解DUs形成机理及治疗提供新策略据。