基于Carnosine类小分子探针研究Pax6的O-GlcNAc糖基化在妊娠糖尿病致胚胎眼畸形的作用

The exact pathophysiology of embryonic eye malformation caused by gestational diabetes mellitus (GDM) remains poorly understood. Recent studies showed that the activtiy of Pax6, one of the key factors in the process of eye development, can be regulated by O-GlcNAcylation under high concentration of glucose environment. This evidence indicates the key role of O-GlcNAcylation of Pax6 in the pathophysiology of eye malformation, which needs to be proved by scientific study. In our preliminary research, carnosine related peptides (CRP) could inhibit the O-GlcNAcylation of Pax6, and restore the damage of chicken embryo eyes caused by high concentration of glucose. Based on the above results, we plan to choose the molecular probes with high specificity from CRP, by which to study the O-GlcNAcylation of Pax6 in pathophysiology of embryonic eye malformation caused by GDM. For such a purpose, we will determine the effects of CRP on the expression of Pax6, and investigate how CRP inhibits the O-GlcNAcylation of Pax6 by employing GDM mice model and chicken embryo model. The pathway mediated by O-GlcNAcylation of Pax6 in the pathophysiology of eye malformation will be also studied using CRP as small molecular probe. This project aims to clarify the pathophysiology of eye malformation caused by GDM, reveal the protective effects of CRP, and provide scientific evidences for the prevention and treatment of eye malformation caused by GDM.

妊娠糖尿病引起胚胎眼睛发育畸形的机理尚不清楚。近来研究发现高糖环境下眼发育关键因子Pax6的活性受O-GlcNAc糖基化调控，提示妊娠糖尿病致胚胎眼发育畸形的机理可能与Pax6的O-GlcNAc糖基化有关，但尚需科学验证。我们前期研究发现Carnosine类活性肽 (CRP) 能抑制Pax6的O-GlcNAc糖基化，且能修复高糖对鸡胚眼发育的损伤。基于上述结果，本项目拟筛选高特异性CRP小分子探针研究Pax6的O-GlcNAc糖基化在妊娠糖尿病致胚胎眼畸形的作用：在妊娠糖尿病小鼠和高糖鸡胚模型上研究CRP对Pax6 的O-GlcNAc 糖基化生物学功能的影响；研究CRP抑制Pax6的 O-GlcNAc糖基化作用途径；研究CRP经由Pax6 的O-GlcNAc 糖基化转导通路的调控机制。旨在阐明妊娠糖尿病致胚胎眼畸形的发生机理，揭示CRP干预机制，为妊娠糖尿病致胚胎眼畸形的防治提供科学依据。

临床研究表明，糖尿病妊娠时母体持续性的高血糖会导致胚胎出现严重畸形，其中常见且最严重的情形是出现神经管畸形（neural tube defects, NTD），进而引起眼睛等器官发育畸形。然而，关于高糖致神经管及眼睛发育畸形的实验研究目前无十分理想的动物模型，其发生机理也尚未阐明。因此，本项目通过建立一个全新的高糖致神经管及眼睛发育畸形的实验模型，深入探讨其内在的分子机制。同时，基于此模型我们研究了内源性小分子活性肽Carnosine对高糖致NTD的干预作用，并进行了深入的机制研究。. 首先，我们采用鸡胚胎来建立高糖致胚胎发育畸形的体内模型，并从多方面验证此模型的有效性和准确性。基于我们建立的高糖诱导鸡胚神经管畸形模型，我们发现了内源性小分子活性肽Carnosine对该模型具有干预作用。 . 其次，本项目研究了 与高糖应激相关的翻译后修饰—O-GlcNAc修饰对Pax3蛋白的影响，探讨此种营养感受调节机制是否参与了高糖诱导的Pax3蛋白降低，以及Carnosine对胚胎Pax3蛋白的修复作用机制。研究结果提示Pax3蛋白的O-GlcNAc异常增多可能参与了高糖诱导NTD的机制，而这也可能是Carnosine修复此发育损伤的作用靶点之一。. 综上，我们采用鸡胚建立了一个高糖诱导胚胎神经管及眼睛发育畸形的新模型，深入地探讨了高糖环境下胚胎NTD的形成机制，为研究糖尿病妊娠导致胚胎畸形的分子机制提供了新的理论依据。同时，我们还证明了内源性小分子活性肽—Carnosine对高糖诱导的神经管畸形的保护作用，为预防和治疗此类代谢相关的胚胎疾病提供了全新的策略与思路。