eIF4F复合物活性下调导致糖尿病创面难愈的分子成因及病理意义

eIF4F complex can promote wound healing by stimulating the translation initiation process of a variety of molecules. Akt-mTOR-4EBP1 signaling pathway regulates the activity of eIF4F complex and its dysfunction has been found to be closely related to the impaired wound healing of diabetic mellitus ulcers (DMU), but its molecular mechanism remains uncovered. In our previous research, we demonstrated that: ① The O-GlcNAc modification of 4EBP1 was enhanced in the skin tissues of diabetic mice; ② The activity of eIF4F complex was reduced in fibroblasts cultured in sustained high glucose condition; ③ The inhibitory effect of high glucose on the proliferation of fibroblasts can be reversed either by restricting the O-GlcNAc modification or by increasing the activity of eIF4F complex; ④ Up-regulation of the activity of eIF4F complex significantly accelerates the skin wound healing in mice. Accordingly, we speculated that high glucose up-regulate the O-GlcNAc modifications of 4EBP1 and Akt, and thereby down-regulate the activity of eIF4F complex, is a new molecular mechanism of the impaired wound healing of DMU. In this project, we intended to systematically explore the effects of high glucose on the post-translation modifications and expression levels of 4EBP1 and Akt, the activity of eIF4F complex and the expression levels of its downstream molecules and the biological behavior of the fibroblasts, therefore provides a scientific basis for a new strategy to use the eIF4F complex as a molecular target for efficiently promoting wound healing of DMU.

eIF4F复合物能通过调控多种促修复分子的翻译起始过程影响创面愈合，已知其上游Akt-mTOR-4EBP1信号通路的异常失活与糖尿病创面难愈密切相关，但机理不明。我们前期研究首次发现：①糖尿病小鼠皮肤组织中4EBP1的O-GlcNAc修饰增强；②持续高糖培养成纤维细胞能够下调eIF4F复合物活性；③限制O-GlcNAc修饰或上调eIF4F复合物活性均能逆转高糖对成纤维细胞增殖的抑制作用；④上调eIF4F复合物活性能加速小鼠皮肤创面愈合。据此我们推测：高糖上调4EBP1和Akt 的O-GlcNAc修饰引发eIF4F复合物活性下调是糖尿病创面难愈的新机制。本项目拟系统探讨高糖对4EBP1和Akt的翻译后修饰方式及水平、eIF4F复合物的活性及其下游分子的表达水平以及细胞生物学行为的影响，为建立以eIF4F复合物为靶点、能高效促进糖尿病创面愈合的治疗新策略提供科学依据。

众所周知，eIF4F复合物控制多种蛋白分子的翻译起始，参与调节多种细胞行为，但其是否参与调控创面愈合尚未见报道。在本项目中，我们发现使用eIF4E进行系统基因治疗能显著促进小鼠皮肤全层缺损的修复，而转染4EBP1阻断eIF4F复合物装配或转染K100阻断Mnk1对eIF4E磷酸化则显著延迟创面愈合。eIF4E局部基因治疗能通过促进肉芽组织生长、加速创面再上皮化和增强血管新生等机制促进小鼠皮肤全层缺损的修复，而创面局部转染4EBP1和K100则能显著抑制创面愈合。体外试验表明转染eIF4E上调eIF4F复合物活性能显著上调多种促创面愈合分子的表达，而转染4EBP1和K100则能显著下调这些分子的表达。此外，转染eIF4E能显著促进HaCaT细胞迁移，而转染4EBP1或K100均能显著抑制HaCaT细胞迁移。这些结果表明eIF4F复合物在调控创面愈合过程中发挥着重要作用。在此基础上我们探讨了糖尿病状态下eIF4F复合物活性的变化及其发生机理。我们发现持续高糖培养能够显著上调细胞内总4EBP1、O-GlcNAc修饰4EBP1以及O-GlcNAc修饰Akt的水平、下调eIF4F复合物活性及多种促创面修复分子的表达、抑制细胞增殖和迁移，但该作用能够被OGT特异性siRNA所逆转。在糖尿病患者和STZ诱导的糖尿病小鼠模型的皮肤组织中均存在多种eIF4F复合物下游分子的表达水平的显著下调，而4EBP1和Akt的O-GlcNAc修饰水平则显著上调，提示在糖尿病状态下eIF4F复合物活性受到抑制。动物实验证实在体转染OGT siRNA能通过增强再上皮化和肉芽组织生长显著促进糖尿病创面愈合。本研究结果不仅发现了eIF4F复合物在调控创面愈合中发挥重要作用，而且证实了eIF4F复合物是促进糖尿病愈合的潜在药物靶点，为建立能高效促进糖尿病创面愈合的治疗新策略提供了实验证据。