bFGF对糖尿病溃疡组织中血管内皮细胞能量代谢关键酶硝化应激的调控作用

The prevalence of diabetes is rising constantly, and the remedy of diabetic skin ulcer has become a difficult problem in clinical. Nowadays, exogenous growth factor bFGF has successfully been applied to treat diabetic skin ulcer and achieved good curative effect. According to former reports, vascular endothelial cell is one of the major targets for bFGF skin ulcer healing. However, the mechanism of signal transmission and network regulation of this process is not yet clear. The former proteomics results showed that bFGF can dramatically decrease the nitration level of ATPSA and ATPSB, which are mainly classified into energy metabolism-related enzymes. Protein nitration may be involved in a variety of functions, possibly including disease initiation and progression, which indicate that bFGF may improve proliferation ability, and finally produce reparative function by regulating the nitration level of these proteins. The purpose of this project is to take further research on the influence of bFGF on the nitration level of these proteins in vascular endothelial cells injured by high glucose, and then to explore relationship between the mechanism of healing function of bFGF and nitrative stress, as well as apoptosis. The achievement of this subject will provide a theoretical basis for bFGF clinical application trials.

糖尿病创面修复是临床亟待解决的难题之一。目前，临床已成功将外源性生长因子bFGF应用于糖尿病创面修复治疗，并取得显著疗效。bFGF促创面修复的主要靶器官之一是与肉芽组织生长密切相关的血管细胞，但其信号传递途径和网络调控作用尚不清楚。前期动物蛋白质组学实验表明bFGF可明显降低给药组创面皮肤中SCOT和ATP合成酶β亚基（ATPSB）的硝化水平，此两种蛋白均为生物体内能量代谢关键酶，而硝化蛋白又可直接影响蛋白质的结构和功能，提示bFGF可能是通过调控SCOT和ATPSB的硝化水平，改善创面能量供应，减少细胞凋亡、提高增殖能力，进而促进创面修复。本课题旨在前期动物实验的基础上进一步研究bFGF对高糖诱导血管内皮细胞中SCOT和ATPSB硝基化修饰酪氨酸位点及其活性的影响，以期深入探索bFGF促糖尿病创面修复机制与硝化损伤的关系。本项研究结果不仅能在理论上指导临床，而且可以直接应用于临床。

糖尿病创面修复是临床亟待解决的难题之一。目前，临床已成功将外源性生长因子bFGF应用于糖尿病创面修复治疗，并取得显著疗效。bFGF促创面修复的主要靶器官之一是与肉芽组织生长密切相关的血管内皮细胞（HUVECs），但其信号传递途径和网络调控作用尚不清楚。本项研究表明，bFGF能降低ROS水平，进而上调蛋白质亚硝基化修饰水平。此外，我们发现bFGF能通过激活AKT，AMPK来增加高糖损伤下HUVECs中eNOS的活性，其中AMPK又在一定程度上对抗ROS的产生，降低了ROS对蛋白质亚硝基化修饰水平的影响。为验证除ROS因素外bFGF在调控高糖诱导下内皮细胞中蛋白质亚硝基化修饰水平变化的关键因素，我们在bFGF组加入小干扰RNA eNOS和iNOS后，则发现eNOS的干扰明显降低了蛋白质的亚硝基化修饰量。我们利用质谱筛选了亚硝基化修饰差异蛋白，发现p65的亚硝基化修饰水平发生显著变化。我们确定GSNO和bFGF能逆转高糖损伤下p65亚硝基化修饰的下降现象，广谱NOS的抑制剂L-NAME能减弱bFGF的作用。考虑到p65是一个转录因子，我们对其进行核质分离，发现GSNO和bFGF组能抑制p65入核，并在荧光定位上验证了这个结论。最后我们对eNOS干扰重新确定了bFGF能通过eNOS对p65进行亚硝基化修饰，从而改变其核定位影响最终的转录水平，进而发挥保护细胞高糖损伤促进创伤修复。