去甲肾上腺素在维持角膜上皮稳态中的作用及其分子机制

Neurotrophic keratitis is a degenerative disease caused by trigeminal nerve injury which resulted in the corneal epithelium detachment, ulcer and even perforation. In clinic, neurotrophic keratitis has been considered as one of the most intractable corneal diseases. However, the mechanism of corneal epithelial detachment after corneal nerves injury and how the corneal nerves regulating the homeostasis of corneal epithelium are still unclear. Our previous study has found that a decrease of norepinephrine (NE) and nicotinamide adenine dinucleotide (NAD+) in the mice after corneal denervation. The depletion of NE or NAD+ in the corneal epithelium impaired the expression of BCL-2 and phosphorylation of AKT and CREB, which resulted in the mitochondrial apoptosis and spontaneous detachment. It suggested that NE secreted by corneal nerves might be important in regulating the biosynthesis of NAD+ in the corneal epithelium and further keeping the homeostasis of corneal epithelium. Based on these results, this project will investigate the functions and molecular mechanisms of NE in maintaining the homeostasis of corneal epithelium by corneal denervation, NE depletion and co-cultured model of corneal epithelium and trigeminal ganglion cells with small molecule compound, RNA interference and gene knockout. It will enrich the clinical theory of corneal wound healing and provide new therapeutic interventions for neurotrophic keratitis.

神经营养性角膜炎是由三叉神经损伤引起的角膜退行性疾病，常引起角膜上皮损伤、溃疡甚至穿孔，是临床上最为棘手的角膜病之一。但对于角膜神经如何调控角膜上皮稳态以及神经损伤后角膜上皮脱落的机制尚不清楚。我们前期研究发现：角膜神经损伤引起小鼠角膜中去甲肾上腺素（NE）及烟酰胺腺嘌呤二核苷酸（NAD+）降低，而耗竭角膜中的NE或NAD+合成可引起角膜上皮细胞内BCL-2下调、AKT与CREB磷酸化水平降低，进而导致角膜上皮细胞通过线粒体途径发生凋亡并自发脱落，提示角膜神经可能是通过NE维持角膜上皮细胞NAD+水平，从而维持角膜上皮的稳态。基于此，本项目拟通过小鼠角膜神经损伤、NE耗竭及角膜上皮与三叉神经节细胞共培养模型，利用小分子化合物、RNA干扰及基因敲除等方法，揭示NE在维持角膜上皮稳态中的作用及其分子机制，丰富角膜损伤修复的基础理论体系，并为神经营养性角膜病提供新的干预治疗方法。

本项目计划通过小鼠角膜神经损伤、去甲肾上腺素（NE）耗竭及角膜上皮与三叉神经节细胞共培养模型，研究NE在维持角膜上皮稳态中的作用及其分子机制。本课题主要在以下几个方面取得了进展：1.发现β肾上腺素能受体阻滞剂滴眼液通过角膜缘干细胞β2肾上腺素能受体（ADRB2）影响角膜上皮稳态，为临床选用β肾上腺素能受体阻滞剂治疗青光眼提供了理论依据；2.发现糖尿病小鼠的角膜上皮刮除后血浆和角膜中NE水平升高，NE通过角膜上皮ADRB2信号通路影响角膜上皮神经营养因子的表达；3.发现烟酰胺磷酸核糖转移酶（NAMPT）在调控糖尿病角膜上皮损伤修复中发挥重要作用，为糖尿病角膜病变的治疗提供新的策略。通过本课题的研究，共发表SCI论文2篇，其中1篇发表于Metabolism-Clinical and Experimental杂志，获两项发明专利授权，在国内专业学术会议发言1次。