Medical College, Qingdao University, Qingdao, China.
State Key Laboratory Cultivation Base, Shandong Provincial Key Laboratory of Ophthalmology, Shandong Eye Institute, Shandong First Medical University & Shandong Academy of Medical Sciences, Qingdao, China.
Invest Ophthalmol Vis Sci. 2019 Aug 1;60(10):3538-3546. doi: 10.1167/iovs.19-26909.
To determine if trigeminal innervations of the corneal epithelium maintains its integrity and homeostasis through controlling the nicotinamide adenine dinucleotide (NAD) content of this tissue.
Corneal denervation of C57BL/6 mice was induced by squeezing the nerve bundles that derive from the trigeminal ganglion and was confirmed by whole-mount corneal nerve staining and the sensation test. The apoptosis of the corneal epithelium was examined by TUNEL assay and annexin V/propidium iodide staining. NAD biosynthesis-related enzymes were analyzed by quantitative PCR, immunofluorescence staining, and Western blotting. FK866, an inhibitor of nicotinamide phosphoribosyltransferase (NAMPT), exogenous nicotinamide mononucleotide (NMN), and NAD+ were used to evaluate the effect of NAD+ on the apoptosis of cultured corneal epithelial cells and epithelial detachment in denervated mice. Protein expression that related to apoptosis and phosphorylation were analyzed by Western blotting.
The denervated mice showed spontaneous corneal epithelial detachment and cell apoptosis accompanied with impaired epithelial NAD+ contents due to low levels of NAMPT. Similarly, inhibition of NAMPT recapitulated epithelial detachment as in denervated mice and induced apoptosis in cultured corneal epithelial cells. The replenishment of NMN or NAD+ partially slowed down corneal nerve fiber degeneration, reduced the epithelial defect in denervated mice, and improved apoptosis induction in FK866-treated cells by restoring the activation levels of SIRT1, AKT, and CREB.
Corneal denervation lowered epithelial NAD+ contents through reducing the expression of NAMPT and caused cell apoptosis and epithelial defects, suggesting that corneal innervations contribute to epithelial homeostasis by regulating NAD+ biosynthesis.
通过控制角膜上皮组织的烟酰胺腺嘌呤二核苷酸（NAD）含量，确定三叉神经对角膜上皮的神经支配是否能维持其完整性和内稳态。
通过挤压源自三叉神经节的神经束，对 C57BL/6 小鼠进行角膜去神经支配，并通过全角膜神经染色和感觉测试进行验证。通过 TUNEL 检测和 Annexin V/碘化丙啶染色检测角膜上皮细胞的凋亡。通过定量 PCR、免疫荧光染色和 Western blot 分析 NAD 生物合成相关酶。使用烟酰胺磷酸核糖转移酶（NAMPT）抑制剂 FK866、外源性烟酰胺单核苷酸（NMN）和 NAD+，评估 NAD+对培养的角膜上皮细胞凋亡和去神经支配小鼠上皮脱落的影响。通过 Western blot 分析与凋亡和磷酸化相关的蛋白表达。
去神经支配的小鼠表现出自发的角膜上皮脱落和细胞凋亡，同时由于 NAMPT 水平降低，上皮 NAD+含量受损。同样，抑制 NAMPT 可重现去神经支配小鼠的上皮脱落，并诱导培养的角膜上皮细胞凋亡。NMN 或 NAD+的补充部分减缓了角膜神经纤维的退化，减少了去神经支配小鼠的上皮缺损，并通过恢复 SIRT1、AKT 和 CREB 的激活水平，改善 FK866 处理细胞的凋亡诱导。
角膜去神经支配通过降低 NAMPT 的表达降低了上皮 NAD+含量，导致细胞凋亡和上皮缺损，表明角膜神经支配通过调节 NAD+生物合成有助于上皮内稳态。
