Objective

Nicotinamide adenine dinucleotide (NAD) is an essential molecule participating in multiple physiological and pathophysiological processes. In diabetic cornea, the NAD⁺-consuming enzyme SIRT1 was down-regulated and contributed to the delayed wound healing. However, the impact of hyperglycemia on corneal NAD⁺ biosynthesis remained elusive. This study was to investigate the relationship of NAD⁺ biosynthesis and the delayed corneal wound healing in diabetic mice.

Methods

Type 1 diabetes mellitus (DM) mice were induced by streptozotocin and corneal epithelial wound healing models were constructed by epithelial scraping. The NAD⁺ contents of corneal epithelium were measured using the NAD/NADH quantification kit. Expression of key enzymes involved in the NAD⁺ biosynthesis in type 1 DM mice and type 2 DM patients were analyzed. The nicotinamide phosphoribosyltransferase (NAMPT)-specific siRNA and the selective inhibitor FK866 were used to achieve the blockade of NAMPT, whereas exogenous NAD⁺ and its precursors were replenished to the corneal epithelial cells and DM mice.

Results

Hyperglycemia attenuated NAD⁺ content and NAMPT expression in the corneal epithelium of both type 1 DM mice and type 2 DM patients. Local knockdown of NAMPT by siRNA or FK866 consistently recapitulated the delayed corneal epithelial wound healing in normal mice. Moreover, NAD⁺ replenishment recovered the impaired proliferation and migration capacity by either FK866 or high glucose treatment in cultured corneal epithelial cells. Furthermore, in DM mice, NAD⁺ and its precursors nicotinamide mononucleotide and nicotinamide riboside also facilitated corneal epithelial and nerve regeneration, accompanied with the recovered expression of SIRT1 and phosphorylated EGFR, AKT, and ERK1/2 in epithelium and corneal sensitivity.

Conclusion

Hyperglycemia-reduced NAD⁺ biosynthesis and contributed to the impaired epithelial wound healing in DM mice. The replenishment of NAD⁺ and its precursors facilitated diabetic corneal wound healing and nerve regeneration, which may provide a novel therapeutic strategy for the treatment of diabetic corneal complications.

中文翻译：

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高血糖降低的NAD ⁺生物合成削弱糖尿病小鼠的角膜上皮伤口愈合

目的

烟酰胺腺嘌呤二核苷酸（NAD）是参与多种生理和病理生理过程的必需分子。在糖尿病性角膜中，NAD ⁺消耗酶SIRT1被下调，并导致伤口愈合延迟。但是，高血糖对角膜NAD ⁺生物合成的影响仍然难以捉摸。本研究旨在探讨NAD ⁺生物合成与糖尿病小鼠角膜伤口愈合延迟的关系。

方法

链脲佐菌素诱导1型糖尿病（DM）小鼠，并通过上皮刮擦构建角膜上皮伤口愈合模型。使用NAD / NADH定量试剂盒测量角膜上皮的NAD ⁺含量。分析了1型DM小鼠和2型D​​M患者参与NAD ⁺生物合成的关键酶的表达。使用烟酰胺磷酸核糖基转移酶（NAMPT）特异性的siRNA和选择性抑制剂FK866来实现对NAMPT的阻断，而将外源性NAD ⁺及其前体补充到角膜上皮细胞和DM小鼠中。

结果

高血糖会减弱1型DM小鼠和2型D​​M患者的角膜上皮中的NAD ⁺含量和NAMPT表达。siRNA或FK866对NAMPT的局部抑制作用一致地概括了正常小鼠延迟的角膜上皮伤口愈合。此外，在培养的角膜上皮细胞中，NAD ⁺补给通过FK866或高葡萄糖处理恢复了受损的增殖和迁移能力。此外，在DM小鼠中，NAD ⁺及其前体烟酰胺单核苷酸和烟酰胺核糖苷也促进角膜上皮和神经再生，并伴有SIRT1和磷酸化EGFR，AKT和ERK1 / 2在上皮和角膜敏感性中的恢复表达。

结论

高血糖症可降低NAD ⁺生物合成，并导致DM小鼠上皮伤口愈合受损。NAD ⁺及其前体的补充促进了糖尿病角膜伤口的愈合和神经再生，这可能为糖尿病性角膜并发症的治疗提供了一种新颖的治疗策略。