The cornea is the anterior physical barrier that protects against pathogen invasion. Various causes, such as trauma, inflammation, and diseases, can lead to irreversible corneal injuries to the normal structure of the epithelium, endothelium, and stroma, eventually resulting in corneal blindness (Barrientez et al., 2019; Wilson, 2023). In the impaired cornea, the wound healing process is promptly initiated by activating multiple cellular activities or signaling cascades. As the outermost structure, epithelial tissue healing is crucial for restoring barrier function and the corneal wound repair process. Importantly, regulating migration and inflammatory responses is vital for improving corneal wound healing (Raghunathan et al., 2017). Although the development of agents or therapies applied in clinical practice is conducive to corneal injury (Dang et al., 2022), our current understanding of the pathogenesis of corneal injury and the underlying mechanism of corneal wound healing remains insufficient. Therefore, further investigation of cellular activities during the corneal wound healing process is needed to identify novel approaches to ameliorate corneal injury.

Nitric oxide (NO) is a free radical whose secretion is controlled by one of the nitric oxide synthase family enzymes, inducible nitric oxide synthase (iNOS) (Anavi and Tirosh, 2020). Optimal concentrations of NO are necessary for cellular activities and trigger wound healing through diverse mechanisms (Schwentker et al., 2002). It has been reported that moderate NO levels are beneficial to the viability of corneal epithelial cells, while excessive NO results in the impairment of epithelial cells, indicating that the corneal wound repair process is closely related to the expression of NO levels (Tummanapalli et al., 2021). Tellios et al. constructed copper-chitosan nanoparticles to supply NO in the corneal epithelium and limbal epithelium, and found that different iNOS levels controlled NO metabolism, which led to a protective or deteriorative role in the wound recovery of epithelium and limbal epithelium cells (Tellios et al., 2018). Indeed, Park et al. identified a positive role for NO in facilitating corneal repair by regulating epithelial cell viability via the MAPK-related pathway (Park et al., 2017b). By contrast, Singh et al. (2023) and Song et al. (2022) suggested that inhibiting iNOS expression by regulating the upstream targets IL-11 or lysozyme improved the corneal repair process. Therefore, as the function of NO in corneal wound repair remains controversial, further exploration of iNOS/NO in corneal damage is needed.

Our study aimed to investigate how iNOS/NO regulates corneal wound healing and its underlying mechanisms. In the corneal injury model of iNOS knockout mice with reduced NO and iNOS expression, we found that iNOS ablation promoted wound healing by facilitating epithelial cell migration, inhibiting inflammatory responses, and activating the Akt pathway. Furthermore, using NO donors to restore NO levels, we confirmed that the protective effects of iNOS ablation were negated by the supplementation with NO. Therefore, we found that NO/iNOS ablation promotes corneal wound healing, providing a novel approach and insight into corneal damage treatment.