Neutrophils can produce many important anti-inflammatory and pro-catabolic lipid mediators, and these mediators can terminate inflammation[11, 12]. In addition, neutrophils are involved in pathogenesis using several mechanisms. For example, neutrophils at the site of lesions release proteases and produce large amounts of reactive oxygen species, leading to tissue damage. This makes the tissue more susceptible to pathogens, which, in turn, develops into chronic inflammation[13]. When the cornea is damaged, neutrophils infiltrate the corneal stroma through the corneal limbal vessels. Their progression through the stroma to the wound area is thought to be facilitated by keratinocyte apoptosis. Neutrophil infiltration is a direct response to injury. However, at the same time, oxidative, hydrolytic, and pore-forming molecules released by neutrophils into the damaged tissue can destroy otherwise healthy host cells[14]. Therefore, pathological examination often reveals neutrophil recruitment around the corneal injury[15].
In this study, we took the differentially expressed genes in corneal scraping, with or without neutrophil closure, and intersected them with neutrophil-related genes to obtain a total of 27 intersected genes. After that, 5 key genes (ITGB2, MMP9, EGF, SERPINE1, and PLAUR) were obtained after the PPI network construction using the MCODE algorithm. Matrix metalloproteinases (MMPs) are a group of zinc endopeptidases that are essential for the breakdown of extracellular matrix components and basement membranes. They play a key role in various physiological and pathological processes, including morphogenesis, wound healing, and inflammation[16]. MMP9 is undetectable in healthy tissues but is highly expressed in cases of inflammation or cancer[17]. MMP9 can delay wound healing by degrading the extracellular matrix associated with normal tissue remodeling processes[18]. MMP9 is the corneal epithelium that produces the major matrix-degrading enzyme. Its activation is one of the key pathophysiological events in corneal ulcers occurring in ocular surface diseases, and it is also thought to be a causative factor in delaying wound healing and re-epithelializing the corneal surface[19]. EGF is a naturally occurring mitogen. Both in vivo and in vitro, receptor activation of EGF induces the synthesis of specific proteins and the proliferation and differentiation of corneal epithelial cells, keratinocytes, and endothelial cells[20]. Therefore, EGF is considered to be a growth factor that is important for the regulation of corneal epithelial homeostasis and wound healing. With topical application of EGF, corneal wounds may heal within hours, and the strength of stromal scars increases, which is a prospect for sutureless surgery[21]. Both MMP9 and EGF are closely associated with neutrophils. It has been shown that EGF is a key factor in epithelial repair and enhances the mechanism of neutrophil accumulation, thus promoting neutrophil defense during acute injuries[22]. It has also been shown that the major peak in MMP9 activity is suspected to be derived from neutrophils[23]. MMP9 was also found to be associated with neutrophil migration, and the inhibition of MMP9 significantly inhibited the number and rate of neutrophil migration[24]. Our results showed that rats' MMP9 and EGF expression was significantly inhibited in neutrophil-occluded corneal scrapings. In addition, we observed high and low neutrophil levels in samples based on median MMP9 and EGF expression values, and the results also showed significantly higher levels of neutrophils in the MMP9 and EGF high expression group than in the MMP9 and EGF low expression group. Other studies showed that MMP-9 expressed by epithelial cells may play an important role in the development of colitis by regulating cell-matrix interactions and wound healing[25]. Furthermore, studies show that tumor-associated neutrophil-derived MMP-9 contributes to tumor angiogenesis and progression associated with cutaneous squamous cell carcinoma[26]. In combination with the present study, it can be shown that corneal injury increases MMP9 expression[27], and MMP9 levels can be suppressed by inhibiting neutrophils. It is suggested that strategies to inhibit MMP9 may have potential therapeutic benefits.
However, the present study is still deficient in that we have not yet observed the effect of neutrophil closure on nerve repair after corneal scraping in mice from a multi-spatial and multi-temporal perspective. The effect of MMP9 inhibition on corneal scraping has not been observed in vitro. We will conduct a more in-depth study on this afterward.
In summary, we used bioinformatic analysis to obtain five core genes about neutrophils, among which MMP9 and EGF were significantly differentially expressed between the two groups, both with and without neutrophil closure. The expression levels of MMP9 and EGF may be closely related to the neutrophil level.