Expression And Effect Of NADPH Oxidase In Alkali Burn-induced Corneal Injury

Purpose: The purpose of this study is to investigate the effect of NADPH oxidases in mouse corneas with Alkali burn.Methods: A mouse model of corneal alkali burn was set up. Immunofluorescence was used to detect the expression of Nox2 and Nox4 both in the human cornea and in the mouse cornea. Then western blot and Real-time PCR were also used to observe the expression of Nox2 and Nox4. NADPH oxidase inhibitors including diphenyleneiodnium(DPI) or Apocynin were administrated by eye drops. Images of CNV on day 1, 3, 7 and 14 after alkali burns treated with eye drops of PBS, DPI and Apocynin were observed by slit lamp. Expression of proinflammatory cytokines such as IL-6, IL-1β, TNF-α; angiogenic factors such as VEGF,MMPs were measured by RT-PCR. Corneal 3-nitrotyrosine expression and inflammatory cell infiltration(CD11b) were determined by immunofluorescence. Expression of 3-nitrotyrosin was examined by western-blot analysis. Corneal ROS was detected with CellROX green regents. Corneal NV was measured and quantified by FITC-dextran corneal angiography.Results: Expression of Nox2 and Nox4 in the cornea was significantly enhanced after alkali injury, detected by immunofluorescence, western blot and real-time RT-PCR. Increased Nox2 expression was mainly colocalized withCD11b+ inflammatory cells in mice with corneal alkali burn. Administration of DPI and Apocynin markedly inhibited alkali burn-induced corneal ROS and 3-nitrotyrosine formation. Meanwhile, DPI and apocynin also greatly attenuated corneal stromal CD11b+ cell infiltrations and the mRNA expression of proinflammatory cytokines such as IL-6, IL-1β in mice with alkali burn. Furthermore, DPI and apocynin obviously degrade the area of corneal neovascularization(CNV) and the mRNA expression of angiogenic factors such as VEGF and its receptors VEGFR1 and VEGFR2 and MMPs in mice with alkali burn.Conclusions: Taken together, our data found that the expression of NADPH oxidase was upregulated in the alkali burned cornea. Inhibition of NADPH oxidases could attenuate alkali burn-induced corneal injuries. It may contribute to a new target for treatment of alkali burned cornea.