SERPINA3K's Protetion Against 4-hydroxynonenal(4-HNE) Induced Corneal Injury And Its Underlving Mechanisms

Purpose:SERPINA3K is a member of the family of serine proteinase inhibitors,it has been recently shown that SA3K has antineovascularization,antiinflammation,and antioxidative stress effects and may have the potential to become a new therapeutic agent to treat ocular disease.Here we further investigated the effects of SERPINA3K on the corneal oxidant injury model developed lately induced by 4-Hydroxynonenal(4-HNE)and its underlying mechanisms.Methods:The 4-HNE induced corneal oxidant stress was applied in cultured Human Corneal Epithelial(HCE)cells to investigate the effects of SERPIN3K on oxidative stress.The following experiments were conducted:CCK-8 assay to detect cell viability;immunofluorescent staining,western blotting and quantitative real-time PCR assay to measure protein levels or gene expressions of key ROS associated factors:3-Nitrotyrosine(3-NT),NADPH oxidase 4(NOX4)and antioxidant enzymes SOD and Catalase as well as nuclear factor(erythroid-derived 2)-like 2(NRF2).The main factors of Wnt/?-catenin signaling pathway:p-LRP6,?-catenin and transcription factor 4(TCF4)were detected to further elucidate the mechanism of SERPINA3K.Wistar rats were applied to establish the animal corneal oxidant injury model.After topically treated by 4-HNE,SERPINA3K was administered in rats eyes.Histological staining and TUNEL staining were conducted to examine sections of rat corneas.Immunofluorescent staining and western blotting were also conducted to detect the level of 3-NT,NOX4,NRF2 and p-catenin.Results:SERPINA3K protected the cell viability in a concentration dependent manner,SERPINA3K decreased the levels of ROS marker:3-NT and ROS generation enzyme:NOX4 and elevated gene expression and protein levels of SOD and Catalase;meanwhile SERPINA3K down-regulated ROS pathway:NRF2 pathway and its downstream factors:NAD(P)H dehydrogenase(quinine)1(NQO1)and heme oxygenase 1(HO1).Furthermore SERPINA3K suppressed the activation of Wnt signaling pathway:p-LRP6,p-catenin and TCF4 in the HCE cells after treated with 4-HNE.In addition,SERPINA3K ameliorated the oxidant injury of rat corneas induced by 4-HNE and down-regulated ROS systems and Wnt/?-catenin pathway.Conclusions:SERPINA3K protected the oxidant damage induced by 4-HNE in cultured Human Corneal Epithelial(HCE)cells in vitro and the cornea of rats in vivo.Its underlying mechanisms were through suppression of ROS system and inhibition of the activated Wnt/?-catenin signaling pathway.On one hand,SERPINA3K suppressed the production of oxidants,inhibited ROS generation,promoted ROS degradation and simultaneously decreased the expression of ROS signaling pathway.On the other hand,SERPINA3K inhibited the levels of main factors of Wnt/?-catenin signaling pathway.Our experimental data further demonstated the antioxidant role of SERPINA3K in cornea with a novel comeal experimental model of oxidative stress,which may contribute to better understanding of the mechanism of SERPINA3K in corneal and to support that SERPINA3K can be served as a potential therapeutic agent to treat corneal diseases.