## 4 Discussion  
Ozone layer depletion causes a continuous and more pronounced exposure of the cornea to UV-B rays, which provokes the formation of ROS, including superoxide anion (O\({}^{2-}\)), hydrogen peroxide (H\({}_{2}\)O\({}_{2}\)), and hydroxyl (OH\(\cdot\)) radicals. ROS overproduction is associated with different ocular disorders, e.g., cataracts, age-related macular degeneration, glaucoma, inflammation, and corneal alteration [52]. The human eye has several defense mechanisms, including enzymes and antioxidants (e.g., glutathione peroxidase and ascorbic acid) which protect ocular tissues against oxidative injury [53]. However, these are gradually reduced during UV-B irradiation, as opposed to the pro-oxidant enzymes such as xanthine oxidoreductase/xanthine oxidase, which remain at normal values or may even increase. Many works demonstrated that PACAP exerts an important antioxidant and anti-apoptotic role in different cell types throughout the body [54; 55; 56]. The present study aimed to investigate the role of the peptide in corneal epithelium protection against UV-B radiation and elucidate the associated molecular mechanisms (Figure 7). Figure 6: (A) JNK activation induced by UV-B at different time points. (B) JNK phosphorylation expression at 6 h. ***** \(p\) < 0.0001 vs. CTRL; ***** \(p\) < 0.0001 vs. UV-B. We first analyzed the endogenous expression of PACAP and its receptors in the corneal epithelium. PACAP and PAC1R are found in the rabbit corneal epithelium, specifically in basal cells. VPAC1R and VPAC2R were observed in the entire length of the epithelial layers as well as in the stroma (Figure 1). To explore the role of PACAP in corneal epithelium, we exposed SIRC cells to UV-B insult. It is widely recognized that UV-B radiation leads to corneal cell apoptotic death due to DNA damage and the production of ROS [47; 48; 49]. Here, we confirmed this evidentially, as well as demonstrating that treatment with PACAP significantly increased cell viability (Figure 2B) by preventing UV-B-induced apoptotic death (Figure 3). These data were also confirmed by the analysis of proteins Bcl-2/Bax. High levels of Bax protein were detected in the UV-B group (Figure 4), consistent with previous findings showing that UV-B irradiation increased pro-apoptotic Bax gene expression [54; 55]. PACAP treatment significantly increased Bcl-2 protein expression and reduced the pro-apoptotic protein Bax in SIRC cells exposed to UV-B insult, confirming the anti-apoptotic mechanism involved in the protective effect of PACAP against UV-B-induced corneal epithelial damage. As previously shown, UV-B irradiation induced ROS formation on the ocular surface [56], leading to DNA and mitochondrial damage, mitochondrial membrane depolarization, lipid peroxidation, and protein denaturation [56]. Here, we showed that UV-B insult induced a time-dependent increase in cellular ROS formation, as well as mitochondrial superoxide anion generation, whereas treatment with PACAP significantly reversed these changes (Figure 5A,B). Therefore, PACAP reduced oxidative stress directly through ROS scavenging and protected cells against UV-B radiation through the modulation of the JNK pathway. ROS induces different pathways, including JNK [9], which serves a central role in the apoptotic event [57; 58]. Accordingly, we showed that UV-B insult triggers the activation of the JNK pathway, whereas PACAP treatment counteracts its phosphorylation (Figure 6). Correspondingly, previous findings show that PACAP exerts important cytoprotective effects by suppressing the JNK pathway [59; 60]. Figure 7: Schematic representation of the protective effects exerted by PACAP on corneal epithelium against UV-B-induced apoptosis via ROS/JNK pathway inhibition. The therapeutic use of PACAP has some limitations due to its poor in vivo stability.