## 3 Results
### Effect of PACAP Treatment on UV-B-Induced Apoptotic Cell Death  
Studies showed that UV-B insult injures the cornea by causing apoptotic cell death [46; 47; 48; 49]. Here, it was found that UV-B rays (20 mJ/cm\({}^{2}\)) significantly decreased cell viability in a time-dependent manner starting from 24 h following UV-B insult (\(\texttt{{}^{{}^{**}}}p<0.0001\) vs. CTRL) (Figure 2A). Therefore, we chose this time point to investigate the effect of PACAP treatment on UV-B-exposed cells' viability. As shown in Figure 2B, the peptide increased cell viability when compared with the UV-B group, confirming its protective role (**** **p** < 0.0001 vs. UV-B). To further analyze the protective effect of PACAP on UV-B-mediated cell damage, the nuclei of SIRC cells exposed to UV-B were stained with Hoechst 33342. As shown in Figure 3, significant fragmentation and nuclear condensation, characteristic of apoptotic death, was observed in the UV-B-exposed group, in comparison with the control (**** **p** < 0.0001 vs. CTRL). When the cells were treated with PACAP, the number of apoptotic nuclei in the UV-B-exposed cells was markedly reduced (**** **p** < 0.0001 vs. UV-B). Figure 1: **(A)** Immunodetection of PACAP **(a)**, PAC1R **(b)**, VPAC1R **(c)**, and VPAC2R **(d)** in rabbit cornea: (1) epithelium, (2) Bowman’s membrane, (3) stroma. Original magnification \(\times\)200, scale bar: 20 \(\upmu\)m. **(B)** Representative immunoblot of PACAP and its receptors in SIRC cells. Figure 2: **(A)** MTT assay performed on SIRC cells at different time points. **(B)** Cell viability evaluated at 24 h on different experimental groups. **** **p** < 0.0001 vs. CTRL; **** **p** < 0.0001 vs. UV-B. Considering that apoptotic cell death is regulated by Bcl-2 family proteins, key mediators, we investigated the role of PACAP to modulate the expression of the anti-apoptotic protein, Bcl-2, and pro-apoptotic protein, Bax. As shown in Figure 4, PACAP markedly increased the expression of Bcl-2 protein in UV-B-exposed cells (# \(p\) < 0.05 vs. UV-B). In contrast, the expression levels of the pro-apoptotic protein Bax, which drastically increased following UV-B exposure (****\(p\) < 0.0001 vs. CTRL), were significantly downregulated by treatment with the peptide (****\(p\) < 0.0001 vs. UV-B). These results confirmed that PACAP inhibits corneal epithelial cells' apoptotic death activated by UV-B ray exposure. Figure 4: Expression of Bcl-2 and Bax proteins at 24 h in different experimental groups. ****\(p\) < 0.0001 vs. CTRL; # \(p\) < 0.05, and ****\(p\) < 0.0001 vs. UV-B. Figure 3: Assessment of cell death using Hoechst 33342 staining. Scale bar: 20 μm. ****\(p\) < 0.0001 vs. CTRL; ****\(p\) < 0.0001 vs. UV-B. PACAP Decreased the Production of ROS Induced by UV-B Rays on Corneal Epithelial Cells via the JNK Pathway Inhibition  
Considering that corneal injuries induced by UV-B are predominantly triggered by oxidative stress [50; 51], we analyzed the time-course of ROS generation in corneal epithelial cells after UV-B radiation exposure (20 mJ/cm\({}^{2}\)) by using the DCFDA assay and the Mito-SOX\({}^{\text{TM}}\) Red fluorescence indicator (Figure 5A,B). The cellular ROS production significantly increased in a time-dependent manner starting from 30 s after UV-B irradiation (****\(p<0.0001\) vs. CTRL). Treatment with the peptide significantly counteracted ROS formation starting from one hour after UV-B irradiation (20 mJ/cm\({}^{2}\) for 30 s) (Figure 5A). Similarly, a time-dependent increase in mitochondrial ROS formation was observed in cells exposed to UV-B irradiation (****\(p<0.0001\) vs. CTRL) (Figure 5B). PACAP treatment strongly reduced UV-B-induced mitochondrial ROS formation at all time points considered. The literature shows that JNK activation is crucial as it represents the signaling pathway downstream of ROS, leading to apoptotic cell death [50; 51].