## 3 Results
### Protein Expression Analysis Using Western Blotting  
Sample apoptosis was assessed at the protein level, as depicted in Figure 5, by specifically examining the expression of apoptosis-related proteins: Bcl-xl, p21 and SOD2. These proteins were selected for Western blot evaluation due to their roles in apoptosis. Bcl-xl, a mitochondrial transmembrane protein from the Bcl-2 family, is pivotal in regulating the intrinsic apoptosis pathway. P21 functions in cell adhesion molecule binding and integrin binding. SOD2 plays a role in electron transfer activity pathways. The protein expressionof these proteins in each experimental groups was visualized as bands on the Western blot (Figure 5) and quantitatively measured as fold changes relative to \(\beta\)-actin.  
By performing ANOVA analysis, significant differential expression was observed for P21 (\(p<0.05\)) and SOD2 (\(p<0.01\)), while the expression of the BCL protein was not significantly different among groups. Furthermore, the interaction among the proteins was evident; BCL expression was significantly correlated with both P21 (\(p<0.01\)) and SOD2 (\(p<0.001\)), while P21 also displayed a significant relationship with SOD2 (\(p<0.001\)). In a detailed analysis, the NIRE group showed a significantly higher Bcl-xl protein expression than the IRE group (1.24-fold, \(p<0.05\)). Compared to the control group, P21 protein expression was downregulated in both the NIR and IRE groups (0.78-fold, \(p<0.05\) for NIR; 0.58-fold, \(p<0.05\) for IRE). Yet, the NIRE group exhibited an elevated p21 expression compared to the IRE group (1.29-fold, \(p<0.05\)). SOD2 expression in the NIR group was noticeably lower than in the control group (0.6-fold, \(p<0.05\)), but its expression was not significantly different in other groups compared to the controls. The NIRE group, however, displayed a reduced SOD2 protein expression relative to the IRE group (0.93-fold, \(p<0.05\)). Taken together, when compared to the IRE group, the NIRE group demonstrated activated expression of p21 and SOD2, both regulators of apoptosis. Moreover, the NIRE group exhibited activated Bcl-xl expression, indicative of its role in apoptosis inhibition.  
Figure 2: Images of gross H & E staining and TUNEL assay. (**A**) An image of H & E staining. (**B**) TUNEL assay for (**1**) control; (**2**) NIR (3 J/cm\({}^{2}\), 808 nm); (**3**) IRE (applied voltage 250 V, pulse width 100 \(\upmu\)s, pulse number 20, electrode distance 2 mm); (**4**) NIRE (NIR + IRE), consisting of the mucosa, submuc, muscular, and serosa layers. Scale bar 400 \(\upmu\)m. (**C**). Apoptosis quantification. Data are shown as the mean \(\pm\) standard deviation (n = 3), and statistical analysis was performed using a two-tailed paired Student’s \(t\)-test (\({}^{\circ}\)\(p<0.05\), \({}^{**}\)\(p<0.01\)) and ANOVA complemented by [PERSON]’s HSD test (\({}^{\circ}\)\(p<0.05\), \({}^{***}\)\(p<0.001\)).  
Figure 4: H & E staining and TUNEL images of the mucosal and submucosal vessels. (**A**) The capillary-like vessels in both the mucosa and submucosal layers maintained their cellular structure in the upper panel.  
Figure 3: H & E staining images of the mucosal, submuc, and muscularis layers. (**1**–**3**) for control; (**4**–**6**) for NIR; (**7**–**9**) for IRE; (**10**–**12**) for NIRE. Scale bar 20–50 μm.  
control samples (**1**,**2**) as well as in NIR-treated samples (**3**,**4**). In contrast, IRE (**5**,**6**) and NIRE (**7**,**8**) samples exhibited fewer cells surrounding the vessels but retained overall vascular structure. Scale bar = 40 \(\upmu\)m. (**B**) Cells within the blood vessels of the mucosa and submucosal layers remained intact in control (**1**,**2**) and NIR-treated samples (**3**,**4**). Conversely, cell apoptosis was seen within the mucosa in both IRE (**5**) and NIRE (**7**) samples. Additionally, perivascular cells in the submuc were non-specific for the TUNEL assay (**6**,**8**). Scale bar = 40 \(\upmu\)m.