Sildenafil pretreatment attenuates apoptosis and intestinal injury in a hypoxia-ischemia-induced NEC-like rat model.

Necrotising enterocolitis (NEC) is a devastating gastrointestinal disease of premature infants, in which hypoxia-induced apoptosis plays a critical role in intestinal epithelial injury. The balance between pro-apoptotic and anti-apoptotic proteins, particularly Bax and Bcl-2, as well as the hypoxia-responsive transcription factor HIF-1α, are key determinant of epithelial survival. Sildenafil citrate, a phosphodiesterase-5 inhibitor, has been shown to improve microcirculation and exert anti-apoptotic effects through nitric oxide-cGMP signalling, but its role in NEC-related intestinal injury remains poorly understood. This study aimed to evaluate the protective effects of sildenafil on apoptosis in a hypoxia-ischemia-induced NEC-like intestinal injury model. A total of 75 Wistar rat pups were randomly assigned to six groups: control, NEC, vehicle (saline+DMSO), and sildenafil-treated groups (1, 5, and 10 mg/kg/day). Sildenafil was administered intraperitoneally for 4 days before NEC induction. NEC was induced by hypoxia (100% nitrogen for 60 s, twice daily for 48 h) and followed by cold stress (10 min at 4 °C). Intestinal tissues were collected for histopathological scoring, caspase-3 immunohistochemistry, and quantitative RT-PCR analysis of HIF-1α, Bax, and Bcl-2 expression. Data were analysed using ANOVA and Kruskal-Wallis tests, with p ≤ 0.05 considered statistically significant. Pups subjected to hypoxia-induced intestinal ischemia exhibited severe villous damage, elevated histological injury scores, and marked upregulation of HIF-1α and Bax, along with downregulation of Bcl-2. Caspase-3 immunoreactivity strongly correlated with histological injury (Spearman's ρ = 0.795, p < 0.001). Sildenafil pretreatment reduced histological damage and apoptosis in a dose-dependent manner. The Sil_10 group showed the lowest injury score (1.06 ± 0.4, p < 0.0001 vs. NEC), significantly reduced Caspase-3 labelling, suppressed Bax and HIF-1α expression, and increased Bcl-2 levels, resulting in a markedly reduced Bax/Bcl-2 ratio compared with NEC (p < 0.0001). Sildenafil attenuates hypoxia-induced, ischemia-dominant NEC-like intestinal injury by modulating apoptotic and hypoxia-related pathways, lowering HIF-1α expression, reducing Bax/Bcl-2 ratio, and preserving epithelial integrity. These findings provide novel histological and molecular evidence for the anti-apoptotic effects of sildenafil in experimental NEC, highlighting HIF-1α modulation as a potential therapeutic strategy.