N-oxalyl-d-phenylalanine ameliorates radiation-induced lung injury by activation of hypoxia inducible factor-mediated pathway

Objective To investigate the radioprotective effects of N-oxalyl-d-phenylalanine (NOFD) against radiation-induced lung injury (RILI), and to determine whether hypoxia inducible factor ( HIF)-related regulation contributes to its protective action. Methods Male C57BL/6 mice were randomized into four groups: Control (non-irradiated + normal saline), IR + NS (irradiated + normal saline), IR + NOFD (irradiated + 5 mg/kg NOFD), and IR + Amifostine (irradiated + 5 mg/kg Amifostine) ( n =5). The in vivo radioprotective potential of NOFD was evaluated in a murine RILI model exposed to a single dose of 15 Gy thoracic X-ray irradiation with head and abdomen shielded. Body weight was recorded daily and histological damage was examined by hematoxylin and eosin (H&E) staining. Oxidative stress markers, including malondialdehyde (MDA), superoxide dismutase (SOD), and catalase (CAT), were measured using corresponding biochemical assay kits. Inflammatory and profibrotic cytokines, including tumor necrosis factor-alpha (TNF-α), interleukin-1 beta (IL-1β), and transforming growth factor-beta 1 (TGF-β1), as well as HIF-related regulatory factors, including hypoxia-inducible factor 1 alpha (HIF1α), prolyl hydroxylase domain protein (PHD), and vascular endothelial growth factor A (VEGF-A), were assessed using ELISA kits. To investigate the role of HIF-related regulation, HIF1A- and HIF2A-deficient human bronchial epithelial BEAS-2B cell lines were generated using CRISPR-Cas9. Intracellular oxidation-sensitive fluorescence, γ-H2AX signal, and cell-cycle distribution were evaluated in irradiated wild-type cells, NOFD-treated wild-type cells, and HIF-deficient cells using immunofluorescence and flow cytometry. mRNA expression of HIF-related and functionally relevant genes was further examined by quantitative real-time PCR (qPCR). Results In a murine RILI model, NOFD effectively alleviated IR-induced body weight loss and lung histopathological damage including alveolar destruction and inflammatory infiltration. Compared with the IR + NS group, NOFD treatment increased SOD and CAT activities (2.11- and 3.42-fold, respectively) and reduced MDA level by 67.60%. NOFD also restored HIF-related regulatory factors in irradiated lung tissues, including a 2.96-fold upregulation of PHD, which reduced HIF1α accumulation by 24.58% and suppressed VEGF-A overexpression by 52.02%. Additionally, NOFD attenuated inflammatory and profibrotic cytokines (TNF-α, IL-1β, TGF-β1). In irradiated bronchial epithelial cells, NOFD pretreatment significantly reduced γ-H2AX signal by 63.72% and ROS accumulation by 51.46% compared to irradiated wild-type BEAS-2B cells. HIF1A-deficient cells showed greater sensitivity than HIF2A-deficient cells, with higher γ-H2AX (1.29-fold) and ROS (2.34-fold) levels relative to irradiated wild-type cells. NOFD reduced γ-H2AX signal and intracellular oxidation-sensitive fluorescence in irradiated wild-type BEAS-2B cells, whereas HIF1A- or HIF2A-deficient cells showed increased radiosensitivity and enhanced radiation-associated G2/M accumulation. Under basal conditions, NOFD increased HO1 and P53 expression by approximately 1.38-fold and 2.83-fold, respectively, while reducing BNIP3 expression by about 25.42%. Conclusion NOFD attenuates radiation-induced lung injury at both the tissue and cellular levels. These findings support that HIF-related regulation is required for NOFD-mediated radioprotection and suggest that NOFD may serve as a potential candidate for the mitigation of RILI.