Active Vitamin D Insufficiency Accelerates Skeletal Aging via Oxidative Stress and p16-Mediated Senescence

BACKGROUND: Vitamin D is essential for skeletal health, but its role in redox homeostasis and cellular senescence during aging in vivo is unclear. We therefore investigated whether active vitamin D insufficiency accelerates bone loss via oxidative stress and senescence pathways. METHODS: Male wild-type (WT) and Cyp27b1 haploinsufficient mice (modeling vitamin D insufficiency) were treated with N-acetylcysteine (NAC) or 1,25-dihydroxyvitamin D₃ [1,25(OH)₂D₃]. Double-mutant p16-/-Cyp27b1+/- mice were used to assess the role of the tumor suppressor protein p16. Mice were maintained until 8 months of age in a specific pathogen-free facility. Outcomes included lifespan (n = variable per group, monitored daily); generation of oxidative stress (determined by serum malondialdehyde [MDA] levels via assay kit); generation of bone reactive oxygen species [ROS] (determined via flow cytometry), development of DNA damage (indicated by 8-hydroxy-2'-deoxyguanosine [8-OHdG] and γ-H2A.X generation and determined via immunohistochemistry and Western blot); and senescence (assessed by generation of β-galactosidase [β-gal], p16, and senescence-associated secretory phenotype [SASP] cytokines as determined via staining, blot, and real-time reverse transcription polymerase chain reaction). Additionally, bone microarchitecture was examined via micro-computed tomography and histomorphometry. Data from at least 5 mice per group were analyzed using unpaired Student's t-test for two-group comparisons and two-way analysis of variance for multi-group comparisons, with significance at p < 0.05. RESULTS: Compared with wild-type controls, Cyp27b1+/- mice showed a significantly shorter lifespan, higher oxidative stress, greater DNA damage, increased senescence markers, and lower trabecular bone volume (all p < 0.05). In Cyp27b1+/- mice, treatment with either N-acetylcysteine or 1,25(OH)₂D₃ significantly improved survival, reduced oxidative stress and DNA damage, attenuated senescence markers, and increased bone volume relative to untreated Cyp27b1+/- mice (p < 0.05 for all relevant comparisons; n = 5 per group). Genetic deletion of p16 in Cyp27b1+/-mice similarly increased bone volume and reduced senescence-associated readouts compared with Cyp27b1+/- controls (p < 0.05; n = 5). CONCLUSIONS: Active vitamin D insufficiency accelerates skeletal aging in vivo through a pathway involving reactive oxygen species-DNA damage-p16/senescence-associated secretory phenotype. Antioxidants, vitamin D repletion, or p16 inhibition rescued bone loss, highlighting redox-senescence axes as potential therapeutic targets for osteoporosis.