Dose-dependent protective effects of punicalagin on steroid-induced osteonecrosis in a rat model

This study aims to evaluate the dose-dependent protective effects of punicalagin (PUN) in a rat model of Steroid-induced osteonecrosis of the femoral head (SONFH).

Twenty-four male Wistar albino rats were randomly assigned to four groups (n = 6 per group): control, osteonecrosis (ON), ON treated with 10 mg/kg PUN, and ON treated with 40 mg/kg PUN. Osteonecrosis was induced using lipopolysaccharide and methylprednisolone. Femoral heads were evaluated using micro-computed tomography (CT), histopathological examination, and immunohistochemical analysis of cleaved caspase-3. Serum oxidative stress and antioxidant parameters including malondialdehyde (MDA), reduced glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT) were measured.

The primary outcome measure, the percentage of empty lacunae, was significantly higher in the ON group compared to controls (p = 0.01), showing a numerical reduction in the 40 mg/kg PUN group. Histopathological evaluation demonstrated a significant reduction in pyknotic cells in the high-dose PUN group compared to the ON group (p < 0.05). Furthermore, PUN treatment reduced the incidence of osteonecrosis (2/6 vs. 6/6; p < 0.01) and improved trabecular architecture. Immunohistochemical analysis showed decreased caspase-3 expression specifically in trabecular bone and adipocytes in the high-dose PUN group (p < 0.01), while downward trends were observed in bone marrow and endothelial cells. Micro-CT analysis confirmed a significant reduction in bone mineral density in the ON group compared to controls (p = 0.02). Regarding oxidative stress, although no statistically significant differences were observed, numerical trends toward reduced lipid peroxidation (p = 0.61) and a strong trend toward enhanced antioxidant capacity (GSH; p = 0.07) with a moderate-to-large effect size were noted.

Punicalagin may exert dose-dependent protective effects against SONFH in rats, primarily associated with reduced apoptotic activity and partial preservation of bone microarchitecture. While systemic oxidative stress markers show numerical improvements, the marked reduction in osteocyte apoptosis and necrotic incidence suggests that PUN represents a promising adjunctive strategy for mitigating steroid-related bone injury.