Immunization with Na⁺/K⁺ ATPase DR peptide prevents bone loss in an ovariectomized rat osteoporosis model

Osteoporosis is characterized by decreased bone strength and microarchitectural deterioration of bone tissue leading to an increase in bone fracture. Here we report a new agent named DR peptide, a conserved sequence of Na⁺/K⁺ ATPase (NKA), can be used to prevent osteoporosis. Our results showed that immunization with DR peptide conjunct with Keyhole limpet hemocyanin (DR-KLH) significantly strengthened trabecular bone and improved bone mineral density of femur and the 5th lumbar in the ovariectomized (OVX) rats when compared with those in OVX rats immunized with KLH alone. To study the underlying mechanisms, anti-DR antibody (DR-Ab) and IgG were purified from the serum of rats immunized with DR-KLH or KLH alone in OVX rats. We found that DR-Ab had no significant effects on the proliferation of osteoclasts precursors, but it alleviated the inhibitory effect of H₂O₂ on the expression of osteogenic marker genes, Runx2, alkaline phosphatase and bone sialoprotein. DR-Ab also reversed the loss of collagen and the reduced bone nodule formation caused by H₂O₂ treatment. Knockdown of NKA with siRNA transfection reduced osteoblast differentiation and rendered the osteoblasts more vulnerable to oxidative challenge. Immunostaining analysis confirmed that NKA expression was reduced in osteoblasts in the femur of OVX rats, but this effect was attenuated by immunization with DR-KLH. Therefore, the protective effects of DR-Ab on osteoblasts might be associated with preservation of NKA level. Taken together, our findings reveal a novel mechanism for the development of osteoporosis and DR peptide and DR-Ab are potential approaches for the treatment of osteoporosis.