Deletion of p16 prevents estrogen deficiency-induced osteoporosis by inhibiting oxidative stress and osteocyte senescence

To investigate whether p16 deletion can prevent osteoporosis caused by estrogen deficiency, we first confirmed that p16 protein expression levels were significantly up-regulated in bony tissue of ovariectomized (OVX) wild-type mice. Eight-week-old wild-type and p16(-/-) mice were then sham-operated or bilateral OVX. After 12 weeks, the bone phenotypes of all models were analyzed by radiography, micro-computed tomography, histology, immunohistochemistry, and molecular biology. The results showed that p16 deficiency could rescue OVX-induced osteoporosis by significantly increased bone mineral density, trabecular bone volume, total collagen positive area, osteoblast number, type I collagen positive area, fibroblast colony-forming unit (CFU-f) and alkaline phosphatase-positive CFU-f with up-regulation of the mRNA expression levels of Alp, Runx2, type I collagen and osteocalcin, and significantly reduced osteoclast surface and the ratio of RANKL/OPG mRNA expression level. Furthermore, we also demonstrated that p16 deletion inhibited OVX-induced oxidative stress and bone cell senescence, such as a significant decrease in reactive oxygen species levels, up-regulation of superoxide dismutase 1 and 2 protein expression levels, and reduction of the percentage of 13-galactosidase-positive osteocytes and p21 protein expression levels in bony tissue. Our results indicate that p16 deletion can prevent estrogen deficiency-induced osteoporosis by inhibiting oxidative stress, osteocyte senescence and osteoclastic bone resorption, stimulating osteogenesis and osteoblastic bone formation. Therefore, this study provides new insights into the potential of p16 as a novel therapeutic target for estrogen deficiency-induced osteoporosis.