Rapamycin-induced autophagy activity promotes bone fracture healing in rats

Abstract

Autophagy is a crucial mediating process for normal bone cell function and metabolism in physiology or pathology. Rapamycin has been demonstrated to induce the autophagy pathway by inhibiting the mammalian target of rapamycin (mTOR) pathway. However, the contribution of autophagy in orthopedic diseases is rarely reported. The aim of the present study was to evaluate the capacity of pharmacologically induced autophagy to modify disease function in a rat model of bone fracture. A femur fracture model was established via surgery in adult male Sprague-Dawley rats. Rapamycin (n=63 rats) or dimethyl sulfoxide (DMSO) vehicle control (n=63 rats) was administered intraperitoneally for 2, 4 and 6 weeks, and 21 randomly selected rats were sacrificed in each group at each time point. X-ray micro-computed tomography and hematoxylin and eosin staining were used to evaluate the extent of fracture healing in each group. The effects of rapamycin on autophagy, mTOR signaling and the expression levels of vascular endothelial growth factor (VEGF) and proliferating cell nuclear antigen (PCNA) were analyzed using immunohistochemistry, immunofluorescence staining and western blot analysis. Rapamycin affected the mTOR signaling pathway in rats following fracture, as indicated by the inhibition of the phosphorylation of ribosomal protein S6, a target of mTOR, and activation of microtubule-associated protein 2 light chain 3, a key marker of autophagy. Histomorphometry and image examination indicated that the number of osteoblasts in each section was significantly (P<0.01) increased in the rapamycin group compared with the control group, and this was associated with a significant (P<0.05) increase in mineralized callus fraction. Furthermore, rapamycin treatment increased the expression levels of VEGF and PCNA in the rat callus tissue. These results suggest that rapamycin may serve a beneficial function in fracture healing, and that the underlying mechanism may involve the activation of autophagy.