Iron overload induced osteocytes apoptosis and led to bone loss in Hepcidin-/- mice through increasing sclerostin and RANKL/OPG

Abstract

Background: Numerous studies have demonstrated that iron overload is a risk factor of osteoporosis. However, there has been no systematic and in-depth studies on the effect of iron overload on osteocytes and its role in iron overload-induced bone loss. Therefore, to address this problem, we carried out in vitro and in vivo studies using MLO-Y4 osteocyte-like cells and Hepcidin^-/- mice as iron overload models.

Methods: (1) MLO-Y4 cells were treated with ferric ammonium citrate (FAC). Intracellular reactive oxygen species (ROS) levels and apoptosis of MLO-Y4 cells were determined by flow cytometry. Western blotting was performed to evaluate the effect of FAC on the expression of sclerostin and RANKL/OPG. (2) The conditioned medium of MLO-Y4 cells after treatment with FAC was collected and used to treat pre-osteoblasts and monocytes. Alkaline phosphatase (ALP) staining and alizarin red (AR) staining were used to evaluate osteogenic differentiation capacity, and tartrate-resistant acid phosphatase (TRAP) staining was performed to demonstrate osteoclast differentiation capacity. (3) In vivo studies included a wild type mouse, Hepcidin^-/- mice, Hepcidin^-/- mice + deferoxamine (DFO), and Hepcidin^-/- mice + N-actyl-l-cysteine (NAC) group. Micro-CT was performed to evaluate the bone mineral density (BMD), bone volume, and bone micro-architecture of the mice, and three bending tests were used to assess bone strength. Histological analysis was used to detect alterations in bone turnover. TUNEL staining and scanning electron microscopy (SEM) were performed to evaluate the apoptosis and morphology of osteocytes. Immunohistochemical staining and Western blotting were used to determine alterations in sclerostin and RANKL/OPG expression levels in mice.

Results: (1) FAC increased intracellular ROS and apoptosis in MLO-Y4 cells, while FAC enhanced the expression of sclerostin and RANKL/OPG in MLO-Y4 cells. (2) Conditioned medium of MLO-Y4 cells inhibited the osteogenic capacity of osteoblasts while stimulating osteoclast differentiation. (3) By increasing oxidative stress, iron overload promotes the apoptosis of osteocytes and undermines the morphology of osteocytes in Hepcidin^-/- mice, further increasing the expression levels of sclerostin and RANKL/OPG in osteocytes, which is considered to be the causative factor for reduced bone formation and enhanced bone resorption. DFO administration reduced iron levels, and NAC treatment decreased oxidative stress in Hepcidin^-/- mice. Therefore, DFO or NAC treatment rescued the decrease in BMD, bone volume, and bone strength and attenuated the deterioration of bone architecture in Hepcidin^-/- mice by attenuating the effect of iron overload on osteocytes.

Conclusion: Osteocyte apoptosis due to increased ROS and resultant sclerostin and RANKL/OPG expression alteration was the main reason for bone loss in Hepcidin^-/- mice. Osteocytes are the main targets for the prevention and treatment of iron overload-induced osteoporosis.

Keywords: Iron; Osteocytes; Osteoporosis; RANKL; Sclerostin.