The Mechanism Of The Effect Of Ferric Ion On Osteoclast Differentiation And Bone Resorption

Objective: These experiments were carried out to explore the role of ferric ion inosteoclast differentiation and bone resorption as well as to elucidate the underlyingmechanism which might be favorable to further clarify the relationship between ironmetabolism and bone metabolism.Methods:1.RAW264.7was taken as the pre-osteoclast and ferric ammonium citrate(FAC) was used as the donor of ferric ion. The cells were treated with differentconcentration of FAC (12μM,25μM,50μM,100μM,200μM) in order to observe theeffect of FAC on cell viability.2. Accoding to the results of experiment1, RAW264.7were treated with FAC (12μM,25μM,50μM) in the presence of20ng/ml RANKL andsubjected to tartrate-resistant acid phosphatase staining. TRAP-positive multinucleatedcells (MNCs) with three or more nuclei were scored as osteoclasts.3.2,7-DCF-DA is acell-permeable dye which could reflect intracellular reactive oxygen species(ROS). Flowcytometric was used to determine the change in intracellular ROS when RAW264.7weretreated with different concentrations of FAC or FAC plus RANKL.4. Total glutathioneand oxidized glutathione (GSSG) were measured using a GSH/GSSH Assay kit afterRAW264.7were treated with FAC and FAC plus RANKL.5. N-actyl-L-cysteine, apotent antioxidant was used to observe if it could produce any influence on FACenhanced osteoclast formation.6.RT-PCR and Western blot were employed to detect thechange in the expression of TRAP、Cathepsin-K、NFATc1after the cells were treatedwith FAC and FAC plus NAC in the presence of RANKL.7.24male ICR mice wererandomly divided into three group (8in each) and were treated intraperitoneally withnormal saline, FAC, or FAC plus NAC respectively. Bone marrow-derived monocyte (BMMs) were separated from the femurs of mice model and were further incubated toobserve the osteoclast formation.8. Longitudinal femoral sections were stained withhematoxylin and eosin (HE) to observe the trabecular structure change.9. Micro-CT wasused to analyse the trabecular and cortical structure changes in femurs of mice model.10.The Ferritin、MDA、8-OH-DG、TRAP-5b、CTX、RANKL/OPG、BALP and BGP inmice serum were determined by enzyme linked immunosorbent assay(ELISA).Results:1. The12μM,25μM,50μM FAC had no direct stimulatory or inhibitoryeffect on RAW264.7proliferation (P>0.05), although higher concentrations did inhibitproliferation (P <0.05).2. FAC (0uM,12μM,25μM,50μM) treatment increased thenumber TRAP-positive MNCs in a dose-dependent manner (P <0.05).3. FAC treatmentincreased intracellular ROS in RAW264.7cells in a dose-dependent manner (P <0.05).Furthermore, ROS levels increased to a greater extent in RAW264.7cells treated withFAC in combination with RANKL than in cells treated with FAC or RANKL alone (P<0.05).4. The addition of FAC to untreated RAW264.7cells led to a significant decreasein both total glutathione and GSH/GSSG ratio (P <0.05), when RAW264.7cells weretreated with both FAC and RANKL, the decreases in the both indices were exacerbated(P <0.05).5.The number of TRAP-positive MNCs in cultures treated with both FAC andNAC was significantly lower than the control group, and slightly greater than theNAC-treated group (P <0.05).6.FAC treatment significantly enhanced RANKL-inducedTRAP、Cathepsin-K、NFATc1mRNA and protein expression, and these effects wereabrogated by the addition of NAC (P <0.05).7. The number of TRAP-positive MNCsformed from the BMMs of the FAC-treated group was significantly greater than that ofcontrol group, and decreased significantly in the presence of NAC (P <0.05).8. HEstaining showed that mice treated with FAC had less trabecular density at distal ends offemurs than mice in placebo group, and NAC treatment attenuated this effect.9.Micro-CT pictures and analysis revealed prominent reduction both in bone trabecularsand cortical bone in the femur of FAC-challenged mice, whereas the reduction was much lower in NAC treated mice.(P <0.05).10. Serum levels of MDA、8-OH-DG、TRAP-5b、CTX and RANKL/OPG increased after FAC treatment and decreased in mice treatedwith NAC (P<0.05). While BALP and BGP in mice serum decreased after FAC treatmentand increased after NAC treatment (P <0.05).Conclusion: Ferric ion treatment enhances osteoclast differentiation and boneresorption both in vitro and in vivo and that this response is associated with iron-inducedgeneration of ROS and oxidative stress. These data also indicate that by reducing boneresorption, antioxidants have the potential to treat osteoporosis caused by iron overload.