Effects Of High Iron Environment On Human Osteoblastic Functions And Effects Of Hepcidin On Intracellular Calcium Of Human Osteoblasts

PartI Effects of high iron environment on human osteoblastic functionsObjective:To investigate the effects of high iron environment (Ferric AmmoniumCitrate) on osteoblastic functions and OPG/RANKL system in vitro.Methods:Human osteoblast cells (hFOB1.19) were incubated in media supplementedwith0–200umol/L of Ferric Ammonium Citrate (FAC). Proliferation viability ofosteoblasts were evaluated by MTT assay at48h. Alkaline phosphatase (ALP) activity wasmeasured using ALP viability kit at10d. Von-kossa staining assay were used to evaluatemineralized bone nodules at15d. The gene and protein expression of OPG and RANKLwas detected by RT-PCR and western blot at48h after treatment with FAC.Results:After treatment with FAC for48h, FAC significantly inhibited cellproliferation (P﹤0.05) at50umol/L, which is more obvious at100umol/L and200umol/LFAC (P﹤0.01). After treatment with FAC for10d, the ALP activity of osteoblasts weresignificantly suppressed by iron overload dose-dependently (P﹤0.01). The number ofmineralized nodules was significantly reduced by FAC at15d. In addition, FAC at100umol/L and200umol/L significantly increased the mRNA and protein expression ofRANKL/OPG (P﹤0.01).Conclusions: High iron environment not only significantly inhibited cell proliferation,differentiation and mineralizaiotn of human osteoblasts, but also increased the mRNA andprotein expression of RANKL/OPG. Therefore, iron overload not only inhibited boneformation directly, but also increased bone resorption indirectly in the pathogenesis ofosteoporosis. PartII Effects of hepcidin on intracellular calcium of human osteoblastsObjective:To study the effects of hepcidin on intracellular calium of humanosteoblasts exposed to normal or high iron environment and explore the underlyingmechanism.Methods:Human osteoblast cells (hFOB1.19) were cultured in normal and differentconcentrations of iron environment (10、50、200umol/L）. After treatment with hepcidinfor24h, flow cytometry was used to detect the fluorescent intensity of human osteoblastspretreated with Fluo-3/AM. In addition, under normal environment, we also observed theeffect of hepcidin (100nmol/L) on human osteoblasts pretreated with10umol/Ldantrolene and20umol/L nimodipine for25min and10min, respectively.Results:In normal environment, fluorescent intensity of intracellular calcium didn’tchange by hepcidin at10nmol/L, started to increase at30nmol/L （P<0.05）, increasedsignificantly at100nmol/L （P<0.01）, no difference was observed by hepcidin at300nmol/L. In10umol/L iron environment, similar results was observed compared to normalenvironment, except for more significantly increased intracellular calcium intensity byhepcidin at30nmol/L （P<0.01）. In200umol/L iron environment, hepcidin increased thefluorescent intensity of intracellular calcium at the concentrations ranging from10nmol/Lto100nmol/L （P<0.01）, no difference was observed by hepcidin at300nmol/L. Inaddition, the increase of intracellular calcium of human osteoblasts by hepcidin (100nmol/L) was completely blocked by pretreatment with either dantrolene or nimodipine.Conclusions: Hepcidin could increase intracellular calcium both at normal and highiron environment when not higher than100nmol/L, which is more obvious in high ironenvironment. Furthermore, these findings indicate that the increase of intracellular calciumis probably due to calcium release from endoplasmic reticulum, which is triggered bycalcium influx.