Effects Of High Iron Environment On Intracellular Iron Homeostasis、Oxidative Stress、Biological Activity Of Human Osteoblasts And Its Interaction

Objective: To observed the effects of high iron environment (Ferric AmmoniumCitrate) on intracellular iron homeostasis and oxidative stress of human osteoblasts, andexplore the relationship of them.Methods: Human osteoblast cells (hFOB1.19) were incubated in mediasupplemented with0–200umol/L of Ferric Ammonium Citrate (FAC). The genesexpression of (transferring receptor,TfR)、(divalent metal transporter1,DMT1) and(ferroportin,FPN1) were detected by RT-PCR at48h; Confocal laser scanning microscopy(CLSM) observed the fluorescence intensity of the iron ions in the osteoblast; Maleicdialdehyde(MDA) content, superoxide dismutase(SOD) and glutathioneperoxidase(GSH-PX) activity were measured with MDA、SOD and GSH-PX commercialkits at48h; Intracellular ROS was measured using the oxidation sensitive dye DCF-DA byflow cytometry at48h after treatment with FAC.Results: After treatment with FAC for48h, the increase of iron concentration inculture environment can up-regulation the expression of FPN1mRNA, anddown-regulation the expression of TfR and DMT1mRNA in hFOB1.19osteoblasts withdoes-dependently and the fluorescence intensity of the iron ions in the osteoblast wassignificantly decreased (P﹤0.05); The lever of MDA was increased significantly withconcentration-dependently, and the activity of SOD and GSH-PX were decreasedsignificantly with concentration-dependently and the levels of Intracellular ROS wasenhanced significantly by FAC treatment (P<0.05);Conclusion: The environment of iron ions affects the iron channel protein of osteoblasts,and the iron regulatory gene can transpire specific changes within a certain range with the iron ion concentration of extracellular increase, resulting in the concentration of intracellular ironions increased. And with the increase of intracellular concentration of iron ions, theintracellular oxidative stress levels along with corresponding increased. Objective: To understood the changes of oxidative stress indicators (reactive species)and biological activity indicators of osteoblast in high iron environment, and to explore therole of reactive species in “iron overload and bone metabolism”.Methods: The osteoblasts were cultured in vitro. The experiment were divided intofour groups treated as follows:(1) cells received with200umol/L ferric ammonium citrate(FAC);(2) a group intervention using2.5mmol/L antioxidant N-acetyl cysteine (NAC);(3)NAC pretreatment1h and using the same concentration of FAC intervention;(4) a groupof normal control. After48h, detect the level of reactive oxygen species (ROS) in eachgroup by flow cytometry; CCK-8assay detect the cell viability; the expression changes ofOPG、BGP and COL1mRNA detected by RT-PCR; Alkaline phosphatase (ALP) activitywas measured using ALP viability kit at10d.Results: The level of reactive oxygen species was significantly different in differentgroups(P<0.05), and the FAC+NAC group was significantly lower than the FAC group,but higher the NAC group; Between groups of active oxygen content and osteoblastactivity, OPG、BGP and COL1mRNA expression ratio of the optical density and alkalinephosphatase activity was negatively correlated was statistically significant(P<0.05).Conclusion: Iron overload can reduce the biological activity and increase the reactivespecies levels of osteoblast, anti-oxidant can reduce the reactive species levels and improvethe biological activity of osteoblast under iron overload environment; Iron overload reducethe biological activity of osteoblast may be associated with reactive oxygen species whichincreased by iron overload.