Abstract: BACKGROUND: Iron overload is an independent factor inducing osteoporosis, but the action mechanism is currently unclear. Therefore, exploring the effects of iron overload on osteoblast-related cells will help to deeply understand the pathogenesis of osteoporosis and provide potential strategies for osteoporosis treatment.
OBJECTIVE: To explore the effects of iron overload environment on osteoblast precursor cell activity, ferroptosis, and osteogenic differentiation.
METHODS: Osteoblast precursor cells (MC3T3-E1 cells) were divided into blank group, iron overload group, fer-1 group, and deferoxamine group. The iron overload group was treated with 300 μmol/L ammonium ferric citrate in the culture medium for 48 hours to simulate the iron overload microenvironment. The cells in fer-1 group and deferoxamine group were pretreated with 5 μmol/L antioxidant fer-1 and 5 μmol/L deferoxamine for 8 hours, respectively, and then added with 300 μmol/L ammonium ferric citrate for 48 hours. CCK-8 assay was used to determine the cell viability. Intracellular reactive oxygen species levels were detected employing a reactive oxygen species fluorescent probe. Changes in mitochondrial membrane potential were monitored with a mitochondrial membrane potential fluorescent probe. Mitochondrial morphology was observed employing transmission electron microscopy. Cellular glutathione levels were measured with a reduced glutathione colorimetric assay kit. Lipid peroxidation levels were assessed with a malondialdehyde colorimetric assay kit. Cellular ferrous ion levels were determined with a ferrous ion colorimetric assay kit. The osteogenic and mineralization capabilities of the cells were verified by alkaline phosphatase staining and alizarin red staining. Collagen secretion ability was detected using Sirius Red staining. The expression of osteogenic/ferroptosis-related genes and proteins was examined through reverse transcription quantitative polymerase chain reaction and western blot analysis.
RESULTS AND CONCLUSION: (1) In an iron-overload environment, the mitochondrial membrane potential of cells decreased and their structure was compromised, with an elevation in intracellular lipid peroxidation levels and a downregulation of genes and proteins associated with ferroptosis resistance. However, pretreatment with fer-1 and deferoxamine led to an increase in mitochondrial membrane potential and partial restoration of morphology, a reduction in intracellular lipid peroxidation levels, and an upregulation of genes and proteins related to ferroptosis resistance. (2) In an iron-overload environment, the levels of cellular alkaline phosphatase, the formation of mineralized nodules, and the synthesis of collagen fibers were all found to be decreased. Pretreatment with fer-1 and deferoxamine was observed to upregulate the expression of osteogenic differentiation in cells. (3) In summary, iron overload could increase intracellular oxidative stress levels, mediate ferroptosis in MC3T3-E1 cells and inhibit osteogenic differentiation, thereby inducing osteoporosis. Therefore, maintaining iron homeostasis and inhibiting osteogenesis-related ferroptosis may be potential strategies to prevent or treat osteoporosis.

Key words: iron overload, osteoporosis, ferroptosis, ferric ammonium citrate, preosteoblastic cells, mitochondrial function, osteogenic differentiation, lipid peroxidation