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[EFFECT OF ACTIVED RAW264.7 INDUCED BY H2O2 ON MIGRATION, PROLIFERATION AND OSTEOGENESIS GENE EXPRESSION OF MC3T3-E1]
Jianqiang Peng1, Zhixin Yi2, Mingxin Wu2
1Department of Spine Surgery, the Eighth Affiliated Hospital of Sun Yat-sen University, Shenzhen Guangdong, 518033, P. R. China.
Objective:
To explore the effect of H2O2-actived RAW264.7 macrophages on the migration, proliferation, and osteogenesis gene expression of MC3T3-E1 in mice.
Methods:
MC3T3-E1 cells and RAW264.7 cells were cultured to the 7th generation. RAW264.7 macrophages were stimulated with 0, 25, 50 and 100 μmol/L H2O2, the cell proliferation rate was detected by MTS at 1, 3, and 6 hours after stimulated, and superoxide dismutase (SOD) content by SOD assay kit at 1 hour after stimulated. The appropriate concentration and action time of H2O2-actived RAW264.7 were obtained. The supernatant of RAW264.7 macrophages stimulated by H2O2 or not was collected at 24 hours. Then, the supernatant was used to culture MC3T3-E1 cells in groups B (not stimulated by H2O2) and C (stimulated by H2O2), and DMEM was used as a control in group A. The migration of MC3T3-E1 cells was detected at 12 and 24 hours by cell scratch test, the proliferation of MC3T3-E1 cells at 24, 48, and 72 hours by MTS assay. MC3T3-E1 cells were cultured with only complete medium in blank control group, with complete medium containing 50 μg/mL vitamin C + 10 nmol/L β sodium glycerophosphate in positive group, normal control group (adding the supernatant not stimulated by H2O2), and experimental group (adding the supernatant stimulated by H2O2). At 3, 7, and 14 days, RT-PCR was used to determine the osteogenesis related mRNA expressions of alkaline phosphatase (ALP), Runx2, osteopontin (OPN), osteocalcin (OC), bone sialoprotein (BSP), and collagen type I (COL-I).
Results:
The results of MTS and SOD assay showed that the appropriate concentration and action time of H2O2-actived RAW264.7 macrophages were 25 μmol/L and 1 hour, respectively. MTS assay showed that the proliferation rate of MC3T3-E1 cells was significant higher in groups B and C than group A (P<0.05), in group B than group C, and significant difference was shown between groups at 2 and 3 days (P<0.05). The cell scratch test indicated that the migration of MC3T3-E1 cells was significantly faster in groups B and C than group A, and in group C than group B at 12 hours (P<0.05); many migrated cells were observed in all scratch sites of groups B and C at 24 hours. When compared with positive control group, the mRNA expressions of ALP, Runx2, OC and BSP in experimental group were significantly down-regulated at 7 and 14 days (P<0.05). When compared blank control group, the mRNA expressions of OPN and COL-I in experimental group were significantly down-regulated at 7 and 14 days (P<0.05).
Conclusions:
The appropriate concentration and action time of H2O2-actived RAW264.7 macrophages are 25 μmol/L and 1 hour. The H2O2-actived RAW264.7 cells can promote MC3T3-E1 cells migration, and suppress MC3T3-E1 cells proliferation and expressions of osteogenesis related genes.
Insights
Hydrogen peroxide-activated RAW264.7 macrophages promote MC3T3-E1 cell migration but suppress proliferation and osteogenesis gene expression. The optimal activation is 25 μmol/L H2O2 for 1 hour, impacting bone formation markers.
Area of Science:
- Cell Biology
- Immunology
- Biochemistry
Background:
- Macrophage activation plays a crucial role in tissue repair and regeneration.
- Understanding macrophage-conditioned media effects on osteoblast precursors is vital for bone healing research.
Purpose of the Study:
- To investigate the impact of hydrogen peroxide (H2O2)-activated RAW264.7 macrophages on MC3T3-E1 cell migration, proliferation, and osteogenesis.
- To determine the optimal concentration and duration for H2O2 activation of RAW264.7 macrophages.
Main Methods:
- RAW264.7 macrophages were stimulated with varying H2O2 concentrations (0-100 μmol/L).
- Cell proliferation and superoxide dismutase (SOD) activity were assessed.
- MC3T3-E1 cells were cultured with conditioned media from stimulated or unstimulated macrophages.
- Cell migration (scratch assay), proliferation (MTS assay), and osteogenesis gene expression (RT-PCR for ALP, Runx2, OPN, OC, BSP, COL-I) were analyzed.
Main Results:
- Optimal H2O2 activation for RAW264.7 macrophages was 25 μmol/L for 1 hour.
- H2O2-activated macrophage supernatant significantly enhanced MC3T3-E1 cell migration.
- Conversely, it suppressed MC3T3-E1 cell proliferation and the expression of key osteogenesis genes (ALP, Runx2, OC, BSP, OPN, COL-I).
Conclusions:
- H2O2-activated RAW264.7 macrophages differentially regulate MC3T3-E1 cell behavior.
- While promoting migration, this activation suppresses osteogenic differentiation, suggesting complex roles in bone remodeling.
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