EFFECT OF ACTIVED RAW264.7 INDUCED BY H<sub>2</sub>O<sub>2</sub> ON MIGRATION, PROLIFERATION AND OSTEOGENESIS GENE EXPRESSION OF MC3T3-E1_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

PENGJianqiang ¹ , YIZhixin ² ^Δ , WUMingxin ² , HUANGAijun ¹ , LINKun ¹ , JINSong ¹ , LILiangping ³ , HUANGSheng ³ , LUOJiaquan ³ ,  ZOUXuenong ³

1. Department of Spine Surgery, the Eighth Affiliated Hospital of Sun Yat-sen University, Shenzhen Guangdong, 518033, P. R. China;
2. Department of Orthopedics, the Third People's Hospital of Huizhou;
3. Guangdong Provincial Key Laboratory of Orthopedics and Traumatology, Department of Spine Surgery, the First Affiliated Hospital of Sun Yat-sen University;

Corresponding author：

ZOUXuenong, Email: zxnong@hotmail.com

Keywords：

Oxidative stress; H₂O₂; MC3T3-E1 cells; RAW264.7 cells; Osteogenesis

DOI：

10.7507/1002-1892.20160234

Video：

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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). Conclusion 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.

Citation： PENGJianqiang, YIZhixin, WUMingxin, HUANGAijun, LINKun, JINSong, LILiangping, HUANGSheng, LUOJiaquan, ZOUXuenong. EFFECT OF ACTIVED RAW264.7 INDUCED BY H₂O₂ ON MIGRATION, PROLIFERATION AND OSTEOGENESIS GENE EXPRESSION OF MC3T3-E1. Chinese Journal of Reparative and Reconstructive Surgery, 2016, 30(9): 1146-1152. doi: 10.7507/1002-1892.20160234 Copy

1.	Pirraco RP, Reis RL, Marques AP. Effect of monocytes/macrophages on the early osteogenic differentiation of hBMSCs. J Tissue Eng Regen Med, 2013, 7(5):392-400.
2.	Chen C, Uludağ H, Wang Z, et al. Macrophages inhibit migration, metabolic activity and osteogenic differentiation of human mesenchymal stem cells in vitro. Cells Tissues Organs, 2012, 195(6):473-483.
3.	Guihard P, Danger Y, Brounais B, et al. Induction of osteogenesis in mesenchymal stem cells by activated monocytes/macrophages depends on oncostatin M signaling. Stem Cells, 2012, 30(4):762-772.
4.	Ekström K, Omar O, Granéli C, et al. Monocyte exosomes stimulate the osteogenic gene expression of mesenchymal stem cells. PLoS One, 2013, 8(9):e75227.
5.	Sims NA, Gooi JH. Bone remodeling:Multiple cellular interactions required for coupling of bone formation and resorption. Semin Cell Dev Biol, 2008, 19(5):444-451.
6.	易志新, 邹学农, 彭建强.巨噬细胞对间充质干细胞成骨分化影响的研究进展.中国脊柱脊髓杂志, 2015, 25(12):1116-1119.
7.	Fridovich I. The biology of oxygen radicals. Science, 1978, 201(4359):875-880.
8.	Poyton RO, Ball KA, Castello PR. Mitochondrial generation of free radicals and hypoxic signaling. Trends Endocrinol Metab, 2009, 20(7):332-340.
9.	Jabs T. Reactive oxygen intermediates as mediators of programmed cell death in plants and animals. Biochem Pharmacol, 1999, 57(3):231-245.
10.	McCubrey JA, Lahair MM, Franklin RA. Reactive oxygen species-induced activation of the MAP kinase signaling pathways. Antioxid Redox Signal, 2006, 8(9-10):1775-1789.
11.	Choe Y, Yu JY, Son YO, et al. Continuously generated H₂O₂ stimulates the proliferation and osteoblastic differentiation of human periodontal ligament fibroblasts. J Cell Biochem, 2012, 113(4):1426-1436.
12.	Raggatt LJ, Wullschleger ME, Alexander KA, et al. Fracture healing via periosteal callus formation requires macrophages for both initiation and progression of early endochondral ossification. Am J Pathol, 2014, 184(12):3192-3204.
13.	Pomari E, Stefanon B, Colitti M. Effect of plant extracts on H₂O₂-induced inflammatory gene expression in macrophages. J Inflamm Res, 2014, 7:103-112.
14.	Mokgobu MI, Cholo MC, Anderson R, et al. Oxidative induction of pro-inflammatory cytokine formation by human monocyte-derived macrophages following exposure to manganese in vitro. J Immunotoxicol, 2015, 12(1):98-103.
15.	崔海燕, 韩晨露, 肖鹏, 等.过氧化氢刺激RAW264.7巨噬细胞促炎细胞因子和趋化因子基因表达.中国生物化学与分子生物学报, 2015, 31(7):734-740.
16.	Brüne B, Dehne N, Grossmann N, et al. Redox control of inflammation in macrophages. Antioxid Redox Signal, 2013, 19(6):595-637.
17.	Ota K, Quint P, Weivoda MM, et al. Transforming growth factor beta 1 induces CXCL16 and leukemia inhibitory factor expression in osteoclasts to modulate migration of osteoblast progenitors. Bone, 2013, 57(1):68-75.
18.	Takeyama K, Chatani M, Inohaya K, et al. TGFβ-2 signaling is essential for osteoblast migration and differentiation during fracture healing in medaka fish. Bone, 2016, 86:68-78.
19.	Zhou FH, Foster BK, Zhou XF, et al. TNF-alpha mediates p38 MAP kinase activation and negatively regulates bone formation at the injured growth plate in rats. J Bone Miner Res, 2006, 21(7):1075-1088.
20.	Kalajzic I, Staal A, Yang WP, et al. Expression profile of osteoblast lineage at defined stages of differentiation. J Biol Chem, 2005, 280(26):24618-24626.
21.	Lu X, Beck GR Jr, Gilbert LC, et al. Identification of the homeobox protein Prx1 (MHox, Prrx-1) as a regulator of osterix expression and mediator of tumor necrosis factor alpha action in osteoblast differentiation. J Bone Miner Res, 2011, 26(1):209-219.
22.	Osta B, Benedetti G, Miossec P. Classical and paradoxical effects of TNF-alpha on bone homeostasis. Front Immunol, 2014, 5:48.
23.	Sonomoto K, Yamaoka K, Oshita K, et al. Interleukin-1β induces differentiation of human mesenchymal stem cells into osteoblasts via the Wnt-5a/receptor tyrosine kinase-like orphan receptor 2 pathway. Arthritis Rheum, 2012, 64(10):3355-3363.
24.	Mumme M, Scotti C, Papadimitropoulos A, et al. Interleukin-1β modulates endochondral ossification by human adult bone marrow stromal cells. Eur Cell Mater, 2012, 24:224-236.

1. Pirraco RP, Reis RL, Marques AP. Effect of monocytes/macrophages on the early osteogenic differentiation of hBMSCs. J Tissue Eng Regen Med, 2013, 7(5):392-400.
2. Chen C, Uludağ H, Wang Z, et al. Macrophages inhibit migration, metabolic activity and osteogenic differentiation of human mesenchymal stem cells in vitro. Cells Tissues Organs, 2012, 195(6):473-483.
3. Guihard P, Danger Y, Brounais B, et al. Induction of osteogenesis in mesenchymal stem cells by activated monocytes/macrophages depends on oncostatin M signaling. Stem Cells, 2012, 30(4):762-772.
4. Ekström K, Omar O, Granéli C, et al. Monocyte exosomes stimulate the osteogenic gene expression of mesenchymal stem cells. PLoS One, 2013, 8(9):e75227.
5. Sims NA, Gooi JH. Bone remodeling:Multiple cellular interactions required for coupling of bone formation and resorption. Semin Cell Dev Biol, 2008, 19(5):444-451.
6. 易志新, 邹学农, 彭建强.巨噬细胞对间充质干细胞成骨分化影响的研究进展.中国脊柱脊髓杂志, 2015, 25(12):1116-1119.
7. Fridovich I. The biology of oxygen radicals. Science, 1978, 201(4359):875-880.
8. Poyton RO, Ball KA, Castello PR. Mitochondrial generation of free radicals and hypoxic signaling. Trends Endocrinol Metab, 2009, 20(7):332-340.
9. Jabs T. Reactive oxygen intermediates as mediators of programmed cell death in plants and animals. Biochem Pharmacol, 1999, 57(3):231-245.
10. McCubrey JA, Lahair MM, Franklin RA. Reactive oxygen species-induced activation of the MAP kinase signaling pathways. Antioxid Redox Signal, 2006, 8(9-10):1775-1789.
11. Choe Y, Yu JY, Son YO, et al. Continuously generated H₂O₂ stimulates the proliferation and osteoblastic differentiation of human periodontal ligament fibroblasts. J Cell Biochem, 2012, 113(4):1426-1436.
12. Raggatt LJ, Wullschleger ME, Alexander KA, et al. Fracture healing via periosteal callus formation requires macrophages for both initiation and progression of early endochondral ossification. Am J Pathol, 2014, 184(12):3192-3204.
13. Pomari E, Stefanon B, Colitti M. Effect of plant extracts on H₂O₂-induced inflammatory gene expression in macrophages. J Inflamm Res, 2014, 7:103-112.
14. Mokgobu MI, Cholo MC, Anderson R, et al. Oxidative induction of pro-inflammatory cytokine formation by human monocyte-derived macrophages following exposure to manganese in vitro. J Immunotoxicol, 2015, 12(1):98-103.
15. 崔海燕, 韩晨露, 肖鹏, 等.过氧化氢刺激RAW264.7巨噬细胞促炎细胞因子和趋化因子基因表达.中国生物化学与分子生物学报, 2015, 31(7):734-740.
16. Brüne B, Dehne N, Grossmann N, et al. Redox control of inflammation in macrophages. Antioxid Redox Signal, 2013, 19(6):595-637.
17. Ota K, Quint P, Weivoda MM, et al. Transforming growth factor beta 1 induces CXCL16 and leukemia inhibitory factor expression in osteoclasts to modulate migration of osteoblast progenitors. Bone, 2013, 57(1):68-75.
18. Takeyama K, Chatani M, Inohaya K, et al. TGFβ-2 signaling is essential for osteoblast migration and differentiation during fracture healing in medaka fish. Bone, 2016, 86:68-78.
19. Zhou FH, Foster BK, Zhou XF, et al. TNF-alpha mediates p38 MAP kinase activation and negatively regulates bone formation at the injured growth plate in rats. J Bone Miner Res, 2006, 21(7):1075-1088.
20. Kalajzic I, Staal A, Yang WP, et al. Expression profile of osteoblast lineage at defined stages of differentiation. J Biol Chem, 2005, 280(26):24618-24626.
21. Lu X, Beck GR Jr, Gilbert LC, et al. Identification of the homeobox protein Prx1 (MHox, Prrx-1) as a regulator of osterix expression and mediator of tumor necrosis factor alpha action in osteoblast differentiation. J Bone Miner Res, 2011, 26(1):209-219.
22. Osta B, Benedetti G, Miossec P. Classical and paradoxical effects of TNF-alpha on bone homeostasis. Front Immunol, 2014, 5:48.
23. Sonomoto K, Yamaoka K, Oshita K, et al. Interleukin-1β induces differentiation of human mesenchymal stem cells into osteoblasts via the Wnt-5a/receptor tyrosine kinase-like orphan receptor 2 pathway. Arthritis Rheum, 2012, 64(10):3355-3363.
24. Mumme M, Scotti C, Papadimitropoulos A, et al. Interleukin-1β modulates endochondral ossification by human adult bone marrow stromal cells. Eur Cell Mater, 2012, 24:224-236.

Chinese Journal of Reparative and Reconstructive Surgery

EFFECT OF ACTIVED RAW264.7 INDUCED BY H₂O₂ ON MIGRATION, PROLIFERATION AND OSTEOGENESIS GENE EXPRESSION OF MC3T3-E1

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EFFECT OF ACTIVED RAW264.7 INDUCED BY H₂O₂ ON MIGRATION, PROLIFERATION AND OSTEOGENESIS GENE EXPRESSION OF MC3T3-E1