The role of glutathione in steroid induced bone marrow mesenchymal stem cells dysfunction_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

DAI Zhipeng ¹ ,  ZHENG Jia ¹ , GAO Yanzheng ¹ , LIU Ke ¹ , YANG Shuhua ² , XU Weihua ²

1. Department of Orthopedics, Henan Provincial People’s Hospital, Zhengzhou Henan, 450003, P.R.China;
2. Department of Orthopedics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan Hubei, 430022, P.R.China;

Corresponding author：

ZHENG Jia, Email: dzp01234@163.com

Keywords：

Osteonecrosis of femoral head; bone marrow mesenchymal stem cells; reactive oxygen species; osteogenesis; adiogenesis; glutathione

DOI：

10.7507/1002-1892.201703129

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Objective To investigate the protective effect of the antioxidant glutathione (GSH) on the steroid-induced imbalance between osteogenesis and adipogenesis in human bone marrow mesenchymal stem cells (BMSCs). Methods The BMSCs were isolated from the proximal femur bone marrow from 3 patients of femoral neck fracture and were separated, cultured, and purificated by density gradient centrifugation and adherent wall methodin vitro. The third generation BMSCs were divided into 5 groups: group A, BMSCs (1×10⁵ cells/mL); group B, BMSCs (1×10⁵ cells/mL)+10 μmol/L dexamethasone; group C, BMSCs (1×10⁵ cells/mL)+10 μmol/L dexamethasone+5 μmol/L GSH; group D, BMSCs (1×10⁵ cells/mL)+10 μmol/L dexamethasone+10 μmol/L GSH; group E, BMSCs (1×10⁵ cells/mL)+10 μmol/L dexamethasone+50 μmol/L GSH. After cultured for 7 days, the reactive oxygen species expression was detected by flow cytometry; the superoxide dismutase (SOD) and Catalase mRNA expressions were determined by RT-PCR; the peroxisome proliferator-activated receptors γ (PPAR-γ), CCAAT/enhancer-binding family of proteins (C/EBP), Runx2, and alkaline phosphatase (ALP) mRNA expressions were evaluated by real-time fluorescence quantitative PCR. After cultured for 21 days, Oil red O staining was used to observe the adipogenesis differentiation of cells, and the expressions of related proteins were detected by Western blot. Results The reactive oxygen species expression in group B was obviously higher than in the other groups, in group C than in groups A, D, and E, and in groups D, E than in group A, all showing significant differences between groups (P<0.05); but there was no significant difference between groups D and E (P>0.05). The oil red O staining positive cells in group B were obviously more than the other groups, and groups C, D, E, and A decreased sequentially, the absorbance (A) values had significant differences between groups (P<0.05). RT-PCR detection showed that the relative expressions of SOD and Catalase mRNA in group B were significantly lower than those in the other groups, while in group C than in groups A, D, and E (P<0.05), but there was no significant difference among groups A, D, and E (P>0.05). Real-time fluorescence quantitative PCR detection showed that the relative expressions of PPAR-γ and C/EBP mRNA in group B were significantly higher than those in the other groups, while in group C than in groups A, D, and E, and in groups D, E than in group A (P<0.05); but there was no significant difference between groups D and E (P>0.05). The relative expressions of Runx2 and ALP mRNA in group B were significantly lower than those in the other groups, while in group C than in groups A, D, and E, and in groups D, E than in group A (P<0.05); but there was no significant difference between groups D and E (P>0.05). Western blot detection showed that the relative expression of PPAR-γ and C/EBP protein in group B was significantly higher than those in the other groups, and groups C, D, E, and A decreased sequentially, all showing significant differences between groups (P<0.05). The relative expression of Runx2 and ALP protein in group B was significantly lower than those in the other groups, and groups C, D, E, and A increased sequentially, all showing significant differences between groups (P<0.05). Conclusions GSH can inhibit the adipogenesis differentiation and enhance the osteogenic differentiation of human BMSCs by reducing the intracellular reactive oxygen species level; and in a certain range, the higher the concentration of GSH, the more obvious the effect is.

Citation： DAI Zhipeng, ZHENG Jia, GAO Yanzheng, LIU Ke, YANG Shuhua, XU Weihua. The role of glutathione in steroid induced bone marrow mesenchymal stem cells dysfunction. Chinese Journal of Reparative and Reconstructive Surgery, 2018, 32(1): 91-98. doi: 10.7507/1002-1892.201703129 Copy

1.	Mirzai R, Chang C, Greenspan A, et al. The pathogenesis of osteonecrosis and the relationships to corticosteroids. J Asthma, 1999, 36(1): 77-95.
2.	Mutijima E, De Maertelaer V, Deprez M, et al. The apoptosis of osteoblasts and osteocytes in femoral head osteonecrosis: its specificity and its distribution. Clin Rheumatol, 2014, 33(12): 1791-1795.
3.	Lin L, Dai SD, Fan GY. Glucocorticoid-induced differentiation of primary cultured bone marrow mesenchymal cells into adipocytes is antagonized by exogenous Runx2. APMIS, 2010, 118(8): 595-605.
4.	Fan L, Liu R, Li J, et al. Low oxygen tension enhances osteogenic potential of bone marrow-derived mesenchymal stem cells with osteonecrosis-related functional impairment. Stem Cells, 2015, 2015: 950312.
5.	代志鹏, 郑稼, 黄遂柱, 等. 激素性股骨头坏死患者骨髓间充质干细胞数、功能及分化能力的变化. 中华实验外科杂志, 2016, 33(3): 813-816.
6.	Ferrelli F, Pastore D, Capuani B, et al. Serum glucocorticoid inducible kinase (SGK)-1 protects endothelial cells against oxidative stress and apoptosis induced by hyperglycaemia. Acta Diabetol, 2015, 52(1): 55-64.
7.	He T, Guan X, Wang S, et al. Resveratrol prevents high glucose-induced epithelial-mesenchymal transition in renal tubular epithelial cells by inhibiting NADPH oxidase/ROS/ERK pathway. Mol Cell Endocrinol, 2015, 402: 13-20.
8.	Shi J, Zuo H, Ni L, et al. An IDH1 mutation inhibits growth of glioma cells via GSH depletion and ROS generation. Neurol Sci, 2014, 35(6): 839-845.
9.	Aoyama T, Goto K, Kakinoki R, et al. An exploratory clinical trial for idiopathic osteonecrosis of femoral head by cultured autologous multipotent mesenchymal stromal cells augmented with vascularized bone grafts. Tissue Eng Part B Rev, 2014, 20(4): 233-242.
10.	Rastogi S, Sankineani SR, Nag HL, et al. Intralesional autologous mesenchymal stem cells in management of osteonecrosis of femur: a preliminary study. Musculoskelet Surg, 2013, 97(3): 223-228.
11.	Parisotto EB, Garlet TR, Cavalli VL, et al. Antioxidant intervention attenuates oxidative stress in children and teenagers with Down syndrome. Res Dev Disabil, 2014, 35(6): 1228-1236.
12.	Huang Q, Gao B, Wang L, et al. Protective effects of myricitrin against osteoporosis via reducing reactive oxygen species and bone-resorbing cytokines. Toxicol Appl Pharmacol, 2014, 280(3): 550-560.
13.	O’Brien CA, Jia D, Plotkin LI, et al. Glucocorticoids act directly on osteoblasts and osteocytes to induce their apoptosis and reduce bone formation and strength. Endocrinology, 2004, 145(4): 1835-1841.
14.	Canalis E, Delany AM. Mechanisms of glucocorticoid action in bone. AnnN Y Acad Sci, 2002, 966: 73-81.
15.	Mody N, Parhami F, Sarafian TA, et al. Oxidative stress modulates osteoblastic differentiation of vascular and bone cells. Free Radic Biol Med, 2001, 31(4): 509-519.
16.	Barbagallo I, Vanella A, Peterson SJ, et al. Overexpression of heme oxygenase-1 increases human osteoblast stem cell differentiation. J Bone Miner Metab, 2010, 28(3): 276-288.
17.	Cremers NA, Lundvig DM, van Dalen SC, et al. Curcumin-induced heme oxygenase-1 expression prevents H2O2-induced cell death in wild type and heme oxygenase-2 knockout adipose-derived mesenchymal stem cells. Int J Mol Sci, 2014, 15(10): 17974-17999.
18.	Mikami T, Ichiseki T, Kaneuji A, et al. Prevention of steroid-induced osteonecrosis by intravenous administration of vitamin E in a rabbit model. J Orthop Sci, 2010, 15(5): 674-677.

1. Mirzai R, Chang C, Greenspan A, et al. The pathogenesis of osteonecrosis and the relationships to corticosteroids. J Asthma, 1999, 36(1): 77-95.
2. Mutijima E, De Maertelaer V, Deprez M, et al. The apoptosis of osteoblasts and osteocytes in femoral head osteonecrosis: its specificity and its distribution. Clin Rheumatol, 2014, 33(12): 1791-1795.
3. Lin L, Dai SD, Fan GY. Glucocorticoid-induced differentiation of primary cultured bone marrow mesenchymal cells into adipocytes is antagonized by exogenous Runx2. APMIS, 2010, 118(8): 595-605.
4. Fan L, Liu R, Li J, et al. Low oxygen tension enhances osteogenic potential of bone marrow-derived mesenchymal stem cells with osteonecrosis-related functional impairment. Stem Cells, 2015, 2015: 950312.
5. 代志鹏, 郑稼, 黄遂柱, 等. 激素性股骨头坏死患者骨髓间充质干细胞数、功能及分化能力的变化. 中华实验外科杂志, 2016, 33(3): 813-816.
6. Ferrelli F, Pastore D, Capuani B, et al. Serum glucocorticoid inducible kinase (SGK)-1 protects endothelial cells against oxidative stress and apoptosis induced by hyperglycaemia. Acta Diabetol, 2015, 52(1): 55-64.
7. He T, Guan X, Wang S, et al. Resveratrol prevents high glucose-induced epithelial-mesenchymal transition in renal tubular epithelial cells by inhibiting NADPH oxidase/ROS/ERK pathway. Mol Cell Endocrinol, 2015, 402: 13-20.
8. Shi J, Zuo H, Ni L, et al. An IDH1 mutation inhibits growth of glioma cells via GSH depletion and ROS generation. Neurol Sci, 2014, 35(6): 839-845.
9. Aoyama T, Goto K, Kakinoki R, et al. An exploratory clinical trial for idiopathic osteonecrosis of femoral head by cultured autologous multipotent mesenchymal stromal cells augmented with vascularized bone grafts. Tissue Eng Part B Rev, 2014, 20(4): 233-242.
10. Rastogi S, Sankineani SR, Nag HL, et al. Intralesional autologous mesenchymal stem cells in management of osteonecrosis of femur: a preliminary study. Musculoskelet Surg, 2013, 97(3): 223-228.
11. Parisotto EB, Garlet TR, Cavalli VL, et al. Antioxidant intervention attenuates oxidative stress in children and teenagers with Down syndrome. Res Dev Disabil, 2014, 35(6): 1228-1236.
12. Huang Q, Gao B, Wang L, et al. Protective effects of myricitrin against osteoporosis via reducing reactive oxygen species and bone-resorbing cytokines. Toxicol Appl Pharmacol, 2014, 280(3): 550-560.
13. O’Brien CA, Jia D, Plotkin LI, et al. Glucocorticoids act directly on osteoblasts and osteocytes to induce their apoptosis and reduce bone formation and strength. Endocrinology, 2004, 145(4): 1835-1841.
14. Canalis E, Delany AM. Mechanisms of glucocorticoid action in bone. AnnN Y Acad Sci, 2002, 966: 73-81.
15. Mody N, Parhami F, Sarafian TA, et al. Oxidative stress modulates osteoblastic differentiation of vascular and bone cells. Free Radic Biol Med, 2001, 31(4): 509-519.
16. Barbagallo I, Vanella A, Peterson SJ, et al. Overexpression of heme oxygenase-1 increases human osteoblast stem cell differentiation. J Bone Miner Metab, 2010, 28(3): 276-288.
17. Cremers NA, Lundvig DM, van Dalen SC, et al. Curcumin-induced heme oxygenase-1 expression prevents H2O2-induced cell death in wild type and heme oxygenase-2 knockout adipose-derived mesenchymal stem cells. Int J Mol Sci, 2014, 15(10): 17974-17999.
18. Mikami T, Ichiseki T, Kaneuji A, et al. Prevention of steroid-induced osteonecrosis by intravenous administration of vitamin E in a rabbit model. J Orthop Sci, 2010, 15(5): 674-677.

Chinese Journal of Reparative and Reconstructive Surgery

The role of glutathione in steroid induced bone marrow mesenchymal stem cells dysfunction

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