EFFECT OF DIFFERENT CONCENTRATIONS OF DEXAMETHASONE ON APOPTOSIS AND EXPRESSION OF FAS/FASL IN HUMAN OSTEOARTHRITIS CHONDROCYTES_Chinese Journal of Reparative and Reconstructive Surgery

Authors：

TU Yihui ¹ , XUE Huaming ¹ , XIA Zhidao ² , CAI Minwei ¹ , LIU Xiaodong ¹ , MA Tong ¹ , ZHANG Changqing ³

1Department of Orthopaedics, Yangpu Hospital, Tongji University, Shanghai, 200090, P.R.China;;
2Botnar Research Centre, Nuffield Department of Orthopaedics, Oxford University;;
3Department of Orthopaedics, SixthPeople’s Hospital, Shanghai Jiaotong University.;

Corresponding author：

Keywords：

Dexamethasone, Osteoarthritis, Human articular chondrocytes, Cell apoptosis, Fas/FasL

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Objective Corticosteroids can destroy the cartilage. To investigate the effect of dexamethasone (Dexa) on the apoptosis and expression of Fas/FasL of human articular chondrocytes (HACs) in vitro so as to explore the mechanism of
pro-apoptotic role of Dexa on HACs. Methods Following full agreement of patients, the cartilage specimens were collected
from the patients with osteoarthritis undergoing knee replacement. The second passage HACs were incubated in cell culture media containing 0.125, 1.25, 12.5, 25, and 50 μg/mL Dexa for 48 hours respectively to determine the optimal concentration of Dexa by MTT. The apoptosis was assessed by TMRE/Hoechst/Annexin V-FITC/7-AAD quadruple staining after culture for 0, 24, and 48 hours. The mRNA expressions of Fas and FasL were determined by real-time quantitative PCR after culture for 48 hours. The protein expressions of Fas and FasL were determined by immunohistochemistry staining analysis after culture for 24 hours and 48 hours. Results The cell inhibitory rate of 25 μg/mL Dexa was significantly higher than that of 50 μg/mL Dexa (P lt; 0.05), and there were significant differences when compared with that at other concentrations of Dexa (P lt; 0.05), so 25 μg/mL Dexa was appropriately selected as an optimal concentration of Dexa. The apoptotic rates of HACs were 5.8% ± 0.3%, 27.0% ± 2.6%, and 36.0% ± 3.1% at 0, 24, and 48 hours, respectively, in a time dependent manner (P lt; 0.05). The expressions of Fas mRNA were (8.93 ± 1.12) × 10—3 in the experimental group and (3.31 ± 0.37) × 10—3 in the control group, showing significant difference (P lt; 0.05). The expressions of FasL mRNA were (5.92 ± 0.66) × 10—3 in the experimental group and (2.31 ± 0.35) × 10—3
in the control group, showing significant difference (P lt; 0.05). The expressions of Fas and FasL proteins showed an increasing tendency with time in the experimental group and the expressions were significantly higher than those in the control group after culture for 24 hours and 48 hours (P lt; 0.05). Conclusion Dexa can induce the apoptosis and significantly upregulate the apoptotic gene expression of Fas/FasL, which can provide the experimental evidence to further investigate the role of Fas/FasL signaling pathway in Dexa-induced HACs apoptosis.

Citation： TU Yihui,XUE Huaming,XIA Zhidao,CAI Minwei,LIU Xiaodong,MA Tong,ZHANG Changqing. EFFECT OF DIFFERENT CONCENTRATIONS OF DEXAMETHASONE ON APOPTOSIS AND EXPRESSION OF FAS/FASL IN HUMAN OSTEOARTHRITIS CHONDROCYTES. Chinese Journal of Reparative and Reconstructive Surgery, 2012, 26(5): 536-541. doi: Copy

1.	Céleste C, Ionescu M, Robin Poole A, et al. Repeated intraarticular injections of triamcinolone acetonide alter cartilage matrix metabolism measured by biomarkers in synovial fluid. J Orthop Res, 2005, 23(3): 602-610.
2.	Madsen SH, Andreassen KV, Christensen ST, et al. Glucocorticoids exert context-dependent effects on cells of the joint in vitro. Steroids, 2011, 76(13): 1474-1482.
3.	Jaramillo MC, Briehl MM, Crapo JD, et al. Manganese porphyrin, MnTE-2-PyP (5+), acts as a pro-oxidant to potentiate glucocorticoid-induced apoptosis in lymphoma cells. Free Radic Biol Med, 2012. [Epub ahead of print].
4.	Chung YJ, Lee JI, Chong S, et al. Anti-proliferative effect and action mechanism of dexamethasone in human medullary thyroid cancer cell line. Endocr Res, 2011, 36(4): 149-157.
5.	Li WZ, Li WP, Huang DK, et al. Dexamethasone and Aβ25-35 accelerate learning and memory impairments due to elevate amyloid precursor protein expression and neuronal apoptosis in 12-month male rats. Behav Brain Res, 2012, 227(1): 142-149.
6.	Zuloaga DG, Carbone DL, Hiroi R, et al. Dexamethasone induces apoptosis in the developing rat amygdala in an age-, region-, and sex-specific manner. Neuroscience, 2011, 199: 535-547.
7.	Ortega-Ortega Y, García-Latorre EA, Lezama RA, et al. Heat shock protein 60 from Klebsiella pneumoniae protects mononuclear cells from apoptotic cell death induced by dexamethasone. Microb Pathog, 2011, 51(5): 352-359.
8.	Silvestrini G, Ballanti P, Patacchioli FR, et al. Evaluation of apoptosis and the glucocorticoid receptor in the cartilage growth plate and metaphyseal bone cells of rats after high-dose treatment with corticosterone. Bone, 2000, 26(1): 33-42.
9.	Hochberg MC, Altman RD, Brandt KD, et al. Guidelines for the medical management of osteoarthritis. Part II. Osteoarthritis of the knee. American College of Rheumatology Arthritis Rheum, 1995, 38(11): 1541-1546.
10.	Dragoo JL, Danial CM, Braun HJ, et al. The chondrotoxicity of single-dose corticosteroids. Knee Surg Sports Traumatol Arthrosc, 2011, [Epub ahead of print].
11.	Braun HJ, Wilcox-Fogel N, Kim HJ, et al. The effect of local anesthetic and corticosteroid combinations on chondrocyte viability. Knee Surg Sports Traumatol Arthrosc, 2011. [Epub ahead of print].
12.	O’Driscoll SW. The healing and regeneration of articular cartilage. J Bone Joint Surg (Am), 1998, 80(12): 1795-1812.
13.	Hossain MA, Park J, Choi SH, et al. Dexamethasone induces apoptosis in proliferative canine tendon cells and chondrocytes. Vet Comp Orthop Traumatol, 2008, 21(4): 337-342.
14.	Chrysis D, Ritzen EM, Sävendahl L. Growth retardation induced by dexamethasone is associated with increased apoptosis of the growth plate chondrocytes. J Endocrinol, 2003, 176(3): 331-337.
15.	Chrysis D, Zaman F, Chagin AS, et al. Dexamethasone induced apoptosis in proliferative chondrocytes through activation of caspases and suppression of the Akt-phosphatidyinositol 3’-kinase signaling pathway. Endocrinology, 2005, 146(3): 1391-1397.
16.	Jia J, Yao W, Guan M, et al. Glucocorticoid dose determines osteocyte cell fate. FASEB J, 2011, 25(10): 3366-3376.
17.	Lotz M, Hashimoto S, Kühn K. Mechanisms of chondrocyte apoptosis. Osteoarthritis Cartilage, 1999, 7(4): 389-391.
18.	Kim HA, Blanco FJ. Cell death and apoptosis in osteoarthritic cartilage. Curr Drug Targets, 2007, 8(2): 333-345.
19.	Lavrik IN. Regulation of death receptor-induced apoptosis induced via CD95/FAS and other death receptors. Mol Biol (Mosk), 2011, 45(1): 173-179.
20.	王玉, 孙黎光, 夏春辉. Caspase介导的Fas凋亡途径. 世界华人消化杂志, 2006, 14(36): 3439-3442.
21.	Naves MA, Pereira RM, Comodo AN, et al. Effect of dexamethasone on human osteoblasts in culture: involvement of β1 integrin and integrin-linked kinase. Cell Biol Int, 2011, 35(11): 1147-1151.
22.	肖娟, 曹谊林, 范华骅, 等. 体外培养软骨细胞凋亡与caspase-3表达的实验研究. 中国修复重建外科杂志, 2003, 17(5): 418-421.
23.	Wang HQ, Yu XD, Liu ZH, et al. Deregulated miR-155 promotes Fas-mediated apoptosis in human intervertebral disc degeneration by targeting FADD and caspase-3. J Pathol, 2011, 225(2): 232-242.
24.	von Rossum A, Krall R, Escalante NK, et al. Inflammatory cytokines determine the susceptibility of human CD8 T cells to Fas-mediated activation-induced cell death through modulation of FasL and c-FLIP (S) expression. J Biol Chem, 2011, 286(24): 21137-21144.

1. Céleste C, Ionescu M, Robin Poole A, et al. Repeated intraarticular injections of triamcinolone acetonide alter cartilage matrix metabolism measured by biomarkers in synovial fluid. J Orthop Res, 2005, 23(3): 602-610.
2. Madsen SH, Andreassen KV, Christensen ST, et al. Glucocorticoids exert context-dependent effects on cells of the joint in vitro. Steroids, 2011, 76(13): 1474-1482.
3. Jaramillo MC, Briehl MM, Crapo JD, et al. Manganese porphyrin, MnTE-2-PyP (5+), acts as a pro-oxidant to potentiate glucocorticoid-induced apoptosis in lymphoma cells. Free Radic Biol Med, 2012. [Epub ahead of print].
4. Chung YJ, Lee JI, Chong S, et al. Anti-proliferative effect and action mechanism of dexamethasone in human medullary thyroid cancer cell line. Endocr Res, 2011, 36(4): 149-157.
5. Li WZ, Li WP, Huang DK, et al. Dexamethasone and Aβ25-35 accelerate learning and memory impairments due to elevate amyloid precursor protein expression and neuronal apoptosis in 12-month male rats. Behav Brain Res, 2012, 227(1): 142-149.
6. Zuloaga DG, Carbone DL, Hiroi R, et al. Dexamethasone induces apoptosis in the developing rat amygdala in an age-, region-, and sex-specific manner. Neuroscience, 2011, 199: 535-547.
7. Ortega-Ortega Y, García-Latorre EA, Lezama RA, et al. Heat shock protein 60 from Klebsiella pneumoniae protects mononuclear cells from apoptotic cell death induced by dexamethasone. Microb Pathog, 2011, 51(5): 352-359.
8. Silvestrini G, Ballanti P, Patacchioli FR, et al. Evaluation of apoptosis and the glucocorticoid receptor in the cartilage growth plate and metaphyseal bone cells of rats after high-dose treatment with corticosterone. Bone, 2000, 26(1): 33-42.
9. Hochberg MC, Altman RD, Brandt KD, et al. Guidelines for the medical management of osteoarthritis. Part II. Osteoarthritis of the knee. American College of Rheumatology Arthritis Rheum, 1995, 38(11): 1541-1546.
10. Dragoo JL, Danial CM, Braun HJ, et al. The chondrotoxicity of single-dose corticosteroids. Knee Surg Sports Traumatol Arthrosc, 2011, [Epub ahead of print].
11. Braun HJ, Wilcox-Fogel N, Kim HJ, et al. The effect of local anesthetic and corticosteroid combinations on chondrocyte viability. Knee Surg Sports Traumatol Arthrosc, 2011. [Epub ahead of print].
12. O’Driscoll SW. The healing and regeneration of articular cartilage. J Bone Joint Surg (Am), 1998, 80(12): 1795-1812.
13. Hossain MA, Park J, Choi SH, et al. Dexamethasone induces apoptosis in proliferative canine tendon cells and chondrocytes. Vet Comp Orthop Traumatol, 2008, 21(4): 337-342.
14. Chrysis D, Ritzen EM, Sävendahl L. Growth retardation induced by dexamethasone is associated with increased apoptosis of the growth plate chondrocytes. J Endocrinol, 2003, 176(3): 331-337.
15. Chrysis D, Zaman F, Chagin AS, et al. Dexamethasone induced apoptosis in proliferative chondrocytes through activation of caspases and suppression of the Akt-phosphatidyinositol 3’-kinase signaling pathway. Endocrinology, 2005, 146(3): 1391-1397.
16. Jia J, Yao W, Guan M, et al. Glucocorticoid dose determines osteocyte cell fate. FASEB J, 2011, 25(10): 3366-3376.
17. Lotz M, Hashimoto S, Kühn K. Mechanisms of chondrocyte apoptosis. Osteoarthritis Cartilage, 1999, 7(4): 389-391.
18. Kim HA, Blanco FJ. Cell death and apoptosis in osteoarthritic cartilage. Curr Drug Targets, 2007, 8(2): 333-345.
19. Lavrik IN. Regulation of death receptor-induced apoptosis induced via CD95/FAS and other death receptors. Mol Biol (Mosk), 2011, 45(1): 173-179.
20. 王玉, 孙黎光, 夏春辉. Caspase介导的Fas凋亡途径. 世界华人消化杂志, 2006, 14(36): 3439-3442.
21. Naves MA, Pereira RM, Comodo AN, et al. Effect of dexamethasone on human osteoblasts in culture: involvement of β1 integrin and integrin-linked kinase. Cell Biol Int, 2011, 35(11): 1147-1151.
22. 肖娟, 曹谊林, 范华骅, 等. 体外培养软骨细胞凋亡与caspase-3表达的实验研究. 中国修复重建外科杂志, 2003, 17(5): 418-421.
23. Wang HQ, Yu XD, Liu ZH, et al. Deregulated miR-155 promotes Fas-mediated apoptosis in human intervertebral disc degeneration by targeting FADD and caspase-3. J Pathol, 2011, 225(2): 232-242.
24. von Rossum A, Krall R, Escalante NK, et al. Inflammatory cytokines determine the susceptibility of human CD8 T cells to Fas-mediated activation-induced cell death through modulation of FasL and c-FLIP (S) expression. J Biol Chem, 2011, 286(24): 21137-21144.

Chinese Journal of Reparative and Reconstructive Surgery

EFFECT OF DIFFERENT CONCENTRATIONS OF DEXAMETHASONE ON APOPTOSIS AND EXPRESSION OF FAS/FASL IN HUMAN OSTEOARTHRITIS CHONDROCYTES

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