Effect of Dexamethasone on Cartilage Degeneration in Rats with Collagen-induced Arthritis

doi:10.12300/j.issn.1674-5817.2022.066

Abstract

Abstract:

Objective To establish a rat model of collagen-induced arthritis (CIA), a rat model of rheumatoid arthritis, and to study the protective effect of dexamethasone (DEX) on cartilage in CIA rats. Methods Thirty Wistar rats were randomly divided into three groups: normal, model, and DEX groups. The CIA model was established by intradermal injection of a white emulsion mixed with 0.1 mL bovine type II collagen and Freund's incomplete adjuvant in equal proportions into the distal part of the rat tail. The dates of the two injections were recorded as days 0 and 7, respectively. After the establishment of the CIA model, DEX (1 mg/kg) was intraperitoneally injected in the treatment group 3 times a week for 4 weeks. After the administration, the rats were euthanized, and the knee cartilages of 10 rats in each group were divided into three parts. The first part was fixed, decalcified, and stained with Alcian blue to observe the pathological changes in the cartilage layer. The second part was used for detection of mRNA expression of matrix metalloproteinase (MMP)-9 and MMP-13, and a disintegrin-like and metalloproteinase with thrombo-spondin motifs (ADAMTS)-4 and ADAMTS-5 by using real-time quantitative PCR analysis in the femoral head cartilage. The third part was used for detection of the expression levels of MMP-9, MMP-13, and ADAMTS-5 using immunohistochemistry in the cartilage layer of knee joint. Results Compared with the model group, the decrease of cartilage area, cartilage thickness, and cartilage cell numbers were significantly inhibited after DEX treatment (P<0.05). The cartilage surface was smooth and the invasion degree significantly improved, which indicated that cartilage destruction was inhibited. After DEX treatment, the mRNA levels of MMP-9, MMP-13, ADAMTS-4, and ADAMTS-5 in the cartilage of CIA rats were decreased (P<0.001), and the protein expression levels of MMP-9, MMP-13, and ADAMTS-5 were also significantly decreased (P<0.05). Conclusion DEX can inhibit the expression of various MMP in CIA rats and has a therapeutic effect on cartilage destruction.

Key words: Rheumatoid arthritis, Collagen induced arthritis, Dexamethasone, Cartilage, Wistar rats

CLC Number:

R-332

Bingxin XU, Kaijian FAN, Tingyu WANG, Huijin CHEN. Effect of Dexamethasone on Cartilage Degeneration in Rats with Collagen-induced Arthritis[J]. Laboratory Animal and Comparative Medicine, 2022, 42(5): 416-422.

Figures/Tables 4

Table 1 Primer sequences for real-time quantitative PCR

基因Gene	上游引物序列Forward primer	下游引物序列Reverse primer
MMP-9	5'-ATCCCTCTATGGACCTCCCAC-3'	5'-AACAAGACTTCTCCCCGCAG-3'
MMP-13	5'-ACCCAGCCCTATCCCTTGAT-3'	5'-TCTCGGGATGGATGCTCGTA-3'
ADAMTS-4	5'-CATCCTACGCCGGAAGAGTC-3'	5'-AAGCGAAGCGCTTGTTTCTG-3'
ADAMTS-5	5'-GCCCACCTAACGGCAAATCT-3'	5'-AGGACACCTGCGTATTTGGG-3'

Figure 1 Inhibitory effect of dexamethasone on degeneration of knee articular cartilage in collagen-induced arthritis ratsNote： A, Alcian blue staining of knee joints of the three groups of rats; the scale bar is 100 μm and magnification is ×100. B, (left to right) Charts of cartilage thickness, cartilage area, and number of cartilage cells in each group. DEX， dexamethasone treatment group. Data are expressed as mean±SEM with 10 rats per group. Compared with the normal group, ***P<0.001; Compared with the model group, #P<0.05, ###P<0.001.

Figure 2 Inhibitory effect of dexamethasone on matrix metalloproteinases transcription in the knee cartilage tissue of collagen-induced arthritis rats detected by real-time quantitative PCRNote： MMP-9 and MMP-13, matrix metalloproteinases-9 and -13; ADAMTS-4 and ADAMTS-5, a disintegrin-like and metalloproteinase with thrombospondin motifs-4 and -5. DEX， dexamethasone treatment group. Data are expressed as mean±SEM with 10 rats per group. Compared with the normal group, **P<0.01, ***P<0.001, ****P<0.000 1; Compared with the model group, ###P<0.001, ####P<0.000 1.

Figure 3 Inhibitory effect of dexamethasone on MMP-9, MMP-13 and ADAMTS-5 expression in the knee cartilage of collagen-induced arthritis rats detected by immunohistochemistryNote：A shows immunohistochemical results (DAB staining; the scale bar is 50 μm and magnification is ×400). Red arrows indicate matrix metalloproteinases-9 (MMP-9), matrix metalloproteinases-9 (MMP-13), and a disintegrin-like and metalloproteinase with thrombospondin motifs--5 (ADAMTS-5) positive chondrocytes, respectively. B shows semi-quantitative results of MMP-9, MMP-13, and ADAMTS-5. Data are expressed as mean±SEM with 10 rats per group. Compared with the model group, #P<0.05, ###P<0.001.

References 22

1	DEANE K D, DEMORUELLE M K, KELMENSON L B, et al. Genetic and environmental risk factors for rheumatoid arthritis[J]. Best Pract Res Clin Rheumatol, 2017, 31(1):3-18. DOI:10.1016/j.berh.2017.08.003 .
2	HARRE U, SCHETT G. Cellular and molecular pathways of structural damage in rheumatoid arthritis[J]. Semin Immunopathol, 2017, 39(4):355-363. DOI:10.1007/s00281-017-0634-0 .
3	SHI W J, TJOUMAKARIS F P, LENDNER M, et al. Biologic injections for osteoarthritis and articular cartilage damage: can we modify disease? [J]. Physician Sportsmed, 2017, 45(3):203-223. DOI:10.1080/00913847.2017.1357421 .
4	BISHNOI M, JAIN A, HURKAT P, et al. Chondroitin sulphate: a focus on osteoarthritis[J]. Glycoconj J, 2016, 33(5):693-705. DOI:10.1007/s10719-016-9665-3 .
5	LORSCHEIDER M, TSAPIS N, UR-REHMAN M, et al. Dexamethasone palmitate nanoparticles: an efficient treatment for rheumatoid arthritis[J]. J Control Release, 2019, 296:179-189. DOI:10.1016/j.jconrel.2019.01.015 .
6	FORMICA F A, BARRETO G, ZENOBI-WONG M. Cartilage-targeting dexamethasone prodrugs increase the efficacy of dexamethasone[J]. J Control Release, 2019, 295:118-129. DOI:10.1016/j.jconrel.2018.12.025 .
7	JENSEN T W, HANSEN M S, HØRSLEV-PETERSEN K, et al. Periarticular and generalised bone loss in patients with early rheumatoid arthritis: influence of alendronate and intra-articular glucocorticoid treatment. Post hoc analyses from the CIMESTRA trial[J]. Ann Rheum Dis, 2014, 73(6):1123-1129. DOI:10.1136/annrheumdis-2012-203171 .
8	许冰馨, 王琪珊, 李钦, 等. 地塞米松对CIA大鼠DRG中炎症因子的抑制和关节疼痛的改善作用[J]. 中国药理学通报, 2020, 36(12):1691-1696. DOI:10.3969/j.issn.1001-1978.2020.12.012 .
	XU B X, WANG Q S, LI Q, et al. Dexamethasone inhibits inflammatory factors in DRG and improves joint pain of CIA rats[J]. Chin Pharmacol Bull, 2020, 36(12):1691-1696. DOI:10.3969/j.issn.1001-1978.2020.12.012 .
9	许冰馨, 王琪珊, 范凯健, 等. 地塞米松改善胶原性关节炎大鼠免疫功能与其血药浓度的相关性研究[J]. 实用药物与临床, 2021, 24(3):198-203. DOI:10.14053/j.cnki.ppcr.202103002 .
	XU B X, WANG Q S, FAN K J, et al. Correlation between dexamethasone's improving immune function and blood concentration in rats with collagen-induced arthritis[J]. Pract Pharm Clin Remedies, 2021, 24(3):198-203. DOI:10.14053/j.cnki.ppcr.202103002 .
10	WU J, FAN K J, WANG Q S, et al. DMY protects the knee joints of rats with collagen-induced arthritis by inhibition of NF-1B signaling and osteoclastic bone resorption[J]. Food Funct, 2020, 11(7):6251-6264. DOI:10.1039/D0FO00396D .
11	FAN K J, WU J, WANG Q S, et al. Metformin inhibits inflammation and bone destruction in collagen- induced arthritis in rats[J]. Ann Transl Med, 2020, 8(23):1565. DOI:10.21037/atm-20-3042 .
12	曾光, 陈芳, 熊新贵, 等. 熊果酸对CIA大鼠关节炎症及骨质破坏的影响[J]. 湖南中医药大学学报, 2013, 33(7):3-7. DOI:10.3969/j.issn.1674-070X.2013.07.001.003.05 .
	ZENG G, CHEN F, XIONG X G, et al. Effects of ursolic acid on arthritis and bone destruction in collagen-induced CIA rats[J]. J Tradit Chin Med Univ Hunan, 2013, 33(7):3-7. DOI:10.3969/j.issn.1674-070X.2013.07.001.003.05 .
13	王琪珊, 范凯健, 许冰馨, 等. DLTH通过调节免疫功能和软骨降解从而抑制RA的作用[J]. 中国药理学通报, 2021, 37(5):624-630. DOI:10.3969/j.issn.1001-1978.2021.05.007
	WANG Q S, FAN K J, XU B X, et al. Study of DLTH inhibiting RA by regulating immune function and cartilage degradation[J]. Chin Pharmacol Bull, 2021, 37(5):624-630. DOI:10.3969/j.issn.1001-1978.2021.05.007
14	YANG C Y, CHANALARIS A, TROEBERG L. ADAMTS and ADAM metalloproteinases in osteoarthritis - looking beyond the 'usual suspects'[J]. Osteoarthr Cartil, 2017, 25(7):1000-1009. DOI:10.1016/j.joca.2017.02.791 .
15	MALEMUD C J. Matrix metalloproteinases (MMPs) in health and disease: an overview[J]. Front Biosci, 2006, 11:1696-1701. DOI:10.2741/1915 .
16	KOVÁCS B, VAJDA E, NAGY E E. Regulatory effects and interactions of the Wnt and OPG-RANKL-RANK signaling at the bone-cartilage interface in osteoarthritis[J]. Int J Mol Sci, 2019, 20(18):4653. DOI:10.3390/ijms20184653 .
17	BURRAGE P S, MIX K S, BRINCKERHOFF C E. Matrix metalloproteinases: role in arthritis[J]. Front Biosci, 2006, 11:529-543. DOI:10.2741/1817 .
18	ZHANG J F, WANG G L, ZHOU Z J, et al. Expression of matrix metalloproteinases, tissue inhibitors of metalloproteinases, and interleukins in vertebral cartilage endplate[J]. Orthop Surg, 2018, 10(4):306-311. DOI:10.1111/os.12409 .
19	冀全博, 张强, 王岩. 带有血小板凝血酶敏感蛋白结构域的解聚素与金属蛋白酶4、5在骨关节炎中作用的研究进展[J]. 中国修复重建外科杂志, 2013, 27(9): 1080-1084. DOI: 10.7507/1002-1892.20130236 .
	JI Q B, ZHANG Q, WANG Y. Research progress of a disintegrin and metalloproteinase with thrombospondin motif 4 and 5 in osteoarthritis[J]. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi, 2013, 27(9):1080-1084. DOI: 10.7507/1002-1892.20130236 .
20	MALFAIT A M, TORTORELLA M D. The elusive DMOAD: Aggrecanase inhibition from laboratory to clinic[J]. Clin Exp Rheumatol, 2019, 37 Suppl 120(5):130-134.
21	MIYOSHI M, LIU S. Collagen-induced arthritis models[J]. Methods Mol Biol, 2018, 1868:3-7. DOI:10.1007/978-1-4939-8802-0_1 .
22	WANG Q S, XU B X, FAN K J, et al. Inflammation suppression by dexamethasone via inhibition of CD147-mediated NF-κB pathway in collagen-induced arthritis rats[J]. Mol Cell Biochem, 2020, 473(1):63-76. DOI:10.1007/s11010-020-03808-5 .

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