地塞米松对胶原诱导性关节炎大鼠模型软骨退化的改善作用

doi:10.12300/j.issn.1674-5817.2022.066

实验动物与比较医学 ›› 2022, Vol. 42 ›› Issue (5): 416-422.DOI: 10.12300/j.issn.1674-5817.2022.066

地塞米松对胶原诱导性关节炎大鼠模型软骨退化的改善作用

许冰馨¹()(), 范凯健¹^,², 王婷玉¹, 陈慧瑾¹()()

^1.上海交通大学医学院附属第九人民医院药剂科, 上海 200011
^2.上海市崇明区精神卫生中心药剂科, 上海 202150

收稿日期:2022-05-16 修回日期:2022-07-27 出版日期:2022-10-25 发布日期:2022-11-04
通讯作者: 陈慧瑾（1971—），女，学士，主管药师，研究方向：药理学、临床药学。E-mail： 69321687@qq.com。ORCID：0000-0002-8835-6620
作者简介:许冰馨（1989—），女，硕士，主管药师，研究方向：药理学。E-mail：15001850704@163.com。ORCID：0000-0002-7442-9630
基金资助:
上海市崇明区科委科技项目“黄芪甲苷抑制炎症分泌防治类风湿性关节炎伴抑郁症的作用及其机制”(CKY2020-15)

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

Bingxin XU¹()(), Kaijian FAN¹^,², Tingyu WANG¹, Huijin CHEN¹()()

^1.Department of Pharmacy, Shanghai Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200011, China
^2.Department of Pharmacy, Mental Health Center, Chongming District, Shanghai 202150, China

Received:2022-05-16 Revised:2022-07-27 Published:2022-10-25 Online:2022-11-04
Contact: CHEN Huijin (ORCID: 0000-0002-8835-6620), E-mail: 69321687@qq.com

摘要/Abstract

摘要：

目的建立胶原诱导性关节炎（collagen-induced arthritis，CIA）大鼠模型，即一种类风湿关节炎大鼠模型，并研究地塞米松对CIA大鼠软骨的保护作用。方法 30只Wistar大鼠随机均分为3组：正常组、模型组和地塞米松组。通过向大鼠尾根部皮内注射0.1 mL牛Ⅱ型胶原与弗氏不完全佐剂等比例混匀而成的白色乳剂建立CIA模型，一共注射2次，分别记为第0天和第7天；模型建立后，给药组腹腔注射地塞米松（1 mg/kg），每周3次，连续4周。给药结束后处死大鼠，每组10只大鼠的膝关节软骨分成3部分：一部分进行固定、脱钙、阿尔新蓝染色，观察软骨层病理变化；另一部分采用实时定量PCR方法检测其股骨头软骨中基质金属蛋白酶（matrix metalloproteinase，MMP）-9、MMP-13、解聚蛋白样金属蛋白酶4（a disintegrin-like and metallo-proteinase with thrombospondin motifs-4，ADAMTS-4）和ADAMTS-5的mRNA表达；最后一部分采用免疫组织化学法检测膝关节软骨层MMP-9、MMP-13和ADAMTS-5的表达水平。结果与模型组相比，经地塞米松治疗后CIA大鼠膝关节软骨面积、厚度和细胞数量下降程度明显被抑制（P＜0.05），软骨表面较光滑且侵袭程度明显改善，提示软骨破坏得到抑制。地塞米松治疗后CIA大鼠软骨中MMP-9、MMP-13、ADAMTS-4和ADAMTS-5 mRNA水平降低（P＜0.001），MMP-9、MMP-13和ADAMTS-5 蛋白表达水平也明显降低（P＜0.05）。结论地塞米松可以抑制CIA大鼠中多种基质金属蛋白酶的表达，对软骨破坏具有治疗作用。

关键词: 类风湿关节炎, 胶原诱导关节炎, 地塞米松, 软骨, Wistar大鼠

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

中图分类号:

R-332

许冰馨, 范凯健, 王婷玉, 陈慧瑾. 地塞米松对胶原诱导性关节炎大鼠模型软骨退化的改善作用[J]. 实验动物与比较医学, 2022, 42(5): 416-422.

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.

图/表 4

表1 实时定量PCR引物序列

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'

图1 地塞米松对胶原诱导性关节炎大鼠膝关节软骨退化的抑制作用注：A为三组大鼠膝关节的阿尔新蓝染色图，比例尺为100 μm，放大倍数为100倍。B图由左往右依次为各组软骨厚度、软骨面积、软骨细胞个数的统计学分析图。DEX即地塞米松治疗组。每组3只大鼠，数据表示为平均值±标准误。与正常组相比，***P＜0.001；与模型组相比，#P＜0.05，###P＜0.001。

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.

图2 实时定量PCR检测地塞米松对胶原诱导性关节炎大鼠膝关节软骨组织中基质蛋白酶mRNA转录的抑制作用注：MMP-9和MMP-13即基质金属蛋白酶9和基质金属蛋白酶13；ADAMTS-4和ADAMTS-5即解聚蛋白样金属蛋白酶4和解聚蛋白样金属蛋白酶5。DEX即地塞米松治疗组。每组4只大鼠，数据表示为平均值±标准误。与正常组相比，**P＜0.01，***P＜0.001，****P＜0.000 1；与模型组相比，###P＜0.001，####P＜0.000 1。

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.

图3 免疫组织化学法检测地塞米松对胶原诱导性关节炎大鼠膝关节软骨中MMP-9、MMP-13和ADAMTS-5蛋白表达的抑制作用注：A图是免疫组织化学的结果（DAB染色，比例尺为50 μm，放大倍数为400倍），红色箭头所指分别为染色的基质金属蛋白酶9（MMP-9）、基质金属蛋白酶13（MMP-13）和解聚蛋白样金属蛋白酶5（ADAMTS-5）阳性软骨细胞。B图是MMP-9、MMP-13和ADAMTS-5在模型组和地塞米松（DEX）组中表达情况的半定量分析；每组3只大鼠，数据表示为平均值±标准误。与模型组相比，#P＜0.05，###P＜0.001。

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.

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地塞米松对胶原诱导性关节炎大鼠模型软骨退化的改善作用

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

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