Effect of Tougu Xiaotong capsule on articular cartilage changes in rat models of osteoarthritis

doi:10.3969/j.issn.2095-4344.2014.49.010

Abstract

Abstract:

BACKGROUND: Tougu Xiaotong capsule is the clinical prescription for the treatment of osteoarthritis, however, its mechanism has not been fully elucidated. Urokinase type plasminogen activator system which participated in the degradation of the extracellular matrix of articular cartilage and hyperplasia of joint synovium plays an important role in the pathological process of osteoarthritis.

OBJECTIVE: To determine the effect of Tougu Xiaotong capsule on urokinase-type plasminogen activator system in knee cartilage tissues of knee osteoarthritis rats.

METHODS: Of 144 Sprague-Dawley rats, 120 rats were randomly made into models of knee osteoarthritis via intra-articular injection of papain, and randomly assigned to model group, Zhuanggu Guanjie Wan group [1.2 g/(kg•d)], low-dose Tougu Xiaotong capsule group [0.092 g/(kg•d)], moderate-dose Tougu Xiaotong capsule group [0.184 g/(kg•d)] and high-dose Tougu Xiaotong capsule group [0.368 g/(kg•d)]. Each group contained 24 rats. Every 2 weeks was considered as a course, with a 2-day interval, totally 4 courses. The remaining 24 normal rats were included in the blank group. After every two courses, a batch of experimental animals was sacrificed. The pathological changes were observed following staining with hematoxylin and eosin. The positive cells of urokinase-type plasminogen activator, urokinase-type plasminogen activator receptor and plasminogen activator inhibitor were measured by immunohistochemistry. The protein levels of urokinase-type plasminogen activator, urokinase-type plasminogen activator receptor and plasminogen activator inhibitor were measured by western blot assay.

RESULTS AND CONCLUSION: Mankin’s score was significantly lower in the Tougu Xiaotong capsule group and Zhuanggu Guanjie Wan group compared with the model group (P < 0.01), in a time-dependent manner. Immunohistochemical staining indicated that the positive cells of urokinase-type plasminogen activator and urokinase-type plasminogen activator receptor were significantly decreased, but plasminogen activator inhibitor was significantly increased in the Tougu Xiaotong capsule group and Zhuanggu Guanjie Wan group in a time-dependent manner. Western blot assay results had an identical trend to immunohistochemistry. These indicated that Tougu Xiaotong capsule showed preventive and therapeutic effects on osteoarthritis by regulating urokinase-type plasminogen activator system.

中国组织工程研究杂志出版内容重点：肾移植；肝移植；移植；心脏移植；组织移植；皮肤移植；皮瓣移植；血管移植；器官移植；组织工程

全文链接：

Key words: tissue construction, osteoarthritis, cartilage

CLC Number:

R318

Wu Guang-wen, Ye Jin-xia, Zheng Chun-song, Chen Wen-lie, Liu Xian-xiang, Ye Hong-zhi . Effect of Tougu Xiaotong capsule on articular cartilage changes in rat models of osteoarthritis [J]. Chinese Journal of Tissue Engineering Research, 2014, 18(49): 7924-7929.

Figures/Tables 4

2.1 骨性关节炎大鼠造模成功结果苏木精-伊红染色观察发现空白组关节软骨细胞排列均匀，无增生，软骨面光滑。模型组关节软骨浅表层大部分缺失，移行层受损，软骨细胞排列不均，并大量簇聚，毛细血管侵入软骨下骨并部分突破潮线。模型组Mankin’s评分显著高于空白组。Mankin’s分数越高表明关节软骨损伤越重。结果说明骨性关节炎大鼠造模成功。 2.2 关节软骨Mankin’s评分苏木精-伊红染色发现经过透骨消痛胶囊和壮骨关节丸治疗后，软骨浅表层比较规则，细胞排列相对均匀。Mankin’s评分：模型组较空白组显著升高(P < 0.01)；透骨消痛胶囊组较模型组降低，中剂量组降低最为显著(P < 0.01)；壮骨关节丸组较模型组显著降低(P < 0.01)。随着治疗时间的延长，Mankin’s评分降低越明显，具有时间依赖性。透骨消痛胶囊中剂量组与壮骨关节丸组比较差异无显著性意义(P > 0.05，见表1。 2.3 免疫组织化学观察关节软骨uPA、uPAR、PAI表达软骨组织中uPA、uPAR及PAI 阳性染色呈棕黄色或棕褐色，主要位于软骨细胞质。空白组uPA、uPAR只有散在的阳性表达，PAI表达较多；模型组着色较深，uPA、uPAR较空白组显著升高，PAI表达降低；透骨消痛胶囊组uPA、uPAR表达降低，PAI表达升高，其中透骨消痛胶囊中剂量组降低或升高最明显，与模型组相比，差异有非常显著性意义；壮骨关节丸组uPA、uPAR表达较模型组显著降低，PAI表达较模型组显著增加。透骨消痛胶囊中剂量组与壮骨关节丸组相比，无明显差异(图1-3)。 2.4 Western blot检测关节软骨uPA、uPAR、PAI表达为进一步验证研究结果，实验运用Western blot进一步检测透骨消痛胶囊干预后uPA、uPAR、PAI蛋白的表达情况，结果发现uPA、uPAR、PAI表达趋势与免疫组织化学结果一致，模型组uPA、uPAR均较空白组显著升高，PAI表达降低。壮骨关节丸组uPA、uPAR表达较模型组低，PAI表达增加；透骨消痛胶囊组uPA、uPAR表达降低，PAI表达升高，具有时间依赖性。透骨消痛胶囊中剂量组与壮骨关节丸组相比，无明显差别。见图4。"

References

[1] 向川.软骨细胞凋亡与骨关节炎[J].国外医学:免疫学分册,2003, 26(2):110-111.
[2] Schwab W,Schulze-Tanzil G,Mobasheri A,et al. Interleukin- lbeta-in duced expression of the urokinase-type plasminogen activator receptor and its co-localization with MMPs in humanarticular chondrocytes. Histol Histopathol. 2004;19(1): 105-112.
[3] Shakibaei M,Schulze-Tanzil G,et al.Expression of the VEGF receptor- 3 in osteoarthritic chondrocytes: stimulation by interleukin-1 beta and association with beta 1-integrins. Histochem Cell Biol.2003;120(3): 235-241.
[4] Lavigne P,BenderdourM,Lajeunesse D,et al.Subchondral and trabecular bone metabolism regulation in canine experimented knee osteoarthritis. Osteoarthritis Cartilage. 2005;13(4):310-317.
[5] 何斌,史晨辉,王永明,等.关节软骨破坏与尿激酶型纤溶酶原激活物的影响[J].中国组织工程研究与临床康复,2007,11(6):15-18.
[6] 林木南,刘献祥.透骨消痛方治疗膝骨性关节炎30例[J].福建中医药,2005,36(4):5-16
[7] 吴追乐,李西海,吴广文,等.透骨消痛胶囊含药血清对软骨细胞线粒体凋亡通路的影响[J].中华中医药杂志,2011,26(2):343-346.
[8] 叶蕻芝,李西海,梁文娜,等.透骨消痛胶囊含药血清对兔软骨细胞增殖影响的研究[J].中国临床药理学与治疗学,2009,14(6): 624-628.
[9] 吴明霞,李西海,吴广文,等.电针抑制SD大鼠骨性关节炎COX2、iNOS、NO表达的实验研究[J].福建中医药大学学报, 2010,20(6): 26-29.
[10] 黄继汗,黄晓晖,陈志扬,等.药理试验中动物间和动物与人体间的等效剂量换算[M].中国临床药理学与治疗学,2004,9(9):1069.
[11] Mankin HJ, Dorfman H, Lippiello L, et al. Biochemical and metabolic abnormalities in articular cartilage from osteoarthritic human hips Ⅱ: Correlation of morphology with biochemical and metabolic data. J Bone Joint Surg(Am). 1971;53(3):523-537.
[12] Soslow RA,Dannenberg AJ,Rush D,et al. Cox-2 is expressed in human pulmonary, colonic, and mammary tumors. Cancer. 2000; 89(12): 2637-2645.
[13] 孟繁杰.uPA/uPAR/PAI系统在膝骨性关节炎中的应用研究进展[J].中国现代医药杂志,2010,12(7):122-124.
[14] Alfano D,Franco P,Vocca I, et al.The urokinase plasminogen activator and its receptor : role in cell growth and apoptosis. Thromb Haemost. 2005;93(2):205-211.
[15] Hilal G, Martel-Pelletier J, Pelletier JP, et al. Abnormal regulation of urokinase plasminogen activator by insulin-like growth factor I in human osteoarthritic subchondral osteoblasts. Arthritis Rheum. 1999;42 (10) : 2112-2122.
[16] Andersen OM, Petersen HH, Jacobsen C, et al. Analysis of a two-domain binding site for the urokinase-type plasminogen activator-plasminogen activator inhibitor-1 complex in low- density-lipoprotein-receptor-related protein. Biochem J.2001; 357( Pti):289-296.
[17] Petersen LC. Kinetics of reciprocal pro-urokinase/ plasminogen activation-stimulation by a template formed by the urokinase receptor bound to poly(D-lysine).Eur J Biochem. 1997;245(2):316-323.
[18] Cerinic MM, Generini S, Partsch G, et al. Synoviocytes from osteoarthritis and rheumatoid arthritis produce plasminogen activators and plasminogen activator inhibitor-1 and display uPA receptors on their surface. Life Sci. 1998;63(6):441-453.
[19] Pap G, Eberhardt R, Rocken C, et al. Exp ression of stromelysin and urokinase type plasminogen activator protein in resection specimens and biopsies at different stages of osteoarthritis of the knee. Pathol Res Pract. 2000;196(4): 219-226.
[20] Massicotte F,Lajeunesse D,BenderdourM,et al.Can altered production of interleukin-lbeta,interleukin-6,transforming growth factor- beta and prostaglandin E(2) by isolated human subchondral osteoblasts identify two subgroups of osteoarthritic patients. Osteoarthritis Cartilage. 2002;10: 491-500.