天然複合物和Indomethacin對關節炎治?效果探討

Combined Effects of Naturocetic Compounds and Indomethacin on the Therapy of Arthritis－in Vitro and in Vivo Studies

李芷殷(Chih-Yin Lee)

指導教授 ： 陳建和 博士 ； 梁有志 博士

臺北醫學大學/醫學院/醫學檢驗生物技術學研究所/碩士(2005年)

摘要

PART1. 骨關節炎（OA）是關節炎中最常發生的疾病，主要的成因是長期的使用和?紀的增長等，並造成關節軟骨的物?性磨損和引起蛋白分解酵素?解軟骨組織，使得軟骨中的膠原蛋白纖維和醣蛋白?解，Matrix metalloproteinases（MMPs）被認為在骨關節炎中是?解軟骨組織過程中重要物質。近?報告指出，在OA 中表現的MMPs mRNA 主要有MMP-1, MMP-3, MMP-9, 和MMP-13 。在本實驗目的，證明Hyaluronan (HA), chondroitin sulfate A（CA）和indomethacin 可以抑制在interleukin-1β (IL-1β ) ?激人?chondrosarcoma SW-1353細胞之下所產生的MMP-3。結果顯示，HA和 CA會隨著劑?的增加有效的抑制MMP-3產生；indomethacin可以有效抑制的MMP-3分?，但?抑制細胞內的表現。此外，NO是軟骨細胞分化和細胞凋亡的重要調節因子之一，並且可經由前發炎激素像是IL-1β所活化產生。結果也顯示，HA和 CA 會隨著劑?的增加，明顯有效的抑制iNOS表現和 NO產生；HA和 CA只抑制NO產生但沒有抑制iNOS表現。結?， HA和 CA可以有效的阻止OA的發展 ，這可再次證明，目前?床使用可以有效治?關節炎。Indomethacin?但減少MMP-3的分?，也藉由抑制NO產生並能夠?低發炎現象，這?有助於治?OA。因此實驗結果提供新的證據印證HA、CA和 indomethacin可以有效幫助OA治?。 PART2. 本篇實驗的目的，研究經由玻?酸（hyaluronan）和indomethacin共同作用治?抗原所引起的關節炎（antigen-induced arthritic ,AIA）兔子實驗動物模式。在玻?酸和indomethacin注射關節中治?的期間，觀察兔子關節的腫脹，血清中C-reactive protein levels (CRP), 和prostaglandin E2 的變化，並且觀察血清中matrix metalloproteinases-3（MMP-3）活性、巨觀和微觀觀察關節的變化。玻?酸和indomethacin會使CRP下?，低劑?的indomethacin結合玻?酸一起治?(Low I-H 5.6 μM indomethacin and hyaluronan)，比單獨玻?酸治??能?低局部的發炎反應。 對 prostaglandin E2 的抑制效果有隨著劑?的增高，在High I-H (High I-H560 μM indomethacin and hyaluronan) 的治?組最明顯。在Western blot analysis 中各組的 MMP-3 表現皆是持續增加的，而在血清中抑制MMP-3效果，在Low I-H的治?組最明顯。巨觀和病??片觀察中，單獨玻?酸、Low I-H 和High I-H 治?皆有相當的治?效果。 結?，此實驗證明在?床前用玻?酸結合 indomethacin 注射關節治?，比單獨服用indomethacin（或MMP inhibitors）治??風濕性關節炎提供?好的?床效果。

關鍵字

美洒辛 ； 玻尿酸 ； 軟骨硫素 ； 關節炎

並列摘要

PART1. Osteoarthritis (OA) is the most prevalent disease of articular joints and is the major cause of disability in the elderly. Pathophysiologic changes occur in OA cartilage due to the excessive expression of cartilage degrading proteinases, the resultant progressive breakdown of collagen fibers, and the degradation of proteoglycan, mainly aggrecan . Matrix metalloproteinases （MMPs）are considered to be important in the chondrolytic processes that contribute to the degenerative changes in OA cartilage. Recent studies have identified the mRNA for some MMPs, such as MMP-1, MMP-3, MMP-9, and MMP-13, in human OA cartilage. In this study, we investigated the inhibitory effects of hyaluronan (HA), chondroitin sulfate A（CA） and indomethacin on the interleukin-1β (IL- 1β)–stimulated expression of MMP-3 in human chondrosarcoma SW-1353. The results showed that HA and CA effectively inhibited MMP-3 expression.. Indomethacin decreased MMP-3 protein secretion, but not inhibited the intracelluar protein expression . Besides, NO is generally believed to be an important mediator of the dedifferentiation and apoptosis of articular chondrocytes by the action of proinflammatory cytokines, such as interleukin- 1β.The results showed that HA and CA significantly inhibited the iNOS expression and nitric oxide production by the dose-dependent mamer. Indomethacin only inhibited nitric oxide production, but not inhibited the iNOS expression. In conclusion, HA and CA could effectively inhibit OA, which supports the clinical use in the treatment. Indomethacin reduced MMP-3 secretion in the mediun and decreased the inflammatory response by inhibition of nitric oxide production were helpful to cure OA. Therefore, our study provides new data on the mechanism of action of these drugs, which could help to explain their clinical efficacy in OA patients. PART2. The combined effect of hyaluronan and indomethacin in the treatment of arthritis using antigen-induced arthritic (AIA) rabbits as a model was evaluated in this study. AIA was monitored by assessing joint swelling, C-reactive protein levels (CRP), and prostaglandin E2 in New Zealand white rabbits for 40 days during the course of special interval treatment by intra-articular injections of hyaluronan and indomethacin. The contralateral knee joint that was not treated served as an internal control. End-point analyses included a matrix metalloproteinases-3 activity assay, and macroscopic and microscopic joint examinations. Hyaluronan (H) and indomethacin (I)-treated rabbits showed a reduction in serum CRP levels with respect to hyaluronan-treated ones. The results of although there was no significant therapeutic effect, an intra-articular injection of low I-H (5.6 μM indomethacin and hyaluronan) was a little more efficient than hyaluronan alone in reducing local inflammation in the knee joint. Clinical assessments of the drug treatments (4 intra-articular injections in the right knee joint) on AIA in rabbits, there were no statistically significant differenc of the experiment, but the effect of indomethacin and hyaluronan on serum CRP, PGE2, and MMP-3 were statistically significant difference. Statistically significant inhibition of serum CRP was only observed in the low I-H group. Inhibition of serum PGE2 was dose-dependent in the low I-H group (13.3% ± 2.9%) and high I-H group (35.9% ± 3.3%). The order of serum MMP-3 inhibition was the low I-H group (-64.6% ± 12.0%) > high I-H group (-75.8% ± 5.0%) > hyaluronan group (-81.7% ± 26.7%). In Western blot analysis of MMP-3, there was a continual increase in serum pro-form MMP-3 protein in all treatment groups (immunization) from days 1 to 40. Macroscopic analysis and histological analysis of the joints, there were improved efficiency in the hyaluronan treatment group , in the low I-H group and high I-H group . Conclusion of this study provides preclinical support for the hypothesis that an intra-articular injection of hyaluronan in combination with indomethacin (and/or MMP inhibitors) might provide substantially greater clinical benefits to rheumatoid arthritis patients than indomethacin (and/or MMP inhibitors) alone at the systemic level than are achievable with current therapies.

並列關鍵字

Indomethacin ； Hyaluronan ； Chondroitin sulfate ； Arthritis

參考文獻

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