Yamada H, Watanabe K, Saito T, Hayashi H, Niitani Y, Kikuchi T, Ito A, Fujikawa K, Lohmander L S
Department of Orthopaedic Surgery, National Defense Medical College, Saitama, Japan.
J Rheumatol. 1999 Mar;26(3):654-62.
To investigate the in vitro effects of 6,7-dihydroxycoumarin (esculetin) on the production of matrix metalloproteinases (MMP) in rabbit articular cartilage, and the in vivo effects of orally administered CPA-926, a prodrug of esculetin, on cartilage destruction in rabbit experimental osteoarthritis (OA).
In vitro studies were performed using rabbit articular cartilage explants. Esculetin 10-100 microM was added to cartilage explants in the presence or absence of interleukin 1alpha (IL-1alpha). Effects of esculetin on cartilage metabolism were assessed. Proteoglycan release into medium was determined by dye precipitation with 1,9-dimethylmethylene blue, synthesis of proMMP-1 (interstitial procollagenase) and proMMP-3 (prostromelysin 1) by Western blotting, and collagen degradation activity using FITC labeled collagen. In vivo experimental OA was induced in the knee joints of 15 Japanese adult white rabbits by partial lateral meniscectomy. Ten rabbits were orally administered 200 or 400 mg/kg/day of CPA-926 from the day of surgery for 14 days. The size of the macroscopic erosive area on the femoral condyle and tibial plateau was measured, and cartilage destruction was histologically evaluated. Collagenolytic activities in synovial fluid were measured using FITC labeled collagen as a substrate.
In vitro, esculetin inhibited the IL-1alpha induced release of proteoglycan into the medium in a dose dependent manner. The collagenolytic activities in cartilage explant medium induced by IL-1alpha were also suppressed with the addition of 33-100 microM esculetin (p = 0.0209 at 33 and 100 microM, p = 0.0202 at 66 microM). Western blotting of cartilage explant medium showed a decrease in the levels of proMMP-1 and proMMP-3 in the medium by treatment with esculetin. In vivo: At 14 days after surgery, the femoral condyle and tibial plateau in the control group showed macroscopic erosions of cartilage. Compared with the control group, the rabbits treated with CPA-926 at the dose of 400 mg/kg exhibited reduction of the size of the erosive area on the tibial plateau (p = 0.009). Histological evaluation indicated protection against the development of destructive changes in the tibial plateau cartilage at a dose of 200 mg/kg (p = 0.0442) and 400 mg/kg (p = 0.0446) of CPA-926.
These results indicate that esculetin inhibits matrix degradation in rabbit joint cartilage explants through the suppression of MMP synthesis, secretion, or activity. Prophylactic administration of its prodrug, CPA-926, appears to provide some protection against cartilage destruction in a short term rabbit experimental OA model.
研究6,7 - 二羟基香豆素（七叶亭）对兔关节软骨基质金属蛋白酶（MMP）产生的体外影响，以及七叶亭前药CPA - 926口服给药对兔实验性骨关节炎（OA）软骨破坏的体内影响。
使用兔关节软骨外植体进行体外研究。在存在或不存在白细胞介素1α（IL - 1α）的情况下，将10 - 100微摩尔的七叶亭添加到软骨外植体中。评估七叶亭对软骨代谢的影响。通过用1,9 - 二甲基亚甲基蓝进行染料沉淀来测定蛋白聚糖释放到培养基中的情况，通过蛋白质印迹法测定前MMP - 1（间质前胶原酶）和前MMP - 3（前基质溶解素1）的合成，并使用异硫氰酸荧光素标记的胶原测定胶原降解活性。通过部分外侧半月板切除术在15只日本成年白兔的膝关节中诱导体内实验性OA。从手术当天起，10只兔子口服200或400毫克/千克/天的CPA - 926，持续14天。测量股骨髁和胫骨平台上宏观侵蚀区域的大小，并对软骨破坏进行组织学评估。使用异硫氰酸荧光素标记的胶原作为底物测量滑液中的胶原溶解活性。
在体外，七叶亭以剂量依赖性方式抑制IL - 1α诱导的蛋白聚糖释放到培养基中。添加33 - 100微摩尔的七叶亭也抑制了IL - 1α诱导的软骨外植体培养基中的胶原溶解活性（33和100微摩尔时p = 0.0209，66微摩尔时p = 0.0202）。软骨外植体培养基的蛋白质印迹显示，用七叶亭处理后培养基中前MMP - 1和前MMP - 3的水平降低。在体内：手术后14天，对照组的股骨髁和胫骨平台出现软骨宏观侵蚀。与对照组相比，以400毫克/千克剂量用CPA - 926治疗的兔子胫骨平台上的侵蚀区域大小减小（p = 0.009）。组织学评估表明，在200毫克/千克（p = 0.0442）和400毫克/千克（p = 0.0446）的CPA - 926剂量下，可防止胫骨平台软骨发生破坏性变化。
这些结果表明，七叶亭通过抑制MMP的合成、分泌或活性来抑制兔关节软骨外植体中的基质降解。其前药CPA - 926的预防性给药似乎在短期兔实验性OA模型中为防止软骨破坏提供了一些保护。
