褪黑素与复方倍他米松治疗大鼠实验性膝骨性关节炎的研究

背景：骨性关节炎（OA）是以关节软骨被侵蚀为特征的关节慢性疾病，由于缺乏血管和神经分布，因此，关节软骨组织的自我修复能力较差。 目的：评估联合应用褪黑素和复方倍他米松对雄性大鼠骨关节炎关节软骨的的影响，从而探讨褪黑素和复方倍他米松的联合应用在骨性节关炎发病机制中的作用。 方法：雄性SPF级SD大鼠40只，通过在大鼠膝关节腔注射4% papain（木瓜蛋白酶）溶液0.2ml制作骨性关节炎模型和采用持续光照的方法建立大鼠去松果体模型。实验随机分为4组：Ⅰ组：正常对照组(n=10)；Ⅱ组：骨性关节炎组（n=10）；Ⅲ组：Ⅱ组 持续24h光照组（n=10）；Ⅳ组：Ⅲ组 给予褪黑素和复方倍他米松治疗（n=10）。成功建立模型1周后，Ⅳ组左膝关节腔注射浓度为20mg/ml褪黑素溶液0.2ml，每周4次，复方倍他米松0.1ml，每周1次，共4周。后应用ELISA测取大鼠2Am和2Pm的血清褪黑素含量，并处死全部老鼠，同时切取股骨髁进行大体观察，之后脱钙制片，行HE和甲苯胺蓝染色观察 (均采用Mankin法进行评分)。 结果：（1）大体观察：Ⅰ组软骨表面光滑，有弹性且边缘规整；Ⅱ组软骨表面凹凸不平，失去原有的光泽，存在软骨软化现象及软骨下骨外露，Ⅲ组较Ⅱ组严重；Ⅳ组软骨软化现象消失及软骨剥脱，软骨下骨外露减少。（2）HE染色观察：Ⅰ组软骨细胞大小一致，排列规整；Ⅱ组软骨细胞排列紊乱，局部可见簇聚现象，Ⅲ组较Ⅱ组严重；Ⅳ组软骨细胞排列较规则，炎性细胞侵润明显较少。（3）甲苯胺蓝染色观察：Ⅰ组：甲苯胺蓝染色均匀, 软骨基质呈淡紫色，细胞核呈深蓝色，关节软骨结构层次清晰，潮线完整，无失染现象；Ⅱ组：软骨细胞排列及层次结构较紊乱，有明显失染现象，部分区域软骨细胞大小不一，可见大量簇聚现象；Ⅲ组：观察结果与Ⅱ组相似，但簇聚现象及基质失染现象较Ⅱ组明显；Ⅳ组：与Ⅱ、Ⅲ组比较，软骨细胞排列及结构较整齐，失染现象减轻，软骨弥散性细胞明显减少。同时，4组HE和甲苯胺蓝染色的Mankin评分比较差异均有统计学意义(P < 0.05)。 结论：20mg/ml褪黑素溶液0.2ml联合7mg/ml复方倍他米松溶液0.1ml关节腔注射治疗4周后能够抑制软骨病变发展。 关键词：骨性关节炎；褪黑素；复方倍他米松；关节软骨； 中图分类号：R684.3 文献标识码：A

The role of melatonin and compound betamethasone in the rat osteoarthritis model

BACKGROUND: Due to lack of the distribution of vessels and nerve, self-repairing capability of articular cartilage tissue is poor after inflammatory erosion. OBJECTIVE: To evaluate the effects of melatonin combined with compound betamethasone on the articular cartilage of osteoarthritis (OA) in rats. METHODS: Thirty Sprague-Dawley rats received intra-articular injection of papain solution for establishing knee OA models. Meanwhile, 20 of them underwent constant intensive light condition for establishing pinealectomy models. Ten rats that under pinealectomy were administered melatonin combined with compound betamethasone. Another 10 normal control rats receiving no treatment served as controls. After 4 weeks of treatment, serum melatonin concentrations at 2 a.m. (highest melatonin concentration within circadian rhythms) and 2 p.m. (lowest melatonin concentration within circadian rhythms) were detected by ELISA. At the same time, all rats were sacrificed to collect femoral condyle cartilage for gross observation. After decalcification and toluidine blue staining, articular cartilage lesions were evaluated based on Mankin scores. RESULTS AND CONCLUSION: After OA model was created, cartilage surface was uneven, lost their luster, the chondrocytes were poorly arranged, severe loss of staining was observed, serum level of melatonin was decreased, and circadian change was unobvious. Constant intensive light condition further aggravated cartilage damage. After treatment by melatonin combined with compound betamethasone, softened cartilage disappeared, there were more regular chondrocytes arrangement, and dispersed chondrocytes and loss of staining were gradually decreased. In addition, there was significant difference in Mankin scores of toludine blue staining among groups (P < 0.05). These findings indicate that melatonin combined with compound betamethasone can restrain the progression of cartilage damage.