褪黑素对大鼠骨性关节炎关节软骨中BMP-2及IL-1β表达的影响

EFFECTS OF MELATONIN ON EXPRESSION OF BONE MORPHOGENETIC PROTEIN 2 AND INTERLEUKIN 1β IN ARTICULAR CARTILAGE OF RAT WITH OSTEOARTHRITIS

目的褪黑素可提高软骨生长因子的表达,刺激软骨基质的合成;通过观察注射褪黑素后大鼠骨性关节炎(osteoarthritis,OA)关节软骨中BMP-2和IL-1β的表达,探讨褪黑素对损伤软骨的防治作用。方法 SPF级4周龄SD雄性大鼠40只,体重120～150g,随机分为4组,每组10只。正常对照组(A组)大鼠不作任何处理。OA模型组(B组)、OA模型/去松果体模型组(C组)、OA模型/去松果体模型/褪黑素治疗组(D组)于大鼠左膝关节腔内注射0.2mL浓度为4%的木瓜蛋白酶溶液,每2天1次,共2周,制备大鼠OA模型。木瓜蛋白酶注射2周后,将C、D组大鼠置于持续24h(光照度为500lx)光照环境中,制备去松果体模型。D组于制备去松果体模型第2天开始于左膝关节腔内注射0.2mL浓度为20mg/mL的褪黑素溶液,每周4次,共4周。在最后1次注射褪黑素1周后,抽取A、B、C组大鼠心尖处血液应用ELISA法检测血清褪黑素水平;之后处死各组动物,取材行大体观察、组织学及免疫组织化学染色观察。结果模型制备后大鼠均存活。A、B、C组相同时间点血清中褪黑素水平比较以及同组各时间点比较,差异均有统计学意义(P<0.05)。A组关节软骨表面光滑平整,有弹性;软骨细胞排列整齐。B、C组关节软骨表面粗糙,局部区域软骨面可见缺损及软骨下骨外露现象;软骨细胞排列及结构较紊乱。D组关节软骨表面较B、C组平整,软骨缺损及外露现象减少;软骨细胞排列及结构较整齐。各组组织学及BMP-2、IL-1β免疫组织化学染色观察Mankin评分及积分吸光度值比较,差异均有统计学意义(P<0.05)。结论持续光照能加速大鼠OA关节软骨的退变。采用0.2mL浓度为20mg/mL的褪黑素溶液于关节腔注射4周后能够延缓大鼠OA的进一步退变,其作用机制可能与上调软骨生长因子BMP-2及下调炎性因子IL-1β表达有关。

Objective Melatonin （MLT） can increase the expression of cartilage-derived growth factor and stimulate the synthesis of cartilage matrix. To investigate the prevention and treatment effects of MLT on damaged cartilage through observing the expressions of bone morphogenetic protein 2 （BMP-2） and interleukin 1β （IL-1β） in articular cartilage of the rats with osteoarthritis （OA）. Methods Forty SPF 4-week-old male SD rats （weighing 120-150 g） were randomly divided into 4 groups （n=10）： normal control group （group A）, OA group （group B）, OA/pinealectomy group （group C）, and OA/ pinealectomy/MLT group （group D）. The rats of group A served as a control without treatment. The rats of groups B, C, and D underwent left knee joint injection of 0.2 mL 4% papain solution 1 time every other day for 2 weeks for establishing OA model. Two weeks after papain injection, the rats of groups C and D were exposed to continuous light for 24 hours （intensity of illumination： 500 lx） for creating pinealectomy models. And at the next day after pinealectomy model establishing, the rats of group D were treated with intra-articular injections of 0.2 mL 20 mg/mL MLT solution 4 times a week for 4 weeks. At 1 week after last MLT injection, the venous blood samples were taken in groups A, B, and C to test the level of serum MLT by ELISA, respectively, and then the specimens of left cartilage of femoral condyle were harvested for macroscopic, histological, and immunohistochemical examinations in 4 groups. Results The OA and pinealectomy models of rats were successfully established, and all rats survived. There were significant differences in the serum MLT level among groups A, B, and C, and among different time points at the same group （P〈0.05）. In group A, articular cartilage surface was smooth and elastic, and chondrocytes arranged regularly. In groups B and C, articular cartilage surface was rough, cartilage defects and subchondral bone exposure were observed in some areas, and chondrocytes arranged irregularly. In group D, cartilage surface was more smooth than that in groups B and C, and the degrees of cartilage defect and subchondral bone exposure decreased with regular arrangment of chondrocytes. There were significant differences in Mankin scores and integral absorbance values among 4 groups （P〈0.05）. Conclusion Exposure to continuous light can accelerate degeneration process of articular cartilage of OA rats. Injections of 0.2 mL MLT solution （20 mg/mL） by intra-articular for 4 weeks can inhibit the progress of cartilage defects. Upregulation of anabolic factor of BMP-2 as well as down-regulation of catabolic factors of IL-1β is associated with cartilage repair in the pathological features of OA.