艾灸疗法影响兔膝骨性关节炎软骨细胞凋亡的效应

Effect of moxibustion therapy on articular chondrocyte apoptosis of osteoarthritis rabbits

目的:观察艾灸疗法对兔膝骨性关节炎软骨细胞凋亡的影响。方法:于2004-03/2005-04在华中科技大学同济医学院和武汉体育学院完成本课题。选取6～8月龄健康新西兰兔36只,按照组间均衡一致的原则,随机抽签法分为3组:正常对照组,骨关节炎模型组,艾灸治疗组,每组12只。骨关节炎模型组,艾灸治疗组动物于左侧或右侧兔膝关节腔内注入16g/L木瓜蛋白酶制备骨性关节炎模型,0.5mL/次,隔3天再注射1次,共注射3次。艾灸治疗组动物于术后6周开始进行艾灸治疗。剪除造模侧膝关节约3cm×3cm区域毛后用点燃的艾条,距后三里(相当于人足三里)、阴阳陵泉、血海四穴高2cm处各悬灸10min,2天1次,共治疗4周。正常对照组,骨关节炎模型组不予治疗。术后10周处死所有动物,切开膝关节腔,先大体观察关节软骨、滑膜的病变。然后切取胫骨平台和股骨内髁的软骨约0.5cm×0.5cm×0.5cm组织块,行苏木精-伊红、Masson三色和甲苯胺蓝染色,通过光镜观察有关的组织形态学变化。采用硝酸还原法测定关节滑液一氧化氮含量。采用原位未端原位标记法和流式细胞仪检测软骨细胞的凋亡率。结果:在模型制备及饲养过程中,所有动物均无死亡,全部纳入结果分析。①大体观察结果:正常组兔膝关节软骨为淡蓝白色,表面光滑而有光泽;造模组关节软骨无光泽,为淡黄白色,软骨表面凸凹不平,出现裂隙;艾灸组关节软骨为淡白色,软骨表面粗糙。②组织形态学结果:正常组苏木精-伊红染色可见关节软骨的细胞致密而均匀,基本无破坏;Masson三色染色正常,软骨胶原蓝绿色;甲苯胺蓝染色均匀无失染。骨关节炎模型组关节软骨破坏严重,软骨表层变薄,粗糙,出现裂隙,部分软骨剥落,形成缺损区,软骨细胞数目明显减少,排列紊乱,四层结构不易分辨;Masson三色染色蓝绿色丢失严重,仅表层软骨尚可保留一些;胺蓝染色仅残存的软骨细胞及簇集的细胞核蓝染,细胞的胞浆呈紫红色。艾灸组苏木精-伊红染色可见关节软骨破坏程度较造模组明显减轻,关节软骨表面有表浅裂隙,软骨细胞排列轻度紊乱,四层结构可以分辨。Masson三色染色全层未钙化软骨保持蓝绿色着染;甲苯胺蓝染色蓝紫色着染均匀。③骨关节炎模型组关节液一氧化氮含量较正常对照组明显升高、艾灸治疗组较正常对照组明显升高,比模型组降低,差异均有显著性意义(P<0.05)。④骨关节炎模型组、艾灸治疗组关节软骨细胞凋亡阳性表达较正常对照组明显升高;艾灸治疗组比骨关节炎模型组明显降低,差异均有显著性(P<0.05)。⑤骨关节炎模型组软骨细胞凋亡率比正常组明显升高,艾灸治疗组比正常对照组明显升高,比模型组明显降低,差异均有显著性(P<0.05)。结论:艾灸疗法能减少关节液一氧化氮的含量,抑制软骨细胞的凋亡,治疗膝骨性关节炎具有有效性和可行性。

AIM： To observe the effect of moxibustion therapy on the articular chondrocyte apoptosis of osteoarthritis （OA） rabbits. METHODS： The experiment was carried out in Tongji Medical College of Huazhong University of Science and Technology and Wuhan Institute of Physical Education from March 2004 to April 2005. A total of 36 adult New Zealand rabbits of 6-8 months old were randomly divided into three groups： normal group （A）, model group （B） and moxibustion group （C）, with 12 rabbits in each. OA was simulated by intra-articular injection of 16 g/L papain in rabbit knee of group B and C, 0.5 mL once, one time every three days and totally for 3 times. The animals of group C were treated with moxibustion from 6 weeks to 10 weeks post-operation when all the rabbits were executed. A piece of 3 cm × 3 cm were cut from lateral knee joint of model rabbits, and then lighted moxa stick was used to perform moxibustion in 2 cm distant from Housanli, Yinlingquan （SP 9）, Yanglingquan （GB 34） and Xueha/（SP 10） for 10 minutes, once every two days for total 4 weeks. Group A and B received no treatment. The lesions of articular cartilage and synovial membrane were observed generally after cutting off the knee articular cavity, and then a cartilage tissue of 0.5 cm × 0.5 cm × 0.5 cm were taken from tibia[ plateau and femoral condylus to conduct hematoxylin-eosin （HE） staining, Masson trichrome stain and toluidine blue （TB） staining. The histomorphological change of chondrocyte was observed by light microscopic; NO content in articular synovia was measured by nitrate reduction method; The chondrocyte apoptosis was detected by TUNEL and flow cytometric assay. RESULTS： All the rabbits were involved in the result analysis without deaths in modeling and feeding.①General observation： The knee cartilage of rabbits in group A was slight white-blue, smooth and sheen. In group B, cartilage became slight white-yellow, fissured, rough and uneven in surface. In group C, cartilage was slight white and rough in surface. ② Histomorphology result： The articular cartilage of group A was compact and well-distributed after HE staining, normal and bluish-green after Masson trichrome stain, and even after TB staining. The articular cartilage of group B was broken seriously, and the surface layer became thin and rough, containing many cracks. Part cartilages peeled off and formed defection and damage area, while the chondrocyte decreased number and arranged in mess, and four layer constructions were not distinguished easily. Only surface layer cartilage remained bluish-green after Masson trichrome stain;The residual chondrocyte and clustered cells presented blue-stain, whereas cellular plasm colored prunosus, The articular cartilage of group C was broken less seriously than that of group B, and articular cartilage surface contained shallow cracks, the chondrocyte arranged tidily, and four layer constructions were distinguished easily. The non-calcified cartilage maintained bluish-blue by Masson trichrome stain, and colored as amethyst by TB staining. ③NO content of group B and C were both apparently higher than that of group A. There was significant difference between group B and group C （P 〈 0.05）. ④ The positive expression of cbondrocyte apoptosis was obviously higher in group B and C than in group A, and also obviously higher in group B than in group C （P 〈 0.05）. ⑤The apoptosis rate was markedly higher in group B and C than in group A, but significantly lower in group C than in group B （P 〈 0.05）. CONCLUSION： Moxibustion therapy can significantly reduce NO content in synovial fluid, and decrease chondrocyte apoptosis of OA models, so it is valid and feasible to achieve therapeutic effect for knee OA.