丹紫康膝冲剂延缓膝骨关节炎模型大鼠软骨退变及软骨下骨异常骨重建的作用机制研究  

Mechanism of Danzi Kangxi Granules on delaying cartilage degeneration and remodeling abnormal subchondral bone in Knee Osteoarthritis rats

作　　者：何花[1] 董大立[2] HE Hua;DONG Dali(The Second Affiliated Hospital of Hunan University of Chinese Medicine,Changsha,Hunan 410005;不详)

机构地区：[1]湖南中医药大学第二附属医院护理部,湖南长沙410005 [2]湖南中医药大学第二附属医院骨伤科,湖南长沙410005

出　　处：《河北中医》2021年第8期1337-1345,共9页Hebei Journal of Traditional Chinese Medicine

基　　金：2018年湖南省教育厅科学研究项目(编号:18C0396);2020年度湖南省中医药科研计划项目(编号:2020077)。

摘　　要：目的探究丹紫康膝冲剂延缓膝骨关节炎(KOA)模型大鼠软骨退变及软骨下骨异常骨重建的可能作用机制。方法将40只SD雄性大鼠随机分为空白组、模型组、双氯芬酸钠组、丹紫康膝冲剂组,每组10只。空白组不做任何处理,其余3组采用Hulth法建立KOA模型。建模成功后,双氯芬酸钠组予5 mg/kg双氯芬酸钠灌胃,丹紫康膝冲剂组予1.67 g/kg丹紫康膝冲剂灌胃,空白组、模型组均予等容积0.9%氯化钠注射液灌胃,每日灌胃1次,连续28 d。采用苏木精-伊红(HE)染色和番红-O-固绿组织染色制作病理切片观察软骨病理损伤情况,并记录病理学退变指标;末端转移酶介导的DIG-dUPT切口末端标记法(TUNEL)观察软骨细胞凋亡情况;酶联免疫吸附法检测外周血白细胞介素1β(IL-1β)、IL-18、肿瘤坏死因子α(TNF-α)、Ⅱ型胶原C端肽(CTXⅡ)、Ⅱ型胶原C前肽(CPⅡ)水平;蛋白免疫印迹法(Western blot)检测B淋巴细胞瘤-2基因(Bcl-2)、Bcl-2相关X蛋白(Bax)、基质金属蛋白酶3(MMP-3)、MMP-13、骨形态发生蛋白7(BMP-7)蛋白表达;取大鼠右侧胫骨近端关节离体标本进行Micro-CT扫描,测量软骨下骨微结构参数骨体积(BV);骨体积分数:骨体积/总体积(BV/TV)、骨小梁平均厚度(Tb.Th)、骨小梁数量(Tb.N)、体积骨密度(vBMD)。结果经HE染色,空白组大鼠软骨滑膜组织完整,细胞排列整齐,无炎症细胞浸润和基质损伤或蜕变。模型组大鼠软骨组织表面凸凹不平,软骨表层变薄,细胞排列不规则,此外可见大量炎症细胞、中性粒细胞、淋巴细胞浸润,而且深部软骨细胞可见明显细胞核压缩,膝关节宽度和关节退变深度显著增加。与模型组比较,丹紫康膝冲剂组大鼠软骨细胞排列均匀,胞核和胞质逐渐恢复正常。经番红-O-固绿染色,空白组大鼠软骨组织染色均匀,结构清晰。模型组大鼠骨关节面不平,软骨钙化,胶原纤维增生,软骨细胞外基质经番红O染色变浅,�Objective To discuss the effect of Danzi Kangxi Granules on delaying cartilage degeneration and remodeling abnormal subchondral bone in Knee Osteoarthritis(KOA)rats.Methods Forty SD rats were randomly grouped,10 rats in each.KOA model was established by Hull method except for Blank group.The gastric irrigation were performed on the four groups,which Blank group and Model group(0.9%sodium chloride injection),Diclofenac Sodium group(5 mg/kg),Danzi Kangxi Granules group(1.67 mg/kg)after successful,once daily for 28 days.Pathological sections were perfromed to observe the pathological damage of cartilage by hematoxylin-eosin(HE)staining and safranin-O-fast green tissue staining andpathological degenerationindicators were recorded.Terminal transferase-mediated DIG-dUPT cut end labeling Method(TUNEL)to observe the apoptosis of chondrocytes;ELISA.Enzyme-linked immunosorbent assay(ELISA)was to detect serum interleukin(IL)-1β,IL-18,tumor necrosis factorα(TNF-α),collagen typeⅡCtelopeptide(CTXⅡ),typeⅡpro-collagen C-propeptide(CPⅡ)levels.The apoptosis of chondrocytes was observed by TUNEL fluorescence staining.Western blot detection of B lymphoma-2 gene(Bcl-2),Bcl-2 related X protein(Bax),matrix metalloproteinase 3(MMP-3),MMP-13,bone morphogenetic protein 7(BMP-7)protein expression;Take the isolated specimen of the proximal joint of the right tibia of the rat for Micro-CT scan to measure the subchondral bone microstructure parameter bone volume(BV);bone volume fraction:bone volume/total volume(BV/TV),average trabecular bone thickness(Tb.Th),number of trabecular bone(Tb.N),volume bone density(vBMD).Results After HE staining,the cartilage and synovial tissue of the blank group was intact,the cells were arranged neatly,and there was no inflammatory cell infiltration and matrix damage or degeneration.In the model group,the cartilage tissue surface is uneven,the cartilage surface layer is thinned,and the cell arrangement is irregular.In addition,a large number of inflammatory cells,neutrophils,and lymphocytes can be se

关 键 词：紫河车 丹参 骨关节炎 膝 大鼠 Sprague-Dawley 模型 动物 软骨 中药疗法 

分 类 号：R684.305.31[医药卫生—骨科学] R285.5[医药卫生—外科学]