摘要
骨性关节炎(Osteoarthritis,OA)是一种严重危害病人生活质量的慢性、退行性关节疾病。其中,膝关节骨性关节炎(KOA)在人体各关节中发病率最高,关节软骨和周围软组织的病变是KOA的两个重要的病理基础。西医治疗目前缺少有效的方法,中医药对这些软组织的作用是肯定的,但目前尚缺乏有说服力的实验研究[1]。本实验采用中西医结合方法,应用光、透射电镜、生化法、免疫组化法等先进手段,从整体(KOA兔)、细胞(凋亡)、分子(SOD、MDA、NO)和基因水平(DNA)系统地研究骨痹消对KOA的治疗作用机理,加大科研力度,对疗效评定进行量化、客观化,使之具有科学性、可比性、可靠性,并阐明“骨痹消”对膝关节周围的软组织(如滑膜等)损伤的治疗作用机制。
目的:探讨骨痹消对实验性兔KOA的治疗作用。
方法:将60只日本大耳白兔随机分为5组(正常对照组、模型组、塞来昔布组、骨痹消组、壮骨关节丸组),每组12只。同等条件下正常饲养1周后,正常对照组12只正常饲养,作为空白对照,其余4组兔均采用伸膝制动OA动物模型法造模,制成兔KOA模型;给药方法:正常对照组和模型组:从造模第1天起经胃灌生理盐水2ml/Bid/d;塞来昔布组:从造模第1天起经胃灌药,2ml/Bid/d(55mg/d);骨痹消组:从造模第1天起经胃灌药,2ml /Bid/d(688mg/d);壮骨关节丸组:从造模第1天起,经胃灌药,2ml/Bid/d(1650mg/d)。
检测指标:
①用生化法,检测五组兔治疗后6周及10周各组血清超氧化物歧化酶(SOD)、丙二醛(MDA)及一氧化氮(NO)水平,并进行统计学分析;
②于治疗后6周及10周取材,进行肉眼大体观察后,分别取胫骨内髁关节软骨及膝前正中部滑膜,固定,脱水,包埋,切片,染色,用光镜、透射电镜对兔膝关节滑膜及软骨组织进行组织形态学观察;
③同时,取膝前正中部滑膜组织,匀浆,取上清液检测滑膜SOD、MDA水平,并进行统计学分析;
④6周及10周后随机取各组兔膝关节软骨,用TUNEL方法进行软骨细胞凋亡的原位检测,采用CIAS-1000型图像分析系统软件分析5组兔软骨细胞AI及Bcl-2与p53基因表达灰度值,探讨其作用机理。
结果:利用公认的伸膝制动OA动物模型法造模,模型成功。通过生化指标检测模型组及各治疗组在6周、10周时兔血清SOD、MDA、NO及滑膜SOD、MDA水平均发生变化。其中,模型组血清和滑膜SOD水平均较低,MDA水平均较高,血清NO水平升高,而各治疗组血清和滑膜SOD活性均有不同程度的增高,MDA和NO水平有不同程度的降低,且组间比较有统计学意义。
通过各组关节软骨的大致肉眼观察,见造模6周后模型组兔关节软骨失去原有光泽,发黄,可见少量糜烂点,滑膜组织增生;10周后软骨明显失去原有光泽,发黄,色泽变暗淡,软骨表面凹凸不平,周围滑膜明显增生。而各治疗组病变均不同程度好转,以骨痹消组最为明显。
通过电镜、光镜观察,模型组电镜下6周时可见细胞固缩,细胞略呈锥形,胞核高度固缩,周围出现空隙,细胞器消失,胞膜破损,基质中有深密度碎屑,10周时出现凋亡小体。光镜下模型组6周软骨细胞排列紊乱,簇集,细胞核固缩,细胞内成分减少,基质内胶原发生轻度纤维变性。10周时细胞簇集明显,核固缩,核溶,基质染色不均,淡染,胶原纤维明显,各组间关节软骨病理学变化评分比较有统计学意义;光镜下模型组6周时滑膜上皮增生,细胞间可见炎细胞浸润,滑膜下局部血管长入。10周时滑膜上皮重度增生,细胞肥大,滑膜下大量肉芽组织增生,大量血管长入,大量炎细胞浸润。骨痹消组、壮骨关节丸组、塞来昔布组在镜下观察,病变较模型组有不同程度的减轻,以骨痹消组最佳。
光镜下,细胞图像分析系统显示:模型组见到p53、Bcl-2的表达增加。骨痹消组、壮骨关节丸组、塞来昔布组Bcl-2的表达随用药时间的延长逐渐增强,而p53的表达逐渐减弱。6周及10周时骨痹消组p53的表达与模型组相比灰度值升高,组间比较具有非常显著性差异(P <0.01)。6周及10周时骨痹消组Bcl-2表达较模型组相比灰度值下降,两组间比较具有非常显著性差异(P <0.01)。
应用TUNEL法进行软骨凋亡细胞的原位检测:显微镜下见模型组软骨细胞的凋亡指数明显增高。治疗后,骨痹消组、壮骨关节丸组、塞来昔布组有不同程度下降,且骨痹消组下降最为明显,10周时,与模型组相比具有非常显著性差异(P<0.01)。
结论:骨痹消可清除机体多余自由基,纠正氧自由基代谢紊乱;促进软骨细胞代谢及软骨修复,维持软骨结构的相对完整;抑制滑膜炎性改变,减少滑膜炎性物质释放入关节腔,阻碍炎性滑膜对软骨的破坏;在一定程度上阻断诱导软骨细胞凋亡的途径,减少软骨细胞的凋亡数量,促进软骨的修复。
Osteoarthritis(OA)is a chronic, retrogressive article disease that seriously does harm to patient quality of life.The incidence of Knee Osteoarthritis(KOA)is the highest in all joints of humanbody, and the pathological changes of arthrodial cartilage and surrounding soft tissues are very important in KOA. At present, Western medicine is lack of effectively therapeutic method.Besides,the effects of the traditional Chinese medicine on soft tissue is firmly, but the persuasively experimental study is still blank. So, by optical microscopy, electron microscopy,biochemistry method, immunohistochemistry method, and other advanced means,the empirical study uses combinative methods of TCM and Western medicine, systematically and comprehensively explore therapeutic effects of gubixiao on KOA. From the whole (the KOA rabbit),cell (Apoptosis), molecule (SOD, MDA, NO) to gene level(DNA),The study enhances the scientific research capability,evaluates therapeutic effects quantificationally,which gives the study scientific nature, comparability,reliability, and at the same time it also illuminates the mechanism of therapeutic effects of“gubixiao”on impaired periarticular soft tissues of knee(for example:synovium).
Objective: To explore the therapeutical effects of gubixiao on KOA of the empirical rabbits.
Methods: 60 Rabbits were randomly divided into five groups:control group,model group,celecoxib group,gubixiao group,zhuangguguanjiewan group,Each group has twelve Rabbits. They are raised in the same environment,one week later,except control group Other four groups of rabbits are made animal model of KOA by extended fixation technique.
medication:From the first day of being fixed,all rabbits are fed the same food and different medicines respectively. control group and model group: normal saline 2ml/Bid. p.o.;celecoxib group, celecoxib liquor 2ml/Bid.p.o.(55mg/d);gubixiao group, gubixiaoliquor 2ml/Bid.p.o.(688mg/d); zhuangguguanjiewan group, zhuangguguanjiewan liquor 2ml/Bid.p.o.(1650mg/d ).
Examination indexes:
①Plasm SOD、MDA and NO of all groups of the rabbits were dividedly measured after the rabbits being treated for 6 weeks and 10 weeks by biochemistry method, and analyzed by spss 11.0 statistic software.
②After being treated for 6 weeks and 10 weeks,the samples are took from arthrodial cartilage of tibia tuberosity and center synovium of knee .At first the samples are observed by naked eye,then they are fixed,dehydrated,embeded,sliced, stained for electron microscopy and optical microscopy observation.
③At the same time,samples from center synovium of knee are homogenated .The clear supernatant liquid are taken to test SOD and MDA level, and then analyzed by spss 11.0 statistic software.
④After being treated for 6 weeks and 10 weeks arthrodial cartilage were taken randomly from five groups and ananlyzed apoptosis incidence (AI) by TUNEL. The expression rate of p53 and Bcl-2 of cartilage cell of all groups of rabbits wered analyzed by CIAS-1000 image analysis system software to explore the mechanism of action.
Results:KOA animal model were made the by extended fixation technique,which is received worldwide. Models are proved to be successed. Biochemical index demonstrates that Plasm SOD,MDA,NO and synovium SOD and MDA of the rabbits change afer being treated for 6 weeks and 10 weeks. Plasm SOD level and synovium SOD level in Model group are lower than that of other groups,but plasm MDA、NO level and synovium MDA level are completely the opposite. Post-treatment the Plasm and synovium SOD activity increase,MDA and NO level decrease differently in degree,and group comparison has statistical significance.
Macroscopic observation indicates that after being treated for 6 weeks,the model rabbits ,articular cartilage lose the original gloss,turn yellow,have few anabrosis dots,and the synovium tissues proliferate.After being treated for 10 weeks,the articular cartilages obviously lose the original gloss,turn yellow and gloom,the cartilage surfaces are uneven,periphery synovium obviously proliferate.But pathological changes of all treating groups are better than that of model group,among which the gubixiao group is the best.
Electron microscopy and optical microscopy observation indicates that after being treated for 6 weeks ,cartilage cell of model rabbits pyknosis,cell is similar to cone,nucleus is highly pyknosised ,cellule is seen surround nucleus,cell organs disappear,cell membrane damaged,and patches are found in matrix under electron microscope.Apoptotic bodies are seen in the samples of model group after being treated for 10 weeks.Under optical microscope,cartilage cell disorder,cluster, swell,karyopyknosis and even dissolved,cellular content decrease,the collagen fibers of matrix are lightly fibronolysis after being treated for 6 weeks,while cartilage cell cluster obviously,karyopyknosis,nucelus dissolve,matrix stain unevenly or lightly,the collagen fibers are obvious after being treated for 6 weeks .Comparison of terms of the scores with cartilage pathological assessment have statistical significance. Under optical microscopy,the changes of synovium of model rabbits are obvious:epitheliums proliferate, phlegmasia cells infiltrate,vasculars invade below synovium after being treated for 6 weeks. After being treated for 10 weeks,the synovium epitheliums proliferate heavily,cells hypertrophy,large quantities of granulational tissues hyperplasia below synovium,a great deal of vasculars invade,a lot of phlegmasia cells infiltrate.Pathological changes of treating groups are lessener in different degree compared with model group,but gubixiao group is significantly lightener compared with that of celexibu group and zhuangguguanjiewan group.
Expression of p53and Bcl-2: Under optical microscope,the cell image analytical system demonstrates that the expression of p53and Bcl-2 of cartilage cell of model group increased; Expression of Bcl-2 increases gradually in gubixiao、celexibu and zhuangguguanjiewan group following treating,while expression of p53 decreases.After being treated for 6 weeks and 10 weeks,the expression of p53 of gubixiao group descend compared with that of model group(P <0.01). While after being treated for 6 weeks and 10 weeks,the expression of Bcl-2 of gubixiao Group significantly increases. Group comparison between gubixiao Group and model group has statistical significance (P <0.01).
TUNEL for apoptosis: Under optical microscope,The AI of the model group is significantly higher than that of other groups.Post-treatment the AI of the treating groups is lower in different degree.Gubixiao group is the lowest. Compared with model group the AI of gubixiao group is obviously lower (P <0.01) after being treated for 10 weeks.
Conclusions:Gubixiao can clear surplus of free radicle, retrieve metabolic disorder of oxyradical;Gubixiao can facilitate metabolism of cartilage cell and help cartilage to recover which can relatively keep the integrity of cartilage structure; Gubixiao can inhibit phlegmasia changing of synovium, reduce phlegmasia liquid delivering into joint cavity,reduce destruction of phlegmasia synovium to cartilage;To some extent, gubixiao can block up path of inducing cartilage cell apoptosis, decrease apoptosis quantity of cartilage cells and help cartilage to recover.
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