摘要
目的:通过骨痹健方用于兔制动法骨关节炎造模进程的对照研究,评估其用于骨关节炎早期预防的疗效和安全性;观察对制动法兔骨关节炎模型造模过程预先、同步、延后应用骨痹健方后关节功能和关节软骨的相应变化及对基质金属蛋白酶-1的调节作用,评估其用于骨关节炎早期预防的最佳时机,为中医中药“治未病”“治欲病”理论用于预防骨关节炎提供实验依据。在本文中也一并对骨关节炎的中医预防策略展开探讨。
方法:1、选用健康新西兰大白兔40只,随机分为四组,以在造模进程中同步使用骨痹健方为一组,以同步使用西药伊索佳(化学名硫酸氨基葡萄糖)为药物对照组,并建立空白对照组和单纯造模对照组,以制动法开始兔骨关节炎造模进程,在30天后观察动物造模关节的临床表现、关节软骨情况和病理变化,并监测各组的血常规、肝肾功能和体重。2、选用健康新西兰大白兔30只,随机分为三组,以在造模进程中同步使用骨痹健方为一组,预先应用骨痹健方为一组,延后应用骨痹健方为一组,以制动法开始兔骨关节炎造模进程,在造模30天后观察动物造模关节的临床表现、关节软骨情况和病理变化,免疫组化方法观察软骨中基质金属蛋白酶-1的表达,并监测各组的血常规、肝肾功能和体重。
结果:1、骨痹健方组的临床表现评分明显优于单纯造模组(P<0.05),与硫酸氨基葡萄糖组没有显著性差异(P>0.05);骨痹健方组关节软骨大体情况和光镜下病理变化评价明显优于模型组,与硫酸氨基葡萄糖组没有明显差异;骨痹健方组Mankin's评分与模型组比较有非常显著性差异(P<0.01),与硫酸氨基葡萄糖组没有显著性差异(P>0.05);各组安全性指标没有明显差异(P>0.05)。空白对照组的临床表现评分、关节软骨大体情况和光镜下病理变化评价及Mankin's评分与其他三组比较均有显著性差异(P<0.05)。
2、预先应用骨痹健方组的临床表现指标中的局部疼痛刺激反应和关节肿胀指标明显优于同步应用骨痹健方组和延后应用骨痹健方组(P<0.05),步态改变和关节活动范围指标明显优于延后应用骨痹健方组,与同步应用骨痹健方组没有显著性差异(P>0.05);同步应用骨痹健方组的各项临床表现指标均明显优于延后应用骨痹健方组(P<0.05);预先应用骨痹健方组软骨大体情况、光镜下病理变化评价、Mankin's评分明显优于同步应用骨痹健方组,同步应用骨痹健方组软骨大体情况、光镜下病理变化评价、Mankin's评分明显优于延后应用骨痹健方组(P<0.05);预先应用骨痹健方组的基质金属蛋白酶-1表达率明显低于同步应用骨痹健方组,同步应用骨痹健方组的基质金属蛋白酶-1表达率明显低于延后应用骨痹健方组(P<0.05)。各组安全性指标没有明显差异(P>0.05)。
结论:1、骨痹健方能显著降低制动法兔骨关节炎动物模型造模进程导致的软骨病理损伤、改善临床表现和组织学评分,以在造模前预先用药组的疗效最为明显。2、骨痹健方能抑制兔骨关节软骨基质金属蛋白酶-1的活化,调节细胞因子,从而抑制了造模进程中软骨基质的降解,以在造模前预先用药组的调节作用最为明显。3、该方虽不能阻止实验性骨关节炎病理进程的发展,但却能延缓其发展过程,早期用药组的疗效优于较晚用药组,并且没有观察到用药过程中实验兔出现血液指标和肝肾功能的明显损害。
Objective:Through the comparison study on the therapy influences, which are brought by Gubijian Formula, in the process of establishing experimental rabbit model of osteoarthritis introduced by the joint immobilization method, its preventive effect of early treatment against osteoarthritis and its safety of medical treatment are evaluated. Through the observations made to changes of joint functions and joint cartilages,and adjustment functions to matrix metalloproteinases-1 , which are corresponding to pre-treated,synch-treated and deferred treatment experimental rabbit groups,during the process of establishing rabbit models of osteoarthritis induced by the joint immobilization method, the evaluation to the best period of the early preventive treatment against osteoarthritis using Gubijian Formula is performed. In the field of osteoarthritis treatment, the thesis provides the experimental basis to the theories of Chinese traditional medicine, which are“To take the preventive treatment is a doctor’s first priority”and“The doctor should pay great attention to people who are in their sub-health conditions”. In this article, the discussion regarding the preventive strategy against osteoarthritis,which originates from the chinese traditional medicine,is also developed.
Designs: 1. Forty healthy New Zealand white rabbits are selected and divided to four groups randomly. As soon as the process of establishing experimental rabbit model of osteoarthritis is started,one group is given the treatment of Gubijian Formula and another group is given the treatment of glucosamine sulfate. These two groups are the medicine -treated control groups. Also, the blank control group and the control group that is only for establishing the model of osteoarthritis are organized. The process of establishing the experimental rabbit model of osteoarthritis is started by adopting the joint immobilization method. After 30 days , the observation is made to the clinical performances of joints that are used to establish the model of osteoarthritis,main situations of joint cartilages and pathological changes under the light microscope,and the monitoring is performed to the blood routine examinations,hepatic and renal functions and weights of each control group. 2. Thirty healthy New Zealand white rabbits are selected and divided to three groups randomly. The synch-treated control group is treated with Gubijian Formula as soon as the process of establishing the model of osteoarthritis is started. The pre-treated control group was treated with Gubijian Formula a few days before the process is started. The last group with the deferred treatment will be treated with Gubijian Formula a few days after the process is started. The process of establishing the experimental rabbit model of osteoarthritis is started by adopting the joint immobilization method. After 30 days, the observation is made to the clinical performances of joints that are used to establish the model of osteoarthritis,main situations of joint cartilages and pathological changes under the light microscope,and the monitoring is performed to the blood routine examinations,hepatic and renal functions and weights of each control group. At the same time,with the immunohistochemical method, the observation is made to the expression of matrix metalloproteinase-1 in cartilages.
Results: 1. The grade of clinical performance gotten from the control group treated with Gubijian Formula is much better than the control group that is only for establishing the experimental model of osteoarthritis(P<0.05),and does not have significant difference with the group treated with the glucosamine sulfate(P>0.05). The evaluation of main situations of cartilages and pathological changes under the light microscope,which is gotten from the control group with the treatment of Gubijian Formula, is much better than the control group with the pure model establishment of osteoarthritis,and does not have the significant difference with the control group treated by the glucosamine sulfate. Mankin's Marks that are gotten from the control group with the treatment o f the Gubijian Formula and the control group with the pure model establishment of osteoarthritis have a significant difference(P<0.01),but Mankin’s Marks of two groups with the Gubijian Formula treatment and the glucosamine sulfate treatment do not have a significant difference(P>0.05). There is no significant difference in the safety indexes of four control groups (P>0.05). Comparing to the other three control groups, there are some significant differences in the blank control group in terms of the clinical performance , situations of cartilages and pathological changes under the light microscope and the Mankin's Mark(P<0.05). 2. The indexes of clinical performance including the indexes of local pain reaction and joint swelling, which is gotten from the pre-treated control group, are much better than the synch-treated and deferred treatment control groups(P<0.05). The indexes of the gait change and motion range of joints, which are gotten from the pre-treated control group, are much better than the deferred treatment group and do not have the significant difference with the synch-treated group(P>0.05).Every index of clinical performance gotten from synch-treated group is much better than the deferred treatment group(p<0.05). The main situations of cartilages,evaluation of pathological changes under the light microscope and Mankin’s Mark,which are gotten from the pre-treated control group,are much better than the synch-treated control group. The main situations of cartilages,evaluation of pathological changes under the light microscope and Mankin’s Mark,which are gotten from the synch-treated control group,are much better than the deferred treatment control group(P<0.05).The expression rate of Matrix Metalloproteinase-1 of the pre-treated control group is much lower than the synch-treated control group(P<0.05). The expression rate of Matrix Metalloproteinase-1 of the synch-treated control group is much lower than the deferred treatment control group(P<0.05).There is no significant difference in the safety indexes of three control groups (P>0.05).
Conclusions: 1. The Gubijian Formula can effectively reduce the pathological injury intensity caused by the process of establishing the experimental rabbit model of osteoarthritis introduced by the joint immobilization method,and improve the clinical performance and histological grade. The control group that was treated with The Gubijian Formula before the model establishment of osteoarthritis has the most obvious medical effect. 2. The Gubijian Formula can control the activation of Matrix Metalloproteinase-1 of rabbits’joint cartilages and adjust the cytokines. Therefore,it can inhibit the egradation of the cartilage matrixes during the process of establishing the model of osteoarthritis. The control group that was treated with The Gubijian Formula before the model establishment of osteoarthritis has the most obvious adjustment function. 3. Although the formula can not prevent the pathological development of the experimental osteoarthritis,it can slow down its progress. The control group that is treated early has better therapeutic effect than the control group that is treated late. No obvious adverse influence is found in the blood indexes and hepatic and renal functions of experimental rabbits.
引文
[1]刘芳,吕志刚.骨关节炎发病机制的分子生物学研究进展.成都体育学院学报,2004,30 (4): 77-82
[2] Clcutini FM,SpectorT D.Osteoarthritis in the aged.Epidemiological issues and optimal management.Drugs Aging,1995,6(5):409-420
[3]王梅,于长隆.老年骨性关节炎.中国临床康复,2002,6 (1): 2 5-27
[4]曾庆馀.加强骨关节炎的研究.中华内科杂志.1995;34(2):75-76
[5]国家中医药管理局.中医病证诊断疗效标准,第1版.南京:南京大学出版社,1999,33-34
[6]国家技术监督局.中华人民共和国标准-中医临床诊疗术语·证候部分,第1版.北京:中国标准出版社,2000,88
[7]王洪图.黄帝内经研究大成.北京:北京出版社,1997,1612
[8]刘健.健脾化湿通络法治疗历节病45例临床研究.安徽中医临床杂志,1999,11 (6) :380～381
[9]王慧敏.骨关节炎研究新进展.江西中医学院学报,1999;11(l):47
[10] Palotie A,Ott J, Elion K.Predisposition to familial osteoarthrosis linked to type collagen gene. Lanet,2003:924-927
[11] Fairbank J,Clinical importance of the intervertebral disc,or back pain for biochemists.Biochem-Soc-Trans.2002 Nov,30(Pt 6): 829-31
[12] Seiler G,Hani H,Staging of lumbar degeneration in nonchondrodystrophic dogs using low-field magnetic resonance imaging,Vet-Radiol-Ultrasound.2003 Mar-Apr, 44(2):179-84
[13] Roughley P,Alini M,Antoniou J,The role of proteoglycans in aging,degeneration and repair.Biochem-Soc-Trans.2002 Nov,30(Pt 6):869-74
[14]王晶,肖德明.性激素与骨关节炎.中华骨科杂志,2001,1(1):50～51
[15]吴昊,查振刚.维生素D受体和骨关节炎.中华风湿病学杂志,2003,7(8):494～497
[16] Spector TD,Hart DJ,Nandra D.Low level increases in serum C-reactive protein are present in early osteoarthritis of the knee and predict progressive disease.Arthritis Rheum,2002,40:723-727
[17] Zhang Y,Glynn RJ,Felson DT.Musculoskeletal disease research:should we analyze the joint or the person.J Rheumatol,1996,23:1130-1134
[18] Guccione AA,Felson DT,Anderson JJ,et al. The effects of specific medical conditions on functional limitations of elders in the Framingham Study. Am J Public Health,2004,84:351-358.
[19]王慧敏.骨关节炎研究新进展.江西中医学院学报,1999;11(l):47
[20]潘碧霞.氧自由基与疾病的基础知识.实用儿科杂志,1993,8(2)67
[21]邓长生,夏冰.炎症性肠病.北京:人民卫生出版社,1998:98-104
[22] Burrage PS , Mix KS , Brinckerhoff CE.Matrix metalloproteinases : role in arthritis.Front Biosci.2006 Jan 1;11:529-43
[23] Moldovan F,Pelletier JP, Modulation of collagenase 3 in human osteoarthritic cartilage by activation of extracellular transforming growth factor beta: role of furin convertase..Arthritis Rheum,2000V43N9:2100-2109
[24]石印玉.透明质酸与骨关节炎.中国中医骨伤科杂志,1999,7(4)56
[25] Sakakibara Y,Effect of high molecular weight sodium hyalu ronate on immobilized rabbit knee. Clin orthop,2004,299,282—292
[26]倪江东.退行性骨关节病患者膝关节液中一氧化氮含量的变化及其意义.湖南医科大学学报,1997,22(4):333
[27]蓝旭,刘雪梅,葛宝丰等.豚鼠原发性骨性关节炎生化研究.中国骨伤2001,14(4):212～213
[28]于顺禄,李德达,李世民等.骨性关节炎研究进展.中国骨伤,2002,15(10):635～637
[29]李晓声,王家让.膝关节骨性关节炎的诊断与治疗概况.医师进修杂志(外科版),2004,5:23～24
[30] Felson DT. Preventing knee and hip osteoarthritis.Bull Rheum Dis,1998,47:l-4
[31]松熊秀明.灸法对胶原诱发关节炎的预防作用[日]明治针灸医学.2002,30:9-19
[32]石印玉,徐荣善,陈友红.养血软坚方治疗膝骨关节炎的临床报告.中国中医骨伤科杂志,1994,2(4):33-36
[33]张伯臾.中医内科学.上海:上海科学技术出版社,1990
[34]凌一揆,颜正华.中药学.上海:上海科学技术出版社,1990,80-274,276
[35]刘健,韩明向.治疗历节病学术经验.安徽中医学院学报,1999,18 (5):45～47
[36]王建忠,曾一林.弃杖散治疗退行性膝关节病临床疗效评价.中医正骨,2O03,15(4):14
[37]汪元,沈鹰.中药熏蒸疗法治疗关节炎30例疗效观察及抗炎机理研究.中医正骨,2O05,17(10):13
[38]孙钢.膝关节骨性关节炎的中医治疗.中国骨伤,2002,15(4):2556
[39]王建中.郝建军.冲洗疗法治疗膝关节骨性关节炎.临床骨科杂志,2003,3(9):1
[40]史宝明,袁培义,郭钧等.膝关节骨性关节炎的冲洗疗法.中华骨科杂志,2000,20(4):1
[41]张金贵.针灸治疗膝关节骨性关节炎60例疗效观察.针灸临床杂志,2003,19(11):21-22
[42]李宁,吴滨,张永铃.艾灸配合运动疗法治疗膝关节骨性关节炎疗效观察.中国针灸,2002,22(11):729-730
[43] Osiri M,Welch V,Brosseau L.Transcutaneous electrical nerve stimulation for knee osteoarthritis.Cochrane Database Syst Rev.2000,(4):CD00282
[44] Ng MM,Leung MC,Pooh DM.The efects of electro-acupuncture and transcutaneous electrical nerve stimulation on patients with painful osteoarthritic knees : a randomized controlled trial with follow-up evaluation J Altem Complement Med.2003 Oct,9(5):641-9
[45]朱汉章.针刀医学在慢性软组织损伤疾病病因病理学方面的新理论.中国针灸. 1995,S2:47-51
[46]胡平安,张利萍.小针刀治疗膝关节骨性关节炎68例.中国中医药科技,2003,10(3):135
[47]张国祥,刘忠文.小针刀治疗膝关节骨性关节炎体会.右江民族医学院学报,1999,(5):839
[48]周殿阁,国谷良,闵志松等.膝关节骨性关节炎患者的肌力分析.中华骨科杂志,2001,21(3):189
[49]俞晓杰,吴毅.膝关节骨关节炎肌肉功能障碍康复治疗.国外医学·骨科学分册,2005,26(2):107
[50]王萍.膝关节骨性关节炎的康复治疗.中国康复医学杂志,2005,20(7):558
[51]查振刚,姚平,吴昊等.硫酸氨基葡萄糖预防兔膝骨关节炎的实验研究.中国新药杂志,2004,l3(9):793-796
[52] Salter RB, Simmonds DF,elal .The biological efect of continuous passive motion on the healing of full-thickness defects in articular cartilage. An experimental investigation in the rabbit. J Bone Joint Surg Am.1980 Dec;62(8):1232-51
[53] O Driscoll SW , Salter RB. The induction of neochondrogenesis in free intra-articular periosteal auto grafts under the influence of continuous passive motion. An experimental investigation in the rabbit. J Bone Joint Surg Am. 1984 Oct; 66(8):1248-57
[54] Kim HK, Kerr RG, el al. Efects of continuous passive motion and immobilization on synovitis and cartilage degradation in antigen induced arthritis. J Rheumatol. 1995 Sep;22(9):1714-21
[55]李舒帆,郭韵,吴春玲,王晓非.康复体操在慢性风湿性疾病关节炎中的作用.中国临床康复.沈阳:2003.3:816
[56] Arnstein P,Caudill M,Mandle CL,et al.Self efficacy as a mediator of the relationship between pain intensity,disability and depression in chronic pain patients.Pain 1999 Apr,80(3):483-91
[57]马克昌,高子范,张灵菊,等.骨碎补对大白鼠骨质疏松模型的影响.中医正骨,1992,4(4):3
[58]常德有,董福慧.骨碎补的研究概况.吉林中医药,2006,6:61-62
[59]任仁安.中药鉴定学.上海:上海科学技术出版社:1986.378
[60]凌一揆.中药学.上海:上海科学技术出版社,1984.171
[61]袁秀荣,常章富.怀牛膝、川牛膝本草考证.中国中药杂志, 2002,27(7):545.
[62]李秀珍.怀牛膝根中游离剂墩果酸及其皂甙的薄层比色测定.中药通报,1988,13(2):37-38
[63]马英,车镇涛,毕开顺等.反相高效液相色谱法测定怀牛膝中羟基促脱皮甾酮的含量.药学学报,2000,35(4):313
[64]方积年,张志花,刘柏年.牛膝多糖的化学研究.药学学报,1990,25(7):526
[65]孙水平,李新华,孙曙光.怀牛膝的药理研究续报.河南中医,1985,5(1):40
[66] Kuroki H, Nakagawa Y, Mori K,et al. Acoustic stiffness and change in plug cartilage over time after autologous osteochondral grafting: correlation between ultrasound signal intensity and histological score in a rabbit model.Arthritis Res Ther. 2004;6(6):492-504.
[67]金光亮.《内经》未病概念与“治未病”理论探讨.北京中医药大学学报. 2006.29(12):804
[68]黄涛,徐传毅,邹季,等.活血止痛汤治疗早期骨性关节炎的实验研究.中医正骨,1999:11(3):3.
[69]韦文武,王大伟.补气活血法治疗膝骨关节炎的实验研究.广西中医药,2004,27(5):46-48
[70]晏雪生,彭亚琴,明安萍.川芍嗦注射液对体外培养软骨影响的实验研究.中国中医骨伤科杂志.2002;10(l):15
[71]吕红斌,岳珍,王嘉芙.四种中药对体外培养的软骨细胞代谢的影响.中国运动医学杂志,1995,14(2): 135-137
[72]高文香,任汉阳.补肾法与活血法治疗兔膝骨关节炎的病理形态学对比研究.中医正骨,2000,6:5-8
[73]胡阿威,张磊,吕建芳,等.复方健骨关节汤治疗骨关节炎的实验研究.药物研究2007:15(2):42-44.
[74]郑福增,程少丹.骨疏康治疗绝经后妇女膝关节功能障碍的疗效观察.中国临床康复.2004:8(17):3322-3323.
[75]潘海东,曲波,王耶.参麦注射液对骨关节炎动物模型血液中白细胞介素-1水平的影响.中国中医骨伤科杂志,2000;8(1):17.
[76]汪青春,石印玉,沈培芝,等.中药对膝骨关节炎小鼠关节软骨IL-1、iNOS基因表达的影响.山西医药杂志,1999:28(5):381-383.
[77]邱贵兴,王桂生.兔膝关节制动引起关节软骨退变的实验研究.中华外科杂志,1987,25(3):175-177.
[78] Cloherty EK,Sultzman LA,Zottola RJ,et al.Net sugar transport is a multistep process.Evidence for cytosolic sugar binding sites in erythrocytes.Biochemistry,1995;34(47):15395～15406.
[79] Kolm-Litty VU. High glucose-induced transforming growth factor Production is mediated by the hexosamine pathway in porcine glomerular mesangial cells. J Clin Invest,1998,101:160.
[80] Sandy JD, Gamett D, Thompson V, et al.Chondrocyte-mediated catabolism of aggrecan: aggrecanase-dependent cleavage induced by interleukin-1 or retinoic acid can be inhibited by glucosamine. Biochem J,1998,355:59-66.
[81] Bassleer C, Rovati L, Franchimont P. Stimulation of proteoglycan production by glucosamine sulfate in chondrocytes isolated from human osteoarthritic articular cartilage in vitro. Osteo-arthritis Cartilage,1998,6:427-434.
[82] Shikhman AR. N-Acetylglucosamine Prevents IL-1β-Mediated Activation of Human Chondrocytes. The Journal of Immunology, 2001,166:5155-5160.
[83] Ma L, Immunosuppressive effects of glucosamine. J Biol Chem, 2002,277:39343-39349
[84] Roden L. Effect of hexosamines on the synthesis of chondroitin sulfuric acid in vitro.Arkiv Kemi,1956,10:345.
[85] Vida1 Y, Plana R, Bizzarri AL, et al. Artieular cartilage pharmaeology. I. In vitro studies on glucosamine and non-steroidal anti-inflammatory drugs. Pharmacol Res Commun,1978,10:557.
[86] Bassleer CY,Henrotin P. In vitro evaluation of drugs proposed as chondroprotective agents. Int J Tissue Reaet, 1992,14:231.
[87] Patwari PB, Kurz JD. Mannosamine inhibits aggrecanase mediated changes in the physical properties and biochemical composition of artieular cartilage. Arch Biochem Biophys, 2000,374:79.
[88] Setnikar I, Cereda MA, Pacini L. Antireactive properties of glucosamine sulfate. Arznei Forsch/Drug Res,1991,41:157.
[89] Setnikar I, Pacini L. Antiarthritic effects of glucosamine sulfate studied in animal models. ArzneimForsch/Drug Res, 1991,41:542.
[90] Bassleer C,Henrotin Y,Franchimont P.In vitro evaluation of drugs proposed as chondroprotective agents.Int J Tissue React,1992;14(5):231～241.
[91] Piperno M,Reboul P,Hellio Le,Graverand MP,et al.Glucosamine sulfate modulates dysregulated activities of human osteoarthritic chondrocytes in vitro.Osteoarthritis Cartilage,2000;8(3):207～212.
[92] Bramono DS,Richmond JC,Weitzel PP,et al.Clin Orthop,2004;428;272-285
[93] Visse R , Nagase H , Matrix metalloproteinases and tissue inhibitors of metalloproteinases:structure,function,and biochemistry.Circ Res,2003;92(8);827- 839
[94] Burrage PS, Mix KS, Brinckerhoff CE.Matrix metalloproteinases: role in arthritis.Front Biosci. 2006 Jan 1;11:529-43.
[95]江蓉星,汪亚强,王洪志.骨关节炎的中医药治疗.成都中医药大学学报,2001,24(4):11-13
[96]高文香,刘元禄.中医药治疗骨关节炎及其研究进展.辽宁中医杂志,2001,25(7):335-38
[97]张景贵,高的,萧文耀.骨性关节炎病理分级与软骨细胞在体外培养中代谢特征关系的研究.兰州医学院学报,2002,28(3):14-16