消瘀散新组方修复膝骨关节炎...软骨损伤及MMP-13表达_崔若琳.pdf

实验动物与比较医学 Laboratory Animal and Comparative MedicineFeb.2023,43(1)DOI:10.12300/j.issn.1674-5817.2022.099消瘀散新组方修复膝骨关节炎模型兔的软骨损伤及MMP-13表达崔若琳1,王 庆2,杨 玲3,冯文昌4,刘智伟1,滕蔚然1,马碧涛1,王乐杨1,秦丽萍1,金立伦1(1.上海交通大学医学院附属新华医院中医科,上海 200092;2.复旦大学附属华山医院中医科,上海 200040;3.同济大学附属上海市第四人民医院老年科,上海 200081;4.湖北省中西医结合医院科教部,武汉 430015)摘要 目的研究消瘀散新组方对膝骨关节炎（knee osteoarthritis，KOA）兔关节软骨的作用及机制。方法将42只6月龄普通级新西兰白兔随机分为正常组、模型组、消瘀散组、消瘀散新组方组，每组10只（另2只用于模型验证）。除正常组外，其他各组兔均按照改良Hulth法对右侧膝关节进行KOA造模。造模5周后，对消瘀散组、消瘀散新组方组动物分别给予相应的贴膏进行膝关节炎治疗，每天1次，每次持续10 h。连续给药治疗2周后，取4组兔膝关节软骨，行大体观察并采用Outerbridge分级法评价各组软骨损伤情况，采用HE染色法在光学显微镜下观察各组兔膝关节软骨、钙化层和软骨下骨病理改变，并采用Mankins法评价软骨退变程度，采用免疫组织化学法在光学显微镜下观察各组兔膝关节软骨组织中基质金属蛋白酶13（matrix metalloproteinase-13，MMP-13）的表达情况。结果关节软骨大体观察后Outerbridge评级比较发现，消瘀散组较模型组的高分级动物数减少（P0.05），消瘀散新组方组比消瘀散组的高分级动物数也减少（P0.05）。HE染色发现4组兔关节软骨的Mankins评分由高到低依次为模型组（10.821.76）、消瘀散组（6.191.23）、消瘀散新组方组（2.641.18）和对照组（0.280.17），各组间差异有统计学意义（P0.05）。免疫组织化学检测发现各组兔关节软骨组织中MMP-13表达阳性率由高到低依次为模型组（67.9013.94）%、消瘀散组（37.1019.16）%、消瘀散新组方组（13.603.10）%和对照组（3.202.39）%，各组间差异有统计学意义（P0.05）。结论消瘀散新组方可修复KOA兔关节软骨损伤，降低软骨组织中MMP-13的表达水平，这可能是其治疗骨关节炎的作用机制之一。关键词 消瘀散新组方；膝骨关节炎；关节软骨；基质金属蛋白酶-13中图分类号 Q95-33；R-332 文献标志码 A 文章编号 1674-5817（2023）01-0030-09Repair Effects of Xiaoyusan New Formula on Cartilage Injury and MMP-13 Expression in Knee Osteoarthritis Model RabbitsCUI Ruolin1,WANG Qing2,YANG Ling3,FENG Wenchang4 LIU Zhiwei1,TENG Weiran1,MA Bitao1,WANG Leyang1,QIN Liping1,JIN Lilun1(1.Xinhua Hospital Affiliated to Shanghai Jiao Tong University School of Medicine Traditional Chinese Medicine Department,Shanghai 200092,China;2.Huashan Hospital Affiliated to Fudan University Traditional Chinese Medicine Department,Shanghai 200040,China;3.Department of Geriatrics,Fourth Peoples Hospital of Shanghai,Tongji University,Shanghai 200081,China;4.Hubei Hospital of Integrated Traditional Chinese and Western Medicine Department of Education and Science,Wuhan 430015,China)Correspondence to:JIN Lilun(ORCID:0000-0001-9840-836X),E-mail:ABSTRACT ObjectiveTo study the effect of Xiaoyusan new formula on the articular cartilage of knee osteoarthritis(KOA)rabbits and its mechanism.MethodsA total of 42 New Zealand white rabbits aged 6 months were randomly divided into normal group,model group,ointment of Xiaoyusan group,and ointment of Xiaoyusan new formula group,with 10 rabbits in each group(the other 2 rabbits were used for 人类疾病动物模型：药学专题 Animal Model of Human Disease:Pharmacology基金项目 上海市科学技术委员会科研计划项目“消瘀散新组方治疗KOA的临床规范化研究”（19401932000）；上海市进一步加快中医药传承创新发展三年行动计划项目“北部区域中医骨关节病专病联盟建设”ZY（2021-2023）-03-02-25 第一作者 崔若琳（1992），女，硕士，主治医师，研究方向：膝骨关节炎的发病机制与中药干预。E-mail：通信作者 金立伦（1967），男，硕士，主任医师，研究方向：膝骨关节炎的发病机制与中药干预。E-mail：。ORCID：0000-0001-9840-836X30实验动物与比较医学 Laboratory Animal and Comparative MedicineFeb.2023,43(1)model validation).Except for the normal group,the right knee joints of all rabbits in the other groups were prepared as KOA models according to the modified Hulth method.After 5 weeks of molding,the rabbits in ointment of Xiaoyusan group,ointment of Xiaoyusan New Formula group were given corresponding ointments for knee arthritis treatment,once a day,each time for 10 hours.After 2-week continuous administration and treatment,the knee joint cartilage of the four groups of rabbits was taken and the cartilage damage of each group was evaluated by Outerbridge grading method.The pathological changes of the cartilage,calcified layer and subchondral bone of the knee joint of rabbits in each group were observed by HE staining method under the light microscope,and the degree of cartilage degeneration was evaluated by Mankins method.The expression of matrix metalloproteinase-13(MMP-13)in the cartilage of rabbit knee joint in each group was deteced by immunohistochemistry.Results After the general observation of articular cartilage,the Outerbridge grading showed that the number of high-grade animals in ointment of Xiaoyusan group was reduced compared with the model group(P0.05),and the number of high-grade animals in ointment of Xiaoyusan new formula group was also reduced(P0.05)compared with ointment of Xiaoyusan group.HE staining showed that Mankins scores of articular cartilage in the four groups ranked from high to low:model group(10.821.76),ointment of Xiaoyusan group(6.191.23),ointment of Xiaoyusan new formula group(2.641.18)and normal group(0.280.17).The difference among four groups was statistically significant(P0.05).Immunohistochemical detection showed that the positive rates of MMP-13 expression in rabbit articular cartilage tissues in each group were(67.9013.94)%of model group,(37.1019.16)%of ointment of Xiaoyusan group,(13.603.10)%of ointment of Xiaoyusan new formula group and(3.202.39)%of normal group,ranking from high to low,and the difference among four groups was statistically significant(P0.05).Conclusion Xiaoyusan new formula can repair articular cartilage degeneration in KOA rabbits and decrease the expression of MMP-13 in cartilage,which may be one of the mechanisms of the treatment.Key words Xiaoyusan new formula;Knee osteoarthrit