文献类型：期刊文献

中文题名：敦煌芮草膏防治大鼠膝骨关节炎的作用机制

英文题名：Mechanism of Dunhuang Ruicao Ointment in Preventing and Treating Knee Osteoarthritis Rats

作者：李亮亮[1];李爽[1];杜籼芹[1];高榕妮[1];陈启启[1];蔺兴遥[1,2]

第一作者：李亮亮

机构：[1]甘肃中医药大学基础医学院,兰州730000;[2]敦煌医学与转化教育部重点实验室,兰州730000

第一机构：甘肃中医药大学基础医学院（敦煌医学研究所）

年份：2024

卷号：44

期号：11

起止页码：1346

中文期刊名：中国中西医结合杂志

外文期刊名：Chinese Journal of Integrated Traditional and Western Medicine

收录：CSTPCD;;北大核心:【北大核心2023】；CSCD:【CSCD2023_2024】；PubMed;

基金：2023年度甘肃中医药大学成果转化培育项目(No.2023CGZH-6);2021年度甘肃省人才发展专项资金项目(No.2021RCXM106);敦煌医学与转化教育部重点实验室开放课题项目(No.DHYX19-14)。

语种：中文

中文关键词：敦煌芮草膏;膝骨关节炎;p38活化蛋白激酶信号通路;机制

外文关键词：Dunhuang Ruicao Ointment;knee osteoarthritis;p38 activated protein kinase signaling pathway;mechanism

摘要：目的 探讨敦煌芮草膏对大鼠膝骨关节炎(KOA)的治疗作用及其机制。方法 将72只SPF级SD大鼠,按随机数字表法分为6组,每组12只。除空白组外,剩余60只大鼠采用关节腔注射木瓜蛋白酶制造KOA模型,并且分为5组,即模型组、双氯芬酸乳膏组(DDE)、按摩组(MGC)、敷药组(DRO)、膏摩组(DRO-M)。除空白组外,各组给予相应药物干预3周。通过CT影像学测定膝关节CT值,测量膝关节肿胀度并进行Lequesne评分,肉眼观察膝关节软骨并进行Pelletier评分,番红固绿、甲苯胺蓝、HE染色观察软骨组织形态结构并进行Mankin评分,ELISA法检测血清中白细胞介素(IL)-1β、转化生长因子-β1(TGF-β1)水平,Western Blot法检测软骨组织中IL-6、p38丝裂原活化蛋白激酶(p38 MAPK)、磷酸化(p)-p38 MAPK、基质金属蛋白酶13(MMP13)、血小板反应蛋白解整合素金属肽酶5(ADAMTS5)蛋白表达。免疫荧光法检测软骨组织MMP13平均荧光强度。结果 与空白组比较,模型组大鼠膝关节肿胀度显著增加(P<0.01);Lequesne评分、膝关节CT值及软骨Pelletier评分均明显升高(P<0.01);膝关节面完整性遭到破坏,在胫骨和股骨内侧形成了骨赘,导致关节腔间隙变窄,软骨微结构出现恶化,骨小梁网结构混乱,软骨厚度减少。HE染色病理结果显示,软骨结构破坏,表面裂隙达辐射层,细胞数量明显减少,形态异常;番红固绿染色中,软骨厚度变薄,基质着色浅;甲苯胺蓝染色中,软骨厚度变薄,基质着色浅,潮线紊乱;大鼠软骨组织甲苯胺蓝、番红固绿染色区域IOD值均降低(P<0.01);血清中IL-1β表达水平升高,TGF-β1表达水平降低(P<0.01);软骨组织中IL-6、p38MAPK、p-p38MAPK、MMP13、ADAMTS5蛋白表达水平均升高(P<0.01);MMP13平均荧光强度增强(P<0.01)。与模型组比较,给药7天后,DRO-M组大鼠膝关节肿胀度及CT值均降低(P<0.05);DRO组及DRO-M组大鼠Lequesne评分降低(P<0.05)。给药14天后,DRO组及DRO-M组大鼠膝关节肿胀度及CT值均降低(P<0.05,P<0.01);各干预组大鼠Lequesne评分均降低(P<0.05,P<0.01),给药21天后,各干预组大鼠膝关节肿胀度、Lequesne评分及CT值均降低(P<0.01,P<0.05)。给药后各干预组关节骨赘生成物减少,关节间隙逐渐恢复正常,软骨结构趋于正常,骨小梁及软骨厚度亦逐渐恢复正常水平。其中DRO组及DRO-M组恢复水平最为显著,DDE组及MGC组恢复效果较差,软骨结构部分缺损,表面有裂纹,色泽较好;DRO组及DRO-M组软骨结构完整,表面较平滑、色泽好、基本无裂纹,接近正常水平。各干预组大鼠软骨Pelletier评分均降低(P<0.01,P<0.05)。HE染色病理结果显示,DDE组及MGC组软骨结构不规则,细胞数量弥漫性增多,形态改变,表现多重潮线;DRO组及DRO-M组软骨结构正常,表面光滑,软骨细胞数量丰富,形态正常,偶见小面积血管翳,结缔组织增生覆盖至软骨表面,潮线较完整。番红固绿染色中,DDE组及MGC组软骨厚度较厚,基质着色深;DRO组及DRO-M组软骨厚度接近空白组水平,基质着色深。甲苯胺蓝染色中,DRO组及DRO-M组表现良好,较模型组着色深,软骨厚度与空白组相似,潮线完整;DDE组及MGC组基质着色较模型组略深,软骨厚度轻微增厚,表现多重潮线。各干预组大鼠软骨组织甲苯胺蓝、番红固绿染色区IOD值均升高(P<0.01,P<0.05)。除MGC组外,各干预组大鼠血清中IL-1β水平均降低,TGF-β1表达水平均升高(P<0.01,P<0.05)。软骨组织中MMP13、p38MAPK、p-p38MAPK、IL-6、ADAMTS5蛋白表达水平均降低(P<0.01,P<0.05);MMP13平均荧光强度均减弱(P<0.01,P<0.05)。结论 敦煌芮草膏具有改善大鼠KOA症状的作用。这种作用机制可能是通过抑制p38MAPK信号通路,进而下调p38MAPK、p-p38MAPK、MMP13、ADAMTS5蛋白表达来实现的。
Objective To study the therapeutic effect and mechanism of Dunhuang Ruicao Ointment(DRO)on knee osteoarthritis(KOA)rats.Methods Totally 72 SPF grade SD rats were randomly divided into 6 groups by random digit table,12 rats in each group.Except those in the blank group,the remained 60 rats were injected with papain(KOA)intra-articularly and then divided into 5 groups,i.e.,model group,diclofenac cream group(DDE),massage group(MGC),DRO dressing group(DRO),and DRO massage group(DRO-M).Except the blank group,rats in the rest groups were administered with corresponding interventions for 3 successive weeks.The CT value of the knee joint was determined by CT imaging,the swelling degree of the knee joint was measured and Lequesne score was performed.The knee cartilage was observed by naked eyes and the Pelletier score was performed.The morphological structures of the cartilage tissue were observed by safranine fast green,toluidine blue and HE staining.The Mankin score was performed.The levels of interleukin-1β(IL-1β),transforming growth factor-β1(TGF-β1)in serum were detected by ELISA.The protein expressions of interleukin-6(IL-6),p38 mitogen activated protein kinase(p38 MAPK),phosphorylated(p)-p38 MAPK,matrix metalloproteinase 13(MMP13),platelet reactive protein disintegrin metallopeptidase 5(ADAMTS5)in cartilage tissue were detected by Western Blot.The average fluorescence intensity of MMP13 in cartilage tissue was detected by immunofluorescence method.Results Compared with the blank group,the swelling degree of knee joint in the model group significantly increased(P<0.01),Lequesne score/knee CT value/cartilage Pelletier score significantly increased(P<0.01).The integrity of the knee joint surface was destroyed,osteophytes were formed in the medial tibia and femur,resulting in the narrowness of the articular space,the deterioration of the cartilage microstructures,the disorder of the trabecular meshwork structure,and the reduction of the thickness of the cartilage.The pathological results of HE staining showed that the cartilage structure was destroyed,the surface fissure reached the radiation layer,the number of cells was significantly reduced,and the morphology was abnormal.In safranine fast green staining,the thickness of cartilage became thinner and the staining of matrix was light.In toluidine blue staining,the thickness of cartilage became thinner,the matrix staining was light,and the tide line was disordered.IOD values in toluidine blue and safranine fast green staining areas of rat cartilage tissue decreased(P<0.01),the expression level of IL-1βin serum increased,the expression level of TGF-β1 decreased(P<0.01),the protein expression levels of IL-6,p38MAPK,p-p38MAPK,MMP13,and ADAMTS5 in cartilage tissue increased(P<0.01),the average fluorescence intensity of MMP13 enhanced(P<0.01).Compared with the model group,the swelling degree and CT value of knee joint in DRO-M group decreased after 7 days of administration(P<0.05).The Lequesne score of rats in DRO group and DRO-M group decreased(P<0.05).After 14 days of administration the swelling degree and CT value of knee joint in DRO group and DRO-M group decreased(P<0.05,P<0.01).The Lequesne score of rats in each intervention group decreased(P<0.05,P<0.01).After 21 days of administration the swelling degree of knee joint,Lequesne score,and CT value of rats in each intervention group decreased(P<0.01,P<0.05).After administration the osteophytes in the joints of the intervention groups decreased,the joint space gradually returned to normal,the cartilage structure tended to be normal,and the trabecular bone and cartilage thickness gradually returned to normal levels.The recovery level of DRO group and DRO-M group was the most significant,and the recovery effect of DDE group and MGC group was the poorest,the cartilage structure were partially defective,with cracks on the surface andgood color. The cartilage structure of DRO group and DRO-M group was complete, the surface was smooth, thecolor was good, and there was basically no crack and close to the normal level. The Pelletier score of rat cartilagein each intervention group decreased (P<0.01, P<0.05). The pathological results of HE staining showed thatthe cartilage structure in DDE group and MGC group was irregular, the number of cells increased diffusely, andthe morphologies changed, showing multiple tidal lines. In DRO group and DRO-M group, the cartilage structurewas normal, the surface was smooth, the number of chondrocytes was abundant, and the morphology wasnormal. A small area of pannus was occasionally seen, the hyperplasia of the connective tissue covered thecartilage surface, and the tide line was relatively complete. In safranine fast green staining the cartilage thicknessin DDE group and MGC group was thicker, and the matrix staining was deep. The thickness of cartilage in DROgroup and DRO-M group was close to the level of the blank group, and the matrix staining was deep. In toluidineblue staining, DRO group and DRO-M group performed well, with deeper staining than the model group. Thethickness of cartilage was similar to that of the blank group, and the tide line was complete. The staining of matrixin DDE group and MGC group was slightly deeper than that of the model group, and the thickness of cartilagewas slightly thickened, showing multiple tidal lines. IOD values of toluidine blue and safranine fast green stainingareas in cartilage tissues of rats in each intervention group increased( P<0.01,P<0.05). Except the MGC group,the serum IL-1β level in each intervention group decreased, and the expression level of TGF- β1 increased(P<0.01,P<0.05). The protein expression levels of MMP13, p38MAPK, p-p38MAPK, IL-6, and ADAMTS5 incartilage tissue decreased( P<0.01,P<0.05). The average fluorescence intensity of MMP13 attenuated( P<0.01,P<0.05). Conclusions DRO improved the symptoms of KOA rats, which might be achieved by inhibitingp38MAPK signaling pathway, and further down-regulating the protein expression levels of p38MAPK, p-p38MAPK,MMP13, and ADAMTS5.