中国中西医结合杂志2024，Vol.44Issue(11)：1346-1355,10.DOI:10.7661/j.cjim.20240412.123
敦煌芮草膏防治大鼠膝骨关节炎的作用机制
Mechanism of Dunhuang Ruicao Ointment in Preventing and Treating Knee Osteoarthritis Rats
摘要
Abstract
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-1β in 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 and good color.The cartilage structure of DRO group and DRO-M group was complete,the surface was smooth,the color was good,and there was basically no crack and close to the normal level.The Pelletier score of rat cartilage in each intervention group decreased(P＜0.01,P＜0.05).The pathological results of HE staining showed that the cartilage structure in DDE group and MGC group was irregular,the number of cells increased diffusely,and the morphologies changed,showing multiple tidal lines.In DRO group and DRO-M group,the cartilage structure was normal,the surface was smooth,the number of chondrocytes was abundant,and the morphology was normal.A small area of pannus was occasionally seen,the hyperplasia of the connective tissue covered the cartilage surface,and the tide line was relatively complete.In safranine fast green staining the cartilage thickness in DDE group and MGC group was thicker,and the matrix staining was deep.The thickness of cartilage in DRO group and DRO-M group was close to the level of the blank group,and the matrix staining was deep.In toluidine blue staining,DRO group and DRO-M group performed well,with deeper staining than the model group.The thickness of cartilage was similar to that of the blank group,and the tide line was complete.The staining of matrix in DDE group and MGC group was slightly deeper than that of the model group,and the thickness of cartilage was slightly thickened,showing multiple tidal lines.IOD values of toluidine blue and safranine fast green staining areas 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 in cartilage tissue decreased(P＜0.01,P＜0.05).The average fluorescence intensity of MMP13 attenuated(P＜0.01,P＜0.05).Conclusion DRO improved the symptoms of KOA rats,which might be achieved by inhibiting p38MAPK signaling pathway,and further down-regulating the protein expression levels of p38MAPK,p-p38MAPK,MMP13,andADAMTS5.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Key words
Dunhuang Ruicao Ointment/knee osteoarthritis/p38 activated protein kinase signaling pathway/mechanism引用本文复制引用
李亮亮,李爽,杜籼芹,高榕妮,陈启启,蔺兴遥..敦煌芮草膏防治大鼠膝骨关节炎的作用机制[J].中国中西医结合杂志,2024,44(11):1346-1355,10.基金项目
2023年度甘肃中医药大学成果转化培育项目(No.2023CGZH-6) （No.2023CGZH-6）
2021年度甘肃省人才发展专项资金项目(No.2021RCXM106) （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) （No.DHYX19-14）
