A Preliminary Study On The Expression Of MMPs And MAPKs Signaling Pathway Kinases In Cartilage Of Osteoarthritic Rabbits

BackgroundOsteoarthritis(OA) is chronic and degenerative disease of joint and is one of the most common diseases.According to statistics,the incidence of OA is the second in the people above 50 years old,only lower than that of heart disease.And the incidence is higher in the people suffering from limbs deformity or joint trauma.Previous researches in the field of OA has characterized that OA is the result of disequilibrium between cartilage degeneration and synthesis controlled by the mechanic and biologic factors.The effect of inflammation mediators,such as interleukin-1(IL-1),is necessary in the cartilage degeneration although cytokines is not enough.It has been certificated that mitogen-activated protein kinases(MAPKs)and nuclear factor-κB(NF-κB) signaling pathways participate in the process IL-1β-induced MMP-1 express and chondrocytes apoptosis.The primary goal of the present study was to establish rabbit OA model and to detect the mRNA and protein expressions of MMP-1/13 and MAPKs.The second goal was to evaluate the effect of ERK1/2 signaling pathway through block it with PD98059.On the base of above results,we discussed the phase difference and relationships between MMPs and MAPKs.PartⅠEstablishment of rabbit osteoarthritis model and expression of MMP-1/-13 in osteoarthritic joint cartilage.Objectives To evaluate the feasibility of animal model of osteoarthritis(OA) by anterior cruciate ligament transaction(ACLT) and excision of partial medial meniscus,and to explore the mRNA and protein expression of matrix metalloproteinase(MMP)-1 and MMP-13 in joint cartilage of rabbit knee at different time after ACLT and partial excision of medial meniscus.Material and methods Thirty rabbits in experimental groups received unilateral ACLT and partial excision of medial meniscus,another 10 rabbits received unilateral arthrotomy as pseudo-operation control.Ten experimental rabbits were sacrificed at 4,8 and 12 weeks after surgery respectively,and 10 pseudo-operation rabbits were sacrificed at 12 weeks after surgery.Knees were harvested to observe the macropathologic changes of joint cartilage.Cartilage near the lesion were obtained,and mRNA and protein ofMMP-1 and MMP-13 were detected by real-time PCR and western blot respectively.Results Cartilage degeneration was obvious at 4 weeks and became more severe at 8 and 12 weeks after operation,while cartilage still remained normal at 12weeks after pseudo-operation.The mRNA and protein expression of MMP-1 and MMP-13 were all lower in normal cartilage,while the mRNA and protein expression of those two proteins increased in 4 and 8 weeks after operation.At 12 weeks after operation,mRNA and protein expression ofMMP-1 still remained high,while mRNA and protein expression of MMP-13 decreased respectively.Conclusions Anterior cruciate ligament transaction(ACLT) with partial excision of medial meniscus is a good method to make osteoarthritis model.MMP-1 and MMP-13,especially MMP-1,are good markers in osteoarthritis study.MMP-13 expression was very different from that of MMP-1 at 12 weeks after operation,which suggest that their upstream signaling pathways are different,and the signaling pathway difference may be before translation because the mRNA and protein expression change of MMP-13 are in parallel.PartⅡExpression of MAPKs in osteoarthritic rabbits' joint cartilageObjectives To examines the expression of MAPKs in osteoarthritic rabbits'joint cartilage on different time after ACLT and partial excision of medial meniscus.Material and methods Thirty rabbits in experimental groups received unilateral ACLT and partial excision of medial meniscus,another 10 rabbits received unilateral arthrotomy as pseudo-operation control.Ten experimental rabbits were sacrificed at 4,8 and 12 weeks after surgery respectively,and 10 pseudo-operation rabbits were sacrificed at 12 weeks after surgery.Knees were harvested to observe the macro pathologic changes of joint cartilage.Cartilage near the lesion was obtained,and mRNA and protein of MAPK(including ERK1/2,P38MAPK and JNK) were detected by real-time PCR and western blot respectively.Results There are no significant changes in mRNA and total protein of MAPK on different time.Compared with that of control group, phospho-ERK1/2 is in high level expression on 4 weeks,8 weeks and 12 weeks postoperation,Phospho-P38 MAPK is in high level expression on 4 weeks postoperation and phospho-JNK is in high level expression on 4 weeks,8 weeks postoperation respectively.Conclusions Activation of MAPKs in osteoarthritic cartilage dependent on not total proteins expression,but proteins phosphorylated. There are phase differences between different MAPK.PartⅢInhibition of ERK-1/2 Pathway Reduce Joint Cartilage Destruction in Rabbit Experimental Osteoarthritis in vivoObjectives Observe the protective effect of PD98059,a selective inhibitor of ERK kinase1/2(MEK-1/2) for joint cartilage in vivo in experimental rabbit osteoarthritis;and to evaluate the contribution of ERK1/2 signaling pathway in the course of OA.Material and methods 30 rabbits receiving unilateral ACLT and excision of partial medial meniscus were randomly separated into 3 groups. From the 4th day post operation,PD98059 or placebo were injected into operated articular cavity,twice a week.The first and second group received 100μM and 40μM PD98059 respectively,and the third group received PBS as placebo.Another group contains 10 rabbits without any prior surgery received PBS as control group.Rabbits were killed at 8 weeks after surgery;knees were harvested to observe the macropathologic changes of joint cartilage.Cartilage near the lesion were obtained, protein of ERK1/2 and phospho-ERK1/2 were detected through Western blot; expression of MMP-1 and MMP-13 were detected through both real-time PCR and Western blot.Results Knees treated with PD98059 showed decreased in joint cartilage destruction compared with those treated with PBS. Phospho-ERK1/2 in joint cartilage treated with 100μM PD98059 depressed obviously.Both mRNA and protein expression of MMP-1 and MMP-13 in all those treated with PD98059 were lower than the placebo control.Results Knees treated with PD98059 showed decreased in joint cartilage destruction compared with those treated with PBS. Phospho-ERK1/2 in joint cartilage treated with 100μM PD98059 depressed obviously.Both mRNA and protein expression of MMP-1 and MMP-13 in all those treated with PD98059 were lower than the placebo control.Conclusions MAPK signaling pathways play active roles in the course of OA.PD98059,a selective inhibitor of MAPK kinase1/2(MEK-1/2) effectively reduces joint cartilage destruction in vivo experimental osteoarthritis of rabbits and the protective effect is positively associated with inhibition of ERK-1/2 activation.