An Experimental Study On The Effects Of MAPKs Signal Pathway In Espressing MMP-1/-13 Of Rabbit Osteoarthritis Chondrocytes

BackgroundOsteoarthritis is a chronic degenerative disease of joint and the most prevalent form of arthritis in ages.OA increase in prevalence with age and is more common in women than in men.Approximately 50 percent of persons over 60 years old and 80 percent of 75 years old persons suffer from this disease.With the coming of aging society,OA is becoming a major cause of disability in elderly people and the social burden and use of medical expense are expect to increase.OA is characterized by degeneration of articular cartilage and it's etiology is not clear. Multiple factors are involved in the pathogenesis of OA,including age, obesity,inflammation,trauma and genetic factor etc.Multiple cytokines are known to affect the progression of OA,in which Interleukin-1(IL-1) and tumor necrosis factor-α(TNF-α) are most important cytokines.IL-1βand TNF-αcan affect synovial fibroblasts and chondrocyte through an autocrine or paracrine manner,stimulate them to secret matrix metailoproteinases(MMPs),nitric oxide(NO),prostaglandin E2(PGE2),etc.They can inhibit progeoglycan and typeⅡcollagen synthesis,cleave different components of the cartilage Extracellular matrix(ECM),induce OA in end.Though inflammatory is not enough to elucidate the mechanism of OA pathogenesis,the major events in OA pathogenesis is correlated with inflammatory cytokines.According to the previous researches,IL-1βand TNF-αcombined with their receptor in cell membranes,then activated the signal transduction pathways that regulate gene expression including increased expressiong of MMPs,free radicals generation and chondrocyteapoptosis and so on.These pathways include mitogen-activated protein kinases(MAPKs) and nuclear factor-κB(NF-κB) signaling pathways.The mechanisms through which IL-1βand TNF-αincrease the expression of MMP-1/-13 and the difference between them have not been fully elucidated.In our study,Quantitative Polymerase Chain Reaction(Real-time PCR) and Western blot analysis are used to observe the different expression of MMP-1/-13 of IL-1βand TNF-αon rabbit articular chondrocytes, evaluate the MAPKs signal pathways protein expression in chondrocytes, then use inhibitors of MAPKs to block IL-1βinduced elevated expression of MMP-1/-13.We examined and compared the signaling pathways required to induce MMP-1 and MMP-13 production,it will provide a basis for further study of MMPs up-regulating mechanism in OA.PartⅠIL-1βand TNF-αon expression of MMP-1/-13 and NO in articular chondrocytes of rabbitObjectives Isolated and cultivated rabbit articular chondrocytes. To observe the effect of IL-1βand TNF-αon expression of MMP-1/-13 and NO in chondrocytes,compared the difference between them.Materials and methods Harvest articular cartilage from New Zealand rabbit knee joints to carry out rabbit chondrocytes culture and identification.IL-1β和TNF-αare used to treat chondrocytes in different time(8h,16h,24h,36).Real-time PCR were carried out to detect the expresion of MMP-1/-13mRNA in chondrocytes,collect culture supernatants to determine the expression of MMP-1/-13 protein by western blot and to detect the NO concentration with Griess method.Results Through cell morphologic and immunocytochemistry identification by inverted microscope,cultured chondrocytes were identified.IL-1βcan induce elevated expression of MMP-1/-13,MMP-1 production accumulated as the time prolonged in 36h,MMP-13 increased and remained in a high level in 36h.TNF-αcannot stimulate either of them production from rabbit chondrocytes.Both IL-1β和TNF-αresult in increasing amounts of NO concentration.Conclusions Isolated and cultured rabbit articular chondrocytes successfully.IL-1βcan induce production of MMP-1 and MMP-13,their increasing trend is different.TNF-αdid not induce either of them in chondrocytes.Both of IL-1βand TNF-αcan stimulate NO expression. PartⅡIL-1βand TNF-αon expression of MAPKs signal transduction protein in articular chondrocytes of rabbitObjective To observe the effect of IL-1βon expression of MAPks signal transduction protein in articular chondrocytes of rabbit.Materials and methods Rabbit articular chondrocytes were cultivated in vitro and randomly divided into the control group,IL-1βtreated group in 15min,30min,45min and 60min groups.Western blot was performed on whole cell extracts to detect respective expression of total ERK1/2, JNK1/2,p38 MAPKs and active form(phosphorylation of MAPK).Results As chondrocytes exposed to IL-1βfor 60mins,the levels of the respective total MAPKs remained constant.IL-1βtime-dependently stimulated the phosphorylation of ERK,p38 and JNK MAPKs in these cells in 15mins.The ERK phosphorylation was sustained up to 60min but phosphor-iNK was peaking at 15min and phosphor-p38 activation levels started declining by 30min,the amount of active p38 and JNK decreased by 60min.The differences among each group and the control groups had statistical significance.Conclusions IL-1βcannot induce the expression of total protein of MAPKs,but increased the phosphor-MAPKs in early time obviously.PartⅢEffect of MAPKs pathways on expression of MMP-1/-13 in articular chondrocytes of rabbit osteoarthritisObjective To observe the expression of MMP-1/-13 in articular chondrocytes of osteoarthritis after inhibition of IL-1β-induced MMP-1/-13 expression by MAPKs inhibitors,to evaluate the effect of different MAPKs signal pathways.Materials and methods Articular chondrocytes of rabbit were cultivated in vitro and randomly divided into negative control group,IL-1βtreated group,other groups were pretreated for 30min with each indicate MAPK inhibitor[ERK inhibitor PD98059(20μM);JNK inhibitor SP600125(μM);p38 inhibitor SB203580(μM)],then subsequently treated with rhIL-1β(10ng/ml) for 24h.Following each treatment,the expression of MMP-1/-13 mRNA was evaluated by real-time PCR.Results IL-1βcan increase the expression of MMP-1/-13 and MAPKs inhibitors inhibited IL-1β-induced MMP-1/-13 expression in chondrocytes. The ERK1/2 pathway inhibitor,PD98059,attained 29.6%(MMP-1) and 39.2% (MMP-13) inhibition of MMP-1/-13 induction in chondrocytes;P38 inhibitor, SB203580 caused 47.7%reduction of MMP-1 and and 37.7%(MMP-13);Inhibitor of JNK,SP600125 achieved 55.4%suppression of MMP-1 and 52.25 of MMP-13 in chondrocytes.P38 inhibitor SB203580 can inhibit the NO expression effectively.Conclusions These results suggest the involvement of MAPKs,in the IL-1 induction of MMPs in chondrocytes.MAPKs signal pathways play an important role in expression of articular chondrocytes of osteoarthritis. For MMP-1 expression,JNK and p38 maybe important,but to MMP-13 the important pathways are ERK and JNK.p38 is involved in the IL-1 induced NO production.