The Disease-modifying Effect Of Dehydroepiandroste Rone On Articular Cartilage In Rabbit With Experimentally Induced Osteoarthritis

Osteoarthritis(OA)is a prevalent and debilitating joint disease for which ageing,obesity and chronic inflammation are known risk factors.The main clinical features of OA involve pain,joint failure and loss of joint architectural integrity.In our country,the morbidity of OA has reached 50%in the population of aged above 60,and this rate has even reached 80%in the population of aged above 75.Due to the hiden symptoms at early stage,the disease usually develops into the advanced stage when patient complain of the joint pain and swelling,and consequently,artificial joint replacement become the only choice.Thus,OA has become a major medical problem that threatens the health and living quality of the elder.Aggrecanses are novel separated enzymes in recent years that belong to the family of a distintegrin and metalloproteinase with thrombospondin motifs(ADAMTS).Recent evidence demonstrates that aggrecanase-1(ADAMTS-4)and aggrecanse-2(ADAMTS-5)play critical roles in aggrecan degradation at early stage of OA.Inhibition of their activity contributes to an attenuated destruction of articular cartilage,thus,retarding disease progress.Tissue inhibitor of mettaloproteinase-3(TIMP-3)has been observed a direct inhibitory effect on the activitiy of ADAMTS-4/5,and has been recognized as a natural endogenous inhihitor for aggrecanses.Based on this rationale,the balance of ADAMTS/TIMP-3 enzyme system is closed related to OA onset and development,which is a potential therapeutic target for interfering,and might bring delight to the new anti-OA agents researches.Dehydroepiandrosterone(DHEA)is a 19-carbon steroid hormone synthesized from pregnenolone.Because of its decline with age,DHEA is known as an ’antidote for aging’,and has protective effects of articular cartilage during OA progress.However,the exact mechanism is still limited.In this study,we investigate the effect of DHEA on knee articular cartilage through four aspects:first,the morphological changes of cartilage in different stages of OA in a surgically-induced rabbit model;second,the effect of DHEA on cartilage in different regions of the knee joint in different stages of OA;third,the modulation pattern of ADAMTS/TIMP-3 enzyme system by DHEA treatment.Our data demonstrate that the structure-modifying efficacy of DHEA against OA is time-dependent and site-specific,the underlying mechanism might be the inhibition of the gene expression of catabolic matrix degrader(like ADAMTS-4/5),protomtion of the gene expression of the anabolic cytokines(like TIMP-3,aggrecan and collage type II).The result of this study contributes our understanding of the complex phathogenesis of OA.Part1 The morphological changeds in rabbit OA modelObjective:To investigated the features of cartilage damage induced by anterior cruciate ligament resection(ACLT)in rabbits.Methods:An ACLT model was used to create a progressive OA model in twenty rabbits.The animals were necropsied at 9 and 16 weeks after operation.The harvested knee joints were evaluated macroscopically.Results:The ACLT animals sacrificed at 9 weeks demonstrated histological features that included extensive partial thickness fissures,loss of the superficial zone layer and empty chondrocyte lacunae within the remaining cartilage with reduced Safranin-O staining,which is similar to the pathological changes in the middle stage of human OA.The animals that were sacrificed at 16 weeks had progressive osteophytosis in the medial knee compartment,as well as partial or full thickness cartilage erosion of the femoral condyle,and to a lesser extent,in the tibial plateau,which is similar to the pathological changes in the terminal stage of human OA.Conclusion:ACLT induced a time-dependent cartilage damage from middle OA to termibal OA.Part 2 The structure-modifying effect of DHEA on different OA stagesObjective:To investigate the cartilage protective pattern of DHEA in middle stage and terminal stage of OA.Methods:Thirty male New Zealand white rabbits were used in this study.An ACLT model was used to create a progressive OA model in twenty rabbits.The animals were treated with DHEA or a placebo and were necropsied at 9 and 16 weeks.Ten rabbits receiving sham operations served as controls.The articular cartilage of the medial femoral condyle(MFC),lateral femoral condyle(LFC),medial tibial plateau(MTP)and lateral tibial plateau(LTP)was evaluated macroscopically and histologically.Results:In the joints of the sham-operated rabbits,few histological changes were detected on the articular surfaces of the femoral condyles and tibial plateaus.ACLT obviously induced erosive changes on the cartilage surfaces.Compared to the placebo group,the macroscopic and Mankin score analyses demonstrated that the DHEA treatment markedly reduced the cartilage lesions and delayed cartilage degeneration in the four regions of the knee at 9 weeks after operation.At 16 weeks,DHEA demonstrated chondroprotective effects on the lateral compartment of the knee compared to the placebo group,whereas the cartilage degeneration at the medial compartment of the knee did not differ among the groups.Conclusion:The disease-modifying efficacy of DHEA aganist OA is time-specific and site-dependent.DHEA could be used as a disease-modifying strategy to limit the progression of OA,especially in the middle stage.Part 3 Effect of DHEA on the balance of ADAMTS/TIMP-3 system in a rabbit modle of osteoarthritisObjective:To observe the effect of intra-articular injection of DHEA on the balance of ADAMTS/TIMP-3 system in a rabbit modle of OA.Methods:Sixty rabbits underwent bilateral ACLT.One knee of each rabbit was treated with 100μmol/L DHEA resolved in the dimethylsulphoxide(experimental group)and the other knee was treated under the same schedule using dimethylsulphoxide(control group)4 weeks after transection,once a week for eight weeks.Twelve weeks after ACLT all rabbits were killed after X-ray assessment and the knee joints were evaluated by gross morphology and histology.The content of hydroxyproline and glycosaminoglycan in cartilage were analyzed according to the kit.The mRNA expression of ADAMTS-4,ADAMTS-5,tissue inhibitor of metalloproteinases-3(TIMP-3),transforming growth factor-pi(TGF-β1),Aggrecan and Collagen Ⅱ in the cartilage was analyzed using reverse transcription polymerase chain reaction(RT-PCR).The protein expression of Aggrecan ARGxx and Collagen Ⅱ in the cartilage was analyzed using western blot.Results:By Mann-Whitney test,Gross morphologic scores on femoral condyle and tibial plateau in control group are significantly higher than experiment group.By unpaired Student’s t test,histological evaluation showed that grade of cartilage damage in the experimental group were less severe than the control group.The content of hydroxyproline and glycosaminoglycan in the experimental group increased significantly compared with the control grou.The mRNA expression of ADAMTS-4 and ADAMTS-5 in the experimental group decreased significantly compared with the control group.The mRNA expression of TIMP-3,TGF-β1,Aggrecan and Collagen Ⅱ in the experimental group increased significantly compared with the control group.The protein expression of Aggrecan ARGxx in the experimental group decreased significantly compared with the control group.The protein expression of Collagen Ⅱ in the experimental group increased significantly compared with the control group.ConslusionDHEA protects against cartilage degradation and inhibits the progression of OA in the experimental model.Down-regulation of ADAMTS-4,ADAMTS-5 and up-regulation of TIMP-3,TGF-β1,Aggrecan and Collagen II in cartilage may be the mechanism of the protective effect of DHEA on OA.