| Objective:To explore the mechanism of platelet rich plasma(PRP)on the cartilage pathological state as well as IL-1? and Wnt/?-Catenin signaling pathway of rabbit knee osteoarthritis(OA)model.Methods:48 clean New Zealand white rabbits were divided into normal group,Sham group,model group and experimental group(PRP group)randomly,12 rabbits for each group(n=12).For normal group,left knee joint remained untreated;for sham group,open the left articular cavity only,without any other treatment;modified Hulth method was used to build a left knee osteoarthritis model for model group and experimental group.One week after modeling,10ml of autologous ear venous blood was taken from the animals of experiment group.0.8ml of PRP was prepared by using Aghaloo method to inject into left articular cavity;for sham group and model group,the articular cavity was injected with normal saline of the equivalent volume in the same method once per week for six consecutive weeks.X-ray radiography was carried out for the left joints of experimental animals,the pathological changes of cartilage were observed with the naked eyes and under microscope and scored respectively,and the ultra-microstructure of chondrocytes were detected with electron microscope.The contents of type II collagen in cartilage and IL-1? in cartilage and synovium were determined by immunohistochemical examination,The contents of IL-1? in synovial fluid and serum were determined by ELISA.while the contents of Wntl mRNA,GSK-3? mRNA,?-catenin mRNA and proteins in Wnt/?-Catenin signaling pathway were detected by real-time quantified PCR and Western Blot respectively.Results:X-ray film observation found no obvious change between normal group and blank control group;for model group,the medial joint space was obviously narrowed,the articular surface was rough,osteophyte was formed,and subchondral bone density was increased;articular degenerative change in experimental group was slighter than model group;Pelletier JP and Mankin's scores of articular cartilage in normal group and blank control group were obviously decreased than model group(p<0.05);Pelletier JP and Mankin's scores of articular cartilage in experimental group was obviously decreased than model group(p<0.05).Transmission electric microscope observation found no obvious pathological change of chondrocytes in normal group and blank control group,and the degree of damage of chondrocytes in the model group was severer than experimental group.Masson and toluidine blue staining detected that the staining percentage and mean average gray value of collagen and proteoglycan in cartilage of normal group and blank control group were remarkably increased respectively than model group(P<0.05);those of experimental group were obviously increased than model group(P<0.05).immunohistochemical examination indicated that the positive cell fraction and the mean integrated optical density of type II collagen in cartilage of normal group and blank control group were obviously increased than model group(P<0.05),while while those of experiment group were obviously higher than model group(P<0.05).IL-1? contents in cartilage,synovial fluid and serum of normal group and blank control group were obviously lower than model group(P<0.05),while IL-1? content of experiment group was obviously lower than model group(P<0.05),and IL-1? in synovial fluid was positively correlated to IL-1? in serum(r2=0.97 02,P=0.0000).Inverted microscope found that osteoarthritis chondrocytes cultured in vitro could maintain cell phenotype stability within two generations.The expression quantities of Wntl,?-catenin mRNA and proteins in chondrocytes in PRP group were obviously decreased than normal saline group(P<0.05);those of in PRP group were slightly increased than inhibitor group(P<0.05),but lower than agonist group(P<0.05);the expression quantities of GSK-3? mRNA and proteins in PRP group were obviously increased than normal saline group(P<0.05);those of in PRP group were obviously decreased than inhibitor group(P<0.05),but remarkably higher than agonist group(P<0.05).Conclusion:Although PRP cannot finally reverse the development process of osteoarthritis,it can alleviate articular cartilage pathological injury,inhibit the degradation of proteoglycan and collagen and slow down the development of osteoarthritis to a certain degree by lowering the level of the major pro-inflammatory cytokine IL-1?,reducing the levels of Wntl and ?-catenin in Wnt/?-Catenin signaling pathway and increasing the level of GSK-3?.Thus,PRP may become a new effective method for the treatment of osteoarthritis. |
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