| Objective: this study aims to establish the rat OA model and observe the expression changes of key proteins of complement system such as C5,MAC,inflammatory factors and cartilage pathological changes after the articular injection of glutamate receptor antagonist MK801,and explore the pathogenesis of peripheral glutamate receptor antagonist to reduce the progression of OA.The study may open a new path for the prevention and treatment of osteoarthritis.Methods:1)Animal model establishment and pathological score: 40 SD rats were randomly divided into four groups: sham operation group,control group,model treatment group and model activation group.A: Sham operation group(SOG,group);B: Model group(MG,group);C: model treatment group(MK801,group);D:model activation group(Glu,group);SD rats in each group were randomly assigned,10 rats in each group.Three groups of MG?MK801?GLU used the method of cutting the medial collateral ligament,anterior cruciate ligament and removing the medial part of meniscus to prepare osteoarthritis(OA)model.In the SOG group,only the knee joint cavity of the affected limb was opened,but the medial collateral ligament and anterior cruciate ligament of the knee joint of the rats were not cut off,and also a part of the medial meniscus was not removed.The model was successfully made four weeks later.MK801 group and Glu group: after the model was established successfully,0.1 m M mk801 100 ul and 1 m M glutamate 100 ul were injected into the left articular cavitie of each group respectively,and 100 ul of physiological saline was injected into the SOG group and CG group.Each group was injected twice a week for two weeks.Six weeks after the modeling,the rats were euthanized in accordance with the rules of animal ethics.The cartilage of the left knee of each group was taken for general observation,Fast green and Safranin-O staining,Western blot and evaluation of the degree of cartilage degeneration and the Mankin's score of histopathology.2)The concentrations of IL-1 ?,TNF-?and MMP13 in serum were measured by ELISA.3)Western blot was used to detect the deposition of MAC,C5 and NF-?B in the cartilage.Results:1)Animal model establishment and pathological score:(1)general observation of left hindlimb knee arthrosis in rats: the cartilage surface of SOG group was smooth and bright,no obvious defect and osteophyte formation was found,and a small amount of light yellow synovial fluid could be seen.In MG group,the color of cartilage was dark yellow,the surface was rough and uneven,part of cartilage was cracked,the medial tibial plateau and the medial femoral condyle had a obvious fibroplasia and osteophyte formation,the synovium was partly hyperplastic,the synovium was yellow turbid and viscous;in Glu group,the joint space was obviously narrowed,a large number of osteophytes were formed,the cartilage surface defect and damage were the most serious in four groups.the joint capsule was obviously increased In MK801 treatment group,cartilage damage and osteophyte formation were lighter than those in Glu group and control group,joint surface was slightly bright,transparent.cartilage was still visible,synovium hyperplasia was lighter,and synovium was clear.(2)The results showed that the surface of cartilage in SOG group was smooth,the shape and quantity of cells had no obvious change,the tide line of cartilage was complete and clear,and the heterochromatism of subchondral bone and cancellous bone was green.The surface of cartilage in MG group and Glu group was not smooth.The surface of cartilage in Glu group was more damaged than that in the control group with the cartilage became thinner.The staining of Safranin-O was light or lost,and the scope was smaller.The fast green staining of collagen fiber was larger,and the capillary could pass through the tide line.The cartilage layer of MK801 group was also thinner,but the degree was lighter than that of Glu group,the cartilage matrix staining was slightly uneven,the degree of loss of staining was light,the cell distribution was more uniform than that of Glu group and MG group,and the tide line was more regular.(3)Mankin's score of cartilage in each group: in MG group,Glu group and MK801 group,the Mankin's score of articular cartilage was increased,and the score in MK801 group was lower than that in MG group and Glu group;in Glu group,the cartilage damage was the most serious,and the Mankin's score was higher than that in MK801 group and MG group.The differences among MG group,Glugroup and MK801 group were statistically significant(P<0.01).2)The levels of IL-1?,TNF-? and MMP13 in serum were measured by ELISA: the levels of IL-1?,TNF-? and MMP13 in control group and glutamic acid group were significantly higher than those in sham operation group(P<0.01);the levels of IL-1?,TNF-? and MMP13 in MK801 group were significantly lower than those in control group and glutamic acid group(P<0.05);the levels of IL-1?,TNF-? and MMP13 in glutamic acid group were significantly higher than those in control group(P<0.01);the levels of IL-1?,TNF-? and MMP13 in control group and MK801 group were significantly higher than those in control group and MK801 group There was no significant difference in the amount of 3.3)Western blot was used to detect the expression of MAC(C5b-9),C5,NF-?B in cartilage: the expression of MAC(C5b-9),C5,NF-?B in control group and Glu group was significantly higher than that in sham operation group.After MK801 treatment,the expression of MAC(C5b-9),C5,NF-?B decreased(P<0.05).Conclusion: In the OA rat model,NMDA receptor antagonist MK801 can reduce the concentration of complement in cartilage,reduce the expression of proinflammatory mediators,and alleviate the pathological changes such as osteophyte formation and cartilage destruction.Complement system plays an important role in the development of OA.NMDA receptor antagonists may affect the development of OA through complement system. |
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