Effect Of Osteoclast In Cartilage/bone Damage Of Mouse Arthritis Models And The Therapeutic Influence Of Low Dose Glucocorticoid

Rheumatoid arthritis (RA) is a common autoimmune and chronic disabling disease characterised by synovitis, destruction of cartilage and bone and, ultimately, loss of joint function.Bone loss is a major unsolved problem in rheumatoid arthritis.RA.The skeletal complications of RA consist of focal erosion of marginal and subchondral bone, juxtaarticular osteoporosis, and generalized bone loss with reduced bonemass. In the past years, the research of RA bone loss mechanisms mainly focus on degradation process matrix of different kinds of proinflammatory cytokines and proteases. Recently, the destruction function of OC and pOC on bone/cartilage in inflammatory state has become a research point at issue.But we still know a little about the function of focal cartilages destruction caused by OC and pOC.Deriving from the monocyte-macrophage lineage in haematopoieticstem cells, Osteoclasts are special multinucleated cells ,which are uniquely capable of lacunar bone resorption. It plays an important role in the bone remodeling. OPG/RANKL/RANK system and M-CSF are the essential molecul in the process of osteoclast's differentiation and maturation. It is proved that proliferative synovial membrane macrophages, synovial fibroblasts and activated lymphocyte can up-regulation expression of RANKL, screted M-CSF by osteoblast, facilitate /support differentiation of monocytes/ macrophages on the effect of pro-inflammatory cytokines (IL-1 TNF-α , IL-6 11 etc) .All kinds of protease in RA synovial fluid participate bone and cartilage destruction.However, the early focal destruction on bone /cartilage first occurred in cartilage-pannus junction (CPJ ) and the accumulation of monocyt/multinucleate cell is found in focal destruction. So in the research of focal cartilage destruction mechanisms, it is to be proved the following questions: wheather the monocyt/multinucleate cell is accumulated that can be differented into OC; wheather it anticipate the focal bone and cartilage damage; wheather it degradated the cartilage matrix by excreting MMPs.Glucocorticoid is an effected anti-inflammatory drug and has been used in therapy of RA for over 50 years, but there is always dispute about it due to the side effect. It is found in recent research that small dose glucocorticoid can delay cartilage destruction process in early RA. It is reported that small dose glucocorticoid can up-regulate the expression of RANKL, superess OPG expression, raise the ratio of RANKL/OPG, facilitate differentiation & maturation of OC as well as induce glucocorticoid-induced osteoporosis (GIO).The following questions are to beproved about the small dose glucocorticoid therapy: Wheather it can prevent the early cartilage destruction in CPJ; Wheather the effect of OC differentiation and form can facilitate the cartilage destruction; What is the best clinical therapy to be taken to reduce the effect of osteoclastogenesis inducing.Based on collagen-induced arthritis (CIA) with collagen II by C57BL/6 mouse, we research osteoclast distributing in CIA mouse's damaged joint and the effect of osteoclast on focal destruction in CPJ. Morever, we set up a model of unilateral adjuvant-arthritis in C57BL/6 mouse. Based on this model, we administer a small dose Prednisone and MTX. We study the effect of a small dose Prednisone and MTX on inflammatory, OC /pOC and cartilage damage .Main experience is as follow:Method:1. The Research on osteoclast in CIA mouse damage joint. We successfully induce CIA animal model by immunization with chicken II collagen and CFA. We observe focal destruction cartilage and histopathology change on HE stained paraffin section of the episode knee joint. To observe the quantity and distributing of TRAP positive cells and expression of MMP-2 9 of monocyt/multinucleate cells in CPJ by enzyme histochemistry and immunohistochemistry. By the above method, we also observe their correlation with the cartilage destruction degree.2. Set up a model of unilateral adjuvant-arthritis induced in C57BL/6 mouse. We inject directly in stifle articular cavity or tibiotarsus joint of C57BL/6 with CFA once a week for 4 weeks so as to induce the mono-articular arthritis model. We make paraffin section of injected side ,contralateral uninjected side joint and normal control joint .Then perform HE staining and Safflower " O " & Masson staining etc, which is cartilage matrix specific Staining. We observe the histology feature and degradation of cartilage matrix .Compare the model with the CIA.To observe the quantity and distributing of TRAP positive cells and expression of RANKL/RANK in CPJ and the focal destruction of cartilage of CIA by enzyme histochemistry and immunohistochemistry. We observe the expression of RANK on TRAP"1" monocyt/multinucleate cells by immunohistochemical staining for RANKcombined with TRAP staining.3. Therapy effect evaluation of MTX and Prednisone in unilateral adjuvant-arthritis model.The mice are random divided into 5 groups : 1.Negative control group; 2.positive control group;3. MTX group;4. Prednisone group;5. MTX combined with Prednisone group.Group 3 are administed MTX . Group 4 are administed Prednisone .Group 5 are administed MTX and Prednisone . Groups are treated for 35 days. We observe the feature of the histology and degradation of cartilage matrix on paraffin section of stifle joint in each group by hematoxylin and eosin (HE) staining and cartilage matrix specific Staining. To observe the quantity and distribution of TRAP positive cells and expression of RANKL/RANK in CPJ and the focal destruction of cartilage of CIA was examined by enzyme histochemistry and immunohistochemistry and look into the expression of RANK on TRAP + monocyt/multinucleate cells by immunohistochemical staining for RANK combined with TRAP staining.Result:1 .A number of monocyt/multinucleate cell cells accumulate in CPJ andfocal destruction cartilage. Some of the cells express TRAP positive and the number increase of TRAP + cells is positive correlation with the carlitilage destruction degree (rs=0.903 , P <0.01); The TRAP+ cells excrete MMP-2> 9 and expression of MMP-2 is higher than MMP-9 in TRAP+ monocyt cells (P <0.05 ). On the contrary, expression of MMP-9 is higher than MMP-2 in TRAP+multinucleate cells (P <0.05).The destruction degree of cartilage is positive correlation with the expression of MMP-2,9 in TRAP+ cells (rs=0.954 , P<0.01) .2. Histopathology of mono-articular arthritis model is similar to CIA mouse. In short period ,we can find destruction of bone and cartilage limited within injection joint. The ration of TRAP~+ cells and expression of RANK, RANKL on osteoclast osteoblast synoviocytes in injection joint is higher than control and contralateral uninjected side joint( P<0.05). The expression of RANK in TRAP+cell and the cells that express RANK and RANK in injection joint is higher than control and contralateral uninjected side joint( P<0.05) .3. We find joint swelling in prednisone group and prednisone combined with MTX is obviously alleviate than MTX group and positive control group in injection joint and uninjection side joint. Ratio of TRAP +cells, expressions of RANK and RANKL and expression of RANK in TRAP+cell in prednisone group and prednisone combined with MTX is obviously higher than MTX group and positive control group in injection joint and uninjection side joint( P<0.05) . But between two groups ,MTX combined with Prednisone and Prednisone, is not changed (P>0.05) . Conclusion:...