Regulation Mechanism Of RKIP In Temporomandibular Osteoarthritis Under Extra Mechanical Stimulations

Osteoarthritis(OA)of tempromandibular joint is a common disease in clinic with the symptom of pain,clicking and limitation of activity,and abnormal load is considered the main cause of this degenerative diseases.Persistent activation of MAPK has close link with OA,and the regulation of MAPK signal pathway has been the hot research topic in these days.However,there are many proteins involved in MAPK.And most studies in this area used exogenousase inhibitor,like chemical inhibitor u0126 and pd98059.Therefore,it is very important to find a key signal molecule in MAPK through endogenousase inhibitor.It was reported that Raf kinase inhibitor protein(RKIP)plays a critical role in the development of OA,which is an improtant negetive modulin of Raf kinease in MAPK pathway.Therefore,we used flexcell 5000 tension system to reveal the relationship between RKIP and OA.Part I:RKIP mediated responses of rat mandibular condylar chondrocytes(Mccs)under cyclic tensile stressObjective:To observe the regulatory effects of RKIP on matrix synthesis of Mccs under cyclic tensile stress.Method:(1)Isolation and identification of rat mandibular condylar chondrocytes(Mccs).Condylar chondrocytes were obtained using two step enzyme digestion method,briefly,digesting tissue blocks with 0.25%trypsin for 30min and 0.2%type? collagenase for 2 hours and identificated by type II collagen staining and toluidine blue staining;(2)Third generation of Mccs were randomly divided into control group and loading group.Cells were cultured with or without CTS for 0,3,6,12,24h respectively prior to CCK8 assay,Real-time PCR and Immunofluorescence.(3)Knock down RKIP expression level of cells by lentiviral transfection technique,and expression of Collagen II and Aggrecan were observed under loading state level.Results:(1)6h of CTS had no effect on cell proliferation,12h and 24h of CTS promoted cell proliferation once the loading were stopped,but inhibited cell proliferation the following three days.(2)6h of CTS increased expression of Collagen ?;3h?6h and 12h of CTS all increased expression Aggrecan;CTS increased expression of MMP13 continuously and TIMP4 in contrast.(3)3h,6h,12h and 24h of CTS increased expression of RKIP(12h group reach the peak);24h of CTS increased expression of nucleus p-ERK.(4)When RKIP was down-regulated in Mccs,expression of Collagen II and Aggrecan caused by 6h of CTS were lower than normal cells.Conclusion:Matrix protein composition of stretched MCCS gradually disordered.It were presented as reduced Aggrecan(used to maintain mechanical property of cells)and increased in both matrix synthesis and(Collagen ?)catabolism(MMP13).An compensatory increased proliferation of chondrocytes was existed.RKIP may play a coordination role in the matrax protein composition change.Part ?:Expression of RKIP in the condylar cartilage in TMJOA model ratsObjective:To study the expression of RKIP in temporomandibular joint osteoarthritis(TMJOA)model rats,to explore and confer the possible effects of RKIP involved in the degradation of the early stage of OA.Methods:Six-week-old female SD rats were divided into two groups:control group,OA group.Each group had 5 rats.Passive mouth opening(3h/day,last for 5days)was used to build up the TMJOA model.Microct was used to evaluate the subchondral bone remodeling.Toluidine blue was used to analyze the structural change of condyle cartilage.Immunohistochemical staining was used to measure the expression of Colagen II and RKIP in the cartilage.Results:Expression of Proteoglycan and Collagen ? decreased in cartilage and bone mineral density of subchondral bone decreased in OA group.RKIP expression level decreased significantly in cartilage of OA group.Conclusions:Consistent mouth opening can cause early stage of TMJOA.The expression of RKIP decreased significantly in the early stage of TMJOA,which indicated that RKIP defection may play an important role in the initial stage of condylar cartilage degradation.