The Role And Mechanism Of CRYAB In The Development Of Osteoarthritis

BackgroundOsteoarthritis(OA)is a common chronic degenerative disease of the elderly.Its main clinical features are degenerative injury of articular cartilage,accompanied by hyperplasia of the edge of the joint and sclerosisofthe subchondral bone.At present,with the increasing aging of population,the incidence of this disease is rising.How to cure osteoarthritis and effectively protect the cartilage has become an urgent problem to be solved in clinic.Chondrocytes,as the sole cell type in the cartilage tissue,play important roles in regulating the homeostasis of cartilage tissue and the metabolism of extracellular matrix of cartilage.CRYAB is a member of the heat shock family with the function of inhibiting protein polymerization induced by oxidation,heat shock and other environmental stresses,thus promoting proliferation and inhibition of apoptosis of cells.It was reported recently that CRYAB played an important role in protecting rat articular chondrocytes against Casein Kinase II inhibition-induced apoptosis.However,the precise roles and the underlying mechanisms of CRYAB in the development of cartilage and the occurrence of OA remain to be expored.Methods33 cases of normal cartilages from femoral fracture patients and 50 cases of human osteoarthritic cartilages were used to determine the relationship between the expression of CRYAB and OA.Real-time PCR and immunohistochemical staining were used to detect the expression level of CRYAB.DMM(destabilization of the medial meniscus,DMM)surgery was performed to construct a mouse model of osteoarthritis.The human umbilical cord mesenchymal stem cells were separated from the fresh umbilical cord,and were used to induce chondrogenic differentiation.The expression of CRYAB during chondrogenic differentiation was detected by Real-time PCR and Western blot.The hind limbs from new born rats at 3,10,20,42 d,were used to determine the expression of CRYAB duringcartilage differentiation with immunohistochemical staining.CRYAB was overexpressed and knockdown in the chondrocytes by virus infection.The effects of CRYAB on the proliferation,cell cycle and apoptosis of primary chondrocytes were determined by CCK8 and flow cytometry,respectively.Western blot was performed to dectet the expression of Bax,Bcl2 and Cleaved Caspase3 after CRYAB overexpression and knockdown.To examine the effect of CRYAB on the metabolism of extracellular matrix of cartilage,the expression of MMP13,ADAMTS5,ACAN,and Col2a1 were detected by Real-time PCR and Western blot,respectively.ResultsThe expression of CRYAB was down-regulated in osteoarthritis cartilage of patients and osteoarthritic mouse.CRYAB expression was up-regulated during chondrogenic differentiation of the induced human umbilical cord mesenchymal stem cells.The expression of CRYAB on cartilages of new born rats was also up-regulated at 3,10,20 and 42 d by immunohistochemical staining.Results of CCK8 assay showed that overexpression of CRYAB promoted the proliferation of primary chondrocytes,and knockdown of CRYAB had the opposite effects.CRYAB had no effect on the cell cycle of primary chondrocytes.However,overexpression of CRYAB inhibited the apoptosis of primary chondrocyte and knockdown of CRYAB promoted chondrocyte apoptosis by Flow cytometry assay.Moreover,overexpression of CRYAB increased the expression of ACAN and Col2a1 and decreased the expression of MMP13 and ADAMTS5 in primary chondrocytes,vice versa.Conclusion(1)The expression of CRYAB was downregulated in the OA patients and OA mouse.(2)The expression of CRYAB was increased during the development of cartilage.(3)CRYAB had no effect on the cell cycle,but inhibited the apoptosis of primary chondrocytes through Bcl2/Bax apoptosis pathway,thereby promoting the proliferation of primary chondrocytes.(4)CRYAB inhibited extracellular matrix degradation to maintain the homeostasis of chondrocytes.Together,our data showed that CRYAB plays key roles in the formation of cartilage and in the occurrence of OA,suggesting that CRYAB may be an effective target for the prevention and treatment of OA.