Mechanism Of Asporin Promoting Osteoarthritis Through The TGF-?1/Smad2/3 Signaling Pathway

Background:Osteoarthritis(OA)is the most common joint disease occurrence in the knee joint,mainly symptoms include knee pain,swelling,stiffness and dysfunction.In recent decades,OA has been a research hotspot,but its pathogenesis is still unclear.A large number of studies have found that asporin,an important component of extracellular matrix proteins belong to the SLRP family,may be involved in the development of OA.but the specific role and mechanism of asporin protein in OA remains unclear.Objective:Firstly,the expression level of asporin in articular cartilage and serum samples of OA patients,articular cartilage of DMM mice and articular cartilage of in vitro osteoarthritis model were detected.Secondly,to further clarify the effect of asporin expression on the pathological changes of OA.Finally,to reveal the relationship between asporin and TGF-?/Smad signaling pathway in articular chondrocytes during the pathological change of OA and to explore the role its molecular mechanism.Providing a new potential target for the prevention and treatment of osteoarthritis.Methods:1.Collecting clinical articular cartilage specimens of OA patients and normal knee joint cartilage specimens of human requiring amputation due to severe trauma,respectively.The expression of asporin in articular cartilage was detected by immunohistochemistry.As well as,serum samples were collected,and detected by enzyme-linked immunosorbent assay.The DMM osteoarthritis model was constructed in mice,detection the expression level of asporin by immunohistochemistry at 4weeks and 8 weeks after DMM surgery.2.The OA model of mouse cartilage transplantation was constructed in vitro,and the expression of asporin in mouse articular cartilage was detected by immunohistochemistry.3.Using ATDC5 cartilage precursor cell line for in vitro experiments,silencing asporin gene by transfection specific siRNA,detecting the expression of extracellular matrix components by RT-qPCR and Western Blot,using the asporin recombinant protein to stimulate ATDC5 cartilage precursor cell line,to test the expression of extracellular matrix components of chondrocytes by RT-qPCR and Western Blot.4.Through the intra-articular injection of DMM mice with lentivirus carrying specific siRNA sequence to mediate the asporin gene silencing on chondrocytes,the expression of cartilage extracellular matrix components was detected by immunofluorescence and immunohistochemistry.5.The TGF-?1 cytokine stimulated ATDC5 cells to observe the effect of asporin on the expression level of TGF-?1 downstream signaling molecule Smad2/3 phosphorylation level as well as the main components of chondrocyte matrix include proteoglycan and type II collagen.Results:1.The expression of asporin in OA patients and mouse osteoarthritic articular cartilage was significantly higher than that in the normal group.At the same time,the expression of asporin in serum was also significantly increased.2.In the OA model of in vitro mouse cartilage transplantation,compared with the control group,the expression of Asporin in the OA group significant higher.3.Compared with the DMM model group,after intra-articular injection of lentivirus,the expression of Asporin protein in chondrocytes was significantly decreased,Aggrecan and COL2 expression was increased,articular cartilage wear was reduced,cartilage thickness was increased,and the pathological changes of arthritis were significantly improved;the expression level of Smad2/3 phosphorylation was increased in articular cartilage.4.After silencing asporin gene by siRNA sequence,the expression of Aggrecan and COLL2 was increased in chondrocytes.The expression of Aggrecan and COLL2 was significantly decreased after stimulation of ATDC5 cartilage precursor cell with asporin recombinant protein.5.Asporin recombinant protein can significantly inhibit the phosphorylation level Smad2/3 downstream of TGF-?1 signaling,and inhibiting the synthesis of Aggrecan and COLL2;Conclusion:1.Asporin expression is up-regulated with the development pathogenesis of osteoarthritis;2.Inhibiting the expression of asporin can promotes the synthesis of extracellular matrix components and ameliorating the pathological progression of osteoarthritis in vitro and vivo studies.3.Asporin inhibits the synthesis of extracellular matrix through the TGF-?/Smad2/3 signaling pathway promote articular cartilage degeneration in experimental Osteoarthritis.