Effect And Mechanism Of SOX4 On Senescence Of Fibroblast-Like Synoviocytes In Patients With Osteoarthritis

Objective: Osteoarthritis(OA)is a kind of chronic degenerative joint disease,with periarticular pain,limited activity,dysfunction as the main clinical manifestations,characterized by reduced articular cartilage matrix damage,osteophyte formation,synovial aseptic inflammation and so on.In recent years,the research on the pathogenesis of osteoarthritis continues.It is generally believed that genetic susceptibility,obesity,early joint trauma,gender,congenital bone or ligament dysplasia are the risk factors,but the most important pathogenic factor is aging.Previous studies on the effect of aging cells on osteoarthritis mainly focused on chondrocytes,while the pathological changes of synovial cells were only considered as secondary changes of osteoarthritis.With the in-depth study of osteoarthritis,there is evidence that synovial cells,like chondrocytes,also participate in the aging burden of joints in the progress of OA,and the removal of these aging cells can reduce the development of osteoarthritis.SOX4 is a transcription factor.It has been reported that SOX4 is highly expressed in inflammatory synovium.It is the target and key mediator of a variety of pro-inflammatory cytokines,and is closely related to the pathogenicity of FLS.However,the relationship between SOX4 and FLS aging and its mechanism have not been reported.This study aims to investigate the role and mechanism of SOX4 in FLS cell senescence in patients with osteoarthritis.Methods :(1)Cellular senescence was measured by the senescence-associatedβ-galactosidas(SA-β-gal)staining,cell cycle analysis by using flow cytometry and the expression of the cell cycle inhibitors p16 and p53 with PCR and western blot methods.(2)Skin fibroblasts(HSF)were used as control to evaluate the senescence of OA-FLS cells,and the senescence of P3 and P8 OA-FLS cells of the same patient was compared.(3)si RN A was used to interfere the expression of SOX4 in OA-FLS cells,and then cell senescence index was detected.The expression of inflammatory cytokines was detected by PCR,and cell proliferation was detected by CCK-8.(4)The supernatant of OA-FLS cells after SOX4 interference was collected.Chondrocytes were cultured in 20% or 50% DMEM medium containing 10% FBS for 72 hours.The levels of cytokines secreted by chondrocytes were detected by PCR,and the supernatant of NC si RN A was used as control.(5)After the OA-FLS cells were treated with Mito Q(400 nmol/L),a scavenger of mitochondrial ROS,for 72 hours,the cell senescence indexes were detected,and the expressions of SOX4 and TGF-β were detected by PCR and western blot.(6)After the OA-FLS cells were stimulated with 2 ng/ml TGF-β for48 hours,the cell senescence indexes were detected,and the levels of p53,p16,SOX4 and inflammatory cytokines were detected by PCR and western blot.Results:(1)Compared with HSF,OA-FLS cells were in the state of senescence.The positive rate of senescence related β-galactosidase staining increased,cell cycle arrest,and the mRNA and protein levels of cell cycle inhibitors p53 and p16 increased;compared with P3 generation of OA-FLS cells,P8 generation of OA-FLS cells showed more senescence,higher positive rate of senescence related β-galactosidase staining,cell cycle arrest,increased protein levels of cell cycle inhibitors p53 and p16,but no significant change in m RNA level.(2)Compared with HSF,OA-FLS highly expressed IL-6,MMP3 and other inflammatory factors,and with the aging degree of OA-FLS increased,the expression of IL-1β,VEGF and other inflammatory cytokines were up-regulated.(3)Compared with chondrocytes cultured with HSF supernatant,the results of 50% OA-FLS supernatant showed that the levels of MMP13 and CCL4 in chondrocytes were increased,and the difference was statistically significant.20% and50% OA-FLS supernatant could inhibit the proliferation of chondrocytes.(4)Compared with NC siRNA treatment,siRNA interference of SOX4 expression in OA-FLS cells significantly alleviated cell senescence,which showed that the positive rate of senescence related β-galactosidase staining decreased,cell cycle arrest was alleviated,and cell cycle inhibitors p53 and p16 decreased the m RNA and protein levels were down regulated,and the m RNA levels of IL-6,MMP3 and other inflammatory cytokines were significantly decreased,the differences were statistically significant.(5)Compared with the control group,Mito Q,a mitochondrial targeted scavenger,decreased the positive rate of senescence related β-galactosidase staining,alleviated cell cycle arrest,and significantly decreased the mRNA and protein levels of p53,p16,SOX4 and TGF-β.(6)Compared with HSF,OA-FLS highly expressed TGF-β m RNA and protein levels,and the differences were statistically significant;compared with P3 generation cells,P8 generation cells had higher TGF-β protein level,which was statistically significant,but the m RNA level was not statistically significant.(7)Compared with the control group,the OA-FLS cells treated with 2 ng/ml TGF-βshowed more senescence,higher positive rate of senescence related β-galactosidase staining,cell cycle arrest,and higher protein levels of p53 and p16,and the results of PCR showed that the m RNA levels of IL-6,MMP3,MMP13,VEGF and other inflammatory cytokines increased after treatment with TGF-β,but the m RNA levels of IL-1β,MMP9 and TNF-α did not change significantly.Conclusion: In this study,we found that SOX4 is highly expressed in OA-FLS cells and plays an important role in their senescence;interference with SOX4 can delay the senescence of OA-FLS cells and alleviate cartilage damage;O A-FLS ROS/TGF-βsignal is over activated and regulates cell senescence through SOX4.