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The expression of LOX-1 in patients with osteoarthritis and its clinical significanceObjective:To evaluate the changes on the levels of LOX-1 and ox-LDL in patients with osteoarthritis and to determine the correlation between LOX-1 and ox-LDL with functional index in patients with osteoarthritis.Methods:OA(n=40)cartilage and synovial fluid(SF)samples were collected at the time of total knee arthroplasty at Shanghai Sixth People's Hospital,Shanghai,China,whereas normal cartilage and SF were obtained from patients(n=40)with no history of OA.The expression level of LOX-1 in osteoarthritic patients was evaluated by quantitative reverse transcription PCR.OX-LDL was detected with SF sample by enzyme-linked immunosorbent assay.Functional assessment of osteoarthritic patients was assessed by the Western Ontario and McMasters University Osteoarthritis Index(WOMAC)scores.Correlation analysis was performed to evaluate the correlation between the level of LOX-1 and ox-LDL with the total WOMAC score and the correlation between the levels of LOX-1 and ox-LDL.Results:The expression level of LOX-1 in articular cartilage of patients with osteoarthritis was significantly higher than those from the control group(0.49±0.11 vs.0.33±0.04;p<0.05).Compared with the level of ox-LDL in the SF samples from the control individuals,the level of ox-LDL in the SF samples from patients with osteoarthritis was significantly increased,and the difference between these two groups showed statistically significance([(64.8±18.3)mU)./ml vs.(34.5±15.7)mU/ml;p<0.05).There was a correlation between LOX-1 expression and total WOMAC score in patients with osteoarthritis(correlation coefficient r=0.805,p<0.05).There was a correlation between the level of ox-LDL of SF samples and the WOMAC total score in patients with osteoarthritis(correlation coefficient r=0.786,p<0.05).There was a correlation between the expression of LOX-1 and the level of ox-LDL in SF samples in patients with osteoarthritis(correlation coefficient r=0.786,p<0.05).Conclusions:LOX-1 and ox-LDL are elevated in patients with osteoarthritis,which reflect the function of patients with osteoarthritis,and a correlation may exist between LOX-1 and ox-LDL.Part ? In vitro evaluation of the role and mechanism of LOX-1 in osteoarthritisObjective:To evaluate the regulation of LOX-1 and ox-LDL on chondrocyte apoptosis and its possible mechanism by establishing an in vitro model of chondrocyte apoptosis.Methods:Human articular chondrocytes at 70-80%confluency were pre-incubated with or without anti-LOX-1 antibody(10 ?g/ml)before processing for vehicle control or TNF-?(50 ng/ml)or ox-LDL(20 ?g/ml)or TNF-?(50 ng/ml)and ox-LDL(20 ?g/ml)co-treatment for 24-48 h.Cell death was assessed by trypan blue staining;cell apoptosis was assessed by flow cytometry;chondrocyte apoptosis was assessed by agarose gel electrophoresis DNA ladder assay.Western-blotting was used to detect the levels of caspase-8 and Caspase-3 and the changes of autophagy marker LC3.The effects of the above-mentioned chondrocyte apoptosis model induced by ox-LDL(20 ?g/ml)and/or TNF-?(50 ng/ml)were further used for evaluated the effects of autophagy inhibitors 3-MA and Atg5 siRNA treatment.Results:TNF-? and ox-LDL alone did not affect the cell death obviously compared to the control(TNF-? vs.ox-LDL vs.Control:4.65±2.21%vs.6.86±4.65%vs.3.60±1.04%,P>0.05),however,combine use TNF-? and ox-LDL could result in significantly increased chondrocyte death and these effects could be reversed by Lox-1 monoclonal antibody(TNF+oxLDL vs.control:66.90±6.98%vs.3.60±1.04%;TNF+oxLDL vs.TNF+oxLDL+Lox-1 Ab:66.90±6.98%vs.11.40±1.50%,P>0.05).LOX-1 neutralization with antibody can reduce the increasing effects on DNA fragmentation in chondrocytes induced by TNF-? and ox-LDL combination and decrease the increasing level of caspase-8 and caspase-3 in chondrocytes induced by TNF-? and ox-LDL combination.Western-blotting and immunofluorescence results showed that the level of autophagy in TNF-? combined with ox-LDL-treated chondrocytes was significantly increased,while LOX-1 antibody neutralization significantly reduced the levels of autophagy marker LC3.Inhibition of autophagy by 3-MA or by Atg5 siRNA could result in a similar effect of LOX-1 antibody neutralization on chondrocytes death induced by TNF-? and ox-LDL combination.Conclusions:Oxidized low density lipoprotein facilitates tumor necrosis factor-?mediated chondrocyte death via its interaction with LOX-1,and autophagy is involved in the mechanisms..Part ? In vivo evaluation of the role and mechanism of LOX-1 in osteoarthritisObjective:To evaluate the regulation of LOX-1 on chondrocytes and its possible mechanism in vivo by establishing an experimental mouse osteoarthritis model.Methods:Osteoarthritis was induced by medial meniscus destabilization(DMM)surgery,and the effect of LOX-1 antibody neutralization on disease progression in osteoarthritis mice was evaluated by systemic injection of LOX-1 neutralizing antibody.Simultaneously,TUNEL staining was used to evaluate the apoptosis of chondrocytes in mouse cartilage tissue,and LC3 staining was used to evaluate autophagy-positive cells to elucidate the relevant mechanisms.Results:Safranin O staining showed that LOX-1 neutralizing antibody treatment significantly reduced the severity of disease in osteoarthritis mice.LOX-1 neutralizing antibody treatment reduced OARSI scores(3.95 ± 0.40 vs.5.80±0.50;p=0.007)and impaired the osteophyte formation(1.30±0.10 vs.1.65±0.10;p=0.02)in osteoarthritic mice.Treatment with LOX-1 neutralizing antibody reduced synovial inflammation and synovitis score in osteoarthritic mice(2.02±0.10 vs.2.70 ±0.28;p=0.02).LOX-1 neutralizing antibody treatment reduced bone mass/tissue volume(BV/TV)ratio in the tibial plateau subchondral bone of osteoarthritic mice,however no significant difference was found.LOX-1 neutralizing antibody treatment could significantly reduce the apoptosis of chondrocytes in osteoarthritic mice.LOX-1 neutralizing antibody treatment significantly reduced the number of LC3+chondrocytes in osteoarthritic mice.Conclusions:LOX-1 neutralizing antibody could significantly reduce the severity of disease in osteoarthritis mice,which is mainly achieved by affecting chondrocyte apoptosis and reducing the number of LC3+ chondrocytes. |
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