| Objective: To investigate the effect of OPC on interleukin-1?(IL-1?)-induced apoptosis in chondrocytes,and to explore the mechanisms underlying the protective effects of OPC.Method:1.Articular cartilage was dissected,cut and grinded from knee of 6-week-old Kunming mice(male or female)under sterile environment,digested with 0.2% type II collagenasefor 6 hours;and filtered using a 74 μm iameter mesh steel filter.The filtrate was centrifuged and supernatant discarded.Chondrocytes were resuspended in DMEM/F12 and cultured in 5% CO2 at 37°C.2.First-generation chondrocytes were seeded onto 96-well plates at a concentration of 105 cells in each well.After adherence chondrocytes co-treated with OPC at final concentration of 0,0.001,0.005,0.010,0.050,0.100,0.500,1.000 and 5.000 mg/m L and with IL-1? at final concentration of 10μg/L.After incubation for 24 hours,cell viability wasdetermined by MTT assay and got the most appropriate concentration.3.First-generation chondrocytes were seeded onto 96-well plates at a concentration of 105 cells in each well.After adherence,three groups(10 wells per group)were made:Control group: treated with 2μL 0.1M PBS and 198μL serum-free DMEM/F12;Apoptosis: group: treated with 10μg/L IL-1?;Administive group: treated with 10μg/L IL-1? plus 0.050 mg/m L OPCAfter incubation for 24 hours and fluorescent probe DCFH-DA added into each well,the ROS levels in chondrocytes were detected using a microplate reader.4.First-generation chondrocytes were divided into 3 groups: Control group: treated with 2μL 0.1M PBS and 198μL serum-free DMEM/F12;Apoptosis: group: treated with 10μg/L IL-1?;Administive group: treated with 10μg/L IL-1? plus 0.050 mg/m L OPC.After incubation for 24 hours,mitochondrial membrane potential(MMP)in chondrocytes was determined using JC-1 kit5.First-generation chondrocytes were divided into 3 groups: Control group: treated with 2μL 0.1M PBS and 198μL serum-free DMEM/F12;Apoptosis: group: treated with 10μg/L IL-1?;Administive group: treated with 10μg/L IL-1? plus 0.050 mg/m L OPC. After incubation for 24 h,chondrocytes were trypsinized,supernatant discarded and resuspended in binding buffer mixed with 10μl Annexin V-FITC and 10μl PI;Assessment of apoptosis was performed by flow cytometry.6.First-generation chondrocytes were divided into 3 groups: Control group: treated with 2μL 0.1M PBS and 198μL serum-free DMEM/F12;Apoptosis: group: treated with 10μg/L IL-1?;Administive group: treated with 10μg/L IL-1? plus 0.050 mg/m L OPC.After incubation for 24 hchondrocytes were trypsinized and washed twice with serum-free DMEM/F12,then centrifugation at 2000 rpm for 5 minutes,cells were pre-fixed with glutaraldehyde and were observed under a transmission electron microscope in Dalian Medical University Electron Microscope Department.Results: 1.using inverted microscope,chondrocytes were transparent and round when seeded.After 2-3days’ adherence cells became triangle,polygon or irregular shape.When chondrocytes grew in cluster,they were in cobblestones appearance.2.The suppression effection of different concentrations of OPC on IL-1?-induced cell death in primary chondrocytes was examined by MTT assay.OPC treatment showed significant protective effects against IL-1?-induced cell death at concentrations of 0.005,0.010,0.050,0.100,0.500,and 1.000 mg/m L(P<0.05).Among these,OPC at a concentration of 0.050 mg/m L appeared to confer the maximal protection Therefore,0.050 mg/m L of OPC was used for the subsequent experiments.3.IL-1? induced a significant increase in ROS levels in the first-generation chondrocytes in fluorescent probe DCFH-DA assay(P<0.05)However,the effect was significantly reversedby co-treatment with OPC,which indicates an antioxidant effect of OPC in chondrocytes.4.The ratio of green and red fluorescence intensity was significantly higher in IL-1? group as compared to that in the control group.However,PC treatment significantly reduced MMP expression in chondrocytes as compared to that in the IL-1? group.5.flow cytometry in Annexin V-FITC/PI assay showed IL-1? treatment was associated with a significant increase in both early and late apoptosis in chondrocytes,as compared to that in the control group,OPC treatment markedly inhibited the early and late apoptosis.6.Control group chondrocytes prolifercation was active and normomorph while more dead cells in apoptosis group.Dose group chondrocytes showed less apoptosis.Conclusion: OPC could protect chondrocytes against IL-1?-induced damage by decreasing ROS content and MMP level. |
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