Study On The ITraq-based Quantitative Proteomics Of AS-IV In IL-1?-induced SW1353 Chondrosarcoma Cells

PART ONE Effect of AS-IV at different concentrations and times on apoptosis of IL-1?-induced human SW1353 chondrosarcoma cellsObjective: To investigate the effects of AS-IV at different concentrations and times on apoptosis in IL-1?-induced human SW1353 chondrosarcoma cells in order to screen the optimal concentration and the best culture time for the subsequent experiments.Methods: The control group and 10 ng·ml-1 IL-1?group were set of being compared.SW 1353 chondrosarcoma cells were pre-treated at 25,50,100,200 and 500?mol·L-1 concentrations AS-IV for 1h,then stimulated with 10 ng·ml-1 IL-1? for 12 h,24h and 48 h separately.Repeat three times.Then the effects of apoptosis in IL-1?-induced SW1353 chondrosarcoma cells were detected using flow cytometry assays and were analyzed furthermore.Results: 1.The FCM results showed that the cell apoptosis in the IL-1?group was increased significantly compared to controls(P<0.05)by flow cytometry assays.2.The effects of AS-IV at 24 h on the cell apoptosis in SW 1353 cells were significantly reduced by compared with 12 h and 48h(P< 0.05).3.Compared with that in 500?mol·L-1 concentrations of AS-IV,the cell apoptosis were statistically significance while IL-1?-induced SW1353 cells were treated with 25,50,100 and 200?mol·L-1 concentrations of AS-IV at time of 24h(P< 0.05),while the apoptosis significantly decreased in 100?mol·L-1 group than other concentrations(P< 0.05).Conclusion: IL-1?activated the apoptosis in SW1353 cells,the time of 24 h have optimal effects on cell apoptosis,and 100 ? mol·L-1AS-IV suppressed the apoptosis in IL-1?-induced SW1353 cells most effectively although 25,50 and 200?mol·L-1 concentrations had the similar effect.Thus the concentration of 100?mol·L-1 AS-IV and the time of 24 h would be used in subsequent experiments.PART TWO THE ITRAQ-BASED QUANTITATIVE PROTEOMICS OF AS-IV IN IL-1?-INDUCED SW1353 CHONDROSARCOMA CELLSObjective: To explore the proteomic changes after IL-1? or AS-IV treatment,a quantitative proteomic analysis using i TRAQ technology was performed to detect the protein expression profiles.In this study,we performed high accuracy LC-MS/MS to quantitatively detect and map proteins in the SW1353 after IL-1? or AS-IV treatment.Methods: The study are divided into 4 groups: control,IL-1?(10 ng·ml-1),100?mol·L-1AS-IV and IL-1?(10 ng·ml-1)combined with 100?mol·L-1AS-IV group.The control group was set of being compared.SW 1353 chondrosarcoma cells were treated at 10 ng·ml-1 concentration IL-1?or 100?mol·L-1 concentration AS-IV for 24 h.While the other was pre-treated at 100?mol·L-1 concentration AS-IV for 1h,then stimulated with 10 ng·ml-1 IL-1 ? for 24 h.The samples were assessed through ultra-performance liquid chromatography-mass spectrometry.The signal peptide analysis,gene chip assay,differential protein analysis and signal pathways were verified by LC-MS/MS.Results:1.There were total 296 differential proteins had been assayed.118 reduced and 8 up-regulated in IL-1 ? group,40 reduced and 32 up-regulated in AS-IV group,and 149 up-regulated and 16 reduced in AS-IV combined with IL-1?-induced group.2.It was showed that both phosphoprotein and acetylation are the top2 differentially expressed proteins in the the SW1353 chondrosarcoma cells after IL-1? or AS-IV treatment.3.Only 26 of 296 differentially expressed proteins have signal peptide,which accounted for 8.78%.4.There were a number of differentially expressed proteins involved metabolic process and cellular process in biological process,binding and catalytic in molecular function,and organelle and cell in cellular component.5.There were 83 differentially expressed proteins localized to 48 metabolic pathways.29 involved in purine metabolism and 5 involved in pyrimidine metabolism,and 7 Biosynthesis of antibiotics are involved in.Furthermore,the tpi1,HEL-S-133 P,SEPN1 and AKR1B10 are also involved in Glycolysis.5 then involved of Inositol phosphate metabolism,while 2 in Ribosome.6.AS-IV up-regulated YAP1 and ACTG1 through Hippo signaling pathway.7.AS-IV up-regulated VTN and COL1A1 through the ECM-receptor interaction pathway.Conclusion: 1.The protective effects of AS-IV on the apoptosis of IL-1?-induced sw1353 cells may be closely related to the intracellular responses and the actions of phosphorylations and acetylations.A large number of differentially expressed proteins are employed for the process of biological processes,molecular functions and cellular localization.2.AS-IV up-regulated the YAP and ACTG1 and it plays a positive role in the apoptosis of human osteoarthritic chondrocytes and protects the cytoskeleton and maintains the morphology of them possibly through modulating the Hippo signaling pathway.3.AS-IV up-regulated the VTN and COL1A1,and it indicates AS-IV reduce the degradation of ECM,promote cell secretion of collagen,repairs the cartilage extracellular matrix through modulating the ECM-receptor interaction pathway.4.The protective effects of AS-IV on cartilage cell apoptosis may be achieved by multi signaling pathways and multi targets.