ITRAQ-based Quantitative Proteomic Of Related Protein Expression In Early Endotoxin Tolerance Induced By Lipopolysaccharide

0bjective: To establish a mouse model of RAW264.7 cells endotoxin tolerance and sepsis.The mechanism of endotoxin tolerance at early stage was explored by analysis of the expressed proteins in the state of endotoxin tolerance and sepsis with iTRAQ technique.Methods: The mouse macrophage strain RAW264.7 was cultured in DMEM medium supplemented with 10%fetal bovine serum which also called complete medium in vitro.The logarithmic phase cells were counted,adjusted to 5×105/ml,inoculated into6 cm culture dishes.Cells were randomly divided into two groups: endotoxin tolerance group(tolerance group)and sepsis group.Two groups were cultured with complete medium for 24 h.Then replacement of fresh medium after 24 h,the endotoxin tolerance group was cultured with 10ng/ml LPS complete medium 3ml as a pretreatment for 24 h.The sepsis group was cultured in complete medium for 24 h.The two groups were replaced with fresh complete medium after 24 h and stimulated with the complete medium containing100ng/ml LPS for 4h and 24 h.After 4h,the cells and the culture supernatant were collected and stored in the refrigerator at-80?.During the experiment,cells morphology was recorded using an inverted microscope.IL-6,TNF-? and IL-10 were detected by ELISA in culture supernatant.The expression of CD16/32 in macrophages membrane was measured by flow cytometry to identify macrophage phenotype.The proteins were isolated from the cells,andiTRAQ technique was used to identify differential proteins,then used bioinformatics analysis including GO,KEGG signaling pathway,proteinprotein interaction network,and veen diagram.After 24 h,then the expression of CD16/32 in macrophages was measured by flow cytometry to identify macrophage phenotype.The cells handling and the samples were taken 3replicates.Collecting and analyzing the data and drawing conclusions.Result:Compared with sepsis group,the tolerance group being pretreated was significantly more activity,the macrophages were more prominent,the adherent was more closely,the surface area becomes larger.The levels of IL-6,TNF-?and IL-10 were evaluated by ELISA: The levels of TNF-?(18273.5±10154.4pg/ml)in tolerance group were significantly lower than sepsis group(133233.7±68955.9pg/ml)(P<0.05).The levels of IL-6(1549.8±399.2pg/ml)in tolerance group were significantly decreased compared with sepsis group(3175.8±959.0pg/ml)(P<0.05).While the level of IL-10 in endotoxin tolerance group(351.1±184.2pg/ml)was slight higher than that in sepsis group(220.3±121.4pg/ml)(P>0.05).Macrophages phenotype was identify by flow cytometry: Cells were stimulated for 4h,the expression of CD16/32 in macrophages from the endotoxin tolerance group(75.33%)was higher than that in sepsis group(49.69%),P<0.05.However cells were stimulated for 24 h,the expression of CD16/32 in macrophages from the endotoxin tolerance group(79.07%)was lower than that in sepsis group(94.27%),P<0.05,the expression of CD16/32 in both sepsis group and endotoxin tolerance group were higher than that in negative control group(54.11%),(P<0.05).A total of 4086 proteins were identified using iTRAQ technique.There were 63 differentially expressed proteins in the endotoxin tolerance group compared with the sepsis group,of which 36 proteins were up-regulated(ratio>1.2)and 27 proteins was down regulated(ratio<0.833).Bioinformatics analysis those proteins: The gene ontologe analysis showed that the biological processes of differential proteins were mainly including the growth and development of organs,the regulation of biological quality,and the response to external stimuli.And the molecular function mainly includes the oxidoreductase activity,receptor binding,DNA binding and oxidoreductase activity.Then the cellular component mainly consist the chromosome,extracellular region,vesicle,cytoplasmic vesicle.KEGG signaling pathway analysis of 63 proteins involved in 112 pathways,of which 27 pathways were important(P<0.05),include TLR signaling pathway,NF-?B signaling pathway,TNF signaling pathway and PPAR signaling pathway.HMGA1,HMGA2,HMGB1 and HMGB2 proteins involved in protein-protein interaction network.There were 35 proteins in the venn diagram which found in the intersection,of which 21 proteins were up-regulated and the other 14 proteins were down regulated.Conclusion: We pretreated the mouse macrophage strain RAW264.7with 10ng/ml LPS as a pretreatment for 24 h and then challenged with 100ng/ml LPS for 4h could induce an early stage of endotoxin tolerance model.In early endotoxin tolerance state,the pro-inflammatory cytokine IL-6,TNF-? level decreased and the anti-inflammatory cytokine IL-10 level was slight increased.ITRAQ technology could be used to identify proteins and screening of earlydifferential proteins,and bioinformatics analysis those proteins which may be associated with early endotoxin tolerance.It may help to sum up protein function,the cell protein components and the possible signaling pathways which may be correlated with endotoxin tolerance.