The Molecular Mechanism Regulated By Deubiquitinase USP14 In Septic Inflammation

[Background]Sepsis is a common clinical syndrome in acute and severe areas.It involves systemic inflammatory reactions,clinical treatment is difficult,and mortality is high.At present,there is still a lack of clear understanding of the pathogenesis and pathophysiological processes of sepsis.After going through many efforts and explorations,specific drugs or therapies for sepsis haven't been found given.people are still confused about the pathogenesis of sepsis.As well as the enormous pressure resulted from clinical treatment,it has extraordinary urgency and important practical significance for people to thoroughly explore the pathogenesis of sepsis and the new direction of septic treatment.The Ubiquitin-proteasome System(UPS)mediates the degradation of most proteins in eukaryotic cells,and it has a variety of biological functions such as regulation of inflammation,cell proliferation,signal transduction,transcription regulation,and apoptosis.At present,studying the mechanism of inflammatory response in immune cells is a new hotspot.The deubiquitinase USP14 is an important regulatory enzyme of intracellular UPS that is involved in the process of ubiquitin-mediated proteolytic degradation of proteins.It has been widely studied in tumors,diseases of the nervous system and aging,but its role in inflammation is rarely reported.Our study will describe the role and molecular mechanisms of USP14 in septic inflammation and provide new potential targets for the treatment of sepsis.[Methods]1.The lipopolysaccharide(LPS)-induced human acute mononuclear leukemia cell THP-1 and mouse-derived macrophage RAW264.7 were used to construct an in vitro inflammatory cell model of sepsis.2.Different concentrations of LPS(0,100,250,500 ng/ml)were given to act on THP-1 cells.After 12,24h,western blotting was used to detect the effect of LPS on the expression of USP14 protein in the cells.3.Different methods were used to block the deubiquitination of intracellular USP14(IU1 inhibited the deubiquitinating enzyme activity of USP14,siRNA interfered with the transcriptional expression of USP14),and then studied the role and molecular mechanism of USP14 in septic inflammation.4.THP-1 inflammatory cell model was established.THP-1 cells were pretreated with USP14 specific inhibitor IU1 for 2 h.Cell viability was measured by MTS,and apoptosis was detected by flow cytometry after Annexin V-FITC/PI double staining;The release amount of pro-inflammatory cytokines(TNF-?,IL-6)in cell culture supernatants was detected by ELISA;the expression level of pro-inflammatory cytokines(TNF-?,IL-6)mRNA was detected by qRT-PCR;The expression of MAPK and NF-?B pathway-associated proteinwas was detected by Western blotting as well as nuclear translocation of NF-?B P65.The nuclear translocation of NF-?B P65 was further observed by immunofluorescence confocal microscopy.5.THP-1 inflammatory cell model was established.THP-1 cells were pretreated with liposome(Lipofectamine 3000)transfected with USP14 siRNA for 48 h.Cell viability was measured by MTS,The expression level of pro-inflammatory cytokines(TNF-a,IL-6)in cell culture supernatants was detected by ELISA;Western blotting was not used to detect the expression of MAPK and NF-?B pathway-related proteins but used to detect the transformation about nuclear translocation of NF-?B P65.6.RAW264.7 inflammatory cell model was established.IU1 was used to pretreat RAW264.7 cells for 2 hours.The activity of USP14 in cells was inhibited.Cell viability was measured by MTS;The amount of release(TNF-?,IL-6)in cell culture supernatantswere was detected by ELISA;qRT-PCR detection of cell pro-inflammatory cytokines(TNF-a,IL-6)mRNA expression;Western blotting was used to detecting MAPK,NF-?B pathway-related protein expression and nuclear translocation of NF-?B P65.[Results]1.LPS-induced THP-1 cells and RAW264.7 cells can successfully construct an in vitro inflammatory cell model of sepsis,LPS group compared with the control group,pro-inflammatory cytokines(TNF-a,IL-6)expression was significantly increased.2.Different concentrations LPS(0,100,250,500 ng/ml)treated with THP-1 cells for 12 and 24 h,compared with the control group,LPS did not affect the expression of USP14 protein in cells.3.IU1 had no significant effect on the survival rate of cells in the range of 100?M.There was no significant difference in apoptosis between the two groups compared with the control group.4.Transfection of siRNA(40nM)for 48 h had little effect on cell viability,and western blotting showed that the protein expression of USP14 was significantly down-regulated,and better knockdown efficiency was obtained.5.IU1 inhibited the activity of USP14.In the inflammatory models of THP-1 and RAW264.7 cells,the expression level of pro-inflammatory cytokines(TNF-a,IL-6)mRNA was significantly decreased by qRT-PCR,its anti-inflammatory effect was the same as ELISA assay.In addition,the phosphorylation of ERK1/2 was reduced,but the phosphorylation levels of JNK and P38 were not significantly changed;at the same time,the phosphorylation of I?B? was reduced,which increased the expression of I?B? and inhibited the expression of nuclear NF-?B p65.6.siRNA interferes with the transcriptional expression of USP14,and can effectively inhibit the release of pro-inflammatory cytokines(TNF-a,IL-6)in the THP-1 cell inflammation model;meanwhile,it also reduces the phosphorylation of ERK1/2 and I?B?.Increasing the expression of I?B? affects the nuclear translocation of NF-?B p65.[Conclusion]Ubiquitin-specific protease 14 regulates LPS-induced inflammation by increasing ERK1/2 phosphorylation and NF-?B activation.