Ubiquitin Ligase NEDD4L And MEKK2 Participate In The Release Of Monocyte Inflammatory Factors In Sepsis

Objective:Through bioinformatics analysis,collection and isolation of peripheral blood mononuclear cells from patients with sepsis,THP-1 cells were used to construct a sepsis cell model,and the relationship between the expression of NEDD4 L and MEKK2 and the release of inflammatory factors in sepsis was explored.Provide evidence for therapeutic targets.Methods:1.Through the GSE28750 dataset of the GEO database,use the R language bioinformatics analysis tool Sangerbox to obtain all the gene expression information in the peripheral blood samples of 10 sepsis patients and 20 healthy patients in the database.Sangerbox3.0 was used to screen differential genes,and KEGG enrichment analysis was used to analyze the related signaling pathways of differential genes.2.Collect 5ML of peripheral blood from 20 patients with sepsis in the EICU of the First Affiliated Hospital of Nanchang University and ten healthy subjects,divide them into normal control group and experimental group,and separate PBMC(human peripheral mononuclear nucleus)by density centrifugation using lymphatic separation fluid.cells),the expression of IL-6,TNF-α,NEDD4 L and MEKK2 was detected by q-PCR,and the correlation between NEDD4 L,MEKK2 and inflammatory factors was analyzed by spearman correlation analysis.3.Human leukemic mononuclear cells(THP-1)were inoculated into T25 culture flasks.When the cells grew to a certain amount,they were stimulated with different concentrations of LPS to construct in vitro cell models.Grouping(1)Control group:no treatment;(2)Experimental group: stimulation with LPS concentration of 50 ng.Q-PCR and other experimental methods were used to detect the expression of NEDD4 L,MEKK2,IL-6,and TNF-α at 6H,12 H,and 24 H.4.Synthesize specific interfering RNA(si-NEDD4L),down-regulate the expression of NEDD4 L in sepsis models,and explore whether NEDD4 L is involved in the inflammatory release of THP-1 cells.The THP-1 cells that have silenced NEDD4 L were added with 50 ng/ml LPS to stimulate 12 H.Q-PCR detection: the expression of IL-6 and TNF-α.5.Upregulation of NEDD4 L by ALCAP2.Q-PCR and Western Blot detected the expressions of NEDD4 L and MEKK2.Transfection of si-NEDD4 L silenced NEDD4 L in THP-1 cells.THP-1 cells were stimulated with 50 ng/ml of LPS.Q-PCR detected the expression of NEDD4 L and MEKK2 at 0H,6H and 12 H.6.Treat the sepsis cell model with S20 and S26 proteasome inhibitors to inhibit the ubiquitin degradation pathway and detect the expression of MEKK2 by Western Blot.Results:1.Multiple sepsis-related genes were screened out by sangerbox3.0,and NEDD4 l meets the criteria for differential genes,and its role in sepsis is worthy of further study.GO and KEGG enrichment analysis found that the pathogenesis of sepsis was related to the MAPK signaling pathway and multiple inflammatory and immune pathways.2.Compared with healthy people,the expression levels of monocytes IL-6,TNF-α,NEDD4 L,and MEKK2 in patients with sepsis were increased(P<0.05).The expression of NEDD4 L in patients with sepsis was negatively correlated with MEKK2,and NEDD4 L was negatively correlated with IL-6 and TNF-α.3.In the sepsis cell model,compared with the control group,the levels of IL-6,TNF-α,NEDD4 L,and MEKK2 in LPS-stimulated THP-1 cells were increased(P<0.05),and NEDD4 L The expression decreased,the expression of MEKK2 increased.The manifestation of IL-6 and TNF-α increased.4.Use si-NEDD4 L to down-regulate the expression of NEDD4 L.Compared with the control group,the expressions of IL-6 and TNF-α in the LPS+Si-NEDD4 L group were significantly increased(P<0.05),indicating that NEDD4 L may be involved in THP-1 release of cellular inflammatory factors.5.Using ALCAP2 to up-regulate NEDD4 L,compared with the control group,the expression of MEKK2,IL-6,and TNF-α decreased,with significant differences(P<0.05).Transferred into interfering RNA,the expression of NEDD4 L was downregulated,the expression of MEKK2 was increased,and IL-6 and TNF-α were increased,with significant differences(P<0.05).It indicated that NEDD4 L might affect the release of inflammatory cytokines by regulating MEKK2.6.Use cycloheximide(CHX)intracellular protein synthesis inhibitor and proteasome inhibitor MG132 to treat sepsis cell models and inhibit the ubiquitination degradation pathway.Compared with the stimulated group,MEKK2 decreased in the group not promoted with MG132,indicating that NEDD4 L may regulate MEKK2 through the ubiquitin-proteasome system.Conclusion:1.In bioinformatics analysis,NEDD4 L is a sepsis differential gene which is related to multiple inflammatory pathways in the pathogenesis of sepsis,among which the MAPK signaling pathway is most closely related2.In sepsis,NEDD4 L may regulate the release of inflammatory factors such as IL-6 and TNF-α in monocytes through ubiquitination and regulation of MEKK2.