UBE2L3 regulates microglia-mediated neuroinflammation after traumatic brain injury via the NF-κB/COX2 pathway
Objective To investigate the role of ubiquitin conjugating enzyme E2L3(UBE2L3)in Ml microglia-associated neuroinflammation following traumatic brain injury,along with its underlying mechanisms.Methods Sprague-Dawley rats were used to construct rat models of traumatic brain injury(TBI)and Sham operation;peptide histology was used to screen the differentially expressed peptides in cortical brain tissues around the traumatic lesions of the Sham group and the TBI group at 3 d.The UBE2L3 protein,which was down-regulated more significantly,was selected as a target.The expression of UBE2L3 in the brain tissues of TBI rats and different BV2 cell models was verified by protein western blotting(WB)and real-time fluorescence quantitative PCR(qPCR)respectively.UBE2L3 plasmid was transfected into BV2 cells and the cells were divided into negative control group(NC)and UBE2L3 overexpression group(OE-UBE2L3).The overexpression of the UBE2L3 gene was identified by qPCR and WB experiments.A lipopolysaccharide(LPS)-induced M1-type microglial neuroinflammation model was constructed,and the experiment was divided into NC group,OE-UBE2L3 group,LPS group and LPS-UBE2L3 group.The effects of UBE2L3 overexpression on microglia polarization and inflammatory factor expression were detected by immunofluorescence staining and qPCR experiments,respectively.The effects of UBE2L3 overexpression on the phosphorylation levels of cyclooxygenase 2(COX2)protein and nuclear factor κB(NF-κB)p65 protein in the NF-κB pathway were analyzed by WB assay.Results The results of peptidomics mass spectrometry analysis showed that the expression levels of peptides contained in UBE2L3 proteins in the brain tissues around the lesions of TBI rats were significantly lower than that of Sham group,with statistical significance(P=0.046).WB verification experiment showed that the expression level of UBE2L3 in BV2 cell neuroinflammation model was down-regulated at 12 h compared with 0 h,with statistical significance(P＜0.01).In the brain tissue of TBI rats,compared with Sham group,UBE2L3 expression level was down-regulated at day 1 after TBI(0.804±0.056 vs.1.394±0.263),and tended to be flat at day 7(0.558±0.024),with statistical significance(all P＜0.01).WB identification experiment showed that UBE2L3 overexpressed cell model was successfully constructed,and UBE2L3 expression level in OE-UBE2L3 group was higher than that in NC group(0.908±0.052 vs.0.362±0.080),the difference was statistically significant(P＜0.01).The experiment of qPCR showed that the expression levels of interleukin(IL)-1β,IL-6 and tumor necrosis factor α(TNF-α)in LPS group were higher than those in NC group,while the expression levels of IL-1β,IL-6 and TNF-α in LPS-UBE2L3 group were lower than those in LPS group,with statistical significance(all P＜0.01).The immunofluorescence experiment showed that the expression level of Ml standard substance iNOS in microglia in LPS group was higher than that in NC group,and the difference was statistically significant(P＜0.001).The WB test of the pathway protein showed that the expression level of COX2 protein in LPS-UBE2L3 group was lower than that in LPS group(0.460±0.280 vs.1.273±0.090),and the difference was statistically significant(P＜0.05).Phosphorylated NF-κB p65 expression level in LPS group was up-regulated compared with NC group(1.738±0.138 vs.0.614±0.192),and down-regulated compared with LPS group(0.924±0.207).The differences were statistically significant(both P＜0.05).Conclusion UBE2L3 can exert an anti-inflammatory function by inhibiting the polarization of microglia to the M1 phenotype after TBI through the modulation of the NF-κB/COX2 pathway,and it may serve as a potential therapeutic target for suppressing neuroinflammation after TBI.
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