泛素结合酶E2L3通过NF-κB/COX2通路调节颅脑创伤后小胶质细胞介导神经炎症的实验研究

UBE2L3 regulates microglia-mediated neuroinflammation after traumatic brain injury via the NF-κB/COX2 pathway

目的探讨泛素结合酶E2L3(UBE2L3)对颅脑创伤(TBI)后小胶质细胞相关神经炎症的作用及其潜在机制。方法采用SD大鼠构建TBI及假手术(Sham)组大鼠模型,通过多肽组学筛选Sham组和TBI组建模3 d时创伤病灶周围皮质脑组织差异表达的肽段,并选取下调显著的UBE2L3蛋白作为研究对象;分别通过蛋白免疫印迹法(WB)和实时荧光定量PCR(qPCR)实验验证TBI大鼠脑组织和不同BV2细胞模型中UBE2L3的表达情况。将UBE2L3质粒转染至BV2细胞中并将细胞分为阴性对照组(NC)、UBE2L3过表达组(OE-UBE2L3),采用qPCR和WB实验鉴定UBE2L3基因的过表达情况。构建脂多糖(LPS)诱导的M1型小胶质细胞神经炎症模型,将细胞分为NC组、OE-UBE2L3组、LPS组和LPS-UBE2L3组,分别通过免疫荧光染色和qPCR实验检测UBE2L3过表达对小胶质细胞极化、炎症因子表达的影响;随后通过WB实验分析UBE2L3过表达对NF-κB通路中COX2蛋白和NF-κB p65蛋白磷酸化水平的影响。结果多肽组学质谱分析结果显示,TBI大鼠病灶周围脑皮质组织中UBE2L3蛋白的表达水平较Sham组降低,差异有统计学意义(P=0.046)。WB验证实验显示,在BV2细胞神经炎症模型中,UBE2L3的表达水平在12 h时较0 h时下调,差异有统计学意义(P<0.01);在TBI大鼠脑组织中,UBE2L3的表达水平在TBI后1 d时较Sham组下调(0.804±0.056对比1.394±0.263),在7 d时(0.558±0.024)表达趋于平缓,差异均有统计学意义(均P<0.01)。WB鉴定实验显示成功构建UBE2L3过表达细胞模型,OE-UBE2L3组UBE2L3表达水平较NC组升高(0.908±0.052对比0.362±0.080),差异有统计学意义(P<0.01)。qPCR实验显示,LPS组白细胞介素1β(IL-1β)、白细胞介素6(IL-6)、肿瘤坏死因子-α(TNF-α)表达水平较NC组均升高,而LPS-UBE2L3组IL-1β、IL-6、TNF-α表达水平较LPS组均下调,差异均有统计学意义(均P<0.01)。免疫荧光实验显示,LPS组小胶质细胞M1型标准物诱导性一氧化氮合酶(iNOS)的表达水平较NC组升高,差异有统计学意义(P<0.001)。通路蛋白WB检测结果显示,LPS-UBE2L3组环氧合酶2(COX2)蛋白的表达水平较LPS组降低(0.460±0.280对比1.273±0.090),差异有统计学意义(P<0.05);LPS组磷酸化的核因子-κB(NF-κB)p65表达水平较NC组上调(1.738±0.138对比0.614±0.192),而LPS-UBE2L3组磷酸化的NF-κB p65表达水平(0.924±0.207)较LPS组下调,差异均有统计学意义(均P<0.05)。结论UBE2L3可通过调控NF-κB/COX2通路抑制TBI后小胶质细胞向M1表型极化而发挥抑制神经炎症功能,可作为TBI后抑制神经炎症的潜在治疗靶点。

Objective To investigate the role of ubiquitin conjugating enzyme E2L3(UBE2L3)in M1 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 factorκB(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 factorα(TNF-α)in LPS group were higher than those in NC group,while the expression levels of IL-1β,IL-6 and TNF-αin 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 M1 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.