丁酸钠通过TLR4/NF-κB信号通路调控脂多糖诱导的BV2小胶质细胞表型极化

Sodium butyrate regulates phenotypic polarization of BV2 microglia through TLR4/NF-κB signaling pathway

目的 研究丁酸钠(sodium butyrate, NaB)对脂多糖(lipopolysaccharide, LPS)诱导BV2小胶质细胞炎症表型的调控作用和分子机制。方法 不同浓度NaB单独及联合LPS干预BV2细胞,采用CCK-8法检测BV2细胞活力的变化,根据结果选择实验浓度。将BV2小胶质细胞分为对照(Control)组、LPS组、LPS+NaB低剂量组(0.125 mmol/L)和LPS+NaB高剂量组(0.25 mmol/L);NaB预处理过夜(17 h)后,采用LPS继续孵育24 h诱导BV2小胶质细胞炎症模型;GRIESS法检测细胞NO释放量,ELISA检测细胞培养液中细胞因子IL-1β、TNF-α和IL-10的含量;RT-qPCR检测细胞iNOS和CD206 mRNA水平;免疫荧光染色法检测细胞iNOS和CD206的平均荧光强度;Western blot检测细胞中TLR4、NF-κB p65、IKB-α和p-IKB-α表达水平,分离胞浆和胞核蛋白,检测NF-κB p65的核转位。结果 GRIESS结果显示,高、低剂量NaB组的NO分泌水平较LPS组低(P<0.05);ELISA结果显示,高、低剂量NaB组中的IL-1β和TNF-α分泌水平较LPS组低(P<0.05),IL-10分泌水平较LPS组高(P<0.05);RT-qPCR结果显示,高、低剂量NaB显著下调了iNOS mRNA的表达(P<0.05),促进了CD206 mRNA的表达(P<0.05);免疫荧光结果显示,高、低剂量NaB可显著减少BV2细胞M1表型标志物iNOS的表达,促进M2表型标志物CD206的表达(P<0.05);Western blot结果显示,与LPS组相比,高、低剂量NaB显著抑制了TLR4、NF-κB p65和p-IKB-α的蛋白表达,同时抑制了NF-κB p65的核转位(P<0.05)。结论 NaB显著减轻LPS诱导的BV2细胞炎症反应,其机制可能与NaB抑制TLR4/NF-κB信号通路、促进BV2细胞由M1表型向M2表型极化有关。

Objective To explore the regulatory role and molecular mechanism of sodium butyrate(NaB)in lipopolysaccharide(LPS)induced inflammatory phenotype in BV2 microglia cells and the molecular mechanism.Methods BV2 cells were treated with different concentrations of NaB alone or combined with LPS.CCK8 assay was used to detect the change of cell viability and to screen the optimal concentration of the drug.Then the BV2 cells were divided into control,LPS group,low-and high-dose NaB groups(0.125 and 0.25 mmol/L).An inflammatory model of BV2 cells were established by pretreatment with NaB for 17 h followed by LPS treatment for 24 h.Griess assay was used to measure the NO content in the supernatant,and ELISA was employed to determine the contents of IL-1β,TNF-αand IL-10 in culture medium.The mRNA levels of iNOS and CD206 were detected by RT-qPCR.Immunofluorescence staining was used to observe the expression of iNOS and CD206.The protein levels of TLR4,NF-κB p65m,IKB-αand p-IKB-αwere detected by Western blotting.Cytoplasmic and nuclear proteins were isolated to detect the nuclear translocation of NF-κB p65.Results Griess assay showed that the secretion of NO in the high-and low-dose NaB groups were lower than that in the LPS group(P0.05).ELISA results indicated that the high-and low-dose NaB groups had lower secretion levels of IL-1βand TNF-α,but high secretion level of IL-10 than the LPS model group(all P0.05).RT-qPCR revealed that high-and low-dose NaB treatment significantly decreased the mRNA expression of iNOS(M1 phenotypic marker)and promoted that of CD206(M2 phenotypic marker)(both P0.05).Immunofluorescence staining presented similar results as RT-qPCR(P0.05).Western blot results displayed that high-and low-dose NaB resulted in significantly inhibited protein expression of TLR4,NF-κB p65 and p-IKB-αas well as nuclear translocation of NF-κB p65 when compared with the LPS group(P0.05).Conclusion NaB significantly reduces LPS-induced neuroinflammatory responses in BV2 cells,which might be associated with its inhibition of TLR4/NF-κB signaling pathway and promotion of BV2 cells shifting from M1 to M2 phenotype.