鹅去氧胆酸抗脂多糖诱导的BV2小胶质细胞炎症反应作用及机制

Anti-inflammatory effect and mechanism of chenodeoxycholic acid on microglia cell BV2 induced by lipopolysaccharides

目的研究鹅去氧胆酸(CDCA)抗脂多糖(LPS)诱导的BV2小胶质细胞炎症反应的作用及其机制。方法BV2细胞与CDCA 25~100μmol·L-1预孵育2 h,加LPS 200 mg·L-1继续培养22 h,采用Griess试剂法检测一氧化氮(NO)含量;Western印迹法检测细胞内环氧合酶2(COX-2)和诱导型一氧化氮合酶(iNOS)蛋白表达水平;RT-PCR法检测细胞肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)、IL-1β和G蛋白偶联受体5(TGR5)mRNA表达水平。BV2细胞与CDCA 25~100μmol·L-1预孵育2 h,加入LPS 200 mg·L-1继续培养1 h,Western印迹法检测NF-κB、NF-κB抑制蛋白α(IκBα)和丝氨酸/苏氨酸蛋白激酶(Akt)磷酸化水平;细胞免疫荧光法观察NF-κB入核情况。结果与正常对照组相比,模型组培养基中NO含量显著增加(P<0.01);COX-2和iNOS蛋白表达水平升高(P<0.05,P<0.01);TNF-α,IL-6和IL-1βmRNA表达显著上调(P<0.01);TGR5 mRNA表达显著下调(P<0.01);并且NF-κB,IκBα和AKT磷酸化水平显著增加(P<0.05,P<0.01),NF-κB入核增多。与模型组相比,CDCA显著减少培养基中NO含量(P<0.01),降低COX-2和iNOS蛋白表达水平(P<0.05,P<0.01);显著下调TNF-α,IL-6和IL-1βmRNA表达(P<0.01);显著上调TGR5 mRNA表达(P<0.01)。与模型组相比,CDCA显著降低NF-κB,IκBα和AKT磷酸化水平(P<0.05,P<0.01),并观察到NF-κB核转位减少现象。结论CDCA可显著抑制LPS诱导BV2细胞炎症反应,其作用机制可能与激活TGR5、抑制Akt/NF-κB信号通路相关。

OBJECTIVE To explore the anti-inflammatory effect and mechanism of chenodeoxycholic acid(CDCA)on lipopolysaccharides(LPS)-induced microglia cell BV2.METHODS BV2 cells were pre-treated with CDCA 25-100μmol·L-1 for 2 h,and then incubated with LPS 200 mg·L-1 for 22 h.Nitric oxide(NO)content in the medium was detected by Griess reagent.Protein expressions of cyclooxygenase-2(COX-2)and induced nitric oxide synthase(iNOS)were examined by Western blotting.Tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),IL-1βand G-protein coupled receptor 5(TGR5)mRNA expressions were evaluated by quantitative PCR.BV2 cells were pre-treated with CDCA for 2 h,and then stimulated with LPS 200 mg·L-1 for 1 h.Phosphorylation of NF-κB,inhibitor of NF-κBα(IκBα)and serine/threonine protein kinase(Akt)was analyzed by Western blotting,while nuclear translocation of NF-κB was examined by immunocytochemistry.RESULTS Compared with the normal control group,LPS stimulation significantly increased the production of NO and the expressions of COX-2 and iNOS in cells of the model group(P<0.05,P<0.01).Meanwhile,LPS stimulation elevated the mRNA expression levels of TNF-α,IL-6,and IL-1β(P<0.01)but decreased that of TGR5(P<0.01).LPS stimulation increased the phosphorylation of NF-κB,IκBα,and Akt(P<0.05,P<0.01),which was accompanied by increased nuclear translocation of NF-κB.Compared with the model group,CDCA significantly reduced the NO content in the medium,and decreased the expression levels of COX-2 and iNOS(P<0.01).It also significantly decreased the mRNA expression levels of TNF-α,IL-6,and IL-1β(P<0.01),but increased that of TGR5(P<0.01).Further study disclosed that CDCA remarkably reduced the phosophorylation of NF-κB,IκBα,and Akt(P<0.05,P<0.01)and nuclear translocation of NF-κB was observed.CONCLUSION CDCA can significantly inhibit LPS-induced inflammation in BV2 cells,which might be related to the inhibition of Akt/NF-κB signaling pathway by activating TGR5.