黄芩苷通过TREM2/TLR4/NF-κB信号通路抑制脂多糖/干扰素γ诱导的BV2细胞炎症反应

Baicalin inhibits LPS/IFN-γ-induced inflammation via TREM2/TLR4/NF-κB pathway in BV2 cells

基于2型髓样细胞触发受体(triggering receptor expressed on myeloid cells 2,TREM2)/Toll样受体4(Toll-like receptor 4,TLR4)/核因子κB(nuclear factor kappaB,NF-κB)信号通路,探讨黄芩苷对脂多糖(LPS)/干扰素γ(IFN-γ)诱导的小胶质细胞炎性活化的作用机制。通过LPS联合IFN-γ干预构建小鼠小胶质细胞(BV2)炎性活化模型,分为正常组、模型组、黄芩苷低剂量组(5μmol·L^(-1))、黄芩苷中剂量组(10μmol·L^(-1))、黄芩苷高剂量组(20μmol·L^(-1))和米诺环素组(10μmol·L^(-1))。CCK-8检测细胞存活率;明场下观察细胞形态;实时荧光定量PCR(quantitative real-time PCR,qPCR)法检测白细胞介素-1β(interleukin-1β,IL-1β)、白细胞介素-4(interleukin-4,IL-4)、诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)、白细胞介素-6(interleukin-6,IL-6)、白细胞介素-10(interleukin-10,IL-10)和精氨酸酶-1(arginase-1,Arg-1)mRNA表达;Western blot法检测肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、IL-1β、TREM2、TLR4、核因子抑制蛋白(inhibitor kappaB alpha,IκBα)、p-IκBα、NF-κB p65、p-NF-κB p65蛋白表达水平;细胞免疫荧光检测NF-κB p65的入核情况。与空白组比较,模型组BV2细胞大多数趋向于促炎症的M1阿米巴形态,IL-1β、IL-6和iNOS mRNA水平显著升高,IL-4、IL-10和Arg-1 mRNA水平显著降低(P<0.01),TNF-α、IL-1β、TLR4、p-IκBα和p-NF-κB p65蛋白表达均明显升高(P<0.01),TREM2蛋白表达水平下降,核内NF-κB p65表达升高;与模型组比较,黄芩苷和米诺环素干预各组BV2细胞形态呈现不同程度的恢复,IL-1β、IL-6和iNOS mRNA水平均明显降低,IL-4、IL-10和Arg-1 mRNA水平升高(P<0.01),TNF-α、IL-1β、TLR4、p-IκBα和p-NF-κB p65蛋白表达均明显降低(P<0.05),TREM2蛋白表达水平升高,核内NF-κB p65的表达降低。以上结果表明,黄芩苷可以通过调节小胶质细胞TREM2-TLR4之间的失衡,抑制下游NF-κB激活,从而促进小胶质细胞从促炎表型向抗炎表型的极化。

This study investigated the mechanism of baicalin on lipopolysaccharide(LPS)/interferonγ(IFN-γ)-induced inflammatory microglia based on the triggering receptor expressed on myeloid cells 2(TREM2)/Toll-like receptor 4(TLR4)/nuclear factor kappaB(NF-κB)pathway.Specifically,LPS and IFN-γwere used to induce inflammation in mouse microglia BV2 cells.Then the normal group,model group,low-dose(5μmol·L^(-1))baicalin group,medium-dose(10μmol·L^(-1))baicalin group,high-dose(20μmol·L^(-1))baicalin group,and minocycline(10μmol·L^(-1))group were designed.Cell viability was detected by CCK-8 assay and cell morphology was observed under bright field.The expression of interleukin-1β(IL-1β),interleukin-4(IL-4),inducible nitric oxide synthase(iNOS),interleukin-6(IL-6),interleukin-10(IL-10),and arginase-1(Arg-1)mRNA was detected by real-time quantitative PCR,the protein expression of tumor necrosis factor-α(TNF-α),IL-1β,TREM2,TLR4,inhibitor kappaB-alpha(IκBα),p-IκBα,NF-κB p65 and p-NF-κB p65 by Western blot,and transfer of NF-κB p65 from cytoplasm to nucleus by cellular immunofluorescence.Compared with the normal group,most of the BV2 cells in the model group tended to demonstrate the pro-inflammatory M1 amoeba morphology,and the model group showed significant increase in the mRNA levels of IL-1β,IL-6,and iNOS,decrease in the mRNA levels of IL-4,IL-10,and Arg-1(P<0.01),rise of the protein expression of TNF-α,IL-1β,TLR4,p-IκBα,and p-NF-κB p65(P<0.01),reduction in TREM2 protein expression,and increase in the expression of NF-κB p65 in nucleus.Compared with the model group,baicalin groups and minocycline group showed the recovery of BV2 cell morphology,significant decrease in the mRNA levels of IL-1β,IL-6 and iNOS,increase in the mRNA levels of IL-4,IL-10,and Arg-1(P<0.01),reduction in the protein expression of TNF-α,IL-1β,TLR4,p-IκBα,and p-NF-κB p65(P<0.05),rise of TREM2 protein expression,and decrease in the expression of NF-κB p65 in nucleus.In summary,these results suggest that baicalin can regulate the imbalance between TREM2 and TLR4 of microglia and inhibit the activation of downstream NF-κB,thus promoting the polarization of microglia from pro-inflammatory phenotype to anti-inflammatory phenotype.