Fig. 5

High concentrations of 17β-Estradiol (hE2) promote microglial response through the NF-κB signaling pathway. A Volcano plot of protein abundances changes in OVX vs. OVX + sE2 mice brians, based on proteomics analysis. B KEGG pathway enrichment analysis of differentially expressed proteins. QNZ is used as an inhibitor of NF-κB activation. The effects of hE2 on CD86 protein expression levels and NF-κB activation are analyzed. C Western blot analysis was used to examine the effects of E2 or QNZ of different concentrations on NF-κB activation in primary microglia. D Typical images of microglia M1 polarization markers (CD86 labeled) and NF-κB signaling pathway (p65) co-located in BV2 cells by immunofluorescence. Bar = 15 μm. E The relative quantification of CD86 and NF-κB-stained positive cells. Nuclear translocation of NF-κB is determined by immunofluorescence microscopy. F Representative band (up) and quantification (down) of protein expression in primary microglia. Typical images of microglia (Iba1-labeled) and NF-κB signaling pathway (IL-1 and TNFα) co-located in BV2 cells by immunofluorescence (G, I). Bar = 15 μm. H Quantification of the mean fluorescence intensity per cell with the indicated number of cells. The mRNA levels of proinflammatory factors IL-1β, IL-6, and TNF-α, and anti-inflammatory factors IL-4, IL-10, and TGF-β are evaluated by qPCR in primary microglia (K) or BV2 cells (J). Student’s t-test is performed to determine the significant difference based on P < 0.05 (*), P < 0.01 (**), and P < 0.001 (***), respectively, in comparison to the control group as indicated by black asterisks or “ns,” to the E2 (High) group as indicated by red asterisks, and to the siRNA negative group as indicated by symbol “#.” ns: no statistical significance. Data are presented as mean ± the standard deviation (SD). Each experiment is repeated independently three