Fig 1: S14G humanin (HNG) ameliorated the inflammation in lung tissues of ALI mice. (A) mRNA level of MPO, mRNA level of IL-6, and mRNA level of TNF-α. (B) The protein level of MPO. (C) The protein level of IL-6. (D) The protein level of TNF-α (n=6, **, P<0.05 vs. Control; &, P<0.05, 0.01 vs. LPS group).
Fig 2: Immunofluorescence analysis of TNF-α and IL-6 expression in hippocampal microglia. A Representative images of TNF-αimmunofluorescence. B Representative images of IL-6 immunofluorescence. C Quantification of mean TNF-α fluorescence intensity (n = 4 mice/group). D Quantification of mean IL-6 fluorescence intensity (n = 4 mice/group). Data are shown as mean ± SEM; ***p ≤ 0.001, ****p ≤ 0.0001
Fig 3: Analysis of microglia-associated, inflammation-related cytokine expression in the hippocampus of LPS-treated mice. A–D RT-qPCR data for M1 markers: A TLR4 (Ctrl group, n = 4 mice; LPS group, n = 5 mice); B IL-1β; C TNF-α; D IL-6 (B-D, n = 5 mice/group). E, F RT-qPCR data for M2 markers (n = 5 mice/group): E IL-10; F IL-4. Data are shown as mean ± SEM; **p ≤ 0.01, ***p ≤ 0.001
Fig 4: Effect of J147 on expression of proinflammatory cytokines in LPS-induced BV2 cells. BV2 cells were pretreated with J147 (2.5 μM) for 2 h before incubation with LPS (1 μg/mL) for 24 h. a–d Effect of J147 on mRNA expression of IL-6 (one way ANOVA, F(3,20) = 3.539, p = 0.0333), IL-1β (one way ANOVA, F(3,20) = 9.469, p = 0.0004), and TNF-α (one way ANOVA, F(3,20) = 5.557, p = 0.0061) in BV2 cells. e Protein expression of IL-6 (one way ANOVA, F(3,20) = 59.12, p < 0.0001) and TNF-α (one way ANOVA, F(3,20) = 46.46, p < 0.0001) was determined using ELISA kits. Columns present the mean ± SEM (n = 6 per group). ANOVA followed by Bonferroni post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001
Fig 5: CD137 stimulation induces M1 polarization in microglia in vitro. A Representative images of TNF-α and Iba1 dual-immunofluorescence in cultured BV2 microglia alternatively treated with IgG1-Fc isotype control (FC) or recombinant CD137-Fc protein (FC-CD137). B Quantification of TNF-α mean fluorescence intensity (n = 11/group). C, D ELISA-based analysis of TNF-α (C) and CD137L (D) levels in culture media from BV2 cells. (n = 3–6/group). E Detection of CD137L mRNA levels in BV2 microglia by RT-qPCR (n = 5/group). F Detection of M1 phenotype markers (TNF-α, IL-1β, and IL-6) in cultured BV2 microglia by RT-qPCR (n = 6/group). G Detection of M2 phenotype markers (IL-10, IL-4, and YM-1) in cultured BV2 microglia by RT-qPCR (n = 3–6/group). Data are shown as mean ± SEM; *p ≤ 0.05; **p ≤ 0.01; ****p ≤ 0.0001
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