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Additional file 1: Fig. S1. Body weight changes in mice receiving radiation or pregabalin. Body weight changes in mice after 14 days of continuous injection of pregabalin (PGB) or saline solution (Con) in RIBI mice. For days 1–4 post-treatment, n = 14–23 mice per group. For days 5–8 post-treatment, n = 10–15 mice per group. For days 9–14 post-treatment, n = 4–9 mice per group. Fig. S2. Microglial body size and CD68 expression changes after radiation in vivo. A Representative confocal images of IBA1 and CD68 co-labeling in the cortex of mice 3, 7, or 14 days after radiation. Red: IBA1, green: CD68. B-C Quantification of the body size of IBA1+ cells and the proportion of CD68+ area / IBA1+ area in the cortex. Data were analyzed by one-way ANOVA followed by the Student’s t-test analysis. All other groups were compared with the control group. n = 4 mice per group and 2–3 slices per mouse for immunofluorescence staining. Data were presented as mean ± SEM, *p < 0.05, **p < 0.01, and ***p < 0.001. Fig. S3. Pregabalin inhibited microglia activation in the cortex of RIBI mice. A Representative images of IBA1 and CD68 co-labeling in the cortex of mice 3 days after radiation. Red: IBA1, green: CD68. B Quantification of the proportion of CD68+ area / IBA1+ area in the cortex of mice 3 days after radiation. C Representative images of IBA1 and CD68 co-labeling in the cortex of mice 7 days after radiation. D Quantification of the proportion of CD68+ area / IBA1+ area in the cortex of mice 7 days after radiation. Data were analyzed by one-way ANOVA followed by the Student’s t-test analysis. All other groups were compared with the indicated group. n = 4 mice per group and 2–3 slices per mouse for immunofluorescence staining. Data were presented as mean ± SEM, *p < 0.05, **p < 0.01, and ***p < 0.001. Fig. S4. Pregabalin inhibited microglia activation in the hippocampus of RIBI mice. A-B Representative confocal images of IBA1 and CD68 co-labeling in the hippocampal CA1 (A) and DG (B) regions of RIBI mice 14 days after pregabalin treatment. Red: IBA1, green: CD68, and blue: DAPI. n = 4 mice per group and 2–3 slices per mouse for immunofluorescence staining. See Fig. 1I-J for statistical data in the main text. Fig. S5. Effect of pregabalin on microglial inflammatory response induced by radiation in vitro. A-D Q-PCR analysis the effects of pregabalin, with different concentration (1 µM, 6.25 µM, 12.5 µM, 25 µM, and 50 µM), on the mRNA levels of inflammatory factors Il-1β, Tnf-α, Cox-2, and iNos in BV2 cells after a single dose of 10 Gy radiation. Data were analyzed by one-way ANOVA followed by the Student’s t-test analysis. All other groups were compared with the indicated group. n = 3 per group for Q-PCR analysis in vitro. Data were presented as mean ± SEM, ns = not significant, **p < 0.01, and ***p < 0.001. Fig. S6. Effect of pregabalin on IL-6 and TNF-α expressions in microglia after radiation. A Representative immunofluorescent images of IL-6 and β-tubulin in BV2 cells among the different groups. Staining with β-tubulin to visualize cytoskeleton and staining with DAPI to visualize nucleus. B The fluorescence intensity data of IL-6 were recorded by confocal microscopy. C Representative immunofluorescent images of TNF-α and β-tubulin in BV2 cells among the different groups. D The fluorescence intensity data of TNF-α were recorded by confocal microscopy. Data were analyzed by one-way ANOVA followed by the Student’s t-test analysis. All other groups were compared with the indicated group. n = 3 per group for immunofluorescence staining in vitro. Data were presented as mean ± SEM, ns = not significant and **p < 0.01. Fig. S7. Pregabalin inhibited microglial inflammatory response not by acting on astrocyte in vitro. A Schematic diagram of BV2 cells incubated with the culture supernatant from astrocyte after different treatment. B-E Q-PCR analysis of Il-1β, Tnf-α, iNos, and Icam-1 mRNA levels in BV2 cells after incubated with the supernatant from pregabalin-treated astrocyte. Data were analyzed by one-way ANOVA followed by the Student’s t-test analysis. All other groups were compared with the indicated group. n = 4 per group for Q-PCR analysis in vitro. Data were presented as mean ± SEM, ns = not significant and *p < 0.05. Fig. S8. Effect of pregabalin on potential chemokines in injured neurons. A-G Q-PCR analysis of Mmp9, Cgrp, Tac1, Cx3cl1, Ccl21, Mmp2, and Ccl2 mRNA levels in neurons after treatment with the different supernatant from BV2 cells. Data were analyzed by one-way ANOVA followed by the Student’s t-test analysis. All other groups were compared with the indicated group. n = 3 per group for Q-PCR analysis in vitro. Data were presented as mean ± SEM, ns = not significant, *p < 0.05, and ***p < 0.001. Fig. S9. Knocking out TLR2/TLR4/RAGE mitigated microglia activation. A-C Schematic diagram of CRISPR/Cas9-mediated TLR2/TLR4/RAGE knockout in BV2 cells and Q-PCR analysis was used to detect the knockout efficiency. D-F Q-PCR analysis of Il-6, Tnf-α, and Cox-2 mRNA levels in activated BV2 cells which were treated with culture supernatant from radiation-injured (activated) or normal (control) neurons for 24 h. Data were analyzed by one-way ANOVA followed by the Student’s t-test analysis. All other groups were compared with the indicated group. n = 3–4 per group for Q-PCR analysis in vitro. Data were presented as mean ± SEM, *p < 0.05, **p < 0.01, and ***p < 0.001. Table S1. List of primers used for RNA analyses. Table S2. List of antibodies used in this study. Table S3. List of gRNA sequences used for CRISPR/Cas9-mediated gene knockout. Table S4. List of oligonucleotide sequences used for plasmid construction. Table S5. List of primers used for the verification of CRISPR/Cas9-mediated gene knockout. |
