Fig. 8
Restoration of O-GlcNAcylation inhibits astrocytes reactivation of AD model mice. a–d Immunostaining (a) and quantification results showed that GlcNAc administration significantly reduced GFAP immunostaining intensity (b), Aβ plaque area (c) and the number of GFAP+Aβ+ cells (d) in the hippocampus region of AD mice compared to Ctrl mice, respectively. 4–5 sections were picked up per animal and n = 5 mice per group. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA analysis followed by Tukey’s multiple-comparison test, F(2, 12) = 8.772 for b, F(2, 12) = 75.82 for c and F(2, 12) = 172.7 for d. e ELISA assay results show that the GlcNAc supplementation increased the levels of UDP-GlcNAc in the supernatants of hippocampal tissues of GlcNAc-treated AD mice compared with saline-treated AD mice. n = 4 mice per group. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA analysis followed by Tukey’s multiple-comparison test, F(2, 9) = 16.13. f–j WB assay (f) and quantification results showed that GlcNAc supplementation significantly restored the level of O-GlcNAcylation (g), and decreased the levels of GFAP (h), IL-1β (i) and TNF-α (j) of AD astrocytes compared to PBS-treated AD cells. n = 3 independent experiments. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA analysis followed by Tukey’s multiple-comparison test, F(2, 6) = 17.08 for (g), F(2, 6) = 24.8 for h, F(2, 6) = 108.5 for i, F(2, 6) = 102.3 for j. k–m ELISA assay results showed that the GlcNAc supplementation increased the level of UDP-GlcNAc (k), and reduced the levels of IL-1β (l) and TNF-α (m) in the supernatants of cultured AD astrocytes compared with PBS-treated AD astrocytes. n = 4 independent experiments for k and n = 3 independent experiments for l, m. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA analysis followed by Tukey’s multiple-comparison test, F(2, 9) = 12.46 for k, F(2, 6) = 100.2 for l, F(2, 6) = 327.7 for m. n Representative images of GFAP and p65 immunostaining with Ctrl and AD adult astrocytes treated with PBS (Ctrl and AD) and GlcNAc (20 μM, AD + GlcNAc) for 72 h, respectively. Scale bar, 50 μm. o Representative images of GFAP and p-p65 immunostaining with Ctrl and AD adult astrocytes treated with PBS (Ctrl and AD) and GlcNAc (20 μM, AD + GlcNAc) for 72 h, respectively. Scale bar, 50 μm. p–r WB assay (p) and quantification results showed that the GlcNAc supplementation did not affect the level of total p65 (q), but significantly increased the protein level of p-p65 (r) in AD astrocytes. n = 3 independent experiments. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA analysis followed by Tukey’s multiple-comparison test, F(2, 6) = 0.6736 for q, F(2, 6) = 100.1 for r. s–u WB assay (s) and quantification results showed that GlcNAc supplementation significantly restored the level of cytoplasmic p65 (t) and reduced the level of p-p65 in nucleus (u). n = 3 independent experiments. Values represent mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA analysis followed by Tukey’s multiple-comparison test, F(2, 6) = 23.04 for t, F(2, 6) = 63.16 for u