Fig. 4
Restoration of O-GlcNAcylation inhibits reactivation and inflammation of cKO astrocytes in vitro and in vivo. a ELISA assay results showed that the level of UDP-GlcNAc significantly decreased in the supernatants of cKO astrocytes compared with Ctrl astrocytes, and the GlcNAc replenishment significantly increased UDP-GlcNAc level of cKO astrocytes. n = 4 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, 9) = 53.04. b Representative images of GFAP and IL-1β immunostaining with adult Ctrl and cKO astrocytes treated with PBS (Ctrl and cKO) and O-GlcNAcylation substrate GlcNAc (20 μM, cKO + GlcNAc) for 72 h, respectively. Scale bar, 50 μm. c–g WB assay (c) and quantification results showed that GlcNAc supplementation significantly restored the decreased level of O-GlcNAcylation (d), and reduced the levels of GFAP (e), IL-1β (f) and TNF-α (g) of cKO astrocytes in vitro. 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) = 59.32 for d, F(2, 6) = 69.6 for e, F(2, 6) = 480.6 for f, F(2, 6) = 788.7 for g. h–j ELISA result showed that GlcNAc supplementation significantly increased the level of UDP-GlcNAc (h), but reduced the levels of of IL-1β (i) and TNF-α (j) in the supernatants of adult cKO 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(3, 8) = 15.49 for h, F(3, 8) = 19.37 for i, F(3, 8) = 19.08 for j. k Representative images of GFAP and O-GlcNAcylation immunostaining with the brain sections of adult Ctrl and cKO mice treated with saline (Ctrl + saline, cKO + saline) and GlcNAc (Ctrl + GlcNAc, cKO + GlcNAc), respectively. Scale bar, 50 μm. l Quantification results of O-GlcNAcylation immunostaining in k showed that GlcNAc supplementation significantly increased the immunostaining intensity of O-GlcNAcylation in the brain of cKO 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(3, 12) = 36.08. m–p WB assay (m) and quantification results showed that GlcNAc supplementation significantly reduced the protein levels of GFAP (n), IL-1β (o) and TNF-α (p) in the hippocampal tissues of cKO mice compared with saline-treated cKO mice. n = 3 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(3, 8) = 65.8 for n, F(3, 8) = 65.61 for o, F(3, 8) = 17.5 for p