Salidroside Inhibits HMGB1 Acetylation and Release through Upregulation of SirT1 during Inflammation

Figure 7

SirT1 overexpression inhibits the acetylation and translocation of HMGB1 induced by LPS in RAW264.7 cells. The cells were transfected with Flag-SirT1 and control plasmids; after 24 h transfection, the cells were treated with LPS for 12 h. (a) Nuclear and cytoplasmic and total proteins were extracted, respectively; the levels of HMGB1 were detected by Western blotting. The histogram showed the relative expression of HMGB1 in nuclear and cytoplasmic protein normalized to lamin B1 or GAPDH, respectively. The experiments were done in triplicate, and data were shown as mean ± SD. compared with the group stimulated without LPS in control plasmid-transfected cells. compared with the group stimulated without LPS in Flag-SirT1-transfected cells. compared with the control plasmid-transfected and LPS-treated cells. (b) After transfection and LPS treatment, the cells in laser confocal dishes were fixed with 4% paraformaldehyde, blocked with 3% BSA, incubated with an anti-HMGB1 antibody, and then incubated with an Alexa Fluor 555 goat anti-rabbit IgG (red) antibody. Cell nuclei were stained with DAPI (blue), and the localization of HMGB1 was visualized by confocal microscopy. (c) The cells were transfected with control and Flag-SirT1 plasmids for 24 h, then stimulated with LPS for 4 h; HMGB1 acetylation was determined by IP. The histogram showed the relative expression of acetylated HMGB1 normalized to total HMGB1. The experiments were done in triplicate, and data were shown as mean ± SD. compared with the control plasmid-transfected group stimulated with LPS.

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