Baicalin Ameliorates Cognitive Impairment and Protects Microglia from LPS-Induced Neuroinflammation via the SIRT1/HMGB1 Pathway

Figure 7

SIRT1 is required for BAI inhibition of LPS-induced HMGB1 release from BV2 microglial cells. BV2 cells were transfected with SIRT1-targeted siRNA-1, siRNA-2, or control siRNA. (a) SIRT1 protein levels in siRNA-1 or siRNA-2-transfected BV2 cells were measured by immunoblotting. (b, c) After transfection for 36 h, cells were exposed to LPS in the presence or absence of BAI for 24 h, and the expression of SIRT1, secreted HMGB1, and intracellular HMGB1 was measured by western blotting. (d, e) After transfection, BV2 cells were cultured in LPS medium alone or with 40 μM BAI for 4 h. Coimmunoprecipitation analysis showed the level of acetylated HMGB1. (f) Cells transfected with SIRT1-targeted siRNA-1 or siRNA-2 for 36 h were exposed to LPS in the presence or absence of BAI for 6 h; immunofluorescent images of the HMGB1 (red) cytoplasmic translocation were observed with confocal scanning microscopy. Scale bar: 10 μM. (g, h) HMGB1 levels in the cytoplasmic and nuclear fractions were analyzed by western blotting. GAPDH and LaminB1 were used as loading controls for cytoplasmic and nuclear fractions, respectively. All the results are presented as of three independent experiments. , , , control vs. LPS or LPS+siRNA-1/2; ^#, ^##, BAI+LPS vs. LPS; ^&, BAI+LPS vs. BAI+LPS+siRNA-1/2.

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