High mobility group box-1 release from H2O2-injured hepatocytes due to sirt1 functional inhibition

Figure 1 Hepatocellular injury in mice treated with a high-fat diet plus single binge alcohol. A: Serum levels of alanine aminotransferase and the levels of H₂O₂ and catalase in C57BL/6 mice fed a control diet for 12 wk or a high-fat diet plus single binge alcohol (n = 12); B: Hematoxylin-eosin stained images (scale bar = 10 μm); C: High mobility group box-1 (HMGB1) staining (scale bar = 20 μm). The black arrow shows HMGB1 translocation; D and E: Western blot analysis for Parp1, Sirt1, and HMGB1 in whole tissue and HMGB1 in the cytoplasm or in nucleus. HMGB1 acetylation was detected by immunoprecipitation; F: The mRNA expression of Parp1 and Sirt1 by real-time PCR and liver NAD⁺ content. All data were presented as mean ± SD. Statistical analysis was done by the Student’s t-test. ^aP < 0.05 vs control, ^bP < 0.01 vs control, ^cP < 0.01 vs control. HFD/etOH: High-fat diet plus single binge alcohol; ALT: Alanine aminotransferase; HMGB1: High mobility group box-1; IP: Immunoprecipitation.

Figure 2 Cytotoxicity increase and high mobility group box-1 release induced by H₂O₂ in BNL CL cells. A and B: BNL CL2 cells cultured with H₂O₂ for 0, 0.5, 2, 8, or 24 h or at different concentrations (0, 100, 500, and 1000 μmol/L); C: 8-hydroxy-2-deoxyguanosine levels in cells treated with 500 μmol/L H₂O₂ or N-acetylcysteine (NAC); D: High mobility group box-1 (HMGB1) content in medium detected by ELISA; E: Cells were treated with 500 μM H₂O₂ for different time intervals HMGB1 content in medium detected by ELISA. Cells were pre-treated with NAC for 24 h, and then incubated with or without H₂O₂ (500 μmol/L) for 8 h. All data are presented as the mean ± SD. Statistical analysis was done by the Student’s t-test. ^aP < 0.05 vs 0 group, ^bP < 0.01 vs 0 group, ^dP < 0.01 vs H₂O₂ group. 8-HOdG: 8-hydroxy-2-deoxyguanosine; NAC: N-acetylcysteine; HMGB1: High mobility group box-1.

Figure 3 H₂O₂ induced high mobility group box-1 translocation and acetylation. A: Confocal microscopy analysis of cells treated with H₂O₂ either for different durations or at different concentrations. (scale bar = 40 μm). The white arrow shows the translocation of high mobility group box-1 (HMGB1) to the cytoplasm; B and C: HMGB1 expression determined by Western blot; D and E: Acetylated HMGB1 determined by immunoprecipitation. All data are presented as the mean ± SD. Statistical analysis was done by the Student’s t-test. ^aP < 0.05 vs 0 group, ^bP < 0.01 vs 0 group. HMGB1: High mobility group box-1.

Figure 4 H₂O₂ treatment inhibits Sirt1 expression and activity. A and B: BNL CL₂ cells were treated with H₂O₂ for 0-24 h at 500 μmol/L. Sirt1 levels determined by Western blot; C and D: BNL CL₂ cells were treated with H₂O₂ at 0-1000 μmol/L for 8 h. Sirt1 levels determined by Western blot; E: Sirt1 deacetylase activity detected by fluorescence spectrophotometry; F: BNL CL₂ cells were treated with H₂O₂ (500 μmol/L) for 4 h, and then SRT1720, a Sirt1 activator, was added into the medium. The supernatants were collected to determinate high mobility group box-1 content by ELISA. All data are presented as the mean ± SD. Statistical analysis was done by the Student’s t-test. ^aP < 0.05 vs 0 group, ^bP < 0.01 vs 0 group or negative control, ^dP < 0.01 vs H₂O₂ group. HMGB1: High mobility group box-1.

Figure 5 Sirt1 inhibits high mobility group box-1 translocation and release. A: Acetylated HMGB1 levels determined by immunoprecipitation. EX527, a Sirt1 inhibitor inhibited sirt1 level and promoted HMGB1 acetylation; B: High mobility group box-1 (HMGB1) expression in the nucleus or cytoplasm determined by Western blot; C: The mRNA and protein levels of Sirt1. BNL CL₂ cells were transfected with control lentiviral vector or Sirt1 shRNA lentiviral vector for 48 h. The mRNA and protein levels of Sirt1 were detected by real-time PCR and Western blot. Multiplicity of infection, refers to the number of lent virus adsorption on the cells and the ratio of the number of cells in culture; D: HMGB1 translocation; E: HMGB1 content in medium determined by ELISA. All data are presented as the mean ± SD. Statistical analysis was done by the Student’s t-test. ^bP < 0.01 vs 0 group or negative control, ^fP < 0.01 vs control lent viral group. MOI: Multiplicity of infection; HMGB1: High mobility group box-1.

Figure 6 Sirt1 activity is inhibited by H₂O₂ through Parp1-NAD⁺. A and B: NAD⁺ contents in cells treated with H₂O₂ for different durations or at different concentrations; C: NAD⁺ contents in cells treated with DIQ, a Parp1 inhibitor; D: Sirt1 activity in cells treated with DIQ and NAM, an NAD⁺ inhibitor; E and F: High mobility group box-1 (HMGB1) acetylation in cells treated with DIQ; G: BNL CL2 cells were treated with DIQ or H₂O₂. And then HMGB1 contents in medium determined by ELISA; H: HMGB1 contents in medium determined by ELISA in cells treated with CP or Parp1⁺ transfection; I: Sirt1 activity of cells with Parp1⁺ transfection. All data are presented as the mean ± SD. Statistical analysis was done by the Student’s t-test. ^bP < 0.01 vs 0 group or negative control, ^dP < 0.01 vs H₂O₂ group, ^hP < 0.01 vs H₂O₂ + DIQ group, ⁱP < 0.01 vs CP group. CP: Control Plasmid.

Figure 7 Sirt1 inhibits Parp1 expression and acetylation in BNL CL₂ cells. A-D: Parp1 expression and acetylation in cells treated with H₂O₂ for different durarions or at different concentrations; E: The mRNA levels of Parp1 in Sirt1 knockdown cells treated with H₂O₂; F and G: Parp1 expression and acetylation in Sirt1 knockdown cells treated with H₂O₂; H and I: Parp1 expression and acetylation in cells treated with SRT1720, a Sirt1 activator. All data are presented as the mean ± SD. Statistical analysis was done by the Student’s t-test. ^aP < 0.05 vs 0 group or negative control, ^bP < 0.01 or negative control, ^dP < 0.01 vs H₂O₂ group, ^fP < 0.01 vs CLv group, ^jP < 0.05 vs shLv group, ^kP < 0.01 vs shLv group.

Figure 8 Imbalance of mutual inhibition between Parp1 and Sirt1 causes high mobility group box-1 translocation in hepatocytes. HFD+etOH: High-fat diet plus ethyl alcohol; HMGB1: High mobility group box-1.