Figure 5.

Redox modulation of HMGB1 in spleen and in drug-intoxicated liver. (A) Western blot probed with anti-CD45, anti-HMGB1, and anti-GAPDH antibodies in reducing conditions (upper panels) or probed with anti-HMGB1 antibody in non-reducing conditions (lower panel) on lysates of spleen and liver isolated from control WT mice. In the lower panel, the upper band corresponds to the fully reduced-HMGB1 (frHMGB1) and the lower band to the disulphide-HMGB1 (dsHMGB1). (B,C) Quantification of total CD45 and HMGB1 protein levels normalized on GAPDH (B), and HMGB1 redox isoforms percentage (C) in spleen and liver lysates. A.U. = arbitrary unit (n = 4 mice/group). (D–G) Drug-induced liver injury (DILI) was induced by i.p. injection of acetaminophen (APAP), 300 mg/kg (body weight). Serum collection and necroscopy were performed at the indicated time points. (D) Representative images of DAPI and Evans Blue (EB) staining, Haematoxylin & Eosin (H&E) staining, and CD45 and HMGB1 immunostaining in liver sections from control mice (Ctrl) and at days 1, 2, 3, and 7 after DILI. Scale bars, 50 μm. (E) Alanine aminotransferase (sALT) and HMGB1 levels in serum before and after APAP injection in mice (n ≥ 5 mice/group). (F) Quantification of total number of intrahepatic leukocytes (IHLs) in control mice and at days 1 and 2 post-APAP injection (n = 4 mice/group). (G) Quantification of HMGB1 redox isoforms percentage, from Western blot assays performed in non-reducing conditions with anti-HMGB1 antibody, in IHLs isolated from control mice and at days 1 and 2 post-APAP injection (n = 4 mice/group). Data represent the means ± SEM and statistical significance was calculated by Student T-test (B), One-way (E,F) and Two-way ANOVA (C,G). ***P < 0.001; ****P < 0.0001; ns, not significant.