Fig. 6.
In vivo inhibition of PDHC and LDH reduces liver injury and increases survival in acute liver failure. Concentrations of (A) acetyl-CoA and (B) lactate in nuclear fractions of livers of mice treated with 80 mg/kg/day of galloflavin (n = 4) compared to mice injected with vehicle (DMSO 40% in saline; n = 5). As controls, levels of the metabolites in nuclear fractions of livers of mice receiving only vehicle are shown (n = 4). Means ± SDs are shown. ANOVA plus Tukey’s post hoc test: *p = 0.00001 for nuclear acetyl-CoA; p = 0.0001 for nuclear lactate. Enzyme activity of (C) PDHC and (D) LDH in nuclear fractions of livers of mice treated with 80 mg/kg/day of galloflavin (n = 4) compared to mice injected with vehicle (n = 5). (E) HDAC activity in nuclear fractions of mice treated with galloflavin (n = 4) compared to controls injected with vehicle (n = 5). As controls, levels of enzyme activities in nuclear fractions of livers of mice receiving only vehicle are shown (n = 5). Means ± SDs are shown. ANOVA plus Tukey’s post hoc test: p = 0.0211 for nPDHC; p = 0.00131 for nLDH; p = 0.0416 for nHDAC. (F) Western blotting for histone H3 (H3) and acetylated histone H3 (H3K9) on livers of mice receiving 80 mg/kg/day of galloflavin or vehicle. β-actin was used as a loading control. (G) Densitometric quantifications of H3K9 over H3 from the Western blotting shown in F; t test; *p <0.05. (H) Representative TUNEL (upper panels) and H&E (lower panels) staining of livers of mice receiving 80 mg/kg/day galloflavin or vehicle. (I) Quantifications of TUNEL-positive cells and (J) necrotic areas on livers of at least three sections from each mouse (at least n = 4 mice per group). Means ± SDs are shown. Likelihood ratio test for generalized linear model: *p = 1.74 × 10−5. ANOVA plus Tukey’s post hoc test; for necrotic area: *p = 0.0571853. Expression levels of (K) Tnf and (L) Il6 in livers of mice receiving 80 mg/kg/day galloflavin or vehicle (at least n = 3 per group). Means ± SDs are shown. ANOVA plus Tukey’s post hoc test: *p = 0.0000444 for Tnf and *p = 0.0026428 for Il6. All determinations in panels A to L were performed 6 h after CD95-Ab. (M) Kaplan-Meier survival curves of mice receiving increasing doses of galloflavin [10 mg/kg/day (n = 24; p = 0.1865), 20 mg/kg/day (n = 20; p = 0.0239), 40 mg/kg/day (n = 13; p = 0.0107), and 80 mg/kg/day (n = 10; p = 0.0173)] or vehicle. The numbers in parenthesis indicate the dose of galloflavin in mg/kg/day. Long-rank test p = 0.0067. Means ± SDs are shown. Ab, antibody; A.U., arbitrary units; nHDAC, nuclear histone deacetylase; nLDH, nuclear lactate dehydrogenase; nPDHC, nuclear pyruvate dehydrogenase complex; n.s, not significant; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.