3-Hydroxyanthralinic acid metabolism controls the hepatic SREBP/lipoprotein axis, inhibits inflammasome activation in macrophages, and decreases atherosclerosis in Ldlr−/− mice

Figures

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  Figure 1. 3-HAA reduces SREBP-2 expression and activity in HepG2 cells. Hepatocytes from the human hepatoma cell line HepG2 were treated overnight with different doses of 3-HAA. (A) SREBP-1 and SREBP-2 mRNA expression [pooled data from six independent experiments (duplicate wells)]. *P < 0.05; Kruskal–Wallis ANOVA with Dunn’s post-test. (B) Representative western blot analysis of SREBP-2 in nuclear protein extracts; histone H4 was used as a loading control (top graph depicts SREBP-2/histone H4 band density ratios). (C) Immunofluorescence staining of SREBP-2 (red); nuclear DAPI staining is shown in blue. (D) ApoB levels in supernatant of HepG2 cells; left panel, representative Superose 6 profile; right panel, area under the curve as percentage of controls (control n = 5, 3-HAA n = 6). AUC, area under the curve. **P < 0.01; Mann–Whitney U test.

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  Figure 2. 3-HAA treatment inhibits the inflammasome in mouse macrophages. Mouse bone marrow-derived macrophages were treated with increasing concentrations of 3-HAA. Macrophages were primed with 10 ng/mL LPS for 6 h, and the inflammasome was then activated with 1 mM ATP. Graphs show IL-1β secretion from cells treated with 3-HAA (A) 0.5 h before LPS (n = differentiated cells from seven individual mice, treated in duplicates) or (B) 30 min before ATP activation (n = differentiated cells from five individual mice, treated in duplicate). **P < 0.01, ****P < 0.0001; Kruskal–Wallis ANOVA with Dunn’s post-test. (C) Representative western blot analysis of cleaved caspase-1 upon treatment with increasing doses of 3-HAA before LPS-priming, except one sample treated before ATP (denoted by §). Vinculin was used as a loading control. Top graph depicts band density ratios between pro-caspase 1/vinculin (open bars), and cleaved caspase-1/vinculin (filled bars).

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  Figure 3. HAAO blockade decreases atherosclerosis and plasma lipid levels. (A) Schematic overview of the kynurenine pathway and the NCR-631 target 3-HAA 3,4-dioxygenase (HAAO). (B) Twelve-week-old Ldlr−/− mice were fed a western diet and treated three times weekly with NCR-631 or 3-HAA (200 mg/kg) for 8 weeks. A PBS group was used as control (PBS, n = 23; NCR-631, n = 10; and 3-HAA, n = 15). En face % lesion area in the aortic roots. Pooled data from two independent experiments are shown. Representative micrographs of each group. (C) Total cholesterol and (D) triglyceride plasma levels (PBS, n = 22; NCR-631, n = 10; and 3-HAA, n = 15). (E) Mice body weight at the end of treatment (PBS, n = 23; NCR-631, n = 10; and 3-HAA, n = 15). *P < 0.05; **P < 0.01; Kruskal–Wallis ANOVA with Dunn’s post-test. IDO, indoleamine-2,3-dioxygenase; TDO, tryptophan dioxygenase; KAT, kynurenine aminotransferase; KA, kynurenic acid.

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  Figure 4. The effects of HAAO blockade in leucocyte infiltration and the inflammasome activation in the artery wall. Twelve-week-old Ldlr−/− mice were fed a western diet and treated three times weekly with NCR-631 (200 mg/kg) or PBS for 8 weeks. (A) Analysis of CD68-positive cell immunostaining in atherosclerotic plaques from NCR-631- or PBS-treated mice (PBS, n = 9; and NCR-631, n = 8). (B) Analysis of CD4-positive immunostaining in atherosclerotic plaques from NCR-631- or PBS-treated mice. Right panels show representative microphotographs. Scale bars = 100 μm. Arrowheads depict CD4+ cells. Data from one independent experiment are shown (PBS, n = 8; and NCR-631, n = 7). (C) Representative western blot analysis of pro- and cleaved caspase-1 expression in aortas from NCR-631- or PBS-treated mice. Vinculin was used as a loading control. Top graph depicts band density ratios between pro-caspase 1/vinculin (left), and cleaved caspase-1/vinculin (right). *P < 0.05; Mann–Whitney U test.

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  Figure 5. HAAO inhibition reduces liver SREBP-2 and HMGCR expression and improves histopathological scores. Analysis of SREBP and target gene mRNA expression in the livers of 12-week-old Ldlr−/− mice fed a western diet and treated three times weekly with NCR-631 (200 mg/kg) or PBS. Graphs show the mRNA levels of (A) SREBP-2 (Srebf-2), (B) its target gene Hmgcr. (C) mRNA levels of SREBP-1 (Srebf-1) and (D) its target gene Scd-1. (A–D; PBS, n = 17; and NCR-631, n = 5). ***P < 0.001; Mann–Whitney U test. (E, F) Semiquantitative histopathological examination of the livers. (E) Percentage steatosis grade, hepatocyte ballooning, and lobular inflammation in NCR-631- and PBS-treated mice. (F) Representative micrographs from H&E stained samples. Scale Bar = 100 μm. (G) Total cholesterol and (H) triglycerides in liver lipid extracts from NCR-631- or PBS-treated mice are shown (G, H; PBS, n = 5; NCR-631, n = 4). AU, arbitrary units. *P < 0.05; Mann–Whitney U test.

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  Figure 6. Effects of NCR-631 treatment on LDL clearance and VLDL output in Ldlr−/− mice. (A) Clearance of injected FITC-LDL (100 μg protein) in Ldlr–/– mice treated for 8 weeks with PBS (n = 11) or NCR-631 (200 mg/kg; n = 5). Data for each individual were normalized to the fluorescence of plasma taken 1 min after injection. No significant differences were observed, two-way ANOVA. (B) Biosynthesis of VLDL was evaluated in Ldlr–/– mice that were treated for 8 weeks with PBS (n = 4) or NCR-631 (n = 5) and injected with tyloxapol to inhibit lipolysis. Data from one independent experiment are shown. Graph shows mean ± SEM plasma triglycerides levels at different times before (0) and after tyloxapol injection. Right side text shows mean ± SEM of triglycerides output rates, and the percentage difference between groups. VLDL, very low-density lipoprotein.