Diminazene Aceturate Stabilizes Atherosclerotic Plaque and Attenuates Hepatic Steatosis in apoE-Knockout Mice by Influencing Macrophages Polarization and Taurine Biosynthesis

Figures

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  Figure 1. Influence of DIZE on atherosclerosis progression. Representative micrographs showing oil-red O-stained atherosclerotic lesions (A,B) and HE-stained necrotic cores (D,E) in the aorta of control and DIZE-treated mice as well as their corresponding quantitative analyses (C,F). mRNA expression of ACE, ACE2, and NEP in the aorta of control and DIZE-treated mice (G). Data are mean ± SEM analyzed using t-test (* p < 0.05 as compared to control; n = 3–11 per group).

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  Figure 2. Impact of DIZE on the stability of atherosclerotic plaques. Immunohistochemical staining of aortic roots showing CD68-positive macrophages (A,B) and smooth muscle α-actin (SMA) (D,E) in control and DIZE-treated mice. Quantitative analysis of the atherosclerotic lesions area occupied by CD68-positive macrophages (C) and smooth muscle cells (F). The plasma levels of total cholesterol, high-density lipoproteins (HDL), low-density lipoproteins (LDL), and triglycerides (TG) (G) in control and DIZE-treated mice. Data are mean ± SEM analyzed using t-test (* p < 0.05 as compared to control mice; n = 4 or 6 per group).

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  Figure 3. Macrophages polarization in atherosclerotic lesions and THP-1 cell culture after treatment with DIZE. Representative immunohistochemical staining of aortic roots showing F4/80 (green), nitric oxide synthase 2 (iNOS)/arginase 1 (red), and 4′6-diamidino-2-phenylindole (DAPI) (blue) co-localization in control (A,D) and DIZE-treated mice (B,E). White arrows indicate M1 (A,B) and M2 (D,E) macrophages, respectively. Quantitative analysis of M1 and M2 contents in the atherosclerotic plaques (C,F). mRNA expression of M1 (IL-1β and TNF-α) (G) and M2 (MRC1, FCER2) (H) markers in THP-1 macrophages cell culture polarized to proinflammatory M1 and anti-inflammatory M2 phenotype after treatment with DIZE. Data are mean ± SEM analyzed using t-test (C,F) or one-way ANOVA with multiple comparisons and Benjamini and Hochberg false discovery rate (FDR) correction (G,H) (* p < 0.05 as compared to control; # p < 0.05 as compared to LPS or IL-4, respectively; n = 3 independent experiments or n = 6 biological replicates per group).

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  Figure 4. Graphs showing the time course response of mesenteric arteries to phenylephrine (A), acetylcholine (B), and DEA-NO (C) from control and DIZE-treated mice. Data are mean ± SEM analyzed using nonparametric Mann–Whitney U test (* p < 0.05 as compared to control mice; n = 4–6 per group).

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  Figure 5. Influence of DIZE on the development of hepatic steatosis. Representative images of livers in con-trol (A) and DIZE-treated mice (B). The figures show hematoxilin and eosin staining (A,B) and quantitative analysis of macrovesicular steatosis (D), triglycerides content in the liver (E) as well as plasma ALT/AST levels (F) in control and DIZE-treated mice. mRNA expression of ACE, ACE2 and NEP in the liver of control and DIZE-treated mice (C) Magnification 40×. Data are mean ± SEM analyzed by using t-test (* p < 0.05 as compared to control mice; n = 3–9 per group).

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  Figure 6. Proteomic analysis in the liver of DIZE-treated mice. The volcano plot of differentially expressed proteins showing the log2 fold change of protein expression vs. p-value in DIZE group compared to control mice (A). Validation of cysteine sulfinic acid decarboxylase (CSAD) expression by Western blot as well as the taurine levels in the liver of DIZE-treated mice (B). Heat map presentation of a hierarchical cluster of significantly changed proteins in the liver of DIZE-treated mice (C) (fold change >1.15 and <−1.15). Bioinformatic analysis by STRING of differentially expressed proteins reveals decreased expression of urea cycle proteins and increased expression of different isoforms of glutathione S-transferase (D). Data are mean ± SEM analyzed using t-test (B) or one-way ANOVA with Benjamini and Hochberg false discovery rate (FDR) correction (A,C) (* p < 0.05 as compared to control mice; n = 4 per group).