1-MNA Ameliorates High Fat Diet-Induced Heart Injury by Upregulating Nrf2 Expression and Inhibiting NF-κB in vivo and in vitro

Figures

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  Figure 1. Effects of 1-MNA on PA-induced Oxidative stress in H9C2 cells. (A) Real-type quantitative polymerase chain reaction analysis of mRNA expression of NNMT in the H9C2 cells.β-actin was taken as an internal control gene. Expression values are expressed as the mean ± SEM. (B) 1-MNA inhibits PA-induced ROS production. Immunofluorescence images of the PA group. The histogram shows the normalized intensity from three independent experiments. (C) qRT-PCR analysis of Nrf2, HO-1, GCLC, and NQO-1 expression. (D) Western blotting analysis of Nrf2, HO-1, GCLC, and NQO-1. β-actin was taken as an internal control gene. Expression is expressed as the mean ± SEM. Differences in expression were analyzed by the unpaired Student′s t-test and one-way ANOVA (n = 3, NS = no significance, *p < 0.05, **p < 0.01, ***p < 0.001).

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  Figure 2. 1-MNA inhibits apoptosis of PA-induced H9C2 cells. (A) Cell and nuclear images by flow cytometry analysis. Data are shown as mean ± SEM from three independent experiments. (B) Western blotting analysis of Caspase-3, Bax, and Bcl-2. The image of the gel shown is a representative picture. (n = 3, NS = no significance, **p < 0.01, ***p < 0.001, ****p < 0.0001).

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  Figure 3. Effects of 1-MNA on PA-induced inflammation in H9C2 cells. (A) Immunofluorescence assay for NF-κB p65 nuclear translocation for NF-κB activity. (B) The mRNA expression of inflammatory cytokines. β-actin was taken as an internal control gene. Expression is expressed as the mean ± SEM. Differences in expression were analyzed by the unpaired Student′s t-test and one-way ANOVA (n = 3, NS = no significance, *p < 0.05, **p < 0.01).

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  Figure 4. Effects of 1-MNA on PA-induced hypertrophy and fibrosis in H9C2 cells. Representative images of H9C2 cells stained with rhodamine-phalloidin from three independent experiments. (A) Representative staining images are shown with the quantitative data for the cell size of 100 randomly selected cells in 3 independent experiments. (B) The mRNA expression of TGF-β. (C) Western blotting analysis of TGF-β, smad2/3, p-smad2/3, and Col-1. β-actin was taken as an internal control gene. The image of the gel shown is a representative image from three independent experiments and intensity were normalized (n = 3, NS = no significance, *p < 0.05, **p < 0.01, ****p < 0.0001).

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  Figure 5. Effects on serum TC, TG, LDL, and body weight following 1-MNA treatment. HFD induces increased (B) serum TG and (D) LDL, which are inhibited by 1-MNA treatment. (A) Body weight and (C) TC were different but not statistically significant. (n = 3, NS = no significance, *p < 0.05, **p < 0.01, ***p < 0.001).

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  Figure 6. 1-MNA administration improved the oxidative stress and inflammation of the heart induced by HFD. (A) Western blotting analysis of IκB expression. Quantification of protein expression shown is normalized to β-actin. (B) qRT-PCR analysis of oxidative stress related gene expression. (C) Western blotting analysis of TNF-α. β-actin was taken as an internal control gene. (D) qRT-PCR analysis of inflammation related gene expression. (E) Western blotting analysis of NRF2, HO-1, and NQO-1. β-actin was taken as an internal control gene. (n = 3, NS = no significance, *p < 0.05, **p < 0.01, ***p < 0.001).

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  Figure 7. 1-MNA amileorated HFD-induced heart injury. (A) Representative images from longitudinal and transverse H&E staining of heart tissues are shown. Data for the ratio of heart weight (HW) to tibia length (TL). (B) 1-MNA administration reduced the indicated gene expression in HFD-induced hearts. (C) Myocardial tissue was colored in red and collagen was colored in blue by Masson staining. Under light microscopy, the myocardial fibers of the control group were arranged neatly, with no obvious breaks or rearrangements. The size of the intercellular space was normal. (n = 5, *p < 0.05, ***p < 0.001, ****p < 0.0001).

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  Figure 8. 1-MNA administration improved the fibrosis and apoptosis of the heart induced by HFD. (A) Heart tissues from each group were individually processed for RNA extraction and qRT-PCR analysis. (B) Western blotting analysis of TGF-β, MMP9, and COL-1.1-MNA administration inhibited HFD-induced cardiomyocyte apoptosis. Images of cardiac tissue sections subject to immunohistochemical staining for Bax. (C) The histogram shows the relative fluorescence density of anti-Bax staining. (D) Images of cardiac tissue sections subject to immunohistochemical staining for Tunel. (n = 5, *p < 0.05, **p < 0.01, ***p < 0.001).

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  Figure 9. Schematic illustration. A schematic illustration for the prevention of 1-MNA from FFA/HFD-induced injury in cardiomyocytes and hearts.