NADPH Oxidase Mediates Oxidative Stress and Ventricular Remodeling through SIRT3/FOXO3a Pathway in Diabetic Mice

Figures

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  Figure 1. Apocynin (APO) improves cardiac function in diabetic mice. (A) C57BL/6 mice were induced with type 2 diabetes by high-fat diet combined with low-dose STZ. The DM+APO group was given APO (40 mg/kg) dissolved in corn oil by daily gavage. (B,C) Blood glucose and body weight in the 3 groups. (D) Representative two-dimensional and M-mode of echocardiographic images. (E) Analysis results of left ventricular ejection fraction (LVEF). (F) Results of hemodynamic studies (SBP, DBP, and MBP). (G) Ratio of heart weight (HW) to tibial length (TL) of mice in each group. Data are mean ± SD; n = 6 mice per group; * p < 0.05, *** p < 0.001.

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  Figure 2. Apocynin improves the ventricular structural and electrical modeling in diabetic mice. (A) Recorded representative epicardial activation mapping of left ventricular (LV) in the 3 groups. (B–D) Mean left ventricular conduction velocity (LVCV), absolute inhomogeneity, and inhomogeneity index of LV between each group. (E) Representative images of HE and Masson’s trichrome staining. (F) Expression of α-SMA among the 3 groups. (G) Quantification of α-SMA in (F). Data are mean ± SD; n = 5–8 mice per group; * p < 0.05, ** p < 0.01.

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  Figure 3. Apocynin decreases reactive oxygen species (ROS) and improves mitochondrial function of diabetic cardiomyopathy. (A,B) Production of ROS in ventricular tissues. Scale bar, 20 μm. (C) Expression of PGC1-α, Mn-SOD, TFAM, and MFN2 among the 3 groups. (D–G) Quantification of PGC1-α, Mn-SOD, TFAM, and MFN2 in (C). Data are mean ± SD; n = 5 mice per group; * p < 0.05, ** p < 0.01.

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  Figure 4. Apocynin regulates SIRT3/FOXO3a signaling pathway in diabetic cardiomyopathy. (A) Protein level of SIRT3 detected by Western blot analysis. (B) Quantification of SIRT3 in (A). (C) Expression of FOXO3a in ventricular tissues. (D) Quantification of FOXO3a in (C). (E) Representative confocal microscopy images of immunofluorescence stained for Ac-FOXO3a and DAPI. (F) Ac-FOXO3a fluorescence intensity quantification. Scale bar, 20 μm. (G) Expression of Ac-MnSOD in ventricular tissues among 3 groups. (H) Quantification of Ac-MnSOD in (G). Data are mean ± SD; n = 5 mice per group; * p < 0.05, ** p < 0.01, *** p < 0.001.

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  Figure 5. Apocynin (APO) attenuates oxidative stress via the SIRT3/FOXO3a pathway in H9c2 cells. (A) H9c2 cells were exposed to normal glucose (NC) or high glucose (HG) or HG with APO for 36 h. Flow cytometry detected H9c2 cells stained with Dihydroethidium (DHE). (B) Quantification of ROS by DHE intensity in (A). (C) Mn-SOD protein level in H9c2 cells detected by Western blot analysis. (D) Quantification of Mn-SOD protein level in (C). (E) The protein levels of SIRT3 and FOXO3a in each group. (F,G) Quantification of SIRT3 and FOXO3a protein level in (E). (H) Ac-FOXO3a fluorescence intensity quantification in (I). Scale bar, 20 μm. (I) Representative confocal microscopy images of immunofluorescence staining for Ac-FOXO3a and DAPI in H9c2 cells exposed HG with or without APO. (J) Expression of SIRT3 and Mn-SOD in NC siRNA and SIRT3 siRNA with or without APO. (K,L) Quantification of SIRT3 and Mn-SOD protein level in (J). Data are mean ± SD; n = 4 for 6 independent experiments; * p < 0.05, ** p < 0.01, *** p < 0.001.

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  Figure 6. Within diabetic ventricles, NADPH oxidase (NOX) is an enzyme that plays a significant part in the development of oxidative stress. Apocynin (APO) can improve myocardial reactive oxygen species (ROS) overproduction and mitochondrial function through recovery of the SIRT3/FOXO3a pathway, thereby reducing the incidence of diabetic cardiomyopathy. It is possible that this substance will be developed into a medication for use in the clinical management of diabetic ventricular remodeling.