Naringenin ameliorates myocardial injury in STZ-induced diabetic mice by reducing oxidative stress, inflammation and apoptosis via regulating the Nrf2 and NF-κB signaling pathways

Figure 1. NG improves hyperglycemia and pathologic damage to cardiac tissue in diabetic mice. (A) Blood glucose of mice from different treatments. Data are presented as the mean ± SD (n = 6). $P < 0.05 vs. corresponding groups in 0 day; **P < 0.05 vs. Ctrl group in 63 days, &P < 0.05 vs. DM group in 63 days. (B) Representative images of HE or Masson staining for each group, light microscope (400 ×). The data are presented as mean ± SD (n = 3). *P < 0.05 vs. Ctrl group; #P < 0.05 vs. DM group.

Figure 2. NG reduces apoptosis of cardiomyocytes in cardiac tissues of diabetic mice. (A) Representative images of TUNEL staining for each group, fluorescence microscope (200×). Data are presented as the mean ± SD (n = 3). *P < 0.05 vs. Ctrl group; #P < 0.05 vs. DM group. (B) Quantitative analysis of the protein expression of Bax and cleaved caspase-3 in cardiac tissues. Data are expressed as mean ± SD (n = 3), *P < 0.05 vs. Ctrl group; #P < 0.05 vs. DM group.

Figure 3. NG inhibites the expression of inflammatory cytokines in cardiac tissues of diabetic mice. (A) Representative immunohistochemical staining of IL-6, TNF-α, and NF-κB p65 in cardiac tissues. Data are expressed as mean ± SD (n = 3), *P < 0.05 vs. Ctrl group; #P < 0.05 vs. DM group. (B) Quantitative analysis of the protein expression of IL-6, TNF-α, and NF-κB p65 in cardiac tissues. Data are expressed as mean ± SD (n = 3), *P < 0.05 vs. Ctrl group; #P < 0.05 vs. DM group.

Figure 4. NG reduces diabetes-induced oxidative stress in cardiac tissues of diabetic mice. (A) Representative images of DHE staining for each group, fluorescence microscope (200×). (B) Quantitative analysis of the number of DHE fluorescence cells in cardiac tissue samples. Data are expressed as mean ± SD (n = 3), *P < 0.05 vs. Ctrl group; #P < 0.05 vs. DM group. (C) The level of malondialdehyde (MDA) from different treatments in cardiac tissue. Data are expressed as mean ± SD (n = 3), *P < 0.05 vs. Ctrl group; #P < 0.05 vs. DM group. (D) The level of superoxide dismutase (SOD) of different treatments in cardiac tissue. Data are expressed as mean ± SD (n = 3), *P < 0.05 vs. Ctrl group; #P < 0.05 vs. DM group. (E) Quantitative analysis of the protein expression of NOX2, HO-1, NQO1, and Nrf2 in cardiac tissues. Data are expressed as mean ± SD (n = 3), *P < 0.05 vs. Ctrl group; #P < 0.05 vs. DM group.

Figure 5. NG reduced HG-induced oxidative stress and apoptosis in H9C2 cells. (A) Representative images of DCFH-DA staining for each group, fluorescence microscope (200×). Data are expressed as mean ± SD (n = 3), *P < 0.05 vs. Ctrl group; #P < 0.05 vs. DM group. (B) Evaluation of apoptosis by flow cytometry using Annexin V staining. Data are expressed as mean ± SD (n = 3), *P < 0.05 vs. Ctrl group; #P < 0.05 vs. DM group. (C) Quantitative analysis of the protein expression of IL-6, HO-1, and NQO1 in H9C2 cells. Data are expressed as mean ± SD (n = 3), *P < 0.05 vs. Ctrl group; #P < 0.05 vs. DM group.

Figure 6. NG regulates the expression of NF-κB and Nrf2 in H9C2 cells under the HG condition. (A) Representative immunofluorescence staining of NF-κB for each group, fluorescence microscope (200×). (B) Quantitative analysis of the NF-κB protein expression in H9C2 cells. Data are expressed as mean ± SD (n = 3), *P < 0.05 vs. Ctrl group; #P < 0.05 vs. DM group. (C) Representative immunofluorescence staining of Nrf2 for each group, fluorescence microscope (200×). (D) Quantitative analysis of the Nrf2protein expression in H9C2 cells. Data are expressed as mean ± SD (n = 3), *P < 0.05 vs. Ctrl group; #P < 0.05 vs. DM group.

Figure 7. Proposed pathway for the cardioprotective effect of NG against diabetes-induced myocardial injury. Hyperglycemia or HG reduces Nrf2 expression and induces NF-κB expression, resulting in oxidative stress and inflammation. NG, a flavonoid abundant in grapefruit, partially attenuates diabetes-induced heart injury by activating the Nrf2 pathway and inhibiting the NF-κB pathway.