Phloretin Prevents Diabetic Cardiomyopathy by Dissociating Keap1/Nrf2 Complex and Inhibiting Oxidative Stress

Abstract

Hyperglycemia induces chronic inflammation and oxidative stress in cardiomyocyte, which are the main pathological changes of diabetic cardiomyopathy (DCM). Treatment aimed at these processes may be beneficial in DCM. Phloretin (PHL), a promising natural product, has many pharmacological activities, such as anti-inflammatory, anticancer, and anti-oxidative function. The aim of this study was to investigate whether PHL could ameliorate the high glucose-mediated oxidation, hypertrophy, and fibrosis in H9c2 cells and attenuate the inflammation- and oxidation-mediated cardiac injury. In this study, PHL induced significantly inhibitory effect on the expression of pro-inflammatory, hypertrophy, pro-oxidant, and fibrosis cytokines in high glucose-stimulated cardiac H9c2 cells. Furthermore, PHL decreased the levels of serum lactate dehydrogenase, aspartate aminotransferase, and creatine kinase-MB, and attenuated the progress in the fibrosis, oxidative stress, and pathological parameters via Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor E2-related factor 2 (Nrf2) pathway in diabetic mice. In additional, molecular modeling and immunoblotting results confirmed that PHL might obstruct the interaction between Nrf2 and Keap1 through direct binding Keap1, and promoting Nrf2 expression. These results provided evidence that PHL could suppress high glucose-induced cardiomyocyte oxidation and fibrosis injury, and that targeting Keap1/Nrf2 may provide a novel therapeutic strategy for human DCM in the future.

Keywords: Keap1; Nrf2; diabetic cardiomyopathy; oxidative stress; phloretin.

Figure 1

The chemical structure of Phloretin (A). MTT assay tests the effect of PHL on H9c2 survival rate after 24 h treatment (B).

Figure 2

PHL reduced hyperglycemia-induced ROS levels through induction of Nrf2 anti-oxidant responses in H9c2 cells. (A) PHL inhibited high glucose-induced ROS generation. H9c2 (1 ^* 10⁶) cells pretreated with PHL (10 μM) for 1 h were incubated with HG (33 mM) for 3 h. DCFH-DA probes loaded and cells were processed by flow cytometry analysis for O₂ level, and mean fluorescence intensity (MFI) value was determined. (B,C) H9c2 (5 ^* 10⁵) cells pretreated with PHL (10 μM) for 1 h and incubated with HG (33 mM) for 6 h. Levels of malondialdehyde (MDA) (B) in lysates prepared from H9c2 cells and enzymatic activity of superoxide dismutase (SOD) (C) as measured using colorimetric assays. (D) H9c2 (1 ^* 10⁶) cells were pre-treated with PHL (10 μM) for 1 h and then incubated with HG (33 mM) for 12 h. The cell lysates were immunoblotted for Nrf2, with GAPDH as a loading control. (E–G) Total RNAs were extracted and the mRNA levels of Nrf2, HO-1, and NQO-1 were detected by RT-qPCR. Cells were treated as in (B). (H) Staining of cultured H9c2 cells with DHE. DHE generates red fluorescence product (ethidium) in the presence of ROS. Cells were treated as in (A). Data are presented as mean ± SEM. ^*P < 0.05, ^**P < 0.01 vs. HG group; ^#P < 0.05, ^##P < 0.01 vs. Control group.

Figure 3

PHL reduced hyperglycemia-induced hypertrophy and fibrosis in H9c2 cells. H9c2 (1 ^* 10⁶) cells were pre-treated with PHL (10 μM) for 1 h and then incubated with HG (33 mM) for 24 h. (A) The cell lysates were immunoblotted for ANP, TGF-β, with GAPDH as a loading control. (B) The cell sizes were detected by Rhodamine Phalloidin/DAPI immunofluorescence staining. Total RNAs were extracted and the mRNA levels of ANP (C), BNP (D), β-MyHC (E), TGF-β (F), collagen-1 (G), and CTGF (H) were detected by RT-qPCR. Data are presented as mean ± SEM. ^*P < 0.05 < 0.01 vs. HG group; ^#P < 0.05, ^##P < 0.01 vs. Control group.

Figure 4

PHL attenuated diabetes-induced cardiac injury. Diabetes mellitus was induced in male C57BL/6 mice by a single intraperitoneal (i.p.) injection of 100 mg/kg STZ and mice with fasting-blood glucose >12 mM were considered diabetes and then diabetic mice were orally treated with phloretin (PHL, 10 mg/kg), or vehicle by gavage once every 2 days for 8 weeks, which was administrated in diabetic mice. Serum levels of LDH (A), CK-MB (B), and AST (C) were determined using indicated kits (Six mice in each group were used for above analysis). (D) Representative images from H&E sections of heart tissues are shown, × 400 amplification. (E–G) The mRNA expression levels of ANP (E), BNP (F), and β-MyHC (G) in myocardial tissues of each group were determined by real-time qPCR. Six mice in each group were used for above analysis. ^*P < 0.05, ^**P < 0.01 vs. STZ-DM1 group; ^##P < 0.01 vs. CON group.

Figure 5

PHL inhibited cardiac oxidative stress and fibrosis in the diabetic myocardium. (A) Representative images for DHE staining using the frozen section of heart tissues as described in Materials and Methods (1000 × magnification). (B) Assessment of cardiac fibrosis by Masson's Trichrome staining (400 × magnification). (C–H) The mRNA expression levels of HO-1 (C), NQO-1 (D), GCLC (E), TGF-β (F), collagen-1 (G), and CTGF (H) in myocardial tissues of each group were determined by real-time qPCR. Six mice in each group were used for above analysis. ^*P < 0.05, ^**P < 0.01 vs. STZ-DM1 group; ^##P < 0.01 vs. CON group.

Figure 6

PHL targeted Keap1 to disassemble the Keap1/Nrf2 complex. (A) Per-residue of top 10 contribution to the binding free energy; (B) Structural analysis of the most 10 contributors of Keap1 to PHL, hydrogen bonds are colored yellow; (C) Alignment of the representative structures between from conventional MD simulation and from GaMD simulation; (D) PCA scatter plot of 200,000 snapshots from GaMD simulation along the first two principal components. (E) Immunoblotting analysis of Nrf2 expression and Co-Immunoprecipitation analysis of Nrf2 and Keap1 complex in lysates prepared from heart tissues. GAPDH was used as a loading control.