Yellow Wine Polyphenolic Compounds prevents Doxorubicin‐induced cardiotoxicity through activation of the Nrf2 signalling pathway

Figures

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  Figure 1. Effect of YWPC on cardiac injury and hypertrophy in rats subjected to DOX intoxication. (A) The study flow diagram for animal experiments. (B) Average body weight change in 4 wk in rats from control group, YWPC group (30 mg/kg/day), DOX groups (18 mg/kg) and DOX + YWPC group (n = 10 per group). (C) Results of heart weight/body weight (HW/BW) in the four groups at fourth week (n = 10). (D) Representative images of global appearance of the heart in different groups. (E) Representative photomicrographs of the heart tissue sections from rats treated with DOX and/or YWPC (H&E staining). The yellow arrow indicates the injured myocardial cell. Bar = 100 μm. (E) WGA staining of left ventricular tissue from rats described in A for determination of cardiac and cardiac myocyte hypertrophy (Bar = 100 μm, n = 10). *P < 0.05, vs. the control; # P < 0.05 vs. the DOX group. CDDO‐EA, CDDO ethyl amide; DOX, doxorubicin; YWPC, Yellow Wine Polyphenolic Compounds

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  Figure 2. YWPC exert cardioprotective effects on DOX‐induced cardiac dysfunction. (A) Anatomic M‐mode echocardiography and corresponding electrocardiographic images of Sprague‐Dawley rat hearts. Results of (B) LVEF, (C) LVFS, (D) LVIDs, and (E) LVIDd in the four groups (n = 10). (F) Mean Heart rate in different treatment groups (n = 10). *P < 0.05, vs. the control; # P < 0.05 vs. the DOX group. DOX, doxorubicin; LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening; LVIDs, left ventricular internal dimension‐systole; LVIDd, left ventricular internal dimension‐diastole; YWPC, Yellow Wine Polyphenolic Compounds

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  Figure 3. YWPC treatment ameliorates DOX‐induced oxidative stress, in vivo. (A) Staining cells with red indicated DHE‐positive cells; DAPI staining (blue) indicated nucleus. Bar = 50 μm. Percentages of DHE‐positive cells of total cells were shown. Cardiac antioxidant enzyme activities of (B) MDA, (C) SOD and (D) catalase were evaluated using in the heart homogenates from indicated groups. Enzyme‐linked immunosorbent assay (ELISA) was used on serum from rats to determine the expression of transforming growth factor‐β1 (TGF‐β1) (E), interleukin‐1β (IL‐1β) (F), and interleukin‐6 (IL‐6) (G). Data are shown as the mean ± standard deviation of 10 rats. *P < 0.05, vs. the control; # P < 0.05 vs. the DOX group. DOX, doxorubicin; MDA, malondialdehyde; SOD, Superoxide Dismutase; YWPC, Yellow Wine Polyphenolic Compounds

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  Figure 4. YWPC prevent DOX‐induced cardiac myocyte apoptosis, in vivo. (A) Transmission electron microscopy showing betterment of mitochondrial ultrastructure in DOX + YWPC group compared to DOX‐treated rats. Bar = 0.2 μm, 50 000× magnification. Corresponding graphs showing mitochondrial diameter and average mitochondrial area in vivo from different treatment groups (n = 10). (B) Representative images of TUNEL staining of the heart tissues. Apoptotic cardiomyocyte nuclei appear brown‐stained and normal nuclei appear blue. (C) Caspase‐3 expression in the different treatment groups were detected via immunofluorescence analysis. (D) Protein expression of Bcl‐2, Bax, Cyt C and cleaved caspase‐3 in the different treatment groups. *P < 0.05, vs. the control; # P < 0.05 vs. the DOX. DOX, doxorubicin; YWPC, Yellow Wine Polyphenolic Compounds

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  Figure 5. YWPC prevent DOX‐induced cardiac myocyte apoptosis, in vitro. (A) Effects of YWPC (50 mg/L for 24 h) on DOX (5 μmol/L for 24 h)‐induced dissipation of mitochondrial membrane potential measured in H9C2 cells loaded with JC‐1 using fluorescence microscopy. Magnification ×400. (B‐C) Effects of YWPC on DOX‐induced cell apoptosis of H9C2 cells, as detected by Flow cytometry. (D) The relative expression of Bcl‐2, Bax and cleaved caspase‐3 in H9C2 cells with or without YWPC treatment under DOX stimulation. *P < 0.05, vs. the control; # P < 0.05 vs. the DOX. DOX, doxorubicin; YWPC, Yellow Wine Polyphenolic Compounds

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  Figure 6. YWPC alleviate DOX‐induced reactive oxygen species (ROS) and down‐regulation of transforming growth factor beta (TGF‐β)/smad3 pathway dependent on Nrf2 status, in vitro. (A) The intracellular ROS production of H9C2 cells under the stimulation of DOX (5 μmol/L for 24 h) with or without pre‐treatment of YWPC (50 mg/L for 24 h) was visualized under a fluorescence microscope with 2′, 7′‐dichlorodihydrofluorescein diacetate (DCFDA) probe. (B) The protein expression level of p‐smad3 and smad3 in H9C2 cells was determined by Western blot using phosphorylated smad3 and smad3 antibodies. (C) DOX treatment for 6 h induced the cytosol accumulation of Nrf2, and YWPC pre‐treatment enhanced the nuclear accumulation of Nrf2 activated by DOX; (D) Knockdown of Nrf2 in H9C2 cells was achieved by specific siRNA and the efficiency validated by Western blot analysis. RT‐qPCR was used to determine the expression levels of Nrf2 target genes, HO‐1 (E), NQO‐1 (F), and GCLM (G) in H9C2 cells managed with DOX and/or YWPC, CDDO‐EA Nrf2 siRNA. (H) The TGF‐β1 expression level in the culture supernatants was analysed by Enzyme‐linked immunosorbent assay (ELISA). *P < 0.05, vs. the control; # P < 0.05 vs. the…

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  Figure 7. YWPC suppress DOX‐induced cardiac fibrosis by inhibiting TGF‐β1/smad3 pathway, in vivo. (A) Masson's trichome staining was used to evaluate collagen deposition in the heart tissue from control group, YWPC group, DOX group, DOX + YWPC group and DOX + CDDO‐EA group. (B) Relative expression of TGF‐β1, Collagen I and α‐SMA were determined by immunohistochemical staining. (C) Collagen volume fraction was quantified (positive area/total area) in groups. (D) Relative expression of TGF‐β1, Collagen I and α‐SMA protein in myocardial tissue were quantified of the results in B. (E) The protein expression of p‐smad3 and smad3 in myocardial tissue was determined by Western blot. *P < 0.05, vs. the control; # P < 0.05 vs. the DOX. CDDO‐EA, CDDO ethyl amide; DOX, doxorubicin; YWPC, Yellow Wine Polyphenolic Compounds

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  Figure 8. Schematic diagram showing the cardioprotective effects of YWPC on DOX‐induced cardiotoxicity. DOX, doxorubicin; YWPC, Yellow Wine Polyphenolic Compounds