Setanaxib (GKT137831) Ameliorates Doxorubicin-Induced Cardiotoxicity by Inhibiting the NOX1/NOX4/Reactive Oxygen Species/MAPK Pathway

Figures

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  FIGURE 1. The protein levels of NOX1 and NOX4 were increased by DOX both in vivo and in vitro. (A) Representative western blot analysis of NOX1 and NOX4 in myocardial tissue. (B,C) Quantification of NOX1 and NOX4 expression relative to the β-actin level (n = 6 per group). (D,E) Representative images of NOX1 and NOX4 expression in myocardial tissue detected by the immunofluorescence. The nuclei were stained with DAPI (Scale bar = 50 µm). (F) Representative western blot analysis of NOX1 and NOX4 of NRCMs treated with DOX of different concentrations for 24 h (G,H) Quantification of NOX1 and NOX4 expression relative to the β-actin level (n = 3). (I) Representative western blot analysis of NOX 1 and NOX 4 of NRCMs treated with DOX for different time. (J,K) Quantification of NOX1 and NOX4 expression relative to the β-actin level (n = 4–5). *p < 0.05, **p < 0.01, ***p < 0.001, n. s., not significant. NOX, NADPH oxidase; DOX, doxorubicin; cTnI, Cardiac troponin I.

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  FIGURE 2. GKT137831 alleviated DOX-induced cardiac dysfunction in vivo and attenuated diminished cell viability in vitro. (A) Representative M-mode echocardiograms. (B) Echocardiographic analysis of LVEF, FS, IVSS, IVSD, LVESD, LVEDD, LVPWS, and LVPWD (n = 5–6 per group). (C,D) Representative images with HE and Masson’s trichrome staining in myocardial tissue (scar bar = 50 µm). (E) NRCMs were stimulated with DOX for 24 h and followed by LDH assay to evaluate the cell viability (n = 4). (F) NRCMs were pretreated with different concentrations of GKT for 1 h before DOX (2 μM) stimulation. The cell viability was evaluated by LDH assay (n = 4). *p < 0.05, **p < 0.01, ***p < 0.001, n. s., not significant. Echo, echocardiography; DOX, doxorubicin; GKT, GKT137831; LVEF, left ventricular ejection fraction; FS, fractional shortening; IVSS, end-systolic inter-ventricular septal wall thickness; IVSD, end-diastolic inter-ventricular septal wall thickness; LVESD, left ventricular end-systolic diameter; LVEDD, left ventricular end-diastolic diameter; LVPWS, left ventricular end-systolic posterior wall; LVPWD, left ventricular end-diastolic posterior wall; LDH, lactic dehydrogenase.

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  FIGURE 3. GKT137831 ameliorated oxidative stress both in vivo and in vitro. (A,B) Representative images of myocardial tissue with DHE staining (scar bar = 50 µm) and fluorescence intensity analysis in different groups (n = 5 per group). (C,D) Representative images of western blotting for 4-HNE and quantifications by densitometric analysis, β-actin was used as a loading control (n = 5 per group). (E,F) Intracellular ROS generation in NRCMs was detected using DCFH-DA probe then evaluated by fluorescence microscopy (scar bar = 200 µm) and fluorescence intensity analysis. NRCMs were pretreated with GKT (5 μM) for 1 h, and then treated with DOX (2 μM) for 24 h. Rosup was used as a ROS positive control (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, n. s., not significant. Abbreviations: DOX, doxorubicin; GKT, GKT137831; DHE, dihydroethidium: 4-HNE, 4-hydroxynonenal: DCFH-DA, dichlorodihydrofluorescein diacetate; AU, arbitrary unit.

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  FIGURE 4. GKT137831 alleviated DOX-induced mitochondrial damage both in vivo and in vitro. (A) Representative transmission electron microscopy image of cardiac mitochondrial ultrastructure. (B) Mitochondrial membrane potential measured with JC-1 staining (scale bar = 50 µm). CCCP was used as a positive control. DOX, doxorubicin; GKT, GKT137831; CCCP, carbonyl cyanide m-chlorophenylhydrazone.

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  FIGURE 5. GKT137831 reduced DOX-induced cardiomyocyte apoptosis in vivo. (A,C) Representative images of TUNEL staining in myocardial tissue (scale bar = 50 μm) and related quantification of TUNEL staining (n = 5 per group). (B) Representative western blot analysis of c-PARP, Bcl-2, BAX, and CC3 in myocardial tissue, and quantifications by densitometric analysis (D,E,F,G), β-actin was used as a loading control (n = 5–7 per group). *p < 0.05, **p < 0.01, ***p < 0.001, n. s., not significant. DOX, doxorubicin; GKT, GKT137831; c-PARP, cleaved PARP; CC3, cleaved caspase 3.

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  FIGURE 6. GKT137831 reduced DOX-induced apoptosis of NRCMs. (A) Representative western blot analysis of c-PARP, Bcl-2, BAX, and CC3 and quantifications by densitometric analysis (C,D,E,F), β-actin was used as a loading control (n = 3). (B,G) Representative images of TUNEL staining in NRCMs (scale bar = 50 μm) and related quantification of TUNEL staining (n = 3). NRCMs were pretreated with GKT (5 μM) for 1 h, and then treated with DOX (2 μM) for 24 h. *p < 0.05, **p < 0.01, ***p < 0.001, n. s., not significant. DOX, doxorubicin; GKT, GKT137831; c-PARP, cleaved PARP; CC3, cleaved caspase 3.

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  FIGURE 7. GKT137831 ameliorated DOX-induced apoptosis by inhibiting the MAPK pathway. (A) Time-dependent effects of DOX on activation of JNK, ERK, and p38 MAPK were evaluated by Western blot. β-actin was used as a loading control. Relative protein expression levels of p-JNK/JNK (E), p-ERK/ERK (F), and p-p38/p38 (G) were quantified (n = 3–4). (B) NRCMs were pretreated with SP600125 (JNK inhibitor) or PD098059 (ERK1/2 inhibitor) or with SB203580 (p38 inhibitor) for 1 h and then treated with DOX for 24 h (H,I) The protein expressions of c-PARP and CC3 were determined by Western blot and quantifications by densitometric analysis (n = 3–4). (C,J) Representative images of TUNEL staining in NRCMs (scale bar = 50 μm) and related quantification of TUNEL staining (n = 3). (D) NRCMs pretreated with GKT or NAC for 1 h, and then treated with DOX for 24 h (K,L,M) The phosphorylation levels of JNK, ERK, and p38 were evaluated by Western blot, and quantifications by densitometric analysis (n = 3–4). *p < 0.05, **p < 0.01, ***p < 0.001, n. s., not significant. DOX, doxorubicin; GKT, GKT137831; NAC, acetylcysteine; c-PARP, cleaved PARP; CC3, cleaved caspase 3.