The role of hesperidin as a cardioprotective strategy against doxorubicin-induced cardiotoxicity: The antioxidant, anti-inflammatory, antiapoptotic, and cytoprotective potentials

Figures

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  Fig. 1.. Showing the effect of DOX and HES on all tested antioxidant enzymes; GPX, CAT, and SOD for all experimental groups. According to Duncan multiple test, the different letters (a–c) are significantly different between treatments at the P < 0.05 level.

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  Fig. 2.. Showing the effect of DOX and HES on all tested cardiac biomarkers; Troponin I (CTnI), CK-Total, CK-MB, LDH and AST for all experimental groups. According to Duncan multiple test, the different letters (a–c) are significantly different between treatments at the P < 0.05 level.

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  Fig. 3.. Showing the effect of DOX and HES on the tested Pro-inflammatory Cytokines; Interferon γ (IFN-γ), Interleukin 1β (IL-1β) and Tumor necrosis factor α (TNF-α) for all experimental groups. According to Duncan multiple test, the different letters (a–c) are significantly different between treatments at the P < 0.05 level.

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  Fig. 4.. A photomicrographs of mature male rat’s heart myocardium of G1; control group. (a) Showing normal and intact cardiomyocyes with normal orientations and directions without any pathological lesions. In longitudinal sections, cardiomyocyes are appeared short, branched, anastomosed forming network with single, central, large oval or ovoid, more euchromatic nucleus and acidophilic sarcoplasm (arrow). (b) Showing that the cardiomyocyes are laterally separated from each other by a considerable amount of highly vascularized connective tissue (arrow). Stain: All: Haematoxylin and Eosin (H&E). Scale bars: a,b =40 μm.

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  Fig. 5.. A photomicrographs of the heart myocardium of the G2 treated with DOX. (a) and (b) Showing sever Zenker’s degenerations of the cardiomyocyes characterized by sever muscular damage with loss of their continuity, loss of cross-striations, fibers swelling, hyper-eosinophilia of their sarcoplasm, hyaline fragmentation of the sarcoplasm with small dark pyknotic nuclei. In addition, the intercellular spaces were increased (arrow). (c) and (d) Showing disarray of the cardiomyocyes characterized with loss of the normal parallel arrangement of the cardiac muscle cells. Stain: All: H&E. Scale bars: All = 40 µm.

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  Fig. 6.. A photomicrographs of the heart myocardium of the G2 treated with DOX. (a) Showing sever Zenker’s necrosis of the cardiomyocyes specially near valves characterized by loss of cell architectures, hypereosinophilic with severe glassy or waxy hyaline degenerations of cardiac muscle sarcoplasm with small dark pyknotic nuclei or completely loss of nuclei in some cells (arrow). (b) Showing severe vacuolization characterized by formation of various scattered vacuoles or vacuole-like structures with variable shape and size within the cardiomyocytes sarcoplasm specially in peri-nuclear area (arrow). (c) Showing congested heart muscle with severe prominent blood vessels dilatation with severe engorgement with RBCs (arrow). (d) Showing thickening of the blood vessels wall. Stain: (a) PAS, (b) and (d) H&E, (c) Blue Masson’s trichrome. Scale bars: All = 40 µm.

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  Fig. 7.. A photomicrographs of the heart myocardium of the G2 treated with DOX. (a) Showing severe thickening of the blood vessels wall, accompanied with severe fibroblasts hyperplasia (arrowhead) with severe fibrosis (excessive collagen fibers productions) (arrow). (b) Showing wide prominent area of hemorrhage (excessive extravasated RBCs) (arrow). (c) and (d) Showing myocarditis characterized by chronic inflammatory cells infiltrations specially lymphocytes and plasma cells with fibrous connective tissue proliferations in between cardiomyocytes (arrow). Stain: (a), (d) Blue Masson’s trichrome, (b), (c) H&E. Scale bars: All = 40 µm, except b = 300 µm.

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  Fig. 8.. A photomicrographs of the heart myocardium of the G3 treated with HES. (a) and (b) Showing normal and intact cardiomyocyes with normal organization and directions without any pathological lesions looks like the control group. Stain: All: H&E. Scale bars: a,b =40 µm.

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  Fig. 9.. A photomicrographs of the heart myocardium of the G4 treated with both DOX and HES. (a) and (b) Showing normal, intact cardiomyocyes with normal directions and orientations looks like normal without any pathological effect, except slightly blood vessels dilatation with congestion (arrow). (c) Showing moderate fibrous connective tissue proliferations surrounding blood vessels (arrow). (d) showing individual extravasated RBCs in between the cardiomyocytes (arrow). Stain: (a) and (b) H&E, (c), (d) Blue Masson’s trichrome. Scale bars: All = 40 µm.

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  Fig. 10.. A photomicrographs of the heart myocardium of the G4 treated with both DOX and HES. (a) Showing mild Zenker’s degenerations of the cardiomyocyes. (b) Showing individual small sized vacuoles within the cardiomyocyes sarcoplasm (arrow). (c) Showing very small focus of inflammatory cells infiltrations between muscle fascicles (arrow). (d) showing individual scattered inflammatory cells between cardiomyocyes specially lymphocytes (arrow). Stain: All: H&E. Scale bars: All = 40 µm.

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  Fig. 11.. A photomicrographs of the immunohistochemical staining of the cardiac myocytes against anti-caspase-3 antibody, in all experimental groups where, the positive signal is mostly expressed in the nuclei of morphologically identifiable apoptotic cells. (a) G1 (control group) demonstrated completely negative expression against anti-caspase-3 antibody within the cardiac myocytes. (b) and (c) G2 treated with DOX revealed strongly positive immuno-reactivity against anti-caspase-3 antibody that was widely expressed in the nucleus of the cardiac myocytes and also within the interfibrillar connective tissue cells confirming widespread of apoptosis. (d) G3 treated with HES claimed negative expression against anti-caspase-3 antibody in the cardiac myocytes resembling the control group. Stain: (a)–(d) Immunohistochemical stain against anti-caspase-3 antibody. Scale bars: a,d = 300 µm, b,c = 40 µm.

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  Fig. 12.. A photomicrographs of the immunohistochemical stain of cardiac myocytes against anti-caspase-3 antibody, (a)–(d) G4 treated with DOX+ HES clarified negative expression against anti-caspase-3 antibody in wide areas of cardiac myocytes (a), but few mild positive immuno- localization against anti-caspase-3 antibody in individual cardiomyocytes were also observed specially around the blood vessels (b)–(d). Stain: (a)–(d) Immunohistochemical stain against anti-caspase-3 antibody. Scale bars: a= 300 µm, b–d=40 µm.