Matrine attenuates oxidative stress and cardiomyocyte apoptosis in doxorubicin-induced cardiotoxicity via maintaining AMPKα/UCP2 pathway

Figures

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  Figure 1. Matrine attenuated doxorubicin (DOX)-induced cardiotoxicity in mice. (A) Quantitative analysis of fraction shortening (FS) obtained from echocardiography (n = 8). (B) and (C) Hemodynamic analysis of mice with or without matrine treatment (n = 8). (D) Statistical results of the heart weight (HW)/tibia length (TL) (n = 12). (E) Body weight of four groups (n = 12). (F) Relative mRNA levels of Anp, Bnp (n = 6). (G) Plasma levels of cTnT, LDH and CK-MB in mice (n = 6). Values represent mean ± SEM. *P< 0.05 versus NS+Vehicle; #P< 0.05 versus DOX+Vehicle.

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  Figure 2. Matrine prevented oxidative damage in response to DOX insult. (A) and (B) Representative DHE and 4-HNE staining images and the statistical results (n = 6). (C) and (D) Representative Western blot and quantitative data (n = 6). (E) The content of MDA, NADPH oxidase activity and total SOD activity in the myocardium (n = 6). (F) Relative mRNA levels of p67phox, Gp91phox and Sod2 (n = 6). Values represent the mean±SEM. *P< 0.05 versus NS+Vehicle; #P< 0.05 versus DOX+Vehicle.

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  Figure 3. Matrine attenuated DOX-induced cardiomyocyte apoptosis. (A) and (B) Representative TUNEL staining images and the quantitative results for apoptosis in heart tissues (n = 6). (C) and (D) Western blot and statistical results in the indicated groups (n = 6). Red arrows indicate TUNEL positive staining. Values represent the mean±SEM. *P< 0.05 versus NS+Vehicle; #P< 0.05 versus DOX+Vehicle.

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  Figure 4. UCP2 was responsible for matrine-mediated beneficial effect on DOX-induced cardiotoxicity. (A) UCP2 expression level in murine hearts with or without DOX or matrine (n = 6; *P< 0.05 versus NS+Vehicle, #P< 0.05 versus DOX+Vehicle). (B)–(G) Western blot and statistical results in the indicated groups (n = 6). (H) Relative mRNA levels of p67phox, Gp91phox and Sod2 (n = 6). (I) The content of MDA, NADPH oxidase activity and total SOD activity in the myocardium (n = 6). (J)–(K) Plasma levels of cTnT and LDH (n = 6). Values represent the mean±SEM. *P< 0.05 versus the matched groups.

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  Figure 5. Knockdown of UCP2 blunted the protective effects of matrine in vitro. (A) Western blot for UCP2 and the quantitative data for H9C2 cells (n = 6). (B) Representative DCFH-DA images and the statistical results. (C) CCK-8 assay for cell viability (n = 5). (D) Representative images of TUNEL in H9C2 cells (n = 5). (E)–(J) Western blot and statistical results (n = 6). (K) The content of MDA, NADPH oxidase activity and total SOD activity in H9C2 cells (n = 5). (L) LDH levels in H9C2 cells (n = 5). Red arrows indicate TUNEL positive staining. Values represent the mean±SEM. *P< 0.05 versus the matched groups. NS indicates no statistical difference.

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  Figure 6. Matrine prevented DOX-induced downregulation of UCP2 via activating AMPKα. (A) and (B) Western blot and statistical results (n = 6). (C)–(F) Western blot and statistical results (n = 6). (G) Statistical results of intracellular ROS production detected by DCFH-DA in H9C2 cells (n = 5). (H) The content of MDA, NADPH oxidase activity and total SOD activity in H9C2 cells (n = 5). (I)–(K) Western blot and statistical results (n = 6). (L) Cell viability by CCK-8 (n = 5). (M) Relative LDH levels (n = 5). Values represent the mean±SEM. *P< 0.05 versus the matched groups. NS indicates no statistical difference.

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  Figure 7. Ampkα deficiency abolished the protective effect of matrine in vivo. (A)–(D) Representative Western blot images and statistical results (n = 6). (E) The content of MDA and total SOD activity (n = 6). (F) Plasma levels cTnT and LDH (n = 6). (G) Heart function evaluated by echocardiography and hemodynamic analysis of mice (n = 8). (H) Statistical results of the HW/TL (n = 12). Values represent the mean±SEM. *P< 0.05 versus the matched groups.