Brain‐derived neurotrophic factor attenuates doxorubicin‐induced cardiac dysfunction through activating Akt signalling in rats

Figure 1. Protein expression of brain‐derived neurotrophic factor (BDNF) and tropomyosin‐related kinase B (TrkB) in Doxorubicin (Dox) and BDNF‐treated rat ventricles. (A and C) Representative western blot bands and statistical results of BDNF protein expression normalized by GAPDH in left and right ventricles. (B and D) Representative western blot bands and statistical results of TrkB protein expression normalized by GAPDH in left and right ventricles. *P < 0.05 versus sham, #P < 0.05 versus Dox, n = 5 each group.

Figure 2. Morphological alterations of LV myocytes in Dox and BDNF‐treated rats. (A–F) Transmission electron microscope images showed the ultrastructure changes of LV myocytes of sham, Dox and BDNF‐treated rats, at magnification 10,000× and 20,000×, respectively, scale bar: 2 μm. (G–I) Representative images of haematoxylin and eosin staining from left ventricles of sham, Dox and BDNF‐treated rats, at magnification 400×, scale bar: 50 μm.

Figure 3. Cardiac apoptosis and interstitial fibrosis of left ventricles in Dox and BDNF‐treated rats. (A–F) TUNEL staining, the images at magnification 200× (scale bar: 50 μm) and 400× (scale bar: 20 μm). (G) Ratio of TUNEL‐positive cells (stained in brown) per field. (H–J) Representative Masson trichrome staining images from left ventricles of sham, Dox and BDNF‐treated rats. The images were magnified 400×, scale bar: 50 μm. (K) Collagen volume fraction was calculated as the ratio of collagen area to total area. **P < 0.01 versus sham; ##P < 0.01 versus Dox, n = 5 each group.

Figure 4. Effect of BDNF on oxidative stress and cardiac function of Dox‐treated rats by echocardiography. (A and D) Superoxide dismutase (SOD) activity, (B and E) malondialdehyde (MDA) level and (C and F) reduced glutathione (GSH) content in left and right rat ventricles. (G) Representative images of cardiac function. (H) Ejection fraction (EF). (I) Fractional shortening (FS). (J) LV end systolic volume (LVESV). (K) LV end diastolic volume (LVEDV). *P < 0.05 versus sham, #P < 0.05 versus Dox, n = 5 each group.

Figure 5. Protein expression of Akt signalling in Dox and BDNF‐treated rats. (A and D) Representative western blot bands and statistical results of phosphorylated Akt (p‐Akt) and total Akt (t‐Akt) of left and right ventricles with GAPDH as internal control. (B and E) Representative western blot bands and statistical results of phosphorylated mammalian target of rapamycin (p‐mTOR) and mTOR of left and right ventricles. (C and F) Representative western blot bands and statistical results of phosphorylated Bad (p‐Bad) and Bad of left and right ventricles. *P < 0.05 versus sham, #P < 0.05 versus Dox, n = 5 each group.

Figure 6. Protein expression of p38 and ERK in Dox and BDNF‐treated left and right rat ventricles. (A and C) Representative western blot bands and statistical results of p‐p38 and p38 of left and right ventricles. (B and D) Representative western blot bands and statistical results of p‐ERK and ERK of left and right ventricles. *P < 0.05 versus sham, n = 5 each group.

Figure 7. BDNF antagonized Dox‐induced toxicity of H9c2 cells. (A) Effect of different concentrations of BDNF including 0.3, 1, 3 μM for 24 hrs on cell viability of H9c2 cells. (B) Effect of different durations of BDNF including 6, 24 and 48 hrs at 1 μM on cell viability of H9c2 cells. (C) Effect of BDNF (200, 400 ng/ml) on Dox (1 μM)‐treated cell toxicity of H9c2 cells. (D) BDNF reduced apoptotic rate in Dox‐induced H9c2 cells. (E) BDNF decreased γH2AX positive cells in Dox‐induced H9c2 cells. (F) Representive images of TUNEL staining showing the apoptotic cells (stained in green) and nucleus (stained in blue with DAPI), scale bar: 100 μM. (G) Representive images of γH2AX staining showing the DNA damage (stained in green) and nucleus (stained in blue with DAPI), scale bar: 100 μM. *P < 0.05, **P < 0.01, versus control, #P < 0.05 versus Dox, n = 5 each group.

Figure 8. BDNF inhibited Dox‐induced apoptosis of H9c2 cells by activating Akt. (A) Annexin V‐FITC/propidium iodide (PI) staining by flow cytometry was performed to detect the apoptosis of H9c2 cells. Early apoptosis (Annexin V‐FITC +/PI −, Q4), late apoptosis (Annexin V‐FITC +/PI +, Q2), and necrosis (Annexin V‐FITC −/PI +, Q1). (B) The quantitative presentation of apoptotic cell population by Annexin V‐FITC/PI staining. *P < 0.05 versus control, #P < 0.05 versus Dox, &P < 0.05 versus +BDNF, n = 3 each group. (C) Western blot analysis of the p‐Akt and t‐Akt. GAPDH served as the internal control. (D) Statistical results of p‐Akt/t‐Akt in H9c2 cells. *P < 0.05 versus control, n = 5 each group. (E) Effect of TrkB‐Fc and Akti on cell viability of BDNF‐treated H9c2 cells. *P < 0.05 versus control, #P < 0.05 versus Dox, and P < 0.05 versus +BDNF, n = 5 each group. (F) The quantitative presentation of apoptotic cell population by Annexin V‐FITC/PI staining. (G) Annexin V‐FITC/PI staining by flow cytometry was performed to detect the apoptosis. *P < 0.05 versus Dox, #P < 0.05 versus +BDNF, n = 3 each group.