Rutaecarpine Inhibits Doxorubicin-Induced Oxidative Stress and Apoptosis by Activating AKT Signaling Pathway

Figure 1. Doxorubicin (DOX) dose-dependently induced cardiac dysfunction and cardiac fibrosis. Mice were intravenously treated with DOX (5 mg/kg) or its solvent once per week for 2 or 4 weeks. (A) Representative M-model echocardiography and the measurement of left ventricular EF% and FS%. The heart rate, left ventricular posterior wall (LVPW), and left ventricular anterior wall (LVAW) at diastole and systole were shown below (n = 6). (B) Representative image of hematoxylin and eosin (H and E) staining and the HW/BW and HW/TL ratios. Scale bar 1 mm. (C) Cardiac myocyte size was evaluated by TRITC-labeled wheat germ agglutinin (WGA) staining, and the cross-sectional area was quantified in the right panel (100 cells counted per heart, n = 3). (D) ANF and BNP mRNA expressions were determined by qRT-PCR in the heart (n = 5). (E) Cardiac fibrosis was detected by Masson's trichrome staining, and the relative fibrotic area was quantified (n = 5, right). Scale bar 50 μm. (F) The collagen I and collagen III mRNA levels in the heart (n = 5). Data are presented as mean ± SEM, and n represents the number of animals per group. *p < 0.05, **p < 0.01, ***p < 0.001 vs. vehicle.

Figure 2. Doxorubicin dose-dependently induced cardiac oxidative stress and apoptosis. Mice were intravenously treated with DOX (5 mg/kg) or its solvent once per week for 2 or 4 weeks. (A,B) The changes of food uptake and body weight after DOX treatment (n = 4). (C) Reactive oxygen species (ROS) generation was measured by dihydroethidium (DHE) staining, and the quantification of fluorescence intensity was shown right (n = 5). Scale bar 200 μm. (D) Representative TUNEL staining in the heart tissues and the TUNEL-positive nuclei were quantified (n = 3, right). Scale bar 100 μm. *p < 0.05, **p < 0.01, ***p < 0.001 vs. vehicle.

Figure 3. Rutaecarpine mitigated DOX-induced cardiac dysfunction and fibrosis. Mice were treated with DOX and then on the same day received intragastric administration of 20 or 40 mg/kg Rut every day for 4 weeks. (A) Representative M-model echocardiography and the measurement of left ventricular EF% and FS%. The heart rate, LVPW, and LVAW at diastole and systole were shown below (n = 6). (B) Representative image of HandE staining and the HW/BW and HW/TL ratios (right, n = 6). Scale bar 1 mm. (C) Cardiac myocyte size was evaluated by TRITC-labeled WGA staining, and the cross-sectional area was quantified in the right panel (100 cells counted per heart, n = 3). (D) ANF and BNP mRNA expressions were determined by real-time quantitative PCR (qRT-PCR) in the heart (n = 5). (E) Cardiac fibrosis was detected by Masson's trichrome staining, and the relative fibrotic area was quantified (n = 5, right). Scale bar 50 μm. (F) The collagen I and collagen III mRNA levels in the heart (n = 5). Data are presented as mean ± SEM, and n represents the number of animals per group. *p < 0.05, **p < 0.01, ***p < 0.001 vs. vehicle; #p < 0.05, ##p < 0.01 vs. DOX group.

Figure 4. Rutaecarpine alleviated DOX-induced oxidative stress and apoptosis. Mice were treated with DOX and then on the same day received 20 or 40 mg/kg Rut (intragastric administration) every day for 4 weeks. (A) ROS generation in the heart was detected by DHE staining, and the quantification of fluorescence intensity was shown right (n = 5). Scale bar 100 μm. (B) Representative TUNEL staining in the heart tissues and the TUNEL-positive nuclei were quantified (n = 3, right). Scale bar 100 μm. (C–E) Relative superoxide dismutase (SOD) activity, glutathione (GSH) content, and enhancement of malondialdehyde (MDA) activity in the heart; data in other groups were normalized to the vehicle group (n = 5). (F) Western blot analysis of p-AKT, Nrf-2, GCLM, HO-1, cleaved caspase-3, Bax, and Bcl-2 in the heart and the relevant quantification was shown right (right, n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 vs. vehicle; #p < 0.05, ##p < 0.01 vs. DOX group.

Figure 5. AKT inhibitor (AKTi) abolished the antioxidant and antiapoptotic effect of rutaecarpine in DOX-treated hearts. Mice were intraperitoneally injected with AKTi (20 mg/kg) every other day for 4 weeks before DOX and Rut administration. (A) ROS generation in the heart was detected by DHE staining, and the quantification of fluorescence intensity was shown right (n = 5). Scale bar 100 μm. (B) Representative TUNEL staining in the heart tissues and the TUNEL-positive nuclei were quantified (n = 3, right). Scale bar 50 μm. (C–E) Relative SOD activity, GSH content, and MDA activity in the heart; data in other groups were normalized to the vehicle group (n = 5). (F) Western blot analysis of p-AKT, Nrf-2, GCLM, HO-1, cleaved caspase-3, Bax, and Bcl-2 in the heart, and the relevant quantification was shown right (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 vs. vehicle; #p < 0.05, ##p < 0.01 vs. DOX group; &p < 0.05 vs. DOX plus Rut group.

Figure 6. AKT inhibitor alleviated the cardio-protective effect of rutaecarpine on DOX-induced heart failure. Mice were intraperitoneally injected with AKTi (20 mg/kg) every other day for 4 weeks from the first day before DOX and Rut administration. (A) Representative M-model echocardiography of the left ventricular EF% and FS%. The heart rate, LVPW, and LVAW at diastole and systole were shown below (n = 5). (B) Representative image of HandE staining and the HW/BW and HW/TL ratios (right, n = 5). Scale bar 1 mm. (C) Cardiac myocyte size was evaluated by TRITC-labeled WGA staining, and the cross-sectional area was quantified in the right panel (100 cells counted per heart) (n = 3, right). Scale bar 100 μm. (D) ANF and BNP mRNA expressions were determined by qRT-PCR in the heart (n = 5). *p < 0.05, **p < 0.01, ***p < 0.001 vs. vehicle; #p < 0.05, ##p < 0.01 vs. DOX group; &p < 0.05 vs. DOX plus Rut group.

Figure 7. The proposed protective mechanism by rutaecarpine on DOX-induced heart failure. Rutaecarpine activated AKT/Nrf-2 signaling pathway, which in turn inhibited DOX-induced oxidative stress and apoptosis through regulating heme oxygenase-1 (HO-1) and GSH cysteine ligase modulatory subunit (GCLM) expression.