A herbal formula, SYKT, reverses doxorubicin‑induced myelosuppression and cardiotoxicity by inhibiting ROS‑mediated apoptosis

Abstract

Doxorubicin (DOX) is an antineoplastic drug widely used for the treatment of various types of cancer; however, it can induce severe side effects, such as myelosuppression and cardiotoxicity. Sanyang Xuedai (SYKT) is a natural medicine originating from an ancient prescription of the Dai nationality in Southwest China. With eight Chinese herbal medicines, including sanguis draconis, radix et rhizoma notoginseng, radix et rhizoma glycyrrhizae and radix angelicae sinensis as the primary ingredients, SYKT has been reported to possess numerous biological functions. The present study investigated whether SYKT can confer protection against DOX‑induced myelosuppression and cardiotoxicity, and explored the potential mechanism involved. Mice were treated with DOX, SYKT or a combination of the two; hematopoietic functions were assessed by measuring the number of peripheral blood cells, cluster of differentiation CD34+/CD44+ bone marrow cells and apoptotic cells. Myocardial enzymes, including aspartate aminotransferase, lactate dehydrogenase, creatine kinase (CK) and its isoform CK‑MB, were assessed using a biochemical analyzer. The apoptotic rate of cardiomyocytes was assessed using flow cytometry. Histopathological analysis was conducted using hematoxylin‑eosin staining. Intracellular reactive oxygen species (ROS) production was evaluated using a dichlorofluorescein intensity assay. The mice treated with DOX exhibited a reduced survival rate, reduced peripheral blood and CD34+/CD44+ cell counts, elevated myocardial enzymes and apoptotic indices in bone marrow cells and cardiomyocytes, all of which were effectively prevented by SYKT co‑administration. Furthermore, bone marrow cells and myocytes from mice treated with DOX demonstrated increased dichlorofluorescein intensity, which was attenuated by SYKT. Notably, SYKT did not interfere with the effects of DOX on tumor volume or the induction of tumor cell apoptosis in tumor‑bearing mice. The present study indicated that SYKT may counteract DOX‑induced myelosuppression and cardiotoxicity through inhibiting ROS‑mediated apoptosis. These findings suggested that SYKT may have potential as a means to counteract the potentially fatal hematopoietic and cardiac complications associated with DOX treatment.

Figure 1.

SYKT reduces DOX-induced mortality in mice. Mice in the SYKT group received SYKT (1.2 ml/kg/d) for 5 days. Mice in the DOX group received a single dose of DOX (20 mg/kg) on day 5. Mice in the SYKT/DOX group received both SYKT and DOX as aforementioned. Control mice received normal saline. Mortality was monitored daily. Mortality among mice treated with DOX alone was significantly higher compared with in mice receiving a combination of DOX and SYKT (P<0.05). DOX, doxorubicin.

Figure 2.

SYKT mitigates the DOX-induced reduction in peripheral blood cell counts. Mice were treated with DOX (3 mg/kg/d, on days 2, 4 and 6) and SYKT (1.2 ml/kg/d, on days 1, 3 and 5) alone or combined. Peripheral blood cell counts were performed at different time points. (A) WBC counts. (B) RBC counts. (C) PLT counts. Data are expressed as the mean ± standard deviation. *P<0.05 vs. control group; ^#P<0.05 vs. DOX group. DOX, doxorubicin; WBC, white blood cell; RBC, red blood cell; PLT, platelet; Cont, control.

Figure 3.

SYKT reduces DOX-induced myelotoxicity. Mice were treated with DOX (3 mg/kg/d on days 2, 4 and 6) and SYKT (1.2 ml/kg/d on days 1, 3 and 5) alone or combined, and the number and apoptotic rate of bone marrow cells were determined. (A) Flow cytometric analysis of CD34⁺ and CD44⁺ cells in bone marrow on day 15. (B) Apoptotic rate of bone marrow cells. The cell distribution was analyzed using dUTP binding and PI uptake. Representative dot plots are shown for one of the six independent experiments. Data are expressed as the mean ± standard deviation. *P<0.05 vs. control group; ^#P<0.05 vs. DOX group. DOX, doxorubicin; CD, cluster of differentiation; dUTP, 2′-deoxyuridine, 5′-triphosphate; PI, propidium iodide; Cont, control group; FITC, fluorescein isothiocyanate.

Figure 4.

SYKT reduces DOX-induced cardiotoxicity. Mice were treated with DOX (3 mg/kg/d on days 2, 4 and 6) and SYKT (1.2 ml/kg/d on days 1, 3 and 5) alone or combined. (A) Serum activity of myocardial enzymes was determined using an automatic biochemical analyzer on day 15. (B) Apoptotic rate of cardiomyocytes. Cell distribution was analyzed using dUTP binding and PI uptake. The results are expressed as dot plots, as represented in one of the six independent experiments. (C) Hematoxylin-eosin staining pattern in cardiac sections. Arrows indicate abnormal ultrastructural changes (as indicated by the loss of the normal radiating pattern of the cell plates) (magnification, ×200). Data are expressed as the mean ± standard deviation. *P<0.05 vs. control group; ^#P<0.05 vs. DOX group. DOX, doxorubicin; AST, aspartate aminotransferase; LDH, lactate dehydrogenase; CK, creatine kinase; dUTP, 2′-deoxyuridine, 5′-triphosphate; PI, propidium iodide; Cont, control group.

Figure 5.

SYKT reduces DOX-induced myelosuppression and cardiotoxicity by inhibiting ROS-mediated apoptosis. Mice were treated with DOX (3 mg/kg/d on days 2, 4 and 6) and SYKT (1.2 ml/kg/d on days 1, 3 and 5) in the presence or absence of vitamin E (0.2 ml bolus dose on days 1, 3 and 5). ROS production and apoptotic rate of bone marrow cells and cardiomyocytes were measured on day 15. DCFDA fluorescence of (A) bone marrow cells and (B) cardiomyocytes was evaluated using flow cytometry. (C) Bone marrow cells and (D) cardiomyocytes apoptosis was assessed using terminal deoxynucleotidyl transferase dUTP nick end labeling staining and flow cytometry. Data are expressed as the mean ± standard deviation. *P<0.05 vs. control group; ^#P<0.05 vs. DOX group. DOX, doxorubicin; DCFDA, 2′,7′-dichlorofluorescin diacetate; Cont, control.

Figure 6.

SYKT does not interfere with the antitumor efficacy of DOX. Mice bearing tumors were treated with DOX (3 mg/kg/d on days 2, 4 and 6) and SYKT (1.2 ml/kg/d on day 1, 3 and 5) alone or combined. Tumor volume and apoptosis were assessed. (A) Tumor volume was measured using Vernier calipers. (B) Tumor cell apoptotic rate was assessed using terminal deoxynucleotidyl transferase dUTP nick end labeling staining and flow cytometry. Data are expressed as the mean ± standard deviation. *P<0.05 vs. control group. DOX, doxorubicin; Cont, control.