Protective effects of valsartan administration on doxorubicin‑induced myocardial injury in rats and the role of oxidative stress and NOX2/NOX4 signaling

Abstract

Clinical application of doxorubicin (DOX) is hampered by its potential cardiotoxicity, however angiotensin receptor blockers could attenuate DOX‑induced cardiomyopathy. The present study tested the hypothesis that simultaneous administration of valsartan (Val) with DOX could prevent DOX‑induced myocardial injury by modulating myocardial NAD(P)H oxidase (NOX) expression in rats. Eight‑week‑old male Sprague‑Dawley rats were randomly divided into control (CON), DOX, and DOX+Val groups. After 10 weeks, surviving rats underwent echocardiography examination, myocardial mRNA and protein expression detection of NOX1, NOX2 and NOX4. H9C2 cells were used to perform in vitro experiments, reactive oxygen species (ROS) production and apoptosis were observed under the conditions of down‑ or upregulation of NOX2 and NOX4 in DOX‑ and DOX+Val‑treated H9C2 cells. Cardiac function was significantly improved, pathological lesion and collagen volume fraction were significantly reduced in the DOX+Val group compared with the DOX group (all P<0.05). Myocardial protein and mRNA expression of NOX2 and NOX4 was significantly downregulated in DOX+Val group compared with in the DOX group (all P<0.05). In vitro, ROS production and apoptosis in DOX‑treated H9C2 cells was significantly reduced by NOX2‑small interfering (si)RNA and NOX4‑siRNA, and significantly increased by overexpressing NOX2 and NOX4. To conclude, Val applied simultaneously with DOX could prevent DOX‑induced myocardial injury and reduce oxidative stress by downregulating the myocardial expression of NOX2 and NOX4 in rats.

Figure 1.

Schematic diagram of experiments. CON, control; DOX, doxorubicin; Val, valsartan.

Figure 2.

Cumulative survival rates of rats post-treatment in each experimental group. CON, control; DOX, doxorubicin; Val, valsartan.

Figure 3.

Representative echocardiographic images and cardiac functional parameters. *P<0.05 vs. CON; ^†P<0.05 vs. DOX. LVEF, left ventricular ejection fraction; LVFS, left ventricular fraction shortening; LVEDD, left ventricular end-diastolic diameter; LVESD, left ventricular end-systolic diameter; CON, control; DOX, doxorubicin; Val, valsartan.

Figure 4.

Myocardial histopathology and protein expression of NOX1, NOX2 and NOX4. (A) Masson staining and CVF (magnification, ×200). (B) Sirius red staining and CVF (magnification, ×200). Collagen stained with Masson staining (blue) and Sirius red staining (red) are significantly increased in the DOX group compared with those in the CON group, which is attenuated in the DOX+Val group. (C) Myocardial protein expression of NOX1, NOX2 and NOX4 detected by western blotting. *P<0.05 vs. CON; ^†P<0.05 vs. DOX.CON, control; DOX, doxorubicin; Val, valsartan; NOX, NAD(P)H oxidase; CVF, collagen volume fraction.

Figure 5.

Cell viability measured by MTT assay. (A) H9C2 cells were treated with various concentrations of DOX for 24 h, DOX resulted in cell death in a dose-dependent manner. Cell viability significantly decreased with 1 µM DOX, so this concentration was chosen as the target dose. (B) H9C2 cells were treated with numerous concentrations of Val, the results revealed that the nine different concentrations of Val did not affect the survival of H9C2 cells within 24 h. (C) Val concentration required to protect against DOX-induced cytotoxicity was calculated by performing a dose-response study in the presence of 1, 5 and 10 µM Val. The cytotoxic effects of DOX were significantly attenuated by pre-treatment with 5 and 10 µM Val. Based on these results, 5 µM Val was selected as the target dose for further study. *P<0.05 vs. CON; ^†P<0.05 vs. DOX; n.s., not significant. CON, control; DOX, doxorubicin; Val, valsartan; NOX, NAD(P)H oxidase.

Figure 6.

Intracellular ROS production in vitro in H9C2 cells post-treatment. (A) Expression of ROS in H9C2 cells was detected by a DCF-DA assay under fluorescence microscope (magnification, ×100). H9C2 cells were treated as follows: i) Control group; ii) H9C2 cells exposed to 1 µM DOX for 24 h; iii) cells exposed to 5 µM Val for 1 h; iv) cells were pre-treated with 5 µM Val for 1 h followed by treatment with 1 µM DOX for 24 h; v) cells were transiently transfected with NOX2-siRNA for 24 h followed by treatment with 1 µM DOX for 24 h; vi) cells were transiently transfected with NOX4-siRNA for 24 h followed by treatment with 1 µM DOX for 24 h; vii) cells were transiently transfected with negative siRNA for 24 h followed by treatment with 1 µM DOX for 24 h; viii) cells were transiently transfected with an empty vector for 24 h; ix) NOX2-OE cells were treated with 1 µM DOX for 24 h; x) NOX4-OE cells were treated with 1 µM DOX for 24 h. (B) Semi-quantitative analysis of ROS production detected by DCF-DA. *P<0.05 vs. control; ^†P<0.05 vs. DOX. DCF-DA, dichlorofluorescein; ROS, reactive oxygen species; DOX, doxorubicin; Val, valsartan; NOX, NAD(P)H oxidase; siRNA, small interfering RNA; OE, overexpressed.

Figure 7.

Apoptosis in vitro in H9C2 cells post-treatment. (A) Apoptosis in H9C2 cells detected by flow cytometry. (B) Cardiomyocyte apoptosis was significantly increased in DOX-treated H9C2 cells compared with the control group, which was significantly reduced by DOX+Val and NOX2-siRNA and NOX4-siRNA, whereas apoptosis in DOX-treated H9C2 cells was further increased following NOX2 and NOX4 overexpression. *P<0.05 vs. control; ^†P<0.05 vs. DOX. DOX, doxorubicin; Val, valsartan; NOX, NAD(P)H oxidase; siRNA, small interfering RNA; OE, overexpressed.