Ursolic acid prevents doxorubicin-induced cardiac toxicity in mice through eNOS activation and inhibition of eNOS uncoupling

Abstract

In addition to the known antitumour effects of ursolic acid (UA), increasing evidence indicates that this molecule plays a role in cardiac protection. In this study, the effects of ursolic acid on the heart in mice treated with doxorubicin (DOX) were assessed. The results showed that ursolic acid improved left ventrical fractional shortening (LVFS) and left ventrical ejection fraction (LVEF) of the heart, increased nitrogen oxide (NO) levels, inhibited reactive oxygen species (ROS) production and decreased cardiac apoptosis in mice treated with doxorubicin. Mechanistically, ursolic acid increased AKT and endothelial nitric-oxide synthase (eNOS) phosphorylation levels, and enhanced eNOS expression, while inhibiting doxorubicin induced eNOS uncoupling through NADPH oxidase 4 (NOX4) down-regulation. These effects of ursolic acid resulted in heart protection from doxorubicin-induced injury. Therefore, ursolic acid may be considered a potential therapeutic agent for doxorubicin-associated cardiac toxicity in clinical practice.

Keywords: apoptosis; cardiac toxicity; doxorubicin; ursolic acid.

Figure 1

Ursolic acid preserves cardiac function in mice treated with doxorubicin in both early and late injury phases. A, Heart echocardiography. Upper panel, Doxorubicin + Ursolic acid group (UA + DOX); lower panel, Doxorubicin group (DOX). LVIDd, left ventricle internal diameter in diastole; LVIDs, left ventricle internal diameter in systole. B, Ursolic acid treatment significantly improved FS at both 7 and 28 days, compared with the Doxorubicin control group (n = 5, *P < 0.05). C, Ursolic acid significantly improved EF at both 7 and 28 days, compared with the doxorubicin control (n = 5, *P < 0.05)

Figure 2

Ursolic acid increases NO production, inhibits ROS generation and decreases CKMB releasing in the mouse heart after doxorubicin treatment. Mice were divided into three groups: Sham group, Doxorubicin group and Doxorubicin + Ursolic acid group. A, DOX significantly decreased NO production compared with the sham (n = 5, **P < 0.05), while ursolic acid significantly increased NO production compared with the doxorubicin control (n = 5, *P < 0.01). B, Ursolic acid significantly inhibited ROS production compared with doxorubicin (n = 5, *P < 0.05). C, Ursolic acid significantly decreased CKMB releasing from the hearts in mice compared with doxorubicin (n = 5, *P < 0.05)

Figure 3

Ursolic acid decreases apoptosis in the heart of mice treated with doxorubicin. A, TUNEL staining of heart samples. a, Sham group; b, Doxorubicin + Ursolic acid group; c, Doxorubicin group (Bar = 20 μm). B, Ursolic acid significantly decreased cardiac cell apoptosis in mice treated with doxorubicin compared with the doxorubicin control (n = 5, *P < 0.01). Apoptotic cell nuclei were stained brown (arrow). C, Western blot detection of cleaved caspase‐3 in the mouse heart after treatment with doxorubicin. Lanes 1 to 3 represent the Sham, Doxorubicin and Doxorubicin + Ursolic acid treatment groups respectively. E. Ursolic acid significantly decreased cleaved caspase‐3 levels in the hearts of mice compared with doxorubicin (n = 5, *P < 0.01)

Figure 4

Ursolic acid inhibits inflammation and fibrosis in the mouse heart after doxorubicin treatment. A, H&E staining of heart. a, Sham group; b, Doxorubicin + Ursolic acid group; c, Doxorubicin group. Ursolic acid overtly decreased inflammatory cell infiltration compared with doxorubicin (Bar = 50 μm; arrows indicate inflammatory cells). B, Masson's staining of heart samples. a, Sham group; b, Doxorubicin + Ursolic acid group; c, Doxorubicin group (Bar = 20 μm). Ursolic acid significantly decreased fibrosis compared with the doxorubicin control (n = 5, *P < 0.01)

Figure 5

Effects of ursolic acid on phosphorylation levels of AKT and eNOS. Protein levels were detected by immunoblot. A, Effects of ursolic acid on AKT and p‐AKT. Lanes 1 to 3 represent the Sham, Doxorubicin and Doxorubicin + Ursolic acid treatment groups respectively. B, doxorubicin significantly decreased AKT phosphorylation compared with sham (n = 5, **P < 0.05), while ursolic acid significantly increased AKT phosphorylation compared with doxorubicin (n = 5, *P < 0.05). C, Effect of ursolic acid on p‐eNOS. Lanes 1 to 3 represent the Sham, Doxorubicin and Doxorubicin + Ursolic acid treatment groups respectively. D, doxorubicin significantly decreased eNOS phosphorylation compared with sham (n = 5, **P < 0.01), while ursolic acid significantly increased eNOS phosphorylation compared with doxorubicin (n = 5, *P < 0.01)

Figure 6

Effects of ursolic acid on eNOS and NOX4. A, Effect of ursolic acid on eNOS. Lanes 1 to 3 represent the Sham, Doxorubicin and Doxorubicin+Ursolic acid treatment groups respectively. B, doxorubicin significantly increased eNOS expression compared with sham (n = 5, **P < 0.05), while ursolic acid significantly increased eNOS expression compared with doxorubicin (n = 5, *P < 0.01). C, doxorubicin significantly increased NOX4 expression compared with sham (n = 5, *P < 0.01), while ursolic acid significantly decreased Nox4 expression compared with doxorubicin (n = 5, *P < 0.01). D, Effect of ursolic acid on the expression of eNOS monomer and dimer. Lanes 1 to 3 represent the Sham, Doxorubicin and Doxorubicin + Ursolic acid treatment groups respectively. E. doxorubicin significantly increased eNOS monomer/dimer ratio compared with sham (n = 3, *P < 0.05), while ursolic acid significantly decreased eNOS monomer/dimer ratio compared with doxorubicin (n = 3, *P < 0.05)

Figure 7

Schematic representation of study outcome. Doxorubicin increased eNOS and NOX4 levels, which results in eNOS uncoupling and decreased eNOS phosphorylation, enhancing ROS production. Meanwhile, ursolic acid increased eNOS production and phosphorylation, and inhibited NOX4 levels, decreasing ROS levels through eNOS activation and reduced eNOS uncoupling