Orosomucoid 1 Attenuates Doxorubicin-Induced Oxidative Stress and Apoptosis in Cardiomyocytes via Nrf2 Signaling

Abstract

Doxorubicin (DOX) is an effective anticancer drug, but its therapeutic use is limited by its cardiotoxicity. The principal mechanisms of DOX-induced cardiotoxicity are oxidative stress and apoptosis in cardiomyocytes. Orosomucoid 1 (ORM1), an acute-phase protein, plays important roles in inflammation and ischemic stroke; however, the roles and mechanisms of ORM1 in DOX-induced cardiotoxicity remain unknown. Therefore, in the present study, we aimed to investigate the function of ORM1 in cardiomyocytes experiencing DOX-induced oxidative stress and apoptosis. A DOX-induced cardiotoxicity animal model was established in C57BL/6 mice by administering an intraperitoneal injection of DOX (20 mg/kg), and the control group was intraperitoneally injected with the same volume of sterilized saline. The effects were assessed after 7 d. Additionally, H9c2 cells were stimulated with DOX (10 μM) for 24 h. The results showed decreased ORM1 and increased oxidative stress and apoptosis after DOX stimulation in vivo and in vitro. ORM1 overexpression significantly reduced DOX-induced oxidative stress and apoptosis in H9c2 cells. ORM1 significantly increased the expression of nuclear factor-like 2 (Nrf2) and its downstream protein heme oxygenase 1 (HO-1) and reduced the expression of the lipid peroxidation end product 4-hydroxynonenal (4-HNE) and the level of cleaved caspase-3. In addition, Nrf2 silencing reversed the effects of ORM1 on DOX-induced oxidative stress and apoptosis in cardiomyocytes. In conclusion, ORM1 inhibited DOX-induced oxidative stress and apoptosis in cardiomyocytes by regulating the Nrf2/HO-1 pathway, which might provide a new treatment strategy for DOX-induced cardiotoxicity.

Figure 1

Effects of doxorubicin (DOX) on C57BL/6 mice. (a) General pictures of the heart. (b) Body weight. (c) Heart weight. (d) Heart weight/body weight. (e, f) Systolic and diastolic functions in both groups (n = 20). (g) B-type natriuretic peptide (BNP) levels in mice detected using polymerase chain reaction (PCR) (n = 10). (h) Malondialdehyde (MDA) levels in both groups (n = 10). (i, j) Myocardial cell size evaluated using wheat germ agglutinin (WGA) staining (n = 5). (k, l) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining (n = 5). Data are expressed as the mean ± standard error of the mean (SEM). ^∗∗P < 0.01.

Figure 2

Downregulation of ORM1 and upregulation of oxidative stress and apoptosis in the DOX-induced cardiomyopathy model in vitro and in vivo. (a) Differentially expressed candidate proteins in control and DOX-treated mice analyzed using LC/MS technology. Low expression is depicted in green, and high expression is depicted in red. (b) mRNA levels of ORM1, Nrf2, and HO-1 in the hearts of mice in the DOX-treated groups (n = 6). (c, d) Western blot analysis of ORM1, Nrf2, HO-1, 4-HNE, and cleaved caspase-3 (n = 6). (e, f) Immunohistochemical staining images of ORM1- and 4-HNE-stained heart sections from control and DOX-treated mice (n = 6). (g) mRNA levels of ORM1, Nrf2, and HO-1 in DOX-treated H9c2 cells (10 μM and 24 h) and the control group (n = 3). (h, i) Western blot analysis of ORM1, Nrf2, HO-1, 4-HNE, and cleaved caspase-3 in DOX-treated H9c2 cells (10 μM and 24 h) and the control group (n = 3). Data are expressed as the mean ±standard error of the mean (SEM). ^∗∗P < 0.01 and ^∗P < 0.05.

Figure 3

ORM1 reduces doxorubicin- (DOX-) induced oxidative stress and apoptosis in H9c2 cells. (a, b) Western blot analysis of ORM1, Nrf2, HO-1, 4-HNE, and cleaved caspase-3. (c) Cell survival rate analysis using the Cell Counting Kit 8 (CCK-8). Cell survival rate is expressed as the optical density (OD) value (% control). (d) Cellular malondialdehyde (MDA) content. (e, f) Fluorescence image (red fluorescence) of reactive oxygen species (ROS) measured using dichlorodihydrofluorescein diacetate (DCFH-DA). (g, h) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining images with calculated apoptosis indices. Data are expressed as the mean ± standard error of the mean (SEM); n = 6. ^∗∗P < 0.01.

Figure 4

Nrf2 knockdown reverses the protective effects of ORM1 in doxorubicin- (DOX-) treated H9c2 cells. (a, b) Western blot analysis of ORM1, Nrf2, HO-1, 4-HNE, and cleaved caspase-3. (c) Cell survival analysis using the Cell Counting Kit 8 (CCK-8). (d) Cellular malondialdehyde (MDA) content. (e, f) Fluorescence image (red fluorescence) of reactive oxygen species (ROS) measured using dichlorodihydrofluorescein diacetate (DCFH-DA). (g, h) Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining images with calculated apoptosis indices. Data are expressed as the mean ± standard error of the mean (SEM); n = 6. ^∗∗P < 0.01 and ^∗P < 0.05.