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

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.