DL-3- n-butylphthalide attenuates doxorubicin-induced acute cardiotoxicity via Nrf2/HO-1 signaling pathway

Abstract

Doxorubicin (DOX) is a widely used chemotherapeutic drug known to cause dose-dependent myocardial toxicity, which limits its clinical potential. DL-3-n-butylphthalide (NBP), a substance extracted from celery seed species, has a number of pharmacological properties, such as antioxidant, anti-inflammatory, and anti-apoptotic actions. However, whether NBP can protect against DOX-induced acute myocardial toxicity is still unclear. Therefore, this study was designed to investigate the potential protective effects of NBP against DOX-induced acute myocardial injury and its underlying mechanism. By injecting 15 mg/kg of DOX intraperitoneally, eight-week-old male C57BL6 mice suffered an acute myocardial injury. The treatment group of mice received 80 mg/kg NBP by gavage once daily for 14 days. To mimic the cardiotoxicity of DOX, 1uM DOX was administered to H9C2 cells in vitro. In comparison to the DOX group, the results showed that NBP improved cardiac function and decreased serum levels of cTnI, LDH, and CK-MB. Additionally, HE staining demonstrated that NBP attenuated cardiac fibrillar lysis and breakage in DOX-treated mouse hearts. Western blotting assay and immunofluorescence staining suggested that NBP attenuated DOX-induced oxidative stress, apoptosis, and inflammation both in vivo and in vitro. Mechanistically, NBP significantly upregulated the Nrf2/HO-1 signaling pathway, while the Nrf2 inhibitor ML385 prevented NBP from protecting the myocardium from DOX-induced myocardial toxicity in vitro. In conclusion, Our results indicate that NBP alleviates DOX-induced myocardial toxicity by activating the Nrf2/HO-1 signaling pathway.

Keywords: DL-3-n-Butylphthalide; Doxorubicin; Myocardial toxicity; Nrf2; Oxidative stress.

Fig. 1

NBP improves DOX-induced cardiac dysfunction and myocardial injury (A) Representative histological images of the HE staining in three groups. (B) Representative echocardiogram images in three groups. (C) Body weight in the indicated groups 7 days after DOX injection (n = 12). (D) The ratio of heart weight to tibia length (n = 12). (E) LVEF in the indicated groups (n = 8). (F) LVFS in the indicated groups (n = 8). (G–I) The LDH, CK-MB, and cTnI levels among groups (n = 6). Values represent mean ± SEM. ****p < 0.0001 vs. Ctrl group; ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001 vs. DOX group.

Fig. 2

NBP inhibited DOX-induced oxidative damage in mice (A) Representative DHE staining images in three groups (n = 4). (B–D) The level of SOD, MDA, and GSH-PX among groups. (E) Representative Western blots of Nrf2, HO-1, NQO1, and SOD2 protein levels in three groups (n = 6). (F) Quantitative analysis of fluorescence intensity of DHE staining. (G–J) Statistical analysis of Nrf2, HO-1, NQO1, and SOD2 protein levels in three groups (n = 6). ****p < 0.0001 vs. Ctrl group; ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001 vs. DOX group.

Fig. 3

NBP attenuated cardiac apoptosis and inflammation in DOX-treated mice (A) Representative TUNEL staining images in heart tissues. (B) The indicated groups showed representative Western blots of BAX, BCL-2, and Cleaved-caspase3. (C) The quantitative results of TUNEL staining (n = 4). (E–F) Statistical results of BAX, BCL-2, and Cleaved-caspase3 protein levels in the indicated groups (n = 6). (G) In three groups, representative Western blots of IL-6, IL-1β, TNF-α, and P–P65 protein levels. (H–J) Statistical analysis of IL-6, IL-1β, TNF-α, and P–P65 protein levels in heart tissues (n = 6). **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. Ctrl group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001 vs. DOX group.

Fig. 4

NBP ameliorates DOX-induced oxidative stress in cardiomyocytes through the Nrf2/HO-1 signaling pathway in vitro (A) Representative TUNEL staining images in H9C2 cells. (B) Cell viability after DOX treatment (n = 5). (C) Quantitative analysis of DHE staining in H9C2 cells. (D–F) SOD, MDA, and GSH-PX levels in H9C2 cell supernatants. (G–K) Representative Western blots and statistical analysis of Nrf2, HO-1, NQO1, and SOD2 protein levels in four groups (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. Ctrl group; ^#p < 0.05, ^##p < 0.01, ^###p < 0.001, ^####p < 0.0001 vs. DOX group; ^&p < 0.05, ^&&p < 0.01, ^&&&p < 0.0001 vs. DOX + NBP group.

Fig. 5

NBP ameliorates DOX-induced apoptosis and inflammation in cardiomyocytes through the Nrf2/HO-1 signaling pathway in vitro. (A) Representative TUNEL staining images in H9C2 cells (n = 3). (B) Representative Western blots of BAX, BCL-2, and Cleaved-caspase3 protein levels in four groups. (C) Quantitative analysis of TUNEL staining in H9C2 cells. (D–F) Statistical analysis of BAX, BCL-2, and Cleaved-caspase3 proteins levels (n = 3). (G–K) Representative Western blots and statistical analysis of IL-6, IL-1β, TNF-α, and P–P65 protein levels in four groups (n = 3). **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. Ctrl group; ^#p < 0.05, ^##p < 0.01 vs. DOX group; ^&p < 0.05, ^&&p < 0.01, ^&&&p < 0.0001 vs. DOX + NBP group.