Nerolidol Attenuates Oxidative Stress, Inflammation, and Apoptosis by Modulating Nrf2/MAPK Signaling Pathways in Doxorubicin-Induced Acute Cardiotoxicity in Rats

Abstract

The clinical usage of doxorubicin (DOX), a potent anthracycline antineoplastic drug, is often limited by its cardiotoxic effects. Thus, for improving usage of DOX, the aim of this study was to assess the cardioprotective effects of nerolidol (NERO) in a rat model of DOX-induced acute cardiotoxicity and examine underlying molecular mechanisms that contribute to these effects. To induce acute cardiotoxicity male albino Wistar rats were injected with single dose intraperitoneal DOX (12.5 mg/kg). The rats were treated with NERO (50 mg/kg, orally) for five days. DOX-injected rats showed elevated levels of cardiac marker enzymes and enhanced oxidative stress markers along with altered Nrf2/Keap1/HO-1 signaling pathways. DOX administration also induced the activation of NF-κB/MAPK signaling and increased the levels and expression of pro-inflammatory cytokines (TNF-α, IL-6, and IL-1β) as well as expression of inflammatory mediators (iNOS and COX-2) in the heart. DOX also triggered DNA damage and apoptotic cell death in the myocardium. Additionally, histological studies revealed structural alterations of the myocardium. NERO treatment exhibited protection against the deleterious results of DOX on myocardium, as evidenced by the restoration of altered biochemical parameters, mitigated oxidative stress, inflammation, and apoptosis. The findings of the present study demonstrate that NERO provides cardioprotective effects against DOX-induced acute cardiotoxicity attributed to its potent antioxidant, anti-inflammatory, and antiapoptotic activities through modulating cellular signaling pathways.

Keywords: acute cardiotoxicity; cardioprotective; doxorubicin; inflammation; nerolidol; oxidative stress; sesquiterpene.

Figure 1

(A–C) Effect of NERO on myocardial injury markers; columns do not share a common symbol (*, **) and differ significantly from each other (* p < 0.05 vs. normal control, ** p < 0.05 vs. DOX control). (D). Histopathology of the myocardium: Normal control rat’s heart has normal architecture of the myocardium; NERO alone treated rat’s heart also shows normal intact muscle fibers without any pathological changes; DOX-injected rat’s heart shows extensive muscle fiber degradation with inflammatory cells; NERO treated DOX-challenged rats shows a reduced muscle fiber degradation without inflammatory cells. CK- creatine kinase; LDH- lactate dehydrogenase; NOR-normal; NERO-nerolidol; DOX- doxorubicin; ND- rats administered with a single intraperitoneal dose of DOX and orally treated with NERO.

Figure 2

(A–D) Effect of NERO on lipid peroxidation and oxidative stress. Lipid peroxidation and antioxidant status in the heart. Each column is mean ± SEM for eight rats in each group; columns do not share a common symbol (*, **) and differ significantly with each other (* p < 0.05 vs. normal control, ** p < 0.05 vs. DOX control). (E,F). Western immunoblot analysis and densitometric analysis for SOD1, SOD2, p67-phox, Nrf2, HO-1, and Keap-1. Values are expressed as mean ± SEM (n = 3); columns do not share a common symbol (*, **) and differ significantly with each other (* p < 0.05 vs. normal control, ** p < 0.05 vs. DOX control). MDA- malondialdehyde; SOD 1- superoxide dismutase 1; SOD 2- superoxide dismutase 2; HO-1-heme oxygenase-1; GSH-reduced glutathione; Nrf 2- Nuclear factor erythroid 2-related factor 2; NOR-normal; NERO-nerolidol; DOX- doxorubicin; ND- rats administered with a single intraperitoneal dose of DOX and orally treated with NERO.

Figure 3

(A–E) Levels/expressions of TNF-α, IL-6, and IL-1β in the myocardium. Each column is mean ± SEM for eight rats in each group for ELISA and 3 rats in each group for immunoblotting. Columns do not share a common symbol (*, **) and differ significantly with each other (* p < 0.05 vs. normal control, ** p < 0.05 vs. DOX control). Representative images of Western immunoblot analysis and densitometric analysis for TNF-α, IL-6, and IL-1β in the myocardium. TNF-α- tumor necrosis factor α; IL-6- interleukin 6; IL-1β- interleukin 1β; NOR-normal; NERO-nerolidol; DOX- doxorubicin; ND- rats administered with a single intraperitoneal dose of DOX and orally treated with NERO.

Figure 4

(A,B) Western immunoblotting and densitometric analysis for iNOS, COX-2, NF-κB-P65, p-NF-κBP65, IκBα and p-IκBα, t-IKKα, p-IKKα, t-JNK, p-JNK, P38, and p-P38. The values are expressed as the mean ± SEM (n = 3); columns do not share a common symbol (*, **) and differ significantly with each other (* p < 0.05 vs. normal control, ** p < 0.05 vs. DOX control).iNOS-inducible nitric oxide synthase; COX-2- cyclooxygenase-2; p-NF-κB-p65- phospho nuclear factor kappa-B-p65; t-JNK- t-Jun-amino-terminal kinase; p-JNK- p-Jun-amino-terminal kinase; p-IKKα- IκB kinase-α; P38-tumor protein; p-P38-phosphorilated p38; NOR-normal; NERO-nerolidol; DOX- doxorubicin; ND- rats administered with a single intraperitoneal dose of DOX and orally treated with NERO.

Figure 5

(A,B). Representative images of Western immunoblot analysis and densitometry for γ-H2AX, P53, Bax, Bcl2, Bcl-xL, active caspase-3, active caspase-9, APAF-1, cleaved PARP, and cytochrome-C. The values are expressed as the mean ± SEM (n = 3); columns do not share a common symbol (*, **) and differ significantly with each other (* p < 0.05 vs. normal control, ** p < 0.05 vs. DOX control). H2AX- H2A histone family member X; Bcl-2- B-cell lymphoma 2; Bax- Bcl2-associated-X- protein; Bcl-xL- B-cell lymphoma extra-large; P53- tumor protein; APAF-1- apoptotic protease activating factor 1; Cyt-C- cytochrome-C; CL-PARP-cleaved poly (ADP-ribose) polymerase; Casp-3-caspase 3; Cl-Casp-3- cleaved caspase-3; Casp-9-caspase 9; Cl-Casp-9- cleaved caspase-9; NOR-normal; NERO-nerolidol; DOX- doxorubicin; ND- rats administered with a single intraperitoneal dose of DOX and orally treated with NERO.