Pristimerin protects against doxorubicin-induced cardiotoxicity and fibrosis through modulation of Nrf2 and MAPK/NF-kB signaling pathways

Abstract

Background/purpose: Pristimerin (Pris) is triterpenoid compound with many biological effects. Until now, nothing is known about its effect on doxorubicin (DOX)-induced cardiotoxicity. Hence, this study investigated the impact of Pris on DOX-induced cardiotoxic effects.

Materials and methods: Rats were treated with Pris 1 week before and 2 weeks contaminant with repeated DOX injection. Afterwards, electrocardiography (ECG), biochemical, histopathological, PCR, and Western blot assessments were performed.

Results: Pris effectively alleviated DOX-induced deleterious cardiac damage. It inhibited DOX-induced ECG abnormities as well as DOX-induced elevation of serum indices of cardiotoxicity. The histopathological cardiac lesions and fibrosis were remarkably improved in Pris-treated animals. Pris reduced hydroxyproline content and attenuated the mRNA and protein expression of the pro-fibrogenic genes. The antioxidant activity of Pris was prominent through the amelioration of oxidative stress parameters and enhancement of antioxidants. Furthermore, Pris enhanced the activation of nuclear factor-erythroid 2 related factor 2 (Nrf2) signaling pathway as it increased the mRNA and protein expression of Nrf2 and Nrf2-dependent antioxidant genes (GCL, NQO1, HO-1). Additionally, the anti-inflammatory effect of Pris was obvious through the inhibition of mitogen activated protein kinase (MAPK)/nuclear factor kappa-B (NF-kB) signaling and subsequent inhibition of inflammatory mediators.

Conclusion: This study provides evidence of the cardioprotective activity of Pris which is related to the modulation of Nrf2 and MAPK/NF-kB signaling pathways.

Keywords: MAPK/NF-kB; Nrf2; cardiotoxicity; doxorubicin; pristimerin.

Figure 1

Impact of Pris on DOX-induced cardiac toxicity. Notes: (A) Chemical structure of Pris. (B) Relative heart weight. (C) ECG variables: (i) ECG recordings; (ii) heart rate; (iii) QTc interval; (iv) ST segment elevation; (v) R wave amplitude. (D) Serum indices of cardiac damage. (i) CK-MB; (ii) LDH; (iii) cTnI; (iv) cTnT. Rats were injected with DOX (2.5 mg/kg) six times over 2 weeks. Pris was administered (0.5, 1 mg/kg, intraperitoneal) once daily for 1 week before and 2 weeks contaminant with DOX injection. Data are means ± standard error (n=8). *P<0.05, **P<0.01, ***P<0.001 vs the control; ^#P<0.05, ^##P<0.01, ^###P<0.001 vs the DOX group (one-way ANOVA). Abbreviations: CK-MB, creatine kinase-MB; cTnI, cardiac troponin I; cTnT, cardiac troponin T; DOX, doxorubicin; ECG, electrocardiography; LDH, lactate dehydrogenase; Pris, pristimerin.

Figure 2

Impact of Pris on DOX-induced cardiac histopathological damage and alteration in hydroxyproline content. Notes: (A) Heart specimen stained by H&E stain where control rats (i) showed normal heart histology while heart specimen of DOX group (ii) showed large areas of coagulative necrosis (circle) (cardiac muscle fragmentation and resorption with loss of striation bands and nuclei) (stars). Pris treated groups (iii) and (iv) showed remarkable attenuation of DOX-induced cardiac damage. There is replacement of injured cardiac muscles with granulation tissue with new vascularization (lightning bolt), collagen deposition which matures to fibrous tissue (arrows) ×400; scale bar 25 µm. Heart specimen stained by MT stain where control rats (v) showed no signs of fibrosis, while DOX group (vi) cardiac muscles showed replacement of injured cardiac muscles, with blue stain indicating increased collagen and fibrous tissue (small arrows) ×200; scale bar 50 µm. Rats pretreated with Pris (vii, viii) showed dose-dependent amelioration of DOX-induced cardiac lesions and fibrosis. (B) Semiquantitative analysis of the severity of histopathological changes in cardiac muscle fibers stained with H&E. (C) Fibrosis percentage in heart muscles in specimen stained with MT. (D) Hydroxyproline content in cardiac tissue. Rats were injected with DOX (2.5 mg/kg) six times over 2 weeks. Pris was administered (0.5, 1 mg/kg, intraperitoneal) once daily for 1 week before and 2 weeks contaminant with DOX injection. Data are means ± standard error (n=8). ***P<0.001 vs the control; ^#P<0.05, ^##P<0.01, ^###P<0.001 vs the DOX group (one-way ANOVA). Abbreviations: DOX, doxorubicin; MT, Masson’s trichrome; Pris, pristimerin.

Figure 3

Effect of Pris on DOX-induced change of mRNA and protein expression of fibrogenic genes. Notes: (A) mRNA expression of (i) TGF-β, (ii) MMP-2, (iii) MMP-9, (iv) TIMP-1, (v) fibronectin-1, and (vi) Col1α1 in cardiac tissue. (B) Western blot for TGF-β, MMP-2, MMP-9, TIMP-1, and their relative protein quantification. β-Actin served as an internal control. Rats were injected with DOX (2.5 mg/kg) six times over 2 weeks. Pris was administered (0.5, 1 mg/kg, intraperitoneal) once daily for 1 week before and 2 weeks contaminant with DOX injection. Data are the mean ± standard error (n=8). *P<0.05, **P<0.01, ***P<0.001 vs the control; ^#P<0.05, ^##P<0.01, ^###P<0.001 vs the DOX group (one-way ANOVA). Abbreviations: Col1α1, collagen type I alpha 1; DOX, doxorubicin; MMP, metalloproteinase; Pris, pristimerin; TGF-β, transforming growth factor-β; TIMP-1, tissue inhibitor of metalloproteinase-1.

Figure 4

Impact of Pris on DOX-induced oxidative stress and depression of antioxidant parameters in cardiac tissue. Notes: (A) 4-HNE; (B) MDA; (C) PC; (D) 8-OHdG; (E) reduced GSH; (F) SOD. Rats were injected with DOX (2.5 mg/kg) six times over 2 weeks. Pris was administered (0.5, 1 mg/kg, intraperitoneal) once daily for 1 week before and 2 weeks contaminant with DOX injection. Data are means ± standard error (n=8). *P<0.05, **P<0.01, ***P<0.001 vs the control; ^#P<0.05, ^##P<0.01, ^###P<0.001 vs the DOX group (one-way ANOVA). Abbreviations: 4-HNE, 4-hydroxynonenal; 8-OHdG, 8-hydroxy-2-deoxyguanosine; DOX, doxorubicin; GSH, glutathione; MDA, malondialdehyde; PC, protein carbonyl; Pris, pristimerin; SOD, superoxide dismutase.

Figure 5

Effect of Pris on DOX-induced alteration of Nrf2 signaling pathway. Notes: (A) (i–v) mRNA expression of Nrf2, GCLc, GCLm, NQO1, and HO-1. (vi) Nrf2 binding capacity. (vii) HO-1 level in the heart. (B) Western blot for Nrf2, GCLc, NQO1, HO-1, and their relative protein quantification. β-Actin served as an internal control. Rats were injected with DOX (2.5 mg/kg) six times over 2 weeks. Pris was administered (0.5, 1 mg/kg, intraperitoneal) once daily for 1 week before and 2 weeks contaminant with DOX injection. Data are the mean ± standard error (n=8). *P<0.05, **P<0.01, ***P<0.001 vs the control; ^#P<0.05, ^##P<0.01, ^###P<0.001 vs the DOX group (one-way ANOVA). Abbreviations: DOX, doxorubicin; GCLc, glutamate-cysteine ligase catalytic subunit; GCLm, glutamate-cysteine ligase modifier; HO-1, hemeoxygenase-1; NQO1, NAD(P) H dehydrogenase quinone; Nrf2, nuclear erythroid 2-related factor 2; Pris, pristimerin.

Figure 6

Effect of Pris on DOX-induced alteration of MAPK/NF-kB signaling pathways. Notes: Western blot showing the protein expression of (A) p-p38 and p38, (B) p-JNK and JNK, (C) p-ERK and ERK, and quantification of the protein intensities. β-Actin was used as an internal control. (D) Specimen of the cardiac tissue in different groups showing different protein expressions of NF-kB p65 via IHC staining and semiquantitative analysis of IHC expressed as OD across 10 different fields for each section; ×400, scale bar 25 µm. (E) NF-kB p65 activation in the cardiac tissue. (F) Western blot for NF-kB p65, p-NF-kB p65, IKKα, phospho-IKKα, p-IkBα, and quantification of the protein intensities. β-Actin was used as an internal control. (G) Cytokine levels in the cardiac tissue. Rats were injected with DOX (2.5 mg/kg) six times over 2 weeks. Pris was administered (0.5, 1 mg/kg, intraperitoneal) once daily for 1 week before and 2 weeks contaminant with DOX injection. Data are the mean ± standard error (n=8). *P<0.05, ***P<0.001 vs the control; ^#P<0.05, ^##P<0.01, ^###P<0.001 vs the DOX group (one-way ANOVA). Abbreviations: DOX, doxorubicin; ERK, extracellular signal-regulated kinase; IHC, immunohistochemical; IKKα, inhibitor of nuclear factor kappa-B kinase alpha; JNK, c-JUN N-terminal kinase; MAPK, mitogen activated protein kinase; NF-kB, nuclear factor kappa-B; p-ERK, phospho-ERK; p-IkBα, phospho-inhibitor of NF-kBp-JNK, phospho-JNK; Pris, pristimerin.

Figure 7

Schematic diagram summarizing the possible cardioprotective mechanisms of Pris against DOX-induced cardiotoxicity. Abbreviations: ERK, extracellular signal-regulated kinase; GCL, glutamate-cysteine ligase; IkB, inhibitor of kappa B; HO-1, heme-oxygenase-1; IL-6, interleukin-6; JNK, c-JUN N-terminal kinase; Keap1, Kelch-like ECH associating protein 1; MAPK, mitogen activated protein kinase; NF-kB, nuclear factor kappa-B; Pris, pristimerin; DOX, doxorubicin; NQO1, NAD(P)H dehydrogenase quinone; NOx, nitric oxide; Nrf2, nuclear erythroid 2-related factor 2; TNF-α, tumor necrosis factor-α.