Taxifolin protects against doxorubicin-induced cardiotoxicity and ferroptosis by adjusting microRNA-200a-mediated Nrf2 signaling pathway

Abstract

The chemotherapeutic agent doxorubicin (Dox) is commonly used to treat various types of cancer, even though it can cause life-threatening cardiotoxicity. Clinically, there is no particularly effective way to treat Dox-induced cardiotoxicity. Therefore, it is imperative to identify compounds that can effectively alleviate Dox-induced cardiotoxicity. Ferroptosis and oxidative stress play a key role in Dox-induced cardiotoxicity, and the inhibition of ferroptosis and oxidative stress could effectively protect against doxorubicin-induced cardiotoxicity. Taxifolin (TAX) is a flavonoid commonly found in onions and citrus fruits. In the present study, we evaluated the effects of TAX on Dox-induced cardiac injury and dysfunction and aimed to explore the mechanisms underlying these effects. Using a mouse model of Dox-induced cardiotoxicity, we administered 20 mg/kg/day of TAX by gavage for 2 weeks. A week after the first use of TAX, each mouse was administered a 10 mg/kg dose of Dox. TAX was first evaluated for its cardioprotective properties, and the outcomes showed that TAX significantly reduced the damage caused by Dox to the myocardium in terms of structural and functional damage by effectively inhibiting ferroptosis and oxidative stress. In vivo, echocardiography, histopathologic assay, serum biochemical analysis and western blotting was used to find the results that Dox promoted ferroptosis-induced cardiomyocyte death, while TAX reversed these effects. In vitro, we also found that TAX alleviated Dox-induced cardiotoxicity by using ROS/DHE staining assay, Cellular immunofluorescence and western blotting. TAX increasing expression of microRNA-200a (miR-200a) which affects ferroptosis by activating Nrf2 signaling pathway. We believe that TAX inhibits ferroptosis and is a potential phytochemical that prevents Dox-induced cardiotoxicity.

Keywords: Cardiotoxicity; Doxorubicin; Ferroptosis; Nrf2; Taxifolin; miR-200a.

Fig. 1

Abbreviations: CK-MB, creatine kinase MB isoenzyme; Doxorubicin, Dox; Hematoxylin-eosin, HE; LDH, lactate dehydrogenase; LVEF, left ventricular ejection fraction; LVFS, leave ventricular fractional shortening; NC, Normal Control; TAX, taxifolin. TAX attenuated Dox-induced cardiotoxicity in vivo. (A) Chemical structure of Taxifolin (B) Schematic protocol for mice treatments (C) Kaplan-Meier survival curves of mice (D) Representative images of each group's echocardiogram. (E–F) LVEF and LVFS, n = 6. (G–I) Body weight, heart weight and heart/Tibia index, n = 6 (J–L) LDH, CK- MB, and cTnI levels in mouse serum, n = 6. (M) Representative images of HE staining, original magnification, ×40, scale bar, 500 μm; original magnification, ×400, scale bar, 50 μm. n = 6. Data are means ± SD, **P < 0.01.

Fig. 2

TAX attenuated Dox-induced ferroptosis in vivo. Abbreviations: DHE, Dihydroethidium; Doxorubicin, Dox; GPX4, Glutathione peroxidase 4; GSH, reduced glutathione; MDA, malondialdehyde; NC, Normal Control; PTGS2, Prostaglandin-endoperoxide synthase 2; SOD, superoxide dismutase; TAX, taxifolin. (A–D) Fe²⁺, MDA, GSH and SOD in vivo, n = 6 (E) Oxidative stress indicator staining, red indicates DHE, original magnification, ×400, scale bar, 50 μm, n = 3. (F) PTGS2 and GPX4 protein expression as assayed by Western blotting in vivo. Unprocessed images of Western blotting were in the supplementary material 1. (G–H) Quantitative analysis of the expression of PTGS2 and GPX4 proteins. The expression levels were normalized to β-actin, n = 3. Data are means ± SD, **P < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

TAX attenuated Dox-induced ferroptosis in vitro. Abbreviations: Ctrl, Control; DHE, Dihydroethidium; Doxorubicin, Dox; GPX4, Glutathione peroxidase 4; GSH, reduced glutathione; LDH, lactate dehydrogenase; MDA, malondialdehyde; PTGS2, Prostaglandin-endoperoxide synthase 2; ROS, Reactive Oxygen Species; SOD, superoxide dismutase; TAX, taxifolin. (A) Cell viability assay in each group, n = 6. (B) LDH, n = 6. (C–F) Fe²⁺, MDA, GSH and SOD in H9c2 cells, n = 6 (G) Oxidative stress indicator staining, green indicates ROS and red indicates DHE, original magnification, ×100, scale bar, 200 μm, n = 3. (H) PTGS2 and GPX4 protein expression as assayed by Western blotting in vitro. Unprocessed images of Western blotting were in the supplementary material 1. (I–J) Quantitative analysis of the expression of PTGS2 and GPX4 proteins. The expression levels were normalized to β-actin, n = 3. Data are means ± SD, **P < 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

TAX activated Nrf2 signal pathway in vivo and in vitro. Abbreviations: Ctrl, Control; Doxorubicin, Dox; GPX4, Glutathione peroxidase 4; HO-1, Heme Oxygenase-1; NQO1, NADPH: Quinone Oxidoreductase 1; Nrf2, Nuclear factor erythroid 2-related factor 2; TAX, taxifolin. (A) Nrf2, HO-1 and NQO1 protein expression as assayed by Western blotting in vivo. Unprocessed images of Western blotting were in the supplementary material 1. (B–D) Quantitative analysis of the expression of Nrf2, HO-1 and NQO1 proteins. The expression levels were normalized to β-actin, n = 3. (E) Nrf2, HO-1 and NQO1 protein expression as assayed by Western blotting in vitro. Unprocessed images of Western blotting were in the supplementary material 1. (F–H) Quantitative analysis of the expression of Nrf2, HO-1 and NQO1 proteins. The expression levels were normalized to β-actin, n = 3. (I) Immunofluorescence staining of Nrf2 in each group, original magnification, ×400, scale bar, 50 μm, n = 3. Data are means ± SD, *P < 0.05; **P < 0.01.

Fig. 5

TAX attenuated doxorubicin-induced ferroptosis via Nrf2 signal pathway in vitro. Abbreviations: Doxorubicin, Dox; GPX4, Glutathione peroxidase 4; GSH, reduced glutathione; HO-1, Heme Oxygenase-1; LDH, lactate dehydrogenase; MDA, malondialdehyde; NC, Normal Control; NQO1, NADPH: Quinone Oxidoreductase 1; Nrf2, Nuclear factor erythroid 2-related factor 2; PTGS2, Prostaglandin-endoperoxide synthase 2; SOD, superoxide dismutase; TAX, taxifolin. (A) Nrf2, HO-1 and NQO1 protein expression as assayed by Western blotting in vitro. Unprocessed images of Western blotting were in the supplementary material 1. (B–D) Quantitative analysis of the expression of Nrf2, HO-1 and NQO1 proteins. The expression levels were normalized to β-actin, n = 3. (E) LDH, n = 6. (F–I) Fe²⁺, MDA, GSH and SOD in vitro, n = 6 (J) PTGS2 and GPX4 protein expression as assayed by Western blotting in vitro. Unprocessed images of Western blotting were in the supplementary material 1. (K–L) Quantitative analysis of the expression of PTGS2 and GPX4 proteins. The expression levels were normalized to β-actin, n = 3. Data are means ± SD, *P < 0.05; **P < 0.01.

Fig. 6

TAX adjusted Nrf2 signal pathway and attenuated ferroptosis by miR-200a. Abbreviations: Doxorubicin, Dox; GPX4, Glutathione peroxidase 4; GSH, reduced glutathione; HO-1, Heme Oxygenase-1; Keap1, Kelch-like ECH-associated protein 1; LDH, lactate dehydrogenase; MDA, malondialdehyde; NC, Normal Control; NQO1, NADPH: Quinone Oxidoreductase 1; Nrf2, Nuclear factor erythroid 2-related factor 2; PTGS2, Prostaglandin-endoperoxide synthase 2; SOD, superoxide dismutase; TAX, taxifolin. (A) The expression levels of Nrf2-related miRNA in H9c2 cells. (B) Nrf2, HO-1 and NQO1 protein expression as assayed by Western blotting in vitro. Unprocessed images of Western blotting were in the supplementary material 1. (C) The expression levels of Keap1 mRNA, n = 6. (D) Cell viability assay in each group, n = 6. (E) LDH, n = 6. (F–H) Quantitative analysis of the expression of Nrf2, HO-1 and NQO1 proteins. The expression levels were normalized to β-actin, n = 3. (I–L) Fe²⁺, MDA, GSH and SOD in vitro, n = 6 (M) PTGS2 and GPX4 protein expression as assayed by Western blotting in vitro. Unprocessed images of Western blotting were in the supplementary material 1. (N–O) Quantitative analysis of the expression of PTGS2 and GPX4 proteins. The expression levels were normalized to β-actin, n = 3. Data are means ± SD, *P < 0.05; **P < 0.01.

Fig. 7

Schematic: Taxifolin protects against doxorubicin-induced cardiotoxicity and ferroptosis by adjusting microRNA-200a-mediated Nrf2 signaling.