Cinnamaldehyde alleviates doxorubicin-induced cardiotoxicity by decreasing oxidative stress and ferroptosis in cardiomyocytes

Figures

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  Fig 1. CA alleviated DOX-induced myocardial injury in vitro and in vivo. H9c2 cells were treated with different doses of DOX (A) or CA (B), and then cell viability was measured using CCK8 after 24 h (n = 3). (C) H9c2 cells were treated with 4 μM of DOX and 100 μM of CA for 24 h, and cell viability was measured using CCK8 after 24 h (n = 3). (D and E) H9c2 cells were treated with 4 μM of DOX and 100 μM of CA for 24 h, and cell death was measured using TUNEL (n = 3). Rats were treated with 15 mg/kg of DOX with or without CA (50 mg/kg, 6 weeks), and serum CK (F) and LDH (G) levels were assessed using commercial kits (n = 5). (H) HE staining was used to assess myocardial injury in rats treated with DOX and CA (n = 5). (I) Masson staining was used to assess myocardial fibrosis in rats treated with DOX and CA (n = 5). qRT-PCR was performed to assess Col1α1 and α-SMA (J) mRNA levels in rats treated with DOX and CA (n = 5). *p<0.05, **p<0.01.

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  Fig 2. CA alleviated DOX-induced myocardial oxidative stress. (A and B) H9c2 cells were incubated with DOX (4 μM) and CA (100 μM), and intracellular ROS levels were measured using the DCFH-DA assay after 24 h (n = 3). MDA (C), SOD (D), GSH (E), and GSH-px (F) levels in cardiac tissues were assessed using commercial kits (n = 5). *p<0.05, **p<0.01.

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  Fig 3. CA activated Nrf2/HO-1 signaling in vitro. Western blot assay (A) and quantification (C) of cytoplasmic Nrf2 protein levels in H9c2 cells treated with DOX (4 μM) and CA (100 μM) (n = 3). Western blot assay (B) and quantification (C) of nuclear Nrf2 protein levels in H9c2 cells treated with DOX (4 μM) and CA (100 μM) (n = 3). (D) qRT-PCR assay of HO-1 mRNA levels in H9c2 cells treated with DOX (4 μM) and CA (100 μM) (n = 3). Western blot assay (E) and quantification (F) of cytoplasmic HO-1 protein levels in H9c2 cells treated with DOX (4 μM) and CA (100 μM) (n = 3). **p<0.01.

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  Fig 4. CA activated Nrf2/HO-1 signaling in vivo. (A and B) The total protein levels of Nrf2 and HO-1 in different groups of cardiac tissues (n = 5) were assessed using western blot assay. (C and D) The nuclear protein levels of Nrf2 in different groups of cardiac tissues (n = 5) were assessed using western blot assay.

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  Fig 5. CA increased cardiomyocyte viability through repressing ferroptosis. (A) Relative iron concentration was assessed using commercial kits in H9c2 cells treated with DOX (4 μM) and CA (100 μM) (n = 3). (B) qRT-PCR assay of Gpx4, Ptgs2, and Acsl4 mRNA levels in H9c2 cells treated with DOX (4 μM) and CA (100 μM) (n = 3). Western blot assay (C) and quantification of Gpx4 (D), Acsl4 (E), and Ptgs2 (F) protein levels in H9c2 cells treated with DOX (4 μM) and CA (100 μM) (n = 3). (G) H9c2 cells were treated with 4 μM of DOX in the presence or absence of CA (100 μM), Fer-1 (1 μM), or Erastin (10 μM) for 24 h, and cell viability was measured with CCK8 (n = 3). *p<0.05, **p<0.01.