Astaxanthin alleviates PM2.5-induced cardiomyocyte injury via inhibiting ferroptosis

Abstract

Background: Long-term exposure of humans to air pollution is associated with an increasing risk of cardiovascular diseases (CVDs). Astaxanthin (AST), a naturally occurring red carotenoid pigment, was proved to have multiple health benefits. However, whether or not AST also exerts a protective effect on fine particulate matter (PM_2.5)-induced cardiomyocyte damage and its underlying mechanisms remain unclear.

Methods: In vitro experiments, the H9C2 cells were subjected to pretreatment with varying concentrations of AST, and then cardiomyocyte injury model induced by PM_2.5 was established. The cell viability and the ferroptosis-related proteins expression were measured in different groups. In vivo experiments, the rats were pretreated with different concentrations of AST for 21 days. Subsequently, a rat model of myocardial PM_2.5 injury was established by intratracheal instillation every other day for 1 week. The effects of AST on myocardial tissue injury caused by PM_2.5 indicating by histological, serum, and protein analyses were examined.

Results: AST significantly ameliorated PM_2.5-induced myocardial tissue injury, inflammatory cell infiltration, the release of inflammatory factors, and cardiomyocyte H9C2 cell damage. Mechanistically, AST pretreatment increased the expression of SLC7A11, GPX4 and down-regulated the expression of TfR1, FTL and FTH1 in vitro and in vivo.

Conclusions: Our study suggest that ferroptosis plays a significant role in the pathogenesis of cardiomyocyte injury induced by PM_2.5. AST may serve as a potential therapeutic agent for mitigating cardiomyocyte injury caused by PM_2.5 through the inhibition of ferroptosis.

Keywords: Astaxanthin; Cardiomyocyte injury; Cardiovascular diseases; Ferroptosis; PM2.5.

Fig. 1

Schematic experimental design and dosage regimen in this study

Fig. 2

Computational pharmacology analysis of PM_2.5 and astaxanthin. A The DEGs between normal mouse cardiomyocyte cells and cardiomyocyte cells after treatment with PM_2.5. B KEGG enrichment analysis of DEGs. C Venn graph showing the numbers of predicted AST targets. D The overlapping targets of AST, FRGs, and DEGs. E The PPI network of overlapping targets. The red nodes represent the core targets

Fig. 3

Astaxanthin attenuates PM_2.5-induced H9C2 cells injury. H9C2 cells were treated with different concentrations of AST (A), PM_2.5 (B), and Fer-1 (C) for 24 h. The cell viability was detected by CCK8 assay. D Cell viability of H9C2 cells treated with PM_2.5, AST and Fer-1. The activities of SOD (E) and LDH (F) in the H9C2 cells treated with PM_2.5, AST and Fer-1. Values are expressed as mean ± SEM (n = 6). *p < 0.05 difference from the control group; **p < 0.001 difference from the control group; ^#p < 0.05 difference from the PM_2.5 exposure group; and ^##p < 0.001 difference from the PM_2.5 exposure group

Fig. 4

Astaxanthin alleviates heart injury induced by PM_2.5 in mice. A–E Hematoxylin and eosin (HE) staining of heart tissue sections from different groups (Scale bar = 50 μm). F The myocarditis score of heart in each group. The activities of Lactate dehydrogenase (LDH) and creatine kinase (CK) activity in heart heart tissue (G, H) and serum (I, J) were measured. Values are expressed as mean ± SEM (n = 6). *p < 0.05 difference from the control group; **p < 0.001 difference from the control group; ^#p < 0.05 difference from the PM_2.5 exposure group; and ^##p < 0.001 difference from the PM_2.5 exposure group

Fig. 5

Effect of astaxanthin on the levels of IL-6, IL-1β and TNF-α in serum and heart. ELISA for TNF-α, IL-1β and IL-6 in heart tissue (A–C) and serum (D–F). Values are expressed as mean ± SEM (n = 6). *p < 0.05 difference from control group; **p < 0.001 difference from the control group; ^#p < 0.05 difference from the PM_2.5 exposure group; and ^##p < 0.001 difference from the PM_2.5 exposure group

Fig. 6

Effect of astaxanthin on the levels of SOD, CAT, GSH and MDA in serum and heart. The activities of SOD, GSH, and CAT in the heart tissue (A–C) and serum (E–G) were detected. Levels of MDA was measured in the heart tissue (D) and serum (H). Values are expressed as mean ± SEM (n = 6). *p < 0.05 difference from control group; **p < 0.001 difference from the control group; ^#p < 0.05 difference from the PM_2.5 exposure group; and ^##p < 0.001 difference from the PM_2.5 exposure group

Fig. 7

Astaxanthin attenuates PM_2.5-induced ferroptosis in H9C2 cells. A Representative fluorescence intensity images of ROS by DCFH-DA obtained by flow cytometry. B Flow cytometric analysis of fluorescence intensity. C Iron content were detected in the H9C2 cells. D–H Western blot bands showing TfR1, FTL, FTH1, GPX4 and SLC7A11 protein expression and the relative signal intensities in H9C2 cells. For quantification, the intensity was normalized to GAPDH and the control was set to 1. Values are expressed as mean ± SEM (n = 6). *p < 0.05 difference from control group; **p < 0.001 difference from the control group; ^#p < 0.05 difference from the PM_2.5 exposure group; and ^##p < 0.001 difference from the PM_2.5 exposure group

Fig. 8

Astaxanthin attenuates PM_2.5-induced ferroptosis in heart. Representative images of fluorescence probe for ROS and its statistical results in heart tissue (A, B). Iron content were detected in the heart tissues (C). Western blots for TfR1, FTL, FTH1, GPX4, and SLC7A11 in heart tissue (D–H). For quantification, protein expression was normalized to GAPDH and the control was set to 1. Values are expressed as mean ± SEM (n = 6). *p < 0.05 difference from the control group; **p < 0.001 difference from the control group; ^#p < 0.05 difference from the PM_2.5 exposure group; and ^##p < 0.001 difference from the PM_2.5 exposure group