Alpha-linolenic acid protects against heatstroke-induced acute lung injury by inhibiting ferroptosis through Nrf2 activation

Abstract

Heatstroke (HS)-induced acute lung injury (ALI) has high morbidity and mortality with no specific therapies. Ferroptosis, a form of programmed cell death driven by lipid peroxidation due to reduced Glutathione Peroxidase 4 (GPX4) activity, is closely linked to HS-induced ALI. This study investigated the effect of alpha-linolenic acid (ALA), a plant-derived ω-3 fatty acid, on ferroptosis in a mouse model of HS-induced ALI. Histopathology analysis found that ALA can attenuate lung injury and improve the 7-day survival rate in mice with HS-induced ALI. In addition, ALA significantly reduced the levels of reactive oxygen species (ROS) and malondialdehyde (MDA), while increasing the level of antioxidant glutathione (GSH). Further analysis showed that ALA upregulated the levels of SLC7A11 and GPX4 by promoting the nuclear translocation of Nrf2. This led to increased GSH synthesis but reduced ROS accumulation, which in turn suppressed ferroptosis and protected the mice against HS-induced ALI. Additionally, the protective effect of ALA was found to be diminished in Nrf2-deficient mice. In summary, ALA inhibits ferroptosis in macrophages by activating the Nrf2/SLC7A11/GPX4 pathway and attenuates HS-induced ALI.

Keywords: GPX4; Heatstroke; Nrf2; SLC7A11; acute lung injury; alpha-linolenic acid; ferroptosis; lipid peroxidation.

Figure 1.

HS induces ALI in mice and macrophage death. (A) Experimental strategy for HS mice in this study. (B) Representative images showing H&E staining of lung sections from different time points. Yellow arrowheads indicate edema, red indicates erythrocyte exudation, blue indicates inflammatory cell infiltration, and black indicates alveolar septa thickening (scale bar = 200 μm). (C) Histopathological scoring of lung injury. (D) Wet/dry ratio of the lung tissue. (E) qRT-PCR analysis of TNF-α, IL-6 and IL-1β in the lung tissue of mice in each group (n ≥ 3). (F, G) Images and semi-quantification of TUNEL and DAPI immunofluorescence staining in the lung tissue of mice (scale bar = 50 μm). (H, I) Images and semi-quantification of immunofluorescence staining for F4/80 and DAPI in the lung tissue of mice (scale bar = 100 μm). (J) qRT-PCR analysis of TNF-α, IL-6, and IL-1β in BMDMs from each group (n ≥ 3). (K, L) The dead cells were detected using the SYTOX fluorescence staining images (scale bar = 100 μm) and semi-quantification analysis in BMDMs. Data are presented as mean ± SD and assessed by one-way ANOVA. *P < 0.05, ** P < 0.01, ***P < 0.001.

Figure 2.

ALA ameliorates HS-induced ALI and inhibits macrophage death. (A) Kaplan–Meier survival analysis for each group (n = 8 per group). (B) Representative images showing H&E staining of lung sections. Yellow arrowheads indicate edema, red indicates erythrocyte exudation, blue indicates inflammatory cell infiltration, and black indicates alveolar septa thickening (scale bar = 200 μm). (C) Lung injury scores. (D) Wet/dry weight ratios of the lungs for each group. (E-G) qRT-PCR analysis of TNF-α, IL-6, and IL-1β mRNA in the lung tissue of mice in each group (n ≥ 3). (H, I) Dead cells were detected using the SYTOX fluorescence staining (scale bar = 100 μm) and semi-quantification analysis in BMDMs. (J-L) qRT-PCR analysis of TNF-α, IL-6, and IL-1β mRNA in BMDMs in each group (n ≥ 3). Data are presented as mean ± SD and assessed by one-way ANOVA. *P < 0.05, ** P < 0.01, ***P < 0.001.

Figure 3.

Ferroptosis occurs in HS-induced ALI and can be inhibited by ALA. (A-E) Representative western blot and quantification analyses of GPX4, SLC7A11, FTH, and ACSL4 protein levels in the lung tissue of mice at different time points after HS. (F) qRT-PCR analysis of GPX4 mRNA in the lung tissue of mice in each group (n ≥ 3). (G-I) GSH, MDA, and iron levels in the lung tissue of mice in each group (n ≥ 3). (J, K) Representative western blot and quantification analyses of GPX4 protein levels in the lung tissue of mice in each group (n ≥ 3). (L) qRT-PCR analysis of GPX4 mRNA in the lung tissue of mice in each group (n ≥ 3). (M–O) GSH, MDA, and iron levels in the lung tissue of mice in each group (n ≥ 3). Data are presented as mean ± SD and assessed by one-way ANOVA. *P < 0.05, ** P < 0.01, ***P < 0.001.

Figure 4.

ALA inhibits HS-induced ferroptosis in macrophages. (A, B) Representative western blot and quantification analyses of GPX4 protein levels in BMDMs (n ≥ 3) after pretreatment with different concentrations of ALA (0, 50, 100, 250 nM). (C) mRNA levels of GPX4 in BMDMs in each group (n ≥ 3). (D, E) Representative immunofluorescence images and semi-quantification analysis of GPX4 (green) staining and cell nuclei (in blue) in BMDMs (scale bar = 25 μm, n ≥ 3). (F, G) Intracellular ROS was measured using the DCFH-DA fluorescent probes and semi-quantification analysis in BMDMs (scale bar = 100 μm, n ≥ 3). (H, I) LPO was measured using the BODIPY 581/591 C11 fluorescent probes and semi-quantification analysis in BMDMs (scale bar = 100 μm, n ≥ 3). Data are presented as mean ± SD and assessed by one-way ANOVA. *P < 0.05, ** P < 0.01, ***P < 0.001.

Figure 5.

ALA inhibits HS-induced ferroptosis in macrophages through the activation of Nrf2. (A-F) Representative western blot and quantification analyses of Nrf2, GPX4, SLC7A11, FTH, and ACSL4 protein levels in BMDMs. (G) Representative immunofluorescence images of Nrf2 (green) staining and cell nuclei (in blue) in BMDMs (scale bar = 2.5 μm). (H-L) Representative western blot and quantification analyses of Nrf2, GPX4, SLC7A11, and HO-1 protein levels in BMDMs from WT and Nrf2^–/– mice. (M–O) qRT-PCR analyses of the relative mRNA levels of Nrf2, GPX4, and SLC7A11 in BMDMs from WT and Nrf2^–/– mice (n ≥ 3). Data are presented as mean ± SD and assessed by one-way ANOVA. *P < 0.05, ** P < 0.01, ***P < 0.001.

Figure 6.

Knockout of Nrf2 exacerbates HS-induced ALI and is not reversed by ALA. (A) Kaplan–Meier survival analysis for each group (n = 12 per group). (B) Representative images showing H&E staining of lung sections. Yellow arrowheads indicate edema, red indicates erythrocyte exudation, blue indicates inflammatory cell infiltrate, and black indicates alveolar septa thickened (scale bar = 200 μm). (C) Lung injury scores. (D) Wet/dry weight ratios of the lungs. (E-G) GSH, MDA, and iron levels in the lung tissue of Nrf2^-/- and WT mice (n ≥ 3). Data are presented as mean ± SD and assessed by one-way ANOVA. *P < 0.05, ** P < 0.01, ***P < 0.001.

Figure 7.

ALA protects against HS-induced ALI by reducing ferroptosis through the activation of the Nrf2/SLC7A11/GPX4 pathway. HS-induced ALI is characterized by increased ROS and LPO accumulation, decreased GSH production, and lower levels of GPX4. ALA attenuated ferroptosis in HS-induced ALI by promoting the translocation of Nrf2 into the nucleus, which enhances the expression of its downstream targets, GPX4 and SLC7A11 (created with BioRender.com).