Polystyrene nanoplastics lead to ferroptosis in the lungs

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Highlights

• •The specific mechanisms of PS-NP-induced pulmonary injury are unclear.
• •A mouse model of lung injury is created based on the real-world NP exposure in human.
• •PS-NP exposure induces ferroptosis in lung tissues and bronchial epithelial cells.
• •HIF-1α inhibition rescues PS-NP-induced ferroptosis in bronchial epithelial cells.
• •PS-NP exposure induces ferroptosis via the HIF-1α/HO-1 signaling pathway in lungs.

Abstract

Introduction

It has been shown that polystyrene nanoplastic (PS-NP) exposure induces toxicity in the lungs.

Objectives

This study aims to provide foundational evidence to corroborate that ferroptosis and abnormal HIF-1α activity are the main factors contributing to pulmonary dysfunction induced by PS-NP exposure.

Methods

Fifty male and female C57BL/6 mice were exposed to distilled water or 100 nm or 200 nm PS-NPs via intratracheal instillation for 7 consecutive days. Hematoxylin and eosin (H&E) and Masson trichrome staining were performed to observe the histomorphological changes in the lungs. To clarify the mechanisms of PS-NP-induced lung injury, we used 100 μg/ml, 200 μg/ml and 400 μg/ml 100 or 200 nm PS-NPs to treat the human lung bronchial epithelial cell line BEAS-2B for 24 h. RNA sequencing (RNA-seq) of BEAS-2B cells was performed following exposure. The levels of glutathione, malondialdehyde, ferrous iron (Fe²⁺), and reactive oxygen species (ROS) were measured. The expression levels of ferroptotic proteins were detected in BEAS-2B cells and lung tissues by Western blotting. Western blotting, immunohistochemistry, and immunofluorescence were used to evaluate the HIF-1α/HO-1 signaling pathway activity.

Results

H&E staining revealed substantial perivascular lymphocytic inflammation in a bronchiolocentric pattern, and Masson trichrome staining demonstrated critical collagen deposits in the lungs after PS-NP exposure. RNA-seq revealed that the differentially expressed genes in PS-NP-exposed BEAS-2B cells were enriched in lipid metabolism and iron ion binding processes. After PS-NP exposure, the levels of malondialdehyde, Fe²⁺, and ROS were increased, but glutathione level was decreased. The expression levels of ferroptotic proteins were altered significantly. These results verified that PS-NP exposure led to pulmonary injury through ferroptosis. Finally, we discovered that the HIF-1α/HO-1 signaling pathway played an important role in regulating ferroptosis in the PS-NP-exposed lung injury.

Conclusion

PS-NP exposure caused ferroptosis in bronchial epithelial cells by activating the HIF-1α/HO-1 signaling pathway, and eventually led to lung injury.

Introduction

In addition to ozone diminution, ocean acidification, and climate alteration, plastic pollution has become a serious environmental problem [1]. Plastic is an inexpensive, lightweight, and durable material that can resist corrosion, and these characteristics have led to its wide usage in various products [2]. Large pieces of plastics can be degraded into microplastics (MPs) with diameters <5 mm or even nanoplastics (NPs) with diameters <1 mm in the environment [3], [4]. Compared to microplastics (MPs), nanoplastics (NPs) can more easily penetrate biological barriers and threaten human health due to their smaller size [5]. The main components of NPs involve polypropylene, polyethylene, and polystyrene [6]. Exposure to polystyrene NPs (PS-NPs) may lead to gastrointestinal toxicity, hepatotoxicity, cardiovascular toxicity, reproductive dysfunction, and neurotoxicity [7].

Gastrointestinal ingestion and dermal infiltration are the primary routes of human exposure to NPs [8]. However, airborne exposure to NPs has been confirmed in recent years, and the pulmonary toxicity of NPs has attracted extensive attention. Due to their very small particle size, NPs are more likely to be suspended in the air and jeopardize respiratory health [9]. A recent in vitro study found that exposure to polyethylene terephthalate nanoplastic particles resulted in toxic effects on A549 cells via oxidative stress [10]. An in vivo study also discovered that PS-NP exposure resulted in structural disorders in the lungs [11]. However, the specific mechanisms are unclear.

Among the factors inducing oxidative stress in cells, lipid peroxidation of the bilayer lipid membrane has become a crucial regulator of cell fate, and excessive lipid peroxidation can result in ferroptosis [12]. Ferroptosis is generally considered a type of regulated cell death (RCD) that is ferrous iron-dependent [13], [14]. Ferroptosis mainly involves reduced glutathione (GSH) production and glutathione peroxidase 4 (GPX4) activity which compromises the normal metabolism of lipid peroxides. Concerning the important role of oxidative stress in the toxic effects of PS-NPs, the mechanism by which pulmonary cells respond to oxidative stress is critical in determining cell fate. Moreover, whether ferroptosis contributes to lung injury induced by PS-NP exposure remains to be clarified.

The potential ferroptotic genes in pulmonary fibrosis are validated by bioinformatics analyses and experiments in a recent study [15]. In this report, KEGG and GO analyses have shown that ferroptotic genes are associated with the Hypoxia-inducible factor-1 (HIF-1) signaling pathway. Therefore, we infer that the HIF-1 signaling pathway may be involved and activated in PS-NP-induced lung injury. HIF-1 has two subunits, including a functional subunit HIF-1α and a structural subunit HIF-1β. HIF-1α has been verified to be closely related to ferroptosis. According to published reports, HIF-1α has different roles in ferroptosis under different biological conditions. For instance, HIF-1α inhibits ferroptosis in gastric cancer [16]. However, HIF-1α facilitates ferroptosis in di-(2-ethylhexyl) phthalate (DEHP)-induced testicular toxicity [17]. Microplastic exposure can lead to pulmonary fibrosis and hypoxia in mice [18]. Nevertheless, in PS-NP-induced lung ferroptosis, whether HIF-1 signaling is triggered and plays a pivotal role remains to be further elucidated.

In our study, we provided foundational evidence to corroborate that ferroptosis and abnormal HIF-1α activity are the main factors contributing to pulmonary dysfunction induced by PS-NP exposure.

Materials and methods

Preparation and characterization of PS-NPs

The 100 and 200 nm PS-NP stock solutions were purchased from Baseline Chromtech Research (Tianjin, P.R.China). We measured the particle size and Z-potential of the 100 and 200 nm PS-NP suspensions. The morphology of the 100 and 200 nm PS-NPs was observed by scanning electron microscopy (SEM, Apreo S HiVac FEI, USA).

Animals and treatments

Eight-week-old C57BL/6 mice were obtained from the Experimental Animal Center of Chongqing Medical University (SCXK, 2018–0003) and kept under the standardized conditions (25 ± 2 ℃, 50 ± 5% humidity, 12-hour light/dark cycle) with access to chow and water ad libitum.

The exposure concentrations were mainly determined based on a previous study [19]. The volume of a microparticle is estimated to be 1 mm³. To estimate PS-NP dosage, we calculated the volume that represented our spherical 100 or 200 nm PS-NP beads using the estimated microparticle volume. We mathematically converted a microparticle to 100 or 200 nm nanoparticles. A single microparticle represented 1.91 × 10¹² 100 nm or 2.39 × 10¹¹ 200 nm nanoparticles ($\frac{{{(10}^6\mathit{nm})}^3}{{\frac{4}{3}\pi r}^3}$, r represents the radius of nanoparticles, r = 50 or 100 nm). Cox et al. [20] reported the human exposure dosage of microparticles via inhalation, and the dosage of daily inhaled nanoparticles was extrapolated from this report. Then, the dosage of daily inhaled nanoparticles in mice was estimated based on a previous report [21]. Eventually, the realistic exposure dose in mice was estimated at 12.5 mg/kg per day. Therefore, we established five groups in this study: the control group, 12.5 mg/kg bw 100 nm PS-NP group (100–12.5), 25 mg/kg bw 100 nm PS-NP group (100–25), 12.5 mg/kg bw 200 nm PS-NP group (200–12.5), and 25 mg/kg bw 200 nm PS-NP (200–25) group. Twenty-five male and twenty-five female mice were randomly divided into each group. The PS-NPs stock solutions were diluted with distilled water and surfactant and sonicated before use. Mice in the control group were administered with distilled water, and mice in the PS-NP exposure groups were administered with 100 nm or 200 nm PS-NPs by intratracheal instillation once per day for 7 consecutive days. On the second day after the last treatment, all mice were anesthetized with phenobarbital sodium, and then the lung tissues were collected. The lung tissues were stored in liquid nitrogen or fixed in 4% paraformaldehyde (PFA).

Ethics statement

All experiments involving animals were conducted according to the ethical policies and procedures approved by the Ethics Committee of Children’s Hospital of Chongqing Medical University (Issue number: CHCMU-IACUC20220323010).

Hematoxylin-eosin and Masson trichrome staining

The fixed lung tissues were dehydrated, paraffin-embedded and sectioned into 4 μm slices. Then, after deparaffinization and dehydration, slices were sequentially stained with hematoxylin-eosin (H&E). Similarly, 4 μm slices were also stained with Masson trichrome reagents according to the manufacturer’s instructions (DC0033, Leagene Biotechnology, P.R.China).

Human lung bronchial epithelial cell line (BEAS-2B) culture and treatment

BEAS-2B cells were cultured in DMEM containing 10% fetal bovine serum (FBS, C04001–500, VivaCell, P.R.China) with penicillin–streptomycin solution. The cells were incubated under a humidified atmosphere of 5% CO₂ at 37 ℃ and randomly separated into the control group, 100 nm groups (100 μg/ml, 200 μg/ml and 400 μg/ml), and 200 nm groups (100 μg/ml, 200 μg/ml, and 400 μg/ml) for subsequent experiments. The PS-NP stock solution was sonicated and diluted with medium before use, and BEAS-2B cells in the PS-NP exposure group were treated with 100 nm or 200 nm PS-NP suspension for 24 h. Cells in the control group were treated with distilled water and medium for 24 h as well.

Ferrostatin-1 (HY-100579, MCE, USA) was used to reverse ferroptosis in BEAS-2B cells. LW6 (HY-13671, MCE, USA), a HIF-1α inhibitor, was used to inhibit the expression of HIF-1α. Inhibitors were added concurrently with PS-NPs and incubated for twenty-four hours.

Cellular and tissue immunofluorescence (IF)

BEAS-2B cells were cultured on coverslips pretreated with polylysine in 24-well plates. The cells were fixed in 4% PFA following treatment. After washing with phosphate-buffered saline (PBS) and blocking with 0.5% bovine serum albumin (BSA), the BEAS-2B cells were incubated with primary antibodies at 4 ℃ overnight. Then, the cells were incubated with Cy3-conjugated secondary antibody and Hoechst 33342 for 1 h and 30 min, respectively.

After deparaffinization and hydration, a citrate solution was used for antigen retrieval of the lung tissue slides. After blocking with 0.5% BSA, the slides were incubated with primary antibodies (dilution at 1:100) against HIF-1α (ab216842, Abcam, USA) or HIF-1β (ab239366, Abcam, USA) at 4 ℃ overnight. Then, they were incubated with Cy3-conjugated antibody and Hoechst 33342 for 1 h and 30 min, respectively.

Finally, images were captured under an A1R confocal microscope (Nikon, Japan).

Immunohistochemistry (IHC) staining

After deparaffinization and hydration, a citrate solution was used for antigen retrieval of the lung tissue slides. After blocking with 0.5% BSA, the slides were incubated with primary antibodies (dilution at 1:100) at 4 ℃ overnight. Then they were incubated with a Rabbit Two-step Detection Kit (PV-9001, ZSGB-BIO, P.R.China), and a chromogenic reaction with 3,30-diaminobenzidine (ZLI-9018, ZSGB-BIO, P.R.China) was performed. Then images were captured under a light microscope (Nikon, Japan).

Transmission electron microscopy (TEM)

BEAS-2B cells were collected after PS-NP treatment and incubated in 3% glutaraldehyde. The TEM protocols were described previously [17]. BEAS-2B cells were observed with a transmission electron microscope (JEM-1400PLUS, Japan) at the Lilai Biomedicine Experiment Center (Chengdu, P.R.China).

Cell viability assay.

BEAS-2B cells were cultured in 96-well plates at a density of 3 × 10³ cells per well. After treatment with PS-NPs for 24 h, CCK-8 reagent (GK10001, GlpBio, USA) was diluted to 10% with FBS-free DMEM and incubated at 37 ℃ for 2 h to evaluate the viability of BEAS-2B cells. The optical density (OD) values were examined at a 450 nm wavelength by using a spectrophotometer.

Cellular ROS detection and calcein-AM/PI staining

BEAS-2B cells were cultured in 12-well plates at a density of 5 × 10⁴ cells per well and then treated with different concentrations of PS-NPs for 24 h. The ROS level was measured by using an H2DCFDA probe according to the manufacturer’s instructions (HY-D0940, MCE, USA). Briefly, the H₂DCFDA probe was diluted to 0.2% with FBS-free DMEM, and 1 ml of solution was added to each well and incubated at 37 ℃ for 30 min. Then, the green fluorescence was observed, and images were captured under a fluorescence microscope (Nikon, Japan).

BEAS-2B cells were cultured in 6-well plates at a density of 1 × 10⁵ cells per well and then treated with different concentrations of PS-NPs for 24 h. BEAS-2B cell death was examined, and calcein-AM/PI staining was performed as described previously [17]. Briefly, diluted calcein-AM and PI reagents (1x) were added to each well and incubated at 37 ℃ for 30 min. Then, the green and red fluorescence was observed, and images were captured under a fluorescence microscope (Nikon, Japan).

Malondialdehyde (MDA), glutathione (GSH) and ferrous iron (Fe²⁺) assay

To measure the levels of MDA and Fe²⁺ in cellular and tissue lysates, an MDA Assay Kit (S0131S, Beyotime, P.R.China), a GSH Assay Kit (E-BC-K030-M, Elabscience, P.R.China) and an Iron Assay Kit (E-BC-K773-M, Elabscience, P.R.China) were used. The specific procedures were performed according to the manufacturers’ instructions and our previous study [17].

Western blot

We extracted whole protein from lung tissues and BEAS-2B cells using RIPA lysis buffer (HY–K1001, MCE, USA) containing a protease inhibitor cocktail (HY–K0010, MCE, USA). A BCA Protein Assay Kit (P0012, Beyotime, P.R.China) was used to measure the concentrations of whole protein. The Western blot protocols were described previously [17]. ImageJ software was used to analyze the relative expression levels of the target proteins. The information and dilution concentrations of the antibodies are listed in Table S1.

Chromatin immunoprecipitation and quantitative PCR (ChIP-qPCR)

The ChIP-qPCR protocols were performed according to the manufacturers’ instructions and our previous study [17]. To analyze the expression level of purified DNA, RT-qPCR was performed (primers shown in Table S2).

RNA sequencing (RNA-seq)

BEAS-2B cells with two replicates in each group were used for RNA-seq. BEAS-2B cells from the control, 100 nm-400 μg/ml, and 200 nm-400 μg/ml groups were selected randomly and sent to Benagen (Wuhan, P.R.China) for RNA-seq. RNA-seq and bioinformatic analyses were performed as previously described [17].

Statistical analysis

Data were analyzed using GraphPad Prism 8.0 (San Diego, USA) software and were expressed as the mean ± standard deviation (SD). Student’s t test was used for comparisons between two groups. For multiple comparisons, one-way analysis of variance (ANOVA) was used. A value of p < 0.05 was considered statistically significant. At least three replicates were performed in all experiments.

Results

Characterization of PS-NPs

First, we verified the characteristics of the 100 nm and 200 nm PS-NPs through SEM. Our results showed the shape (Supplemental Fig. 1A), size (Supplemental Fig. 1B and C), and zeta potential (Supplemental Fig. 1D) of PS-NPs. The diameters were nearly 100 and 200 nm.

Fig. 1.

Histopathological changes in the lungs after PS-NP exposure. (A) Protocols used to construct the PS-NP-exposed mouse model. (B) Gross morphology of the lungs from control, 100 nm and 200 nm PS-NP-exposed mice (white circles indicate atelectasis or pulmonary hemorrhage). (C) H&E staining of lung tissues after PS-NP exposure for seven days. H&E staining showed substantial perivascular lymphocytic inflammation in a bronchiolocentric pattern (arrowhead). (D) Masson trichrome staining indicated significant collagen deposits and foamy macrophages (arrowhead) in the alveolar space. (100–12.5, 12.5 mg/kg bw 100 nm PS-NPs; 100–25, 25 mg/kg bw 100 nm PS-NPs; 200–12.5, 12.5 mg/kg bw 200 nm PS-NPs; 200–25, 25 mg/kg bw 200 nm PS-NPs).

PS-NP-induced lung toxicity.

To investigate the lung injury induced by PS-NPs, adult mice were given PS-NPs by intratracheal instillation for 7 consecutive days (Fig. 1A). There was no significant difference in body weight between the control and PS-NP exposure groups (Table S3). Compared with the control group, we observed gross atelectasis and pulmonary hemorrhage after PS-NP treatment (Fig. 1B). H&E staining demonstrated predominantly lymphocytic inflammation in a bronchiolocentric pattern (Fig. 1C). In addition, substantial perivascular lymphocytic inflammation was also observed. To assess pulmonary fibrosis caused by PS-NP exposure, we performed Masson trichrome staining. We found more collagen deposits in the PS-NP treatment group than in the control group in a dose-dependent manner. Clusters of foamy macrophages were also observed in the alveolar space (Fig. 1D). Furthermore, sexual difference was not observed in the lung injury induced by PS-NP exposure, either.

PS-NP exposure compromised the viability of BEAS-2B cells

Based on our histopathological findings, PS-NP exposure mainly caused critical damage to bronchioles. Therefore, we used bronchial epithelial cells to further explore the in vitro toxicity and mechanism of PS-NP treatment. BEAS-2B cells were treated with 100 nm or 200 nm PS-NPs, and cell viability was measured to verify the optimum exposure concentrations (Fig. 2A). Using TEM, we confirmed that the PS-NPs were taken up by BEAS-2B cells (Fig. 2B), and the viability of BEAS-2B cells treated with 100 μg/ml PS-NPs was significantly decreased (Fig. 2C and D). Thus, we selected 100, 200, and 400 μg/ml PS-NPs for subsequent experiments. Additionally, calcein-AM/PI staining indicated that PS-NP exposure accelerated the death of BEAS-2B cells in a dose-dependent manner (Fig. 2E and F).

Fig. 2.

The toxic effects of PS-NPs on BEAS-2B cells. (A) The PS-NP exposure and cell viability measurement protocols. (B) TEM indicated the uptake of PS-NPs in BEAS-2B cells (arrowhead). (C) The viability of BEAS-2B cells after 100 nm PS-NP exposure. (D) The viability of BEAS-2B cells after 200 nm PS-NP exposure. (E) Calcein AM/PI staining of BEAS-2B cells after 100 nm PS-NP exposure. (Red and green fluorescence indicate dead and live cells, respectively) (F) Calcein AM/PI staining of BEAS-2B cells after 200 nm PS-NP exposure. *p < 0.05, **p < 0.01, ***p < 0.001, n.s., not significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Transcriptome analysis of PS-NP-exposed BEAS-2B cells

To clarify the underlying reasons and mechanisms of cellular injury induced by PS-NP exposure, we employed RNA-seq. The differentially expressed genes (DEGs) between the control and 400 μg/ml 100 nm or 200 nm PS-NP-exposed BEAS-2B cells were isolated. There were 104 DEGs between the control and 400 μg/ml 100 nm PS-NP groups. 75 DEGs were upregulated, and 29 DEGs were downregulated. There were 141 DEGs between the control and 400 μg/ml 200 nm PS-NP groups. A total of 102 DEGs were upregulated, and 39 DEGs were downregulated (Supplemental Fig. 2A and B). Gene Ontology (GO) enrichment analysis found that DEGs were enriched in biological processes associated with lipid biosynthesis, lipid metabolism, and iron ion binding (Supplemental Fig. 2C and D). Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis revealed that DEGs were enriched in fatty acid metabolism and biosynthesis pathways (Supplemental Fig. 2E and F). Gene set enrichment analysis (GSEA) indicated that the ferroptosis pathway was activated (Supplemental Fig. 2G). These results suggested that PS-NP exposure might lead to ferroptosis in BEAS-2B cells.

PS-NP exposure induced ferroptosis in BEAS-2B cells

Based on our previous RNA-seq and bioinformatic analysis, we performed subsequent experiments to verify PS-NP-induced ferroptosis in BEAS-2B cells. Compared with the control group, the level of GSH was decreased (Fig. 3A and G), and the levels of Fe²⁺ (Fig. 3B and H) and MDA (Fig. 3C and I) were elevated significantly after 100 nm or 200 nm PS-NP exposure. Furthermore, cellular ROS levels increased significantly after PS-NP treatment in a dose-dependent manner (Fig. 3D and J). Moreover, we found the upregulation of FTH1, SLC7A11, TF, HO-1, and ACSL4 and downregulation of GPX4 (Fig. 3E and K) after PS-NP exposure. Additionally, using TEM, we observed that there were fewer mitochondrial cristae and that the outer membranes were denser after PS-NP exposure (Fig. 3F and L). In summary, these findings showed that PS-NP exposure induced ferroptosis in BEAS-2B cells.

Fig. 3.

PS-NP exposure induced ferroptosis in BEAS-2B cells. (A) The GSH level decreased after 100 nm PS-NP exposure. (B) Ferrous iron was overloaded after 100 nm PS-NP exposure. (C) The MDA level increased after 100 nm PS-NP exposure. (D) Cellular ROS increased in BEAS-2B cells after 100 nm PS-NP exposure. (E) The expression levels of ferroptotic proteins after 100 nm PS-NP exposure. (F) The mitochondrial morphology of BEAS-2B cells after 100 nm PS-NP exposure (white arrows indicate that there were fewer mitochondrial cristae and that the outer membranes were denser). (G) The GSH level decreased after 200 nm PS-NP exposure. (H) Ferrous iron was overloaded after 200 nm PS-NP exposure. (I) The MDA level increased after 200 nm PS-NP exposure. (J) Cellular ROS increased in BEAS-2B cells after 200 nm PS-NP exposure. (K) The expression levels of ferroptotic proteins after 200 nm PS-NP exposure. (L) The mitochondrial morphology of BEAS-2B cells after 200 nm PS-NP exposure (white arrows indicate that there were fewer mitochondrial cristae and that the outer membranes were denser). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Ferrostatin-1 rescued PS-NP-induced ferroptosis in BEAS-2B cells

To clarify that ferroptosis was the major RCD phenotype after PS-NP exposure (400 μg/ml), we used ferrostatin-1 (Fer-1, 1 μM) for further experiments. Fer-1 treatment successfully mitigated the compromised cell viability (Supplemental Fig. 3A and H) and GSH level (Supplemental Fig. 3C and J) in BEAS-2B cells. In addition, Fer-1 administration alleviated the increased levels of MDA (Supplemental Fig. 3B and I), ferrous iron (Supplemental Fig. 3D and K), and cellular ROS (Supplemental Fig. 3E and L) in BEAS-2B cells. Calcein-AM/PI staining also showed that PS-NP-induced cell death was rescued by Fer-1 treatment (Supplemental Fig. 3F and M). Moreover, we found that Fer-1 treatment restored the altered expression of FTH1, GPX4, SLC7A11, TF, HO-1, and ACSL4 caused by PS-NP exposure (Supplemental Fig. 3G and N). These results suggested that ferrostatin-1 rescued PS-NP-induced cellular ferroptosis.

PS-NP exposure led to ferroptosis through the HIF-1α/HO-1 signaling pathway

HIF-1 has two subunits, the functional subunit HIF-1α and the structural subunit HIF-1β. To verify whether the HIF-1 signaling pathway was activated, we measured the expression of HIF-1α, HIF-1β, and their upstream PHD complex. Following PS-NP treatment, the expression levels of PHD1, 2, and 3 were decreased, indicating the stabilization of HIF-1α. As expected, HIF-1α and HIF-1β levels increased dramatically (Fig. 4A and E). Since HIF-1α is a transcription factor, we also examined the localization of HIF-1α and HIF-1β using IF. We found that HIF-1α (Fig. 4B and F) and HIF-1β (Fig. 4C and G) translocated into the nucleus after PS-NP exposure. These results suggested that PS-NP exposure activated HIF-1 signaling in BEAS-2B cells.

Fig. 4.

Hmox1 transcription was regulated by HIF-1α. (A) The expression levels of HIF-1α, HIF-1β, and the PHD complex in BEAS-2B cells after 100 nm PS-NP exposure. (B) The intracellular location of HIF-1α in BEAS-2B cells after 100 nm PS-NP exposure. (C) The intracellular location of HIF-1β in BEAS-2B cells after 100 nm PS-NP exposure. (D) ChIP-qPCR results of HIF-1α levels in BEAS-2B cells after 100 nm PS-NP exposure (400 μg/ml). (E) The expression levels of HIF-1α, HIF-1β, and the PHD complex in BEAS-2B cells after 200 nm PS-NP exposure. (F) The intracellular location of HIF-1α in BEAS-2B cells after 200 nm PS-NP exposure. (G) The intracellular location of HIF-1β in BEAS-2B cells after 200 nm PS-NP exposure. (H) ChIP-qPCR results of HIF-1α levels ChIP-qPCR in BEAS-2B cells after 200 nm PS-NP exposure (400 μg/ml). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

To our knowledge, HO-1 is encoded by the Hmox1 gene. HO-1 is a critical target of HIF-1α, and upregulation of HO-1 can augment ferrous iron levels. Thus, we speculated that PS-NP exposure enhanced HIF-1α/Hmox1 binding and promoted ferroptosis in the lungs. Based on the ChIP-qPCR results, we observed that HIF-1α/Hmox1 binding was enhanced by PS-NP exposure (400 μg/ml, Fig. 4D and H).

HIF-1α inhibition rescued PS-NP-induced ferroptosis in BEAS-2B cells

To identify whether PS-NP-induced ferroptosis was regulated by HIF-1α, we used LW6, a HIF-1α inhibitor, to suppress HIF-1α expression in BEAS-2B cells. We found that LW6 (10 μM) successfully inhibited the expression of HIF-1α in BEAS-2B cells (Fig. 5A and I). The reduced cell viability caused by PS-NP exposure (400 μg/ml) was reversed due to HIF-1α inhibition (Fig. 5B and J). Moreover, the abnormal levels of lipid peroxidation (Fig. 5C and K), GSH (Fig. 5D and L), Fe²⁺ (Fig. 5E and M), and ROS (Fig. 5F and N) due to PS-NP exposure were rescued after HIF-1α inhibition. HIF-1α inhibition also alleviated cell death due to PS-NP exposure (Fig. 5G and O). Furthermore, HIF-1α inhibition reversed the increased HO-1 expression level (Fig. 5H and P). Overall, PS-NP exposure might lead to ferroptosis in BEAS-2B cells in a HIF-1α-dependent manner.

Fig. 5.

Inhibition of HIF-1α rescued PS-NP-induced ferroptosis in BEAS-2B cells. (A) LW6 treatment (10 μM) significantly reduced the upregulation of HIF-1α following 100 nm PS-NP exposure (400 μg/ml). (B) The compromised cell viability due to 100 nm PS-NP exposure was reversed following HIF-1α inhibition. (C) The increased MDA level due to 100 nm PS-NP exposure was reversed following HIF-1α inhibition. (D) The reduced GSH level due to 100 nm PS-NP exposure was alleviated following HIF-1α inhibition. (E) The overload of ferrous iron due to 100 nm PS-NP exposure was reversed following HIF-1α inhibition. (F) The increased cellular ROS in BEAS-2B cells after 100 nm PS-NP exposure was abrogated following HIF-1α inhibition. (G) Cell death due to 100 nm PS-NP exposure was mitigated following HIF-1α inhibition. (H) The expression levels of ferroptotic proteins after 100 nm PS-NP exposure with/without LW6 treatment. (I) LW6 (10 μM) treatment significantly reduced the upregulation of HIF-1α following 200 nm PS-NP exposure (400 μg/ml). (J) The compromised cell viability due to 200 nm PS-NP exposure was reversed following HIF-1α inhibition. (K) The increased MDA level due to 200 nm PS-NP exposure was reversed following HIF-1α inhibition. (L) The reduced GSH level due to 200 nm PS-NP exposure was alleviated following HIF-1α inhibition. (M) The overload of ferrous iron due to 200 nm PS-NP exposure was reversed following HIF-1α inhibition. (N) The increased cellular ROS in BEAS-2B cells after 200 nm PS-NP exposure was abrogated following HIF-1α inhibition. (O) Cell death due to 200 nm PS-NP exposure was mitigated following HIF-1α inhibition. (P) The expression levels of ferroptotic proteins after 200 nm PS-NP with/without LW6 treatment. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n.s., not significant.

PS-NP exposure induced ferroptosis in the lungs through the HIF-1 signaling pathway

To confirm our in vitro findings, we examined ferroptosis in the lung tissues of mice after PS-NP exposure. Following 100 nm or 200 nm PS-NP exposure, the level of GSH was decreased (Fig. 6A), and the levels of Fe²⁺ (Fig. 6B) and MDA (Fig. 6C) were increased significantly. Additionally, the expression levels of FTH1, SLC7A11, TF, HO-1, and ACSL4 were enhanced, and the level of GPX4 was reduced significantly (Fig. 6F).

Fig. 6.

PS-NP exposure induced ferroptosis by activating HIF-1 signaling in the lungs. (A) The GSH level in lung tissues decreased after PS-NP exposure. (B) Ferrous iron in lung tissues was overloaded after PS-NP exposure. (C) The MDA level in lung tissues increased after PS-NP exposure. (D) IHC results indicated enhanced expression of HIF-1α after PS-NP exposure in lung sections. (E) IHC results indicated enhanced HIF-1β expression after PS-NP exposure in lung sections. (F) The expression levels of ferroptotic proteins and the HIF-1 signaling pathway after PS-NP exposure. (G) IF results indicated enhanced expression of HIF-1α after PS-NP exposure in lung sections. (H) IF results indicated enhanced HIF-1β expression after PS-NP exposure in lung sections. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (100–12.5, 12.5 mg/kg bw 100 nm PS-NPs; 100–25, 25 mg/kg bw 100 nm PS-NPs; 200–12.5, 12.5 mg/kg bw 200 nm PS-NPs; 200–25, 25 mg/kg bw 200 nm PS-NPs).

Subsequently, we also measured the levels of HIF-1 signaling pathway proteins in the lung tissues of mice after PS-NP exposure. These results were consistent with our in vitro findings. Western blot analysis showed that the expression levels of HIF-1α and HIF-1β were increased significantly (Fig. 6F). IF and IHC analyses revealed that HIF-1α (Fig. 6D and G) and HIF-1β (Fig. 6E and H) were also enhanced after PS-NP exposure.

Discussion

As a result of their characteristics, such as high stability, easy production and material diversity, plastics are widely used in our daily life. Plastics can be degraded into MPs and NPs and released into our environment, leading to contamination of the ocean, soil, and atmosphere [22], [23], [24]. Several studies have demonstrated that NP exposure has a variety of adverse effects on human health [7], [25]. Recently, the toxic effects of NPs on the respiratory system via inhalation have attracted increasing attention. Recently published studies have verified that NPs cause lung injury through oxidative stress and the inflammatory response [26], [27], [28]. However, the underlying mechanisms need further clarification.

In this study, we established a PS-NP-induced lung injury model in mice based on a recent study that simulated realistic nanoparticle exposure in humans [19]. We found that PS-NP exposure led to substantial perivascular lymphocytic inflammation and considerable collagen deposits in the lung tissues. Notably, we found that lung injury in the 200 nm PS-NP exposure group was more critical than that in the 100 nm PS-NP exposure group. According to published reports, the toxicity on human placental cells induced by the smaller size of PS-NPs was greater [29], but the neurotoxicity induced by the smaller size of PS-NPs was weaker [30]. This may be due to the preferential cellular distribution of different-sized nanoparticles, which leads to different toxicities.

Based on the findings in the literature, nanoparticle exposure leads to lung injury mainly through oxidative stress and inflammation [26], [31], [32], but the specific mechanisms of lung injury are still unknown. In our study, we performed RNA-seq to investigate the potential mechanisms. GO analysis revealed that the DEGs in BEAS-2B cells after PS-NP exposure were enriched in the lipid metabolism process, which is an important biomarker for ferroptosis [33]. Furthermore, GSEA showed that the ferroptosis pathway was activated in mice after PS-NP exposure. Thus, we inferred that ferroptosis might be the main factor in PS-NP-induced lung injury. In addition, by using ferrostatin-1, a specific ferroptosis inhibitor, we found that PS-NP-induced ferroptosis was successfully reversed. For the first time, we experimentally confirmed that PS-NP exposure resulted in ferroptosis in the lungs.

Ferroptosis is a type of RCD caused by excessive oxidative stress and is associated with ferrous iron overload and lipid peroxidation. Numerous genes and pathways related to ferroptosis have been verified [34], [35], [36]. In our study, GPX4, which is an important antioxidant molecule that inhibits ferroptosis, was downregulated in mice following PS-NP exposure. The decline in GPX4 can lead to pulmonary fibrosis [37], which was consistent with the observed critical collagen deposits caused by PS-NP exposure in our study. Notably, FTH1, as an important iron storage protein, was upregulated in mice after PS-NP exposure in our study. FTH1 upregulation potentially occurred to resist ferroptosis and bind more free ferrous iron for compensation. In addition, the level of SLC7A11, an antiporter, was also increased following PS-NP treatment, and we speculated that upregulation of SLC7A11 produced more GSH to mitigate lipid peroxidation in our lung injury model.

HIF-1α, as a transcription factor, allows cells to adapt to hypoxic conditions. Its stabilization and activity are regulated by the PHD complex [38], E3 ubiquitin ligase [39], and ROS [40]. Additionally, HIF-1α has been confirmed to regulate ferroptosis. In the process of intestinal ischemia/reperfusion-induced ferroptosis of lung epithelial cells, HIF-1α expression increases [41]. However, HIF-1α expression decreased in the process of clockophagy-mediated ferroptosis [42]. These results illustrate that HIF-1α plays different roles in ferroptosis under various biological conditions. In our study, during PS-NP-induced lung ferroptosis, HIF-1α expression increased significantly, and ferroptosis of BEAS-2B cells was rescued after HIF-1α inhibition, indicating that HIF-1α activity promotes PS-NP-induced ferroptosis in the lungs.

As one of the downstream targets of HIF-1α, HO-1 can not only protect against oxidative stress but also promote ROS generation by releasing free ferrous iron [43], [44], [45]. In our study, we found an overload of ferrous iron, and upregulation of HO-1 was caused by enhanced HIF-1α expression after PS-NP exposure. However, this phenomenon was reversed by HIF-1α inhibition. Therefore, we inferred that the HIF-1α/HO-1 signaling pathway possibly regulated the surplus of released ferrous iron and aggravated oxidative stress in the lungs following PS-NP exposure. These results were consistent with the ferroptosis of testicular somatic cells induced by DEHP exposure [17].

We acknowledge the limitations of this study. First, although we estimated the real-world exposure dosage of PS-NP, the exposure duration in this study was relatively acute. Therefore, chronic pulmonary injury due to long-term exposure to PS-NPs needs to be investigated in the future. Second, we only used bronchial epithelial cells for in vitro experiments, and whether PS-NP exposure led to ferroptosis in other pulmonary cells is unknown. Further single-cell RNA-seq should be performed to assess other pulmonary cells. Third, with the sole use of HIF-1α inhibitors, off-target effects might have existed. HIF-1α knockdown or knockout should be conducted to investigate the individual role of HIF-1α in PS-NP-induced lung ferroptosis.

Conclusions

Based on our findings, PS-NP exposure may induce ferroptosis in the lungs and bronchial epithelial cells. In particular, PS-NP exposure leads to ferroptosis through the HIF-1α/HO-1 signaling pathway. These novel findings shed light on the concerns of PS-NP-induced lung injury, and further investigations based on long-term/chronic exposure are needed.

Compliance with Ethics Requirements:

All Institutional and National Guidelines for the care and use of animals (fisheries) were followed.

All animal experiments were approved by the Ethics Committee of Children’s Hospital of Chongqing Medical University (Issue number:CHCMU-IACUC20220323010).

Funding

This study was supported by grants from the Chongqing Medical University Program for Youth Innovation in Future Medicine in 2022 (No. W0204), National Natural Science Foundation of China (Grant No. 81801521), Clinical Innovation Project of Children’s Hospital of Chongqing Medical University (CHCMU-XJS-2022-56), and the Guangdong Basic and Applied Basic Research Foundation (No. 2022A1515012576).

CRediT authorship contribution statement

Yuhao Wu: Methodology, Software. Junke Wang: Investigation, Methodology. Tianxin Zhao: Methodology, Software. Mang Sun: Conceptualization. Maozhu Xu: Investigation, Data curation, Methodology. Siyi Che: Investigation, Data curation, Methodology. Zhengxia Pan: Supervision. Chun Wu: . Lianju Shen: Funding acquisition.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary material

The following are the Supplementary data to this article: