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Introduction

Hyperbilirubinemia, one of the most common disorders in newborns, is characterized by an imbalance between bilirubin production and elimination (1). Aberrant accumulation of bilirubin may cause bilirubin encephalopathy, which is the most serious complication of neonatal hyperbilirubinemia that can lead to neonatal death (2–4). Survivors often have neurological sequelae, including learning disabilities, movement disorders, mental retardation and cerebral palsy (5). Neonatal bilirubin encephalopathy causes 4.8% of all pediatric hospitalization and its mortality is estimated to be 16.1% in a multicenter study in China (6). Although a number of treatment options have been employed for the treatment of neonatal hyperbilirubinemia, including phototherapy and exchange transfusion (7), neurological damage may still occur, suggesting that several other factors contribute to increased neurotoxicity (8). Hemolysis and its major byproduct, ferrous ion (Fe2+), are among the factors that serve important roles in the development of neurotoxicity (9,10); however, the underlying mechanism remains unclear.

In both developing and developed countries, hemolysis is one of the risks factors for hyperbilirubinemia (11). During excessive hemolysis, increased amounts of iron are released from damaged red blood cells. Iron enters the endothelial cells that form the blood-brain barrier via transferrin receptor-mediated endocytosis, or it is independently transported as non-protein-bound iron (12). A significant increase in non-protein-bound iron levels and an imbalance between pro-oxidant and antioxidant systems is observed in neonatal hemolytic diseases (10,13).

Ferroptosis, a recently identified form of programmed cell death that differs from apoptosis and necrosis, is driven by iron-dependent accumulation of lipid hydroperoxides (14). Dysregulation of iron homeostasis, glutathione (GSH) depletion and lipid peroxidation are closely linked to cell sensitivity to ferroptosis (15). Iron homeostasis imbalance has been implicated in ferroptosis, which may lead to pathological conditions in the central nervous system (16,17). Studies have reported that ferroptosis is involved in numerous neurodegenerative diseases, such as Alzheimer's disease (16,18), Parkinson's disease (19), ischemic stroke (20), intracerebral hemorrhage (21) and traumatic brain injury (22). These conditions lead to increased hemoglobin release and iron overload, resulting in an abnormal increase in lipid peroxidation, which is the main cause of secondary injury and neuronal ferroptosis (23). Hemolytic hyperbilirubinemia also results in an increased release of hemoglobin and iron (11).

Moreover, specific features of ferroptosis, such as increased products of lipid peroxidation, are similar to those that appear in iron-mediated neurological dysfunction caused by neonatal hemolytic diseases (24). In a rat model of hemolytic hyperbilirubinemia-induced brain damage (HHIBD), comparing to the control group, the level of malondialdehyde (MDA) increased and GSH decreased in brain tissues of the treatment group, closely resembling the biochemical characteristics of ferroptosis (25,26). It is therefore hypothesized that ferroptosis may contribute to the pathogenesis of neuronal dysfunction induced by neonatal hemolytic hyperbilirubinemia (8). However, the contribution of ferroptosis to hemolytic HHIBD remains unclear. The present study, utilizing both in vitro and in vivo assays, assessed the role of ferroptosis in HHIBD.

Materials and methods

Modeling neonatal hemolytic hyperbilirubinemia in rats

A total of 60 male Sprague-Dawley (SD) rats (aged 10–12 days; 24–28 g) were purchased from the Laboratory Animal Center, Fujian Medical University (Fuzhou, China). Rat pups were housed in the same standard laboratory polycarbonate cage with their mothers at a suitable temperature (21±2°C) and humidity (50±10%) with 12/12-h light/dark cycles to maintain their circadian rhythm. Maternal milk was used to feed the rat pups, while adult female rats had free access to standardized granular food (Beijing Keao Xieli Feed Co., Ltd.) and tap water. The 10–12 day old rat pups were randomly divided into the control, phenylhydrazine (PHZ) and deferoxamine (DFO) + PHZ groups, with 12 rats in each group.

To establish bilirubin encephalopathy secondary to hemolysis, 75 mg/kg PHZ (Sigma-Aldrich; Merck KGaA) was injected intraperitoneally once daily for 2 days, based on previous reports (26,27). Rats in the DFO + PHZ group were injected intraperitoneally with DFO (100 mg/kg; cat. no. D9533; Sigma-Aldrich; Merck KGaA) 1 h prior to administration of PHZ (28,29) whilst rats in the PHZ group received the same volume of saline. Control rats received the same volume of saline in parallel. Subsequently, 24 h after the last dosing, all animals were anesthetized intraperitoneally with 100 mg/kg pentobarbital sodium and then euthanized by decapitation to collect trunk blood and brain tissue. A total of 0.5 ml blood from each rat was collected and centrifuged at 1,500 × g at 4°C for 10 min and then preserved at −80°C for later biochemical analysis. Brain tissue was immediately collected, placed in liquid nitrogen for 5 min, and then stored at −80°C for future analysis. All treatments were performed with care and rat suffering was minimized. During the experiment, none of the rats reached any of the following humane endpoints: Inability to drink the maternal milk, labored breathing, inability to stand or no response to external stimuli. Death was verified by monitoring the cessation of breathing and heartbeat and pupil dilation. No animals were found dead before the end of the experiment.

Cell culture and drug treatment

The highly differentiated PC12 cell line, derived from a pheochromocytoma of the rat adrenal medulla, resembles neurons and is widely used in the study of neurological diseases in vitro (30,31). PC12 cells (Wuhan Boster Biological Technology, Ltd.) were cultured in Dulbecco's Modified Eagle Medium (cat. no. C11995500BT; Gibco; Thermo Fisher Scientific, Inc.), supplemented with 10% fetal bovine serum (cat. no. SV30207.03; HyClone; Cytiva) and 100 U/ml penicillin-streptomycin (cat. no. V900929; Sigma-Aldrich; Merck KGaA). Cells were cultured at 37°C as monolayers in a humidified atmosphere containing 5% CO2. Based on MTT cell viability assay results (Fig. S1) and the level of superoxide dismutase (SOD), MDA and reactive oxygen species (ROS) in the PC12 cells exposed to 0, 200 and 400 µM ferric ammonium citrate (FAC; cat. no. F5879; Sigma-Aldrich; Merck KGaA) for 24 h (Fig. S2), 400 µM FAC treatment for 24 h was selected for subsequent experiments, to assess the mechanism of iron overload-induced neurotoxicity in vitro. PC12 cells were pretreated with 10 µM ferrostatin-1 (Fer-1; cat. no. SML0583; Sigma-Aldrich; Merck KGaA) or 100 µM DFO for 1 h at 37°C and exposed to 400 µM FAC.

Measurement of serum hemoglobin, hematocrit and bilirubin levels

Serum hemoglobin and hematocrit levels were assessed using an automated analyzer (Coulter LH 780; Beckman Coulter, Inc.). Another automated analyzer (Architect ci16200 Integrated System; Abbott Pharmaceutical Co. Ltd.) was used to measure the serum levels of total bilirubin, indirect bilirubin and direct bilirubin using a diazo reagent-based method (32). All procedures strictly followed the manufacturers' instructions.

Measurement of S100 calcium-binding protein B (S100B) and serum neuron-specific enolase (NSE) levels

Serum levels of S100B were assessed using the Rat S100B ELISA kit (cat. no. E-EL-R0868; Elabscience Biotechnology, Inc.), and those of serum NSE were assessed using the Rat NSE ELISA Kit (cat. no. E-EL-R0058c; Elabscience Biotechnology, Inc.) according to the manufacturer's instructions.

Measurement of SOD activity, MDA and reduced GSH/oxidized glutathione disulfide (GSSG) levels

A total of 15 mg brain tissue from six rats in each group was placed into 200 µl ice-cold 10% radioimmunoprecipitation assay lysis buffer (RIPA; cat, no. P0013E; Beyotime Institute of Biotechnology). Brain samples were homogenized using a tissue homogenizer (cat. no. KZ-III-F; Wuhan Servicebio Technology Co., Ltd.) and centrifuged at 1,000 × g for 10 min at 4°C. The supernatant was then transferred into microcentrifuge tubes. For cell extracts, PC12 cells were seeded in the 6-well plate at a density of 50–60% for 24 h, and then exposed to FAC. A total of 200 µl RIPA lysis buffer were added to plate to homogenize the sample and centrifuged at 1,000 × g for 10 min at 4°C, waiting for next measurement.

SOD activity and total GSH/GSSG detection kits (cat. nos. A001-3-1 and A061-1-1; Nanjing Jiancheng Bioengineering Institute) were used to assess SOD and GSH/GSSG levels, respectively. Tissue iron detection kits (cat. no. A039-2-1; Nanjing Jiancheng Bioengineering Institute) were used to assess tissue iron levels. An MDA detection kit (cat. no. S0131S; Beyotime Institute of Biotechnology) was used to assess MDA levels. All assays were conducted in triplicate according to the manufacturers' protocols.

Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis

Proteomic analysis of separate triplicate brain tissue samples from the control and PHZ groups was performed by tandem mass tag (TMT) labeling (Cat. no. A44520; Thermo Fisher) (33) and liquid chromatography-tandem mass spectrometry (Q Exactive™ HF-X; Thermo Fisher) (34). The detection was conducted by multiple reaction monitoring (MRM) in negative ionization mode. The electrospray voltage was 2.1 kV. The m/z scan range was 350–1600 for the full scan, and intact peptides at a resolution of 120,000 were detected in the Orbitrap. We set the automatic gain control (AGC) at 3E6, and the fixed first mass was 100 m/z. The nitrogen gas temperature was at 300°C with a flow rate of 5 l/min and nebulizer pressure was 40 psi. The differentially abundant protein (DAP) screening parameters were adjusted to P<0.05 and log2 fold change=0, KEGG enrichment analysis was performed using ClueGO (v2.5.6; http://apps.cytoscape.org/apps/cluego) (35). The databases used were KEGG_20.05.2019 (genome.jp/kegg/pathway.html) and Gene Ontology (GO) (https://www.geneontology.org/)_Biological Process-EBI-Uniprot-GO Annotation_20.05.2019 (https://david.ncifcrf.gov/home.jsp). The GO term ‘fusion mode’ was selected according to the P-value <0.05.

Protein-protein interaction network analysis

The number of differential protein sequences in the protein database (Maxquant: v1.6.15.0; http://www.maxquant.org/) screened according to a 1.2 expression difference multiplesin PHZ group vs. Control group, and then compared with those in the protein network interaction database of the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING; v. 11.0; http://cn.string-db.org/), with a confidence score of >0.7 (high confidence). The differential protein interaction was analyzed, and the R package (Dev-version: 0.4; christophergandrud.github.io/networkD3/) ‘networkD3’ function was used to visually display the differential protein interaction network.

Cell viability assay

In the FAC model, PC12 cells were seeded in 96-well plates for 24 h at a density of 1×104 cells/well. Cells were then treated with different concentrations of FAC (0, 100, 200, 400 and 800 µM, 1 and 5 mM) for 3, 6, 12 and 24 h. The viability of PC12 cells was assessed using MTT (cat. no. F5655; Sigma-Aldrich; Merck KGaA). Briefly, after the aforementioned treatments, MTT (5 mg/ml) was added to the 96-well plate. The plate was incubated at 37°C for 4 h to allow the formazan crystals to form. The medium was then discarded and 100 µl dimethyl sulfoxide was added to dissolve the formazan. Absorbance was measured at a detection wavelength of 570 nm using a plate reader (Thermo Fisher Scientific, Inc.).

Measurement of lipid oxidation using confocal laser scanning microscopy

After PC12 cells were seeded in the 35 mm confocal dish (cat. no. FCFC016; Beyotime Institute of Biotechnology; China) at a density of 50 %, the cells were treated with FAC as aforementioned for 24 h, and then were rinsed with phosphate-buffered saline (PBS) three times. To visualize neuronal lipid oxidation, 10 µM BODIPY 581/591 C11 (cat. no. D3861; Thermo Fisher Scientific, Inc.) was added to each well and incubated at 37°C for 0.5 h and stained with 1 µg/ml DAPI for 5 min at 37°C. Cells were rinsed with PBS three times and imaged using a confocal laser scanning microscope (SP5; Leica Microsystems GmbH) (36). The argon laser (excitation, 488 nm; emission, 500–535 nm) was used to detect oxidized lipids, whereas the white light laser (excitation, 561 nm; emission, 573–613 nm) was used to detect non-oxidized lipids.

Measurement of ROS levels

PC12 cells were seeded in the 6-well plates for 24 h at a density of 2×105 cells/well and then were treated with FAC for 24 h as aforementioned. Subsequently, cells were stained with the ROS indicator 2′,7′-dichlorodihydrofluorescein diacetate (cat. no. S0033S; Beyotime Institute of Biotechnology) for 30 min at 37°C. Subsequently, the cells were washed with PBS three times and digested with 0.25 % trypsin (cat. no. C0201S; Beyotime Institute of Biotechnology) at 37°C for 2 min. Finally, labelled cells were detected with a flow cytometer (LSRFortessa™ X-20; BD Biosciences) and analyzed by Flow v10.9 (BD Biosciences) (37).

Detection of intracellular free iron using fluorescence microscopy

FerroOrange (cat. no. F374; Dojindo Laboratories, Inc.) was used to detect intracellular Fe2+. PC12 cells were seeded in the 12-well plates for 24 h at a density of 2×105 cells/well and then were treated with 400 µM FAC as aforementioned., They were rinsed with PBS and stained with 1 µM FerroOrange at 37°C for 0.5 h. The cells were rinsed with PBS three times and imaged under a fluorescence microscope (38).

Transmission electron microscopy (TEM)

PC12 cells were seeded in a 10 cm dish for 24 h at a density of 1×106 cells/well and then exposed to FAC as aforementioned. Subsequently, cells were fixed in 2.5% glutaraldehyde (diluted in 0.1 µM PBS; pH 7.4) at 25°C for 24 h and then post-fixed in 1% osmium tetroxide (dissolved in 0.1 µM PBS; pH 7.4) at 25°C for 60 min. After dehydration with ethanol (50, 70, 80, 90 and 100%), samples were embedded by resin (cat. no. 45347; Merck) with different condition (37°C for 12 h; 45°C for 12 h; 60°C for 12 h) and ultrathin sectioning (50 nm). Then, the samples were stained with uranyl acetate at room temperature for 30 min and stained with lead citrate at room temperature for 8 min. Digital images were captured using TEM (FEI Tecnai G2 F30; Thermo Fisher Scientific, Inc.) (39).

Western blotting

Proteins were extracted from brain tissue using RIPA lysis buffer (cat. no. P0013E; Beyotime Institute of Biotechnology) supplemented with phenylmethylsulfonyl fluoride and then denatured at 95°C in 5X protein loading buffer (cat. no. P0286; Beyotime Institute of Biotechnology) for 10 min. The concentration of protein in sample were measured by Enhanced BCA Protein Assay Kit (cat. no. P0009; Beyotime Institute of Biotechnology) according to the manufacturer's instructions. An equal amount of protein samples (20 µg/lane) were added to per lane and then were separated on 10% gels by SDS-PAGE. Proteins were then transferred to 0.2 µm PVDF membranes and blocked with 5% non-fat milk for 60 min at room temperature. PVDF membranes were incubated overnight at 4°C with primary antibodies against the following: Rabbit acyl-coenzyme A synthetase long-chain family member 4 (ACSL4; 1:1,000; cat. no. A16848; ABclonal Biotech Co., Ltd.); ferritin heavy chain (FTH; 1:1,000; cat. no. A19544; ABclonal Biotech Co., Ltd.); transferrin receptor 1 (TFRC; 1:1,000; cat. no. A5865; ABclonal Biotech Co., Ltd.); ferroportin 1 (FPN1; 1:1,000; cat. no. A14884; ABclonal Biotech Co., Ltd.); divalent metal transporter 1 (DMT1; 1:1,000; cat. no. A10231; ABclonal Biotech Co., Ltd.); iron regulatory protein 2 (IRP2; 1:1,000; cat. no. A6382; ABclonal Biotech Co., Ltd.); GAPDH (1:5,000; cat. no. A19056; ABclonal Biotech Co., Ltd.); xCT (1:1,000; cat. no. ab175186; Abcam); GSH peroxidase 4 (GPX4; 1:1,000; cat. no. ab125066; Abcam) and ferroptosis suppressor protein 1 (FSP1; 1:1,000; cat. no. AVARP09054_P050; Aviva Systems Biology, Corp). After washing by TBS buffer with 0.1% Tween-20 (cat. no. ST1727; Beyotime Institute of Biotechnology), PVDF membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (1:8,000; cat. no. 31460; Thermo Fisher) at 37°C for 60 min. Enhanced chemiluminescence detection was performed (cat. no. 1705061; BIO-RAD) (40) and ImageJ software (version 1.53; National Institutes of Health) was used to semi-quantify the protein bands.

Statistical analysis

Data are presented as mean ± standard error of the mean. All analyses were conducted using GraphPad Prism 8.0 (GraphPad Software; Dotmatics). Significance of differences between two groups was analyzed using two-way independent-sample t-test. For three or more groups, one-way analysis of variance followed by Tukey's post-hoc test was used to determine significance. P<0.05 was considered to indicate a statistically significant difference.

Results

Successful establishment of the hemolytic hyperbilirubinemia model

To establish a hemolytic hyperbilirubinemia model of neonatal SD rats, PHZ was used to increase erythrocyte turnover via hemolysis (Fig. S3A). In the PHZ group, the appearance of the neonatal rats and their brains was more jaundiced than that of the control group (Fig. S3B and C). PHZ group had significantly decreased hematocrit and hemoglobin levels compared with the control group (Fig. S3D and E). Moreover, the serum levels of three types of bilirubin (total, direct and indirect bilirubin) significantly increased in the PHZ group compared with that of the control group (Fig. S3F-H), indicating that the hemolytic hyperbilirubinemia model was established successfully.

Subsequently, changes in the brain injury markers NSE and S100B in the PHZ group were assessed. Compared with those in the control group, serum S100B and NSE levels were significantly increased in the PHZ group (Fig. 1A and B). Simultaneously, the concentration of iron significantly increased in the brain tissue of the PHZ group compared with that of the control group (Fig. 1C). Disturbances in oxidative balance caused by hemolytic hyperbilirubinemia were then assessed. SOD, which serves an important function in the first line of antioxidant defense, significantly decreased in the brains of the PHZ group compared with that of the control group (Fig. 1G). Compared with the control group, the GSH and GSH/GSSG levels significantly decreased in the PHZ group. However, there was no difference in the level of GSSG between the two groups (Fig. 1D-F). Similarly, the level of lipid peroxidation, as assessed by detecting the levels of MDA, significantly increased in the PHZ group compared with that of the control group (Fig. 1H). Overall, these results suggested that hemolytic hyperbilirubinemia could disrupt the redox balance in rat brains, resulting in brain injury. However, the specific mechanism of action remained unclear. Therefore, mass spectrometry analysis was performed to evaluate the potential molecular mechanisms of hemolytic hyperbilirubinemia.

Figure 1. Hemolytic hyperbilirubinemia-induced brain damage in neonatal Sprague-Dawley rats. Serum levels of (A) S100B and (B) neuron-specific enolase. (C) Tissue iron levels in the brain. Redox imbalances assessed by measuring levels of (D) GSH, (E) GSSG, (F) GSH/GSSG, (G) SOD and (H) MDA. n=6. *P<0.05; **P<0.01; ***P<0.001. S100B, S100 calcium-binding protein B; GSH, glutathione; GSSG, glutathione disulfide; SOD, superoxide dismutase; MDA, malondialdehyde; PHZ, phenylhydrazine; ns, not significant.

Ferroptosis participates in brain damage induced by hemolytic hyperbilirubinemia

Proteomic analysis of brain tissue samples from the control and PHZ groups were performed in triplicate using TMT labelling to evaluate whether ferroptosis is involved in HHIBD. KEGG analysis using ClueGO software demonstrated the enrichment of ferroptosis-related proteins in the PHZ group compared with those in the control group. When the DAPs screening parameters were adjusted to P<0.05 and log2 fold change=0, compared with the control, a total of 26 ferroptosis-associated DAPs were demonstrated to be enriched in the PHZ group (Fig. 2A). These DAPs were also involved in other metabolic pathways, including mineral absorption, glutathione metabolism, and fatty acid biosynthesis. The STRING database was then used to determine the protein-protein interaction network (Fig. 2B; confidence score >0.7) among the screened ferroptosis-associated DAPs.

Figure 2. Kyoto Encyclopedia of Genes and Genomes pathway and PPI network analysis of differentially abundant proteins enriched in ferroptosis (A) Kyoto Encyclopedia of Genes and Genomes pathway analysis of differentially abundant proteins using ClueGO software. (B) PPI network analysis associated with hemolytic hyperbilirubinemia-induced brain damage. Search Tool for the Retrieval of Interacting Genes/Proteins 11.0 database was used for PPI analysis with a confidence score of >0.7 (high confidence). The network nodes correspond to interacting proteins and the edges represent the predicted functional associations. Each of the colored lines represents different evidence for each interaction: Red, gene fusions; green, gene neighborhood; blue, gene co-occurrence; purple, experimentally determined; yellow, text mining; light blue, from curated databases. PPI, protein-protein interaction.

To evaluate the hypothesis that ferroptosis has a role in HHIBD, ferroptosis-associated proteins were assessed using western blotting. As it was previously demonstrated that the iron concentration in the brain tissue of the PHZ group significantly increased compared with that of the control group (Fig. 1C), protein-controlled iron import and export were assessed. The levels of the iron transporters TFRC (Fig. 3A and B) and DMT1 (Fig. 3A and C), which represent important components of ferroptosis that regulate iron levels, significantly increased in the PHZ group compared with that of the control group, whilst FPN1 levels did not significantly change (Fig. S4A and B). The results also demonstrated that levels of the iron storage protein FTH significantly increased in the PHZ group compared with that of the control group (Fig. 3D and F). Furthermore, the levels of IRP2, which is involved in the regulation of iron metabolism, significantly increased in the brain in the PHZ group compared with that of the control group (Fig. 3D and E). ACSL4 is a specific biomarker and driver of ferroptosis (41), while FSP1 has been identified as key molecules in independent pathways associated with ferroptosis inhibition (42). ACSL4 levels significantly increased and FSP1 levels significantly decreased in the PHZ group compared with that of the control group (Fig. 3G-I). The level of GPX4, an inhibitor of ferroptosis that converts lipid hydroperoxides into non-toxic lipid alcohols, was also demonstrated to have significantly increased after the onset of hemolytic hyperbilirubinemia in the PHZ group compared with that of the control group (Fig. S4A and C). By contrast, the level of xCT, an important antioxidant defense molecule, did not change significantly (Fig. S4A and D).

Figure 3. Changes in the expression of ferroptosis-associated proteins in the brain following treatment with PHZ. (A) Expression levels of TFRC and DMT1 determined using western blotting and semi-quantitative analysis of the protein expression of (B) TFRC and (C) DMT1. (D) Expression levels of IRP2 and FTH determined using western blotting and semi-quantitative analysis of the protein expression of (E) IRP2 and (F) FTH. (G) Expression levels of ACSL4 and FSP1 determined using western blotting and semi-quantitative analysis of the protein expression of (H) ACSL4 and (I) FSP1. n=6. *P<0.05. PHZ, phenylhydrazine; TFRC, transferrin receptor 1; DMT1, divalent metal transporter 1; IRP2, iron regulatory protein 2; FTH, ferritin heavy chain; ACSL4, acyl-coenzyme A synthetase long-chain family member 4; FSP1, ferroptosis suppressor protein 1.

Effect of the neuroprotective ferroptosis inhibitor DFO on HHIBD

DFO and its function as an iron chelator and specific inhibitor of ferroptosis (28,29), was used to further assess the function of ferroptosis in HHIBD. Compared with that in the PHZ group, DFO did not significantly influence the levels of hematocrit, hemoglobin and bilirubin in the DFO + PHZ group (Fig. S5). However, DFO pretreatment significantly reduced the levels of NSE and S100B, compared with that of the PHZ only group, indicating that brain injury was alleviated (Fig. 4A and B). DFO mitigated the imbalanced iron levels in the brain (Fig. 4C), thereby partially alleviating the redox imbalance compared with the PHZ group. The levels of GSH and SOD were increased following treatment with DFO (Fig. 4D-G), whereas MDA levels were significantly reduced, compared with that of the PHZ only group (Fig. 4H). There was no difference in the level of GSSG between the DFO + PHZ groups and the PHZ group (Fig. 4E).

Figure 4. DFO pretreatment alleviates hemolytic hyperbilirubinemia-induced brain damage in neonatal Sprague-Dawley rats. Serum levels of (A) S100B and (B) neuron-specific enolase. (C) Levels of iron in the brain. Redox imbalances assessed by measuring the levels of (D) GSH, (E) GSSG, (F) GSH/GSSG, (G) SOD and (H) MDA. n=6. *P<0.05; **P<0.01; ***P<0.001. DFO, deferoxamine; S100B, S100 calcium-binding protein B; GSH, glutathione; GSSG, glutathione disulfide; SOD, superoxide dismutase; MDA, malondialdehyde; PHZ, phenylhydrazine; ns, not significant.

The effect of DFO on the proteins related to ferroptosis was then evaluated. The protein levels of DMT1 and TFRC, which regulate iron transport in and out of cells, were decreased following DFO treatment compared with those of the PHZ group (Fig. 5A-C), whereas the FPN1 level was not significantly altered (Fig. S6A and B). Similarly, DFO restored the levels of IRP2 and FTH proteins compared with those of the PHZ group (Fig. 5D-F). Moreover, the altered levels of the key proteins in the ferroptosis pathway, ACSL4 and FSP1, were decreased following DFO treatment (Fig. 5G-I). However, the protein level of xCT did not significantly change following DFO pretreatment (Fig. S6A and D). Furthermore, compared with the PHZ group, the GPX4 level decreased in the DFO + PHZ group (Fig. S6A and C).

Figure 5. Ferroptosis is mitigated in neonatal Sprague-Dawley rats following DFO pretreatment. (A) Protein expression levels of TFRC and DMT1 determined using western blotting and semi-quantitative analysis of the protein expression of (B) TFRC and (C) DMT1. (D) Protein expression levels of IRP2 and FTH determined using western blotting and semi-quantitative analysis of protein expression of (E) IRP2 and (F) FTH. (G) Protein expression levels of ACSL4 and FSP1 determined using western blotting and semi-quantitative analysis of the protein expression of (H) ACSL4 and (I) FSP1. n=6. *P<0.05. DFO, deferoxamine; TFRC, transferrin receptor 1; DMT1, divalent metal transporter 1; IRP2, iron regulatory protein 2; FTH, ferritin heavy chain; ACSL4, acyl-coenzyme A synthetase long-chain family member 4; FSP1, ferroptosis suppressor protein 1; PHZ, phenylhydrazine.

Iron overload promotes ferroptosis in PC12 cells

Pretreatment with Fer-1 or DFO was demonstrated to antagonize the reduction in cell proliferation induced by FAC (Fig. 6A). The results also demonstrated that FAC exposure significantly increased MDA and reduced SOD activity levels and induced excessive generation of ROS compared with those in control group (Fig. 6B-E). Furthermore, FAC treatment also increased the Fe2+ content in PC12 cells, as indicated by significantly higher FerroOrange signals compared with those observed in control cells (Fig. 6F and G). Changes in the mitochondria were also demonstrated, with FAC treatment resulting in a reduction in mitochondrial volume and increased membrane density (Fig. 6H). Furthermore, the level of lipid peroxidation assessed using confocal laser scanning microscopy demonstrated that FAC significantly increased lipid peroxidation compared with that in control cells (Fig. 6I and J). However, pretreatment with 10 µM Fer-1 and/or 100 µM DFO effectively attenuated the aforementioned damage induced by FAC treatment.

Figure 6. PC12 cells were pretreated with 100 µM DFO or 10 µM Fer-1 for 1 h and then exposed to 400 µM FAC for 24 h. (A) Cell viability was assessed using an MTT assay. Level of (B) SOD activity and (C) MDA in PC12 cells. (D) Level of reactive oxygen species in PC12 cells assessed using 2′,7′-dichlorodihydrofluorescein diacetate via flow cytometry and (E) quantified. (F) Concentrations of free iron assessed using FerroOrange staining (scale bar, 100 µm). (G) Relative FerroOrange fluorescence. (H) Mitochondrial morphology observed using transmission electron microscopy (scale bar, 1 µm, upper images; 500 nm, lower images). (I) Confocal microscopy images of the oxidized and non-oxidized variants of lipid peroxides stained using a BODIPY C11 probe (scale bar, 50 µm). (J) Relative fluorescence intensity of oxidized variants of lipid peroxides. n=3. *P<0.05; **P<0.01; ***P<0.001. DFO, deferoxamine; Fer-1, ferrostatin-1; FAC, ferric ammonium citrate; SOD, superoxide dismutase; MDA, malondialdehyde; N, nucleus; M, mitochondria.

Discussion

In the present study, the results demonstrated that iron accumulation and ferroptosis occur in rats with HHIBD. DFO treatment restored the normal expression of ferroptosis-associated proteins (IRP2, FTH, TFRC, DMT1, ACSL4 and FSP1) and the levels of ferroptosis-associated biochemical markers (MDA, GSH, SOD and tissue iron).

Intraperitoneal injection of PHZ can induce hemolysis to increase the concentration of unconjugated bilirubin (25,26). Thus, compared with exogenous administration of bilirubin and Gunn rat (43,44), the PHZ-induced rat model provides a reproduction of the states of bilirubin encephalopathy secondary to hemolytic disease. According to previous data, the highest permeability of the blood-brain barrier occurs on the 10th day of life of a newborn rat pup (45). Thus, 10- to 12-day-old rats were administered PHZ and it was demonstrated that the total bilirubin level significantly increased, whereas the hemoglobin and hematocrit levels significantly decreased compared with the control group. Notably, the total bilirubin level exceeded the accepted hyperbilirubinemia threshold (>51.3 µM) in rats (26). It was also confirmed that the neonatal rats developed HHIBD by measuring the NSE and S100B levels.

Extensive research has been conducted on the potential mechanisms of bilirubin neurotoxicity in vitro and in vivo, including studies assessing oxidative stress (46), endoplasmic reticulum stress (47,48), inflammation (49), autophagy (50), apoptosis (51,52) and abnormal synthesis of both DNA and protein (53). However, the underlying mechanisms of neurotoxicity induced by bilirubin remain unclear and require further investigation. The contribution of other hemolysis products in the development of neurotoxicity have also not been well studied. Moreover, excess ferrous ion can accumulate in the basal ganglia and cause damage, and the basal ganglia is also a target site of bilirubin neurotoxicity (54).

Accumulation of peroxidized lipids is a typical characteristic of ferroptosis (55). For example, in the present study, the level of MDA was significantly increased in the PHZ group compared with the control group. Furthermore, GSH has been indicated to exhibit neuroprotective effects against bilirubin-induced cytotoxicity (56). Brain tissue GSH is reported to be significantly reduced following the induction of HHIBD in rats (26,27). Moreover, in the current study, it was demonstrated that HHIBD resulted in the downregulation of GSH levels and SOD activity and the upregulation of MDA levels. DFO, an iron-chelating agent and ferroptosis inhibitor, has been reported to induce antioxidant activity and reduce oxidative damage (57). Accordingly, pretreatment with DFO reversed the aforementioned changes. In addition, compared with the PHZ group, DFO pretreatment was demonstrated to significantly reduce the levels of NSE and S100B in the DFO + PHZ group, while the hemolysis related indicators such as hematocrit, hemoglobin and bilirubin were not alleviated. Thus, DFO pretreatment could alleviate HHIBD.

ACSL4 is considered to be a specific biomarker and driver of ferroptosis. Low expression of ACSL4 mediates glioma cell proliferation by inhibiting ferroptosis (58). In an animal model of acute brain injury, ACSL4 levels increased significantly, and knockout of ACSL4 reduced brain injury by inhibiting ferroptosis (59,60). In the present study, the expression of ACSL4 was significantly upregulated in the rat HHIBD model, which further demonstrated that ferroptosis may serve an important role in HHIBD and provides a possible intervention target for further study. Additionally, FSP1 acts as an oxidoreductase, restraining the propagation of lipid peroxides and preventing ferroptosis (61). Moreover, the expression of FSP1 was decreased in the brain tissue of rats with hemolytic hyperbilirubinemia in the present study, thereby suggesting excess lipid peroxidation in the rat model.

Another biochemical characteristic of ferroptosis is iron accumulation (62). Iron overload and an increase in lipid peroxidation products have been reported in infants with hemolytic diseases (63). These findings indicate that iron toxicity may serve a vital role in the pathobiology of severe hemolytic diseases. Results from the present study demonstrated that the level of iron was significantly elevated in the brain tissue of rats with HHIBD, which is consistent with the aforementioned studies (8,25). Intracellular iron levels are controlled by highly regulated mechanisms involving proteins, such as the iron importer TFRC, iron storage protein ferritin and FPN1 (64,65). IRP2 is the main regulator of iron metabolism and maintains intracellular iron homeostasis (66). When intracellular iron is insufficient, IRP2 can promote iron transport into cells by inhibiting the degradation of TFRC (67). TFRC is considered a positive regulator of ferroptosis, as it promotes iron intake (68,69). In the present study, it was demonstrated that iron overload caused by hemolytic hyperbilirubinemia leads to ferroptosis and serves an important role in HHIBD, based on the upregulated expression of IRP2 and TFRC in combination with the increased total iron content.

Ferritin consists of FTH and light chain subunits and is considered to be the pivotal protein for storing and dislodging excess iron to reduce cell damage and stress (64). Autophagic degradation of ferritin, mediated by lysosomes, has been reported to increase intracellular Fe2+ levels and promote ferroptosis (70). However, it has been reported that ferroptosis induced by iron overload is accompanied by the upregulation of FTH expression under certain pathological conditions, such as traumatic brain injury and cerebral ischemia/reperfusion injury (71). Moreover, the present study demonstrated that the level of FTH significantly increased in the PHZ group, whilst this change was restored in the DFO + PHZ group. In summary, FTH may serve a key role in ferroptosis in HHIBD, which requires further exploration.

To further evaluate the role of ferroptosis in the neurotoxicity of iron overload, a FAC model was established in vitro. FAC, which has been widely used to induce iron overload, has high solubility and the ability to convert into ferrous ion (72). Typical mitochondrial features of ferroptosis include shrinking, increased membrane density and reduced or absent cristae (73,74). In the present study, ferroptosis-specific mitochondrial morphological changes in PC12 cells, induced by FAC, were demonstrated using TEM. In the FAC-induced PC12 cell damage model, Fer-1 and DFO pretreatment antagonized the increase in lipid peroxides, demonstrated using confocal laser scanning microscopy. These findings further demonstrated that iron overload could lead to neuronal damage via ferroptosis.

The present study has certain limitations. Firstly, histological analysis was not performed to visually detect the tissue damage caused by HHIBD. Secondly, recent studies have pointed out that activation of inflammation, including the activation of multiple inflammation-related signaling pathways, can lead to ferroptosis (75,76). However, plasma TNF-α, IFN-γ, IL-2 or IL-10 levels were not assessed in the animal model. In future studies, the role of inflammatory reactions in hyperbilirubinemia-induced brain damage and the interplay between inflammation and ferroptosis should be evaluated. However, higher levels of iron and MDA and lower levels of GSH and SOD activity were measured in the brain tissues of the PHZ group compared with those in the control group. In addition, HHIBD resulted in increases in the expression of the ferroptosis-related proteins ACSL4, FTH, TFRC and DMT1 and a reduction in the expression of FSP1. KEGG pathway enrichment analysis demonstrated that the differentially expressed rat brain proteins between the control and PHZ groups were significantly enriched in ferroptosis, GSH metabolism and fatty acid biosynthesis pathways. Pretreatment with DFO alleviated redox imbalance and lipid peroxidation-mediated HHIBD. PC12 cells treated with FAC showed shrinking mitochondria, high mitochondrial membrane density, and increased lipid reactive oxygen species and intracellular ferrous iron, which were antagonized by pretreatment with ferrostatin-1 or DFO.

To the best of our knowledge, this is the first study to elucidate the role of ferroptosis in HHIBD in vivo. The present study demonstrated the vital role of ferroptosis in HHIBD, and iron overload was demonstrated to trigger ferroptosis potentially via the activation of lipid peroxidation in the brain. These findings demonstrate that ferroptosis is involved in HHIBD and shed light on the potential underlying molecular mechanisms of HHIBD. Furthermore, the present study provides new insights into candidate proteins that are potentially involved in ferroptosis in the brain during hemolytic hyperbilirubinemia.

Supplementary Material

Supporting Data

Acknowledgements

The authors would like to thank Professor Junjin Lin and Professor Linying Zhou (Public Technology Service Center, Fujian Medical University, Fuzhou, China) and Dr Liping Zhu, Dr Xudong Zhuang and Dr Xinrui Wang (NHC Key Laboratory of Technical Evaluation of Fertility Regulation for Non-Human Primate, Fujian Maternity and Child Health Hospital, Fuzhou, China) for their technical assistance.

Funding

The present study was supported by the Natural Science Foundation of Fujian Province (grant no. 2020J01327), the Fujian Provincial Health Technology Project (grant no. 2020GGB017), the Joint Funds for the Innovation of Science and Technology, Fujian Province (grant no. 2020Y9143), the Open Project of the Key Laboratory of Environment and Health of Fujian Medical University (grant no. GWGXZD-202002) and Fujian Maternity and Child Health Hospital (grant no. YCXM 20-08).

Availability of data and materials

Mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the iProX partner repository with the dataset identifier PXD041604. Other datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Authors' contributions

HL and JZ confirm the authenticity of all the raw data. HL and LX conceived and designed the study. JZ, XL, SL, GL, JT, JL, CZ and SW performed the experiments. JZ and XL collected and analyzed the experimental data. JZ and XL prepared the first version of the manuscript. XL, SL, GL, JT, JL, CZ, SW and LX provided critical comments on the revision of the manuscript. HL and LX revised and finalized the manuscript. All authors have read and approved the final manuscript.

Ethics approval and consent to participate

The present study was approved by the Institutional Animal Care and Use Committee of Fujian Maternity and Child Health Hospital (approval no. 2020KY093; Fuzhou, China).

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References