Hyperbilirubinemia, one of the most common clinical neonatal diseases, is characterized by the accumulation of bilirubin (a waste product of heme metabolism) in the bloodstream, which can be further classified as conjugated or unconjugated hyperbilirubinemia (UHB) [1]. In particular, UHB affects approximately 85 % of newborns within the postnatal life. Moreover, severe cases of UHB may further develop into hyperbilirubinemia encephalopathy, which is one of the principal causes of neonatal mortality and handicap in middle- and low-income countries [2,3]. A multicenter epidemiological study showed that cases of neonatal bilirubin encephalopathy reached 4.8 % of the total number of admitted children, with a mortality rate of 16.1 % [4]. Additionally, 30 % of newborns with bilirubin encephalopathy often experience irreversible brain damage [5], and develop nerve sequelae, including epilepsy [6], autism [7,8], and attention deficit hyperactivity disorder [9]. Hence, it is of great importance to improve our knowledge of the molecular pathogenesis of bilirubin-induced brain damage.

Ferroptosis, a recently discovered cell death, has been implicated in various pathological conditions [10]. Iron-dependent lipid peroxidation, iron load, and mitochondrial damage are the dominant characteristics of ferroptosis [11]. Ferroptosis is involved in brain damage in acute central nervous system diseases, including cerebral hemorrhage [12,13], traumatic brain injury [14,15], and ischemic stroke [16,17]. Brain trauma and hemorrhage increase the release of hemoglobin (an oxidized form of heme rich in iron), iron overload, and abnormal increases in lipid reactive oxygen species (ROS), which induce ferroptosis. Population-based studies revealed an augment of iron overload and lipid peroxidative products in newborns with hemolytic diseases, including hyperbilirubinemia [18,19]. Previously published studies have proposed that high levels of bilirubin increase oxidative stress, lipid peroxidation and reduce glutathione (GSH) content in nerve cells [20,21]. These biochemical characteristics of hyperbilirubinemia are markedly similar to those of ferroptosis. However, the crosstalk between bilirubin-induced brain damage and ferroptosis is yet to be uncovered, and the precise mechanisms involved in this crosstalk require urgent exploration.

N6-methyladenosine (m6A) methylation, a predominant modification of eukaryotic messenger RNAs (mRNAs), is the most crucial post-transcriptional mechanism. The formation of m6A is spatially balanced and controlled by methyltransferases, demethylases, and m6A binding proteins. Methyltransferase proteins include Wilms tumor 1-associating protein, methyltransferase-like 3 (METTL3), and methyltransferase-like 14. On the contrary, fat mass and obesity (FTO)-associated protein and alkB homolog 5 (ALKBH5), RNA demethylases, can remove m6A modifications. Moreover, m6A binding proteins consist of heterogeneous nuclear ribonucleoprotein A2/B1, YTH-domain-containing protein 1/2 (YTHDC1/2), YTH domain family 1/2/3 that recognize the m6A motif that affects RNA metabolism [22]. The m6A modification regulates ferroptosis in various disorders. Inhibition of METTL3 reduces ferroptosis suppressor protein 1 (FSP1) expression by m6A modification, thereby inhibiting ferroptosis induction and promoting tumor growth [23]. ALKBH5-mediated m6A modification restrain solute carrier family 7-member 11 (SLC7A11) to promote ferroptosis in colorectal cancer cells [24]. Additionally, the YTHDC2 protein binds to the m6A motif of SLC7A11 mRNA to promote its degradation, thereby decreasing antioxidant GSH synthesis and lipid peroxidation and inducing ferroptosis [25]. In contrast, YTHDC2 recognizes the 3′ UTR m6A site of the transcription factor HOXA13, decreasing SLC3A2 expression and promoting ferroptosis [26]. Given the critical function of m6A in the modulation of RNA metabolism and its dynamic properties, we hypothesized that m6A modification modulates ferroptosis during the pathogenesis of hyperbilirubinemia encephalopathy. Therefore, revealing the post-transcriptional mechanism of m6A-regulated ferroptosis may provide novel clinical therapeutic targets of hyperbilirubinemia.

Herein, in our study, we aimed to explore the molecular mechanism of m6A modification in ferroptosis in hyperbilirubinemia-induced oxidative damage. Our data suggested that m6A modification has crucial function in ferroptosis regulation, which may be identified as the critical post-transcriptional mechanism in the pathogenies of hyperbilirubinemia. These findings could open avenues for the effective prevention and clinical treatment of bilirubin-induced brain damage.