The liver is one of the vital organs in the body and plays a vital role in the detoxification of foreign substances, secretion of bile for digestion, metabolic functions of various nutrients, and regulation of iron metabolism. The liver is not only the major iron storage site but also where the iron regulation hormone hepcidin is synthesized (Nicolas et al., 2001; Sikorska et al., 2016). Systemic and cellular iron homeostasis is sustained through several main iron-related proteins, including iron importer, exporter and storage proteins. Iron is taken up into cells via transferrin receptor 1 (TfR1) and sequestered by ferritin light chain (Ft-L) (Hentze et al., 2010) or exported out of cells via ferroportin (Fpn). Iron homeostasis is controlled through regulation of duodenal iron absorption, macrophage iron release and hepatocyte iron storage, which is mainly carried out by the iron-related proteins above (Hentze et al., 2010; Ganz 2011).

Acute liver injury (ALI) often develops rapidly and may involve drug-induced liver failure or cholestasis. Animals that develop ALI show enhanced generation of hepatic ROS and enhanced lipid peroxidation with the formation of lipid peroxides. ALI may be caused by viruses, drugs or toxins. Among the various toxins, a thiono-sulfur-containing compound, thioacetamide (TAA), has been used extensively in the development of animal models of ALI (Chu et al., 2001; Bruck et al., 2007) because it induces lipid peroxidation, oxidative stress, and inflammation, ultimately causing functional hepatocyte death and liver dysfunction (Muller et al., 1991; Grek and Arasi 2016; Thawley 2017).

Ferroptosis, which is a recently identified novel form of regulated cell death, proceeds differently from apoptosis, necrosis, and autophagic cell death (Dixon et al., 2012). It is mediated by the iron-dependent oxidative degeneration of lipids and leads to mitochondrial shrinkage, with increased mitochondrial membrane density and outer mitochondrial membrane rupture (Xie et al., 2016). It has been reported that excess cellular iron is the main driver of the Fenton reaction and the production of reactive oxygen species (ROS) (He et al., 2020). Moreover, ferroptosis was demonstrated to be induced by phospholipid peroxidation associated with free iron-mediated Fenton reactions (Hadian and Stockwell 2020). Thus, we hypothesized that TAA-induced ALI is mediated by excess iron, causing substantial oxidative stress/lipid peroxidation and further inducing ferroptosis in hepatocytes, which finally results in liver injury and dysfunction. In this study, iron and ferroptosis were identified as the cornerstones for detecting the mechanism of ALI. TAA was administered intraperitoneally to induce the ALI model in mice. We found that iron content and mobilization were significantly enhanced, as well as liver dysfunction, while protein expression of the cystine/glutamate antiporter SLC7A11 (xCT) and glutathione peroxidase 4 (GPX4) in the liver was decreased in the TAA-induced ALI model. However, both deferoxamine (DFO) and ferrostatin-1 (Fer-1) suppressed the liver dysfunction induced by TAA by reducing iron accumulation in the liver but did not affect xCT or GPX4 expression. Our data suggest that decreasing the iron content in the liver may be a potential therapeutic strategy for ALI.

TAA induced liver dysfunction, suppressed anti-ferroptosis-related protein expression and enhanced iron-related protein expression in the liver.

Injection of TAA induced prominent inflammation and vacuolar degeneration in the liver within 3 days compared with the control group (Figure 1A). TAA induced obvious liver dysfunction, which was reflected by increased plasma ALT and AST levels in the TAA model (Figures 1B,C, p < 0.001). In addition, TAA significantly reduced anti-ferroptosis-related protein (xCT) expression but not GPX4 expression compared to the control (Figures 1D–F). In addition, TAA induced a significant increase in iron-related proteins, including TfR1, Fpn and Ft-L expression, compared to the control (Figures 1G–J, p < 0.05 or p < 0.01).

Both Fer-1 and DFO suppressed TAA-induced liver dysfunction but did not affect anti-ferroptosis-related protein expression in the liver.

In comparison with the control, injection of TAA induced a profound increase in plasma levels of ALT and AST concomitant with a marked increase in the plasma LDH level. Pre-administration of Fer-1 for 3 days significantly decreased TAA-induced alterations in the plasma ALT, AST and LDH levels compared with the TAA group (Figures 2A–C, p < 0.001). TAA significantly reduced xCT but not GPX4 expression in the livers of mice. However, pre-administration of Fer-1 did not affect xCT or GPX4 expression in the liver compared to TAA-treated mice (Figures 2D–F).

Administration of DFO for 3 days with TAA treatment significantly attenuated the TAA-induced increase in plasma ALT, AST and LDH (Figures 3A–C, p < 0.001). In addition, administration of DFO did not alter xCT or GPX4 expression induced by TAA in the liver compared to TAA-treated mice (Figures 3D–F).

TAA is a potent toxicant that causes oxidative stress and further induces liver damage, which is highly similar to human acute liver damage (Zargar et al., 2019). The TAA-induced liver injury model has been widely studied for its biochemical and histological effects in animals and is usually used to establish liver injury models (Rahman and Hodgson 2003). However, administration of TAA to mice causes either chronic/acute liver failure or cirrhosis depending on the dose and duration of TAA exposure (Shapiro et al., 2006). In previous studies, high-dosage TAA is known to induce lipid peroxidation and oxidative stress, and the pathological effects are mainly limited to ALI rather than causing direct damage to other organs (Muller et al., 1991; Mladenovic et al., 2012). Thus, according to previous studies (França et al., 2019; Han et al., 2019; Liu et al., 2021), we administered 250 mg/kg/day TAA to mice via intraperitoneal injection for 3 days to establish the ALI model in our present study.

Although TAA-induced ALI was primarily provoked by oxidative stress (Zhan et al., 2019), whether it is partially an iron-dependent process is still uncertain. To investigate whether iron disorder is involved in liver dysfunction, we evaluated liver function and liver iron content in TAA-induced ALI mice. Because the liver is the major extraerythrocyte storage organ for iron and most of the iron within the liver is stored in Ft-L, which is often used to indicate iron levels (Ishizaka et al., 2005; Galaris and Pantopoulos 2008). TAA not only significantly induced iron mobilization, which was reflected as increased transferrin receptor 1 (TfR1), but also increased iron storage, which was reflected as increased ferritin-L (Ft-L) expression in the liver. These results were consistent with those of other studies suggesting that TAA induces iron accumulation in the liver of ALI mice (Ackerman et al., 2015). However, we also noticed that TAA induced the increase of ferroportin (Fpn) expression in the liver, which usually facilitate iron efflux to lower tissue iron. Actually, iron is tightly controlled at cell and systemic levels to prevent both deficiency and overload under physiological conditions. Iron regulatory proteins post-transcriptionally control genes encoding proteins that modulate iron transport, use and storage and are themselves regulated by iron (Gao et al., 2019). Thus, the elevated Fpn may represent an endogenous effort to maintain iron homoeostasis (Anderson and Frazer 2017). It is also understandable that both Fer-1 and DFO decreased iron content, while reducing Fpn in the liver after TAA exposure. Intracellular iron can be stored in the protein shell of ferritin (Ft) as a crystalline core of ferric (Fe³⁺) ions, and the iron must first be released from the core to catalyze oxidative reactions (Skaper 2019). Thus, Ft is able to restrict the availability of iron to participate in redox active iron species in the cytosol. However, more ferritin subunits are synthesized using translational and transcriptional mechanisms in response to increased cell iron (Badu-Boateng and Naftalin 2019). Increased intracellular ferritin may occur in response to oxidative stress provoked by accumulated iron, which could be result of various stressors such as UV light, increased temperature, and inflammation (Vile and Tyrrell 1993; Tisma et al., 2009). Consistent with previous findings, we found that TAA significantly induced iron increase in the liver and also promoted Ft-L expression.

Although ferroptosis was reported to be involved in the pathogenesis of many injury models (Skouta et al., 2014; Liu et al., 2020), it is still unclear whether ferroptosis is involved in the TAA-induced ALI model. In addition, iron accumulation is considered to be the main cause of ferroptosis initiation (Li et al., 2020). This prompted us to wonder whether TAA induces iron accumulation and ultimately initiates ferroptosis in the liver. The synthetic antioxidant Fer-1 was reported to act as a ferroptosis inhibitor via a reductive mechanism to prevent damage to membrane lipids, thereby inhibiting ferroptosis (Dixon et al., 2012). The iron chelator DFO is widely used to reduce iron in tissue after injection or oral administration (Buss et al., 2003). In the present study, we introduced Fer-1 and DFO to investigate whether ferroptosis occurred because these agents can decrease iron content to achieve an anti-ferroptotic effect in the TAA-induced ALI model. We found that both Fer-1 and DFO rescued liver dysfunction and inhibited iron content and accumulation in the liver. However, it was previously reported that Fer-1 can attenuate oxidative, iron-dependent cancer cell death by blocking cystine import and glutathione production (Skouta et al., 2014). Our data demonstrated that Fer-1 and DFO did not play anti-ferroptotic roles by affecting cystine import or glutathione production in a TAA-induced ALI model. This result may suggest that Fer-1 plays an anti-ferroptotic role similar to DFO in the liver in the ALI model and that the mechanism involves chelation of hepatic iron. Moreover, a recent study demonstrated that Fer-1, in the presence of ferrous iron, produces the most relevant anti-ferroptotic effect by forming a complex with iron (Miotto et al., 2020).

Ferroptosis is a multi-step regulated cell death that is characterized by excessive iron accumulation and lipid peroxidation (Yu et al., 2020). Lipid peroxide accumulation is mainly through the xCT and GPX4-dependent mechanisms (Kim et al., 2021). The process of ferroptosis is also dependent on intracellular iron because the accumulation of iron acts as a catalyst for converting peroxides into free radicals (Kim et al., 2021). Oxidative degradation of lipids occurs when there is depletion of the antioxidant glutathione and a loss of activity of the lipid repair enzyme GPX4. Lipid peroxidation then leads to cell membrane denaturation (Cao and Dixon 2016). The results were also consistent with other studies, which confirmed that ferroptosis can be induced by a loss of activity of xCT and suppression of GPX4, followed by accumulation of lipid reactive oxygen species (ROS) (Ma et al., 2008; Yang and Stockwell 2016; Latunde-Dada 2017). However, both anti-ferroptotic proteins xCT and GPX4 were not modulated by Fer-1 investigated. It was concluded that the outcome of TAA-induced acute liver injury may depend on the level of hepatic iron concentration and iron overload may exacerbate the injury. However, it’s not entirely clear that whether Fer-1 or DFO block iron mobilisation into the liver instead. Thus, more study is necessary to investigate their effects on the expression of molecules that play key roles in iron transport, use and storage.

In addition, the study revealed HID-fed mice had developed a significant iron increase. In addition, HID did not increase the protein expression of TfR1 and Fpn in the same magnitude of TAA. Indeed, HID even downregulated TfR1 compared to NID. Similar with previous research (Weiss et al., 2018), the decreased TfR1 after HID may also represent endogenous feedback to maintain the iron balance. Unlike TfR1 level, Ft-L expression was significantly enhanced by HDI in the liver, which directly indicated increased iron levels. We further found that HID did not increase the levels of hepatic damage biomarkers (ALT, AST and LDH), and this diet was not inducing the same acute liver damage as TAA. However, previous study has revealed that an increase in hepatic iron concentration might exacerbate TAA-induced live injury (Ackerman et al., 2015). We confirmed that TAA aggravated the damage to the liver with iron overload. We further revealed that Fer-1 still had an anti-ferroptotic effect on TAA-induced liver ferroptosis by reducing the iron content in high iron-fed ALI mice. All these results provide new evidence of the anti-ferroptotic effect of Fer-1 in the TAA-induced ALI model, the mechanism of which is mediated by the ability of Fer-1 to reduce iron content and iron accumulation in the liver (Figure 7).

Taken together, these findings indicate a role of TAA-induced iron deposition and ferroptosis in the pathogenesis of ALI model. Ferroptosis inhibitor Fer-1 plays a role in rescuing liver injury and dysfunction by reducing liver iron levels and inhibiting TAA-induced ferroptosis in the liver. Similar to DFO, Fer-1 might be a useful reagent to reverse liver dysfunction under iron overload conditions.