Overview of ferroptosis and pyroptosis in acute liver failure

Abstract

In this editorial, we comment on the article by Zhou et al published in a recent issue. We specifically focus on the crucial roles of ferroptosis and pyroptosis in acute liver failure (ALF), a disease with high mortality rates. Ferroptosis is the result of increased intracellular reactive oxygen species due to iron accumulation, glutathione (GSH) depletion, and decreased GSH peroxidase 4 activity, while pyroptosis is a procedural cell death mediated by gasdermin D which initiates a sustained inflammatory process. In this review, we describe the characteristics of ferroptosis and pyroptosis, and discuss the involvement of the two cell death modes in the onset and development of ALF. Furthermore, we summarize several interfering methods from the perspective of ferroptosis and pyroptosis for the alleviation of ALF. These observations might provide new targets and a theoretical basis for the treatment of ALF, which are also crucial for improving the prognosis of patients with ALF.

Key Words: Acute liver failure; Ferroptosis; Pyroptosis; Glutathione peroxidase 4; Gasdermin D

Core Tip: In this review we describe the characteristics of ferroptosis and pyroptosis, and discuss the involvement of the two cell death modes in the onset and development of acute liver failure (ALF). Furthermore, we summarize several interfering methods from the perspective of ferroptosis and pyroptosis for the alleviation of ALF. These observations might provide new targets and a theoretical basis for the treatment of ALF, which is also crucial for improving the prognosis of patients with ALF.

INTRODUCTION

Acute liver failure (ALF) is a severe clinical syndrome characterized by massive hepatocyte necrosis and acute liver damage in a short period of time with a mortality rate as high as 30%[1]. When ALF occurs, liver function declines sharply, which fails to fulfill the organ’s basic physiological actions. Clinically, the main manifestations of ALF are jaundice, ascites, and hepatic encephalopathy[2]. The common causes of ALF include pharmacological liver injury, hepatitis virus, ischemia, and autoimmunity, which account for nearly 80% of the ALF cases[3]. For example, acetaminophen overdose triggers the formation of NOD-like receptor protein 3 (NLRP3) inflammasome, resulting in pyroptosis and ultimately leading to ALF[4]. In addition, acute hepatitis B (AHB) virus infection is the main cause of ALF in many countries[5]. A recent study indicated that 6.6% of patients with AHB developed ALF[6].

Previous studies have established that the primary mode of cell death involved in ALF is apoptosis[7]. As a form of cell-autonomous programmed death, apoptosis triggers a series of reactions by activating the caspase family of proteins, leading to intracellular protein degradation and the elimination of damaged or no longer needed cells[8]. Rutherford et al[9] demonstrated that apoptosis was markedly activated in ALF, with notable elevations of apoptosis markers such as tumor necrosis factor-alpha, hepatocyte growth factor (HGF), interleukin-6, and receptor interacting protein kinase 3 (RIPK3). Notably, the elevated levels of HGF and RIPK3 could serve as a confirmatory diagnostic marker and a poorer clinical prognosis for patients suffering from ALF, respectively[10].

Studies over the past 10 years have garnered widespread attention related to two emerging modes of cell death, ferroptosis and pyroptosis, in ALF pathogenesis. Ferroptosis is a form of cell death that relies on iron ions (Fe²⁺), characterized by the peroxidation of polyunsaturated fatty acids on the cell membrane[11]. Fe²⁺ promotes the generation of lipid peroxides, leading to excessive reactive oxygen species (ROS) production, which further disrupts the integrity of the cell membrane and triggers cell death[12,13]. It is worth noting that GSH peroxidase 4 (GPX4) functions as a crucial enzyme in the ferroptosis pathway, which catalyzes the decomposition of lipid peroxides. When GPX4 activity is inhibited, lipid peroxides accumulate in the cell membrane, ultimately culminating in the onset of ferroptosis[14]. Pyroptosis is a form of cell death with inflammatory characteristics, typically manifested by the formation of numerous small vesicular structures known as pyroptotic bodies[15]. Pyroptotic bodies mediate the cleavage of gasdermin D (GSDMD) by caspase proteins to release its N-terminal fragment. The N-terminus of GSDMD is the main effector of pyroptosis, which is released and inserted into the cell membrane to form holes, leading to rupture of the cell membrane and the release of cell contents, ultimately triggering the onset of pyroptosis[16,17]. These released substances can activate immune cells and trigger severe inflammatory responses[18]. Additionally, the released proinflammatory mediators may further initiate a sustained inflammatory process and create a vicious cycle[19]. The intense inflammatory response may aggravate liver tissue damage and promote the further development of ALF.

Ferroptosis and pyroptosis represent two different modes of cell death, with an increasing body of evidence indicating a potential correlation between them. The release of damage-associated molecular patterns (DAMPs) from plasma membrane pores may be a common feature of ferroptosis, pyroptosis, and necroptosis[20]. Furthermore, the released DAMPs triggered by ferroptosis may promote pyroptosis and necroptosis. GPX4 is a key enzyme in ferroptosis and has also been demonstrated to be implicated in the negative modulation of pyroptosis[21]. Interestingly, GPX4 has been observed to induce a range of other forms of cell death, including apoptosis, autophagy, necroptosis, and pyroptosis[22]. This suggests the possibility of a complex interconnection between ferroptosis and other forms of programmed cell death. It has been demonstrated that iron-activated ROS can induce pyroptosis through the Tom²0-Bax-caspase-GSDME pathway, and GPX4 can inhibit the activity of caspase-1, thereby preventing the process of pyroptosis[23]. Additionally, GPX4 functions as a regulator of NOD-, LRR-, and NLRP3-mediated pyroptosis in kidney injury[24]. The interaction between pyroptosis and ferroptosis in epilepsy may be associated with toll-like receptor 4 (TLR4)-mediated neuroinflammation[25]. Ferroptosis has been demonstrated to induce neuroinflammation and the release of a considerable number of inflammatory factors. Among these factors, IL-1β can activate the HMGB1/TLR4 signaling pathway, thereby increasing the production of NLRP3 and IL-1β. This process serves to amplify the inflammatory response by inducing pyroptosis.

In the article by Zhou et al[26] entitled ‘Silent information regulator sirtuin 1 ameliorates acute liver failure (ALF) via the p53/glutathione peroxidase 4/gasdermin D axis’, the authors demonstrated that ferroptosis and pyroptosis are key modes of hepatocyte death in the progression of ALF. Therefore, it is crucial to conduct a comprehensive analysis of the regulatory roles of ferroptosis and pyroptosis in ALF. This will not only enhance our understanding of the pathological process of liver failure but also potentially provide theoretical support for the development of new treatment strategies.

MECHANISMS OF FERROPTOSIS AND PYROPTOSIS IN ALF

The mechanisms underlying the occurrence and development of ALF have been described in previous publications[3,10,11,27]. Here, we mainly focus on the roles of ferroptosis and pyroptosis in ALF.

The involvement of ferroptosis in ALF

Ferroptosis has been shown to play important roles in several pathological processes associated with liver diseases[12,14], including ALF[28], which is regulated by lipid peroxidation and iron accumulation[29]. The key process in ferroptosis is the Fenton reaction, in which Fe²⁺ converts lipid peroxides to ROS[30]. As a key regulator of ferroptosis, GPX4 is the only GSH peroxidase isoform capable of regulating lipid peroxidation and ROS levels[31], which could catalyze the decomposition of toxic peroxides depending on the conversion of GSH to oxidized GSH[32]. Additionally, the glutamate/cystine antitransport system (System Xc) is another important regulator of ferroptosis, the inhibition of which would promote the ferroptosis process[33]. It has been indicated that p53 could induce ferroptosis by inhibiting System Xc activity and reducing cystine entry into the cell[34]. Moreover, solute carrier family 7 member 11 (SLC7A11) has been proved to be a crucial upstream regulator of ferroptosis[35], the down-regulation of which inhibits the cysteine metabolic pathway, leading to a reduction in intracellular cystine levels and an indirect inhibition of GPX4 activity[36]. Jiang et al[34] found that p53-mediated transcriptional repression of SLC7A11 is critical for ROS-induced ferroptosis. Another study revealed that G3BP stress granule assembly factor 1 (G3BP1) inhibits the entry of p53 protein into the nucleus and reduces SLC7A11 transcription and hepatocyte ferroptosis during ALF[37]. Also, hepatitis B virus X protein exacerbates ALF by promoting iron-mediated cell death through EZH2/H3K27me3-mediated inhibition of SLC7A11[38]. In addition, nuclear factor erythroid 2-related factor 2 (Nrf2) is also an important regulator of ferroptosis[39], and its activation inhibits H₂O₂-induced cellular ferroptosis by modulating the GPX4 pathway[14]. Collectively, these findings suggest that ferroptosis plays a significant role in the development of ALF.

The involvement of pyroptosis in ALF

Pyroptosis, another cell death mode, also triggers significant hepatocyte death in liver failure[40]. In the pathogenesis of ALF, GSDMD can be cleaved by caspase-1 into inflammatory GSDMD-N in the hepatocyte cytoplasm[41]. GSDMD-N recognizes phospholipids and inserts into the cell membrane, facilitating membrane pore formation and pyroptotic cell death[42]. Additionally, GSDMD-N up-regulates the release of inflammatory mediators from macrophages recruited by monocyte chemotactic protein 1 and its receptor CC chemokine receptor-2[43]. This leads to an amplified inflammatory response, which in turn exacerbates ALF. NLRP3 could stimulate pyroptosis by inducing caspase-1 and GSDMD activation[18]. Furthermore, HMGB1 induces the formation of NLRP3 inflammasome by activating the TLR4/MyD88/NF-κB signaling pathway, thereby causing pyroptosis[44]. Studies have shown that IL-10 inhibits NLRP3 expression[45], suggesting a potential role for IL-10 in alleviating ALF. CD38, a type II transmembrane protein, is an important NAD-dependent enzyme, and it has been shown that CD38 stimulated TLR4-NLRP3-GSDMD-mediated pyroptosis and aggravated liver injury[46]. The occurrence of pyroptosis is influenced by multiple factors, which in turn impacts the progression of ALF, suggesting that the inhibition of pyroptosis could potentially alleviate ALF.

The regulation of deacetylases on ferroptosis and pyroptosis in ALF

In the study by Zhou et al[26], the authors suggested that SIRT1 activation reduced hepatic injury and inflammatory responses by reducing the p53 acetylation level and further suppressing the expression of ferroptosis and pyroptosis-related proteins GPX4 and GSDMD. SIRT1 is one of the important deacetylases, which could remove acetyl groups from histones and non-histone proteins. As protein acetylation is a crucial post-translational modification, we here review the regulation of deacetylases on ferroptosis and pyroptosis, and further on ALF.

SIRT1, which regulates the onset and progression of ALF by affecting ferroptosis and pyroptosis pathways[47] has received significant attention in recent years. The inhibitory effect of SIRT1-mediated deacetylation on ferroptosis is exerted through two major pathways: The deacetylation of p53 to suppress p53-dependent downregulation of SLC7A11[48], and the other pathway is the deacetylation of Nrf2 to promote Nrf2-mediated upregulation of GPX4[49]. In addition, SIRT1 could reduce the activation of NLRP3 inflammasome induced by IL-1β, thereby inhibiting pyroptosis[50]. Besides SIRT1, other deacetylases, such as HDAC6 and HDAC2 also have crucial actions in the regulation of ferroptosis and pyroptosis. For instance, studies have demonstrated that inhibition of HDAC6 expression reduced the expression of NLRP3 inflammatory vesicles and NF-κB, thereby affecting pyroptosis and ferroptosis in ALF, respectively[51,52]. Furthermore, downregulation of HDAC2 alleviates cell pyroptosis in ALF by regulating the acetylation level of the ULK1 K68 site[53].

In summary, it can be concluded that deacetylases could alleviate ALF by inhibiting both the ferroptosis and pyroptosis process. These results may provide new therapeutic targets for alleviating ALF.

CONCLUSION

ALF is a severe liver disease in clinical practice, which is accompanied by the rapid death of massive hepatocytes and multiple inflammatory outbreaks. Ferroptosis and pyroptosis are important cell death modes in ALF. Ferroptosis is the result of increased intracellular ROS due to iron accumulation, GSH depletion, and decreased GPX4 activity[54]. Pyroptosis is a procedural cell death mediated by the gasdermin family, which is dependent on the cleavage of GSDMD to the active GSDMD-N fragment by the NLRP3-induced caspases, and further triggering cell membrane perforation and an inflammatory storm[42]. Therefore, suppressing the ferroptosis and pyroptosis process has great potential in the treatment of ALF.

To date, some agents with iron chewing or GPX4 regulating effects have shown ferroptosis-inhibitory functions. As iron ion accumulation is an essential prerequisite for ferroptosis, iron chewing agents can be used to reduce the accumulation and deposition of iron in the organ and further mitigate the ferroptosis procedure. Iron chelators such as deferoxamine, deferiprone and deferasirox have been proved to weaken lipid peroxidation by restricting the Fenton reaction, thereby inhibiting ferroptosis and alleviating ALF development[55]. GPX4 is a critical regulator of ferroptosis, thus enhancing the expression or the activity of GPX4 could inhibit this cell death mode. It was indicated that selenium supplementation could increase the level of GPX4 and inhibit ferroptosis as selenium is required for the synthesis of GPX4[56]. Multiple studies have shown that SIRT1 alleviates the inhibitory effects of p53 on GPX4 and SLC7A11 by reducing the level of p53 acetylation, which is a potential target for the treatment of ferroptosis-related diseases[57,58].

To date, a significant number of inhibitors and drugs targeting pyroptosis-related proteins have shown therapeutic potential in preclinical studies. As the activation of NLRP3 serves as the initiating point of pyroptosis, NLRP3-specific inhibitors have emerged as promising therapeutic targets for the treatment of pyroptosis, with MCC950 and the natural compound kaempferol currently undergoing clinical trials[59]. A study also pointed out that metformin, a "magic drug for diabetes treatment", could reduce inflammatory responses and cellular pyroptosis by inhibiting the NF-κB-NLRP3 pathway[60]. In addition, caspase inhibitors such as Vx765 and Z-YVAD-FMK block the binding of caspases to GSDMD and further inhibit pyroptosis[61]. Research has shown that the tricarboxylic acid cycle intermediate fumarate acts as a pyroptosis inhibitor by succinylating GSDMD to prevent its binding to caspases[62].

Although several candidates could be selected to regulate the critical steps in ferroptosis and pyroptosis, we still face many problems in clinics. For example, many existing inhibitors may interfere with normal metabolism pathways due to their nonspecific activity. The poor absorption rate of some drugs is another problem which needs to be resolved. With advances in science and technology, the use of big data and artificial intelligence methods to screen existing FDA-approved drugs may identify new ferroptosis and pyroptosis modulators. Existing studies have revealed that there is crosstalk between ferroptosis and pyroptosis through complex feedbacks, and the specific mechanism of the two cell death modes is still largely unknown, and the in-depth study of these pathways may provide targets and a theoretical basis for the development of ferroptosis and pyroptosis-targeted drugs.