In this study, we identified the hub genes related with ferroptosis and explored the potential pathogenesis of ferroptosis in ACLF. 35 DEGs were screened from the cross set of GSE139602 and FerrDb, including 16 down-regulated genes and19 up-regulated genes. GO enrichment suggested that the ferroptosis-related DEGs were mostly distributed in peroxisomal membrane and microbody membrane, and involved in antioxidant activity, and transaminase activity. They mediate processes such as response to nutrient and toxic substance. The GO enrichment circle map demonstrated that ACSL4, NQO1, SESN2, TXNRD1, and CDKN1A were involved in multiple biological processes. These genes likely play a more critical role in ACLF. KEGG enrichment analysis indicated that these genes are mainly involved in biosynthesis of amino acids pathways, peroxisomes, fluid shear stress and atherosclerosis pathways.
Ferroptosis is a new type of cell death characterized by lipid-ROS accumulation due to intracellular iron overload[1]. Glutathione(GSH)/glutathione peroxidase (GPX4) is the classical regulatory pathway of ferroptosis[15]. Increasing evidence has shown that ferroptosis is one of the main triggers for the exacerbation of several liver diseases[5, 16-18]. The liver, the organ of iron recirculation and storage, and synthesis of iron-containing enzyme, plays a vital role in iron metabolism. Iron overload can cause abnormalities in the mitochondrial oxidative phosphorylation pathway in hepatocytes, which can produce large amounts of ROS [19]. When the level of ROS exceeds the clearance level of the body's antioxidant system, it can oxidize unsaturated fatty acids on cell membranes and organelle membranes and directly or indirectly damage the structural function of hepatocytes, contributing to the progression of ferroptosis in liver cells[20].
The biosynthesis of amino acids pathway is crucial in the ferroptosis mechanism[21-25].GSH is synthesized from three key amino acids, cysteine, glutamate, and glycine[26, 27]. Both glycine and cysteine can be produced through the metabolic axis from glucose to serine. As an essential amino acid for cells, serine is required not only for protein biosynthesis, but also for the biosynthesis of numerous intracellular molecules include GSH[28]. Sufficient cysteine contributes to the maintenance of redox homeostasis in cells and protects against oxidative death[18, 29]. Therefore, the metabolites and enzymes that convert serine and cysteine to GSH are essential to inhibit the occurrence of ferroptosis in liver cells.
HRAS, TXNRD1, NQO1, PSAT1, and SQSTM1 were simultaneously identified as top hub genes. The RT- qPCR results suggested that the expression levels of HRAS, TXNRD1, NQO1, and SQSTM1 were obviously lower, while PSAT1 was higher in ACLF group than that of control group,confirming the reliability of bioinformatics analysis. Among them, the gene expression of PSAT1, TXNRD1, and HRAS all affect the amino acid biosynthesis pathway in cells. PSAT1 is precisely a critical regulator in the serine synthesis pathway(SSP). A study found that PSAT1, one of the class-V family of pyridyl aldehyde phosphate-dependent aminotransferase, catalyzes the second step of SSP [30]. Specifically, a significant increase in the expression of key enzymes such as PSAT1 caused elevated levels of serine and its downstream products[31].PSAT1 expression increased the proportion of GSH through SSP in cells, which increased oxidative stress tolerance and thus inhibited the occurrence of ferroptosis[32]. Therefore, PSAT1 gene expression should be decreased, And the suppressed PSAT1 would decrease its enzymatic activity, which would affect SSP to reduce GSH synthesis and eventually cause ferroptosis in cells. However, our results showed that PSAT1 gene expression was highly expressed in the ACLF group, which is inconsistent with previous studies. In this regard, we speculate that the upregulation of PSAT1 may result from negative feedback of the enzyme activity decrease of PSAT1 in ACLF. Simultaneously, TXNRD1 is a critical regulator that may involve cysteine depletion-induced cellular ferroptosis. The TXNRD1 gene encodes thioredoxin reductase 1, a key antioxidant selenoprotease that regulates cellular redox homeostasis[33]. Some reports have suggested that the thioredoxin system regulates the dithiol/disulfide bond balance in proteins through disulfide bond reductase activity, and can defend against oxidative stress[34-36]. A study[37] indicated that TXNRD1 was obviously upregulated in chronic myeloid leukemia cells after cysteine depletion, and its TXNRD1 activity was significantly reduced, suggesting that the expression of the TXNRD1 gene was increased in order to maintain the activity of TXNRD1 enzyme in cells. Subsequently, ferroptosis occurred on the usage of TXNRD1 inhibitor after cysteine depletion. Therefrom, it is reasonable to believe that when the gene expression of TXNRD1 is down-regulated or the activity of TXNRD1 is inhibited, cysteine is depleted, eventually leading to ferroptosis. HRAS, referred to as oncogenic RAS with NRAS and KRAS, is one of the most frequently mutated driver proto-oncogenes in cancer. On the one hand, oncogenic RAS abnormally rewires metabolic pathways leading to ROS generation[38]. And then ROS promotes the accumulation of oxidative by-products, decreasing the threshold for cancer cells to undergo ferroptosis. On the other hand, oncogenic RAS also establishes ROS scavenging mechanisms to inhibit cellular senescence and promote tumor formation[39]. Oncogenic RAS promotes the transport of intracellular glutamate to the outside of the cell by affecting the system xc-transporter, and at the same time takes the extracellular cystine into the cell and converts it into cysteine for the synthesis of GSH[40]. In summary, PSAT1 affects the serine synthesis pathway, TXNRD1 is involved in the cysteine depletion pathway, and HRAS affects the xc-transporter system to influence cysteine and glumate synthesis, all three of which affect the synthesis of GSH, resulting in intracellular redox imbalance and promoting lipid peroxidation to generate ROS, thus causing ferroptosis.
In addition, the present study reveals that SQSTM1 and NQO1 may mediate ferroptosis by targeting the NRF2 pathway. SQSTM1, as known as p62, is an autophagy receptor[41]. Sun et al.[42] provided the first evidence that activation of the p62-Keap1-NRF2 pathway protects hepatocellular carcinoma cells from ferroptosis. Irion metabolism and lipid peroxidation are modified by the NRF2-regulated genes NQO1, FTH1, and HO1, contributing to the inhibition of ferroptosis. Another study[43] also found that in the presence of high SQSTM1 expression, ferroptosis could be prevented by promoting nuclear transfer of NRF2 and increasing heme oxygenase-1 expression. Knocking down SQSTM1 inhibited NRF2 expression and led to growth inhibition with increased ferroptotic events including a reduction in GSH, an increase in lipid ROS, as well as an increase in iron levels. Daiha Shin’s study [44] indicated that cell viability was reduced and cellular lipid ROS levels were increased when SQSTM1 was inhibited; this was reversed by ferroptosis inducer(ferrostatin-1). NQO1 is a protective antioxidant agent and regulates the oxidative stress in the chromatin [45]. Interestingly, the NRF2-NQO1 axis represents a protective mechanism against ROS[46]. Thus, we infer that high SQSTM1 expression may activate the NRF2/ NQO1 signaling pathway through binding to Keap1 against ferroptotic events including lipid ROS production, GSH depletion, iron accumulation, and lipid peroxidation, thus protecting hepatocytes from ferroptosis. (Fig.9).
There are some limitations to our study. We used two different gene databases for screening DEGs, but these data were mainly derived from published literature, and it is unclear whether there are still some genes that have not been discovered or have not been brought to human attention that also play a common role. Currently, the insufficient sample size may lead to bias in the final gene validation results. In this regard, we expect that we can further investigate the mechanisms and specific signaling pathways of ferroptosis in ACLF by collecting data with larger sample size.