Hepatic steatosis is a hallmark of non-alcoholic fatty liver disease, which is currently the most prevalent chronic liver disease globally. This disease is strongly associated with obesity and metabolic syndrome. Most abnormalities of hepatic lipid metabolism are the result of an imbalance in lipid synthesis and metabolism, leading to excessive accumulation of lipids and subsequent hepatocyte dysfunction. NAFLD symptoms include simple steatosis, cirrhosis, non-alcoholic steatohepatitis, and hepatocellular carcinoma[15]. The incidence and mortality rates of NAFLD are increasing annually and shifting toward a younger age group. According to a mathematical model, the epidemic of obesity and diabetes is expected to increase the incidence of NAFLD, and the death rate from associated illnesses will double[16]. Therefore, research on the pathophysiology, relevant biomarkers, initial therapy, and prevention of NAFLD is urgently needed.
Hepatocyte death is a central mechanism of various liver injuries. It involves several cell death mechanisms, such as autophagy, cell scorching, and programmed necrosis, in addition to apoptosis and necrosis[17, 18]. The liver is highly vulnerable to oxidative damage, and most liver disorders are characterized by excessive iron accumulation[19]. NAFLD is closely related to the mechanism of ferroptosis lipid peroxidation, and ferroptosis suppression has been shown to substantially alleviate NAFLD[7]. Therefore, ferroptosis could be an essential target for treating and preventing NAFLD. In this study, we used bioinformatics to analyze the genetic differences in ferroptosis between NAFLD and normal samples. We screened the diagnostic markers among them, explored the molecular pathogenesis of ferroptosis in NAFLD, and validated these genes in an established NAFLD mouse model. Our findings provide valuable clinical references for the prevention and treatment of NAFLD.
In our study, we evaluated the following six DEGs involved in ferroptosis in NAFLD: EGR1, IL6, JUN, SOCS1, ZFP36, and PTGS2. The AUC values of the ROC curves for these genes were higher than 0.8, indicating their reliable accuracy and specificity in distinguishing NAFLD cases from healthy samples. Since human liver samples are difficult to obtain, we induced NAFLD in C57BL/6 mice by feeding them an HFD to further validate the expression of these genes. This model shows similar characteristics to the development of human NAFLD and is widely used for metabolic studies[20]. Our results indicated that the expression of EGR1, IL6, NR4A1, and SOCS1 was significantly lower in the NAFLD mice than in the controls, confirming the predictions made by our analysis.
EGR1 is a transcription factor primarily involved in tissue damage, immune response, and fibrosis[21]. The primary characteristic of EGR1 is its highly conserved, GC-rich nucleotide sequence in the zinc finger structural domain, which is crucial for tissue healing and inflammation[22]. Both EGR1 and ROS are elevated in the ischemia-reperfusion paradigm, and a positive feedback signaling pathway exists between them[23]. This suggests that EGR1 is implicated in oxidative stress-induced damage and that ferroptosis is a critical component of redox imbalance in NAFLD. Hence, the emergence of ferroptosis in NAFLD may be relevant to EGR1. By targeting and suppressing the expression of miR-15a-5p, the knockdown of EGR1 can increase the protein expression of GPX4[24], which is the primary endogenous inhibitor of ferroptosis[25]. This implies that low levels of EGR1 can contribute to the disease by preventing ferroptosis. Our findings revealed that EGR1 is lowly expressed in NAFLD mice, suggesting this may be an organismal defense mechanism. By controlling ferroptosis, an EGR1/GPX4 axis may, in part, regulate the development of NAFLD.SOCS1 belongs to the cytokine signaling repressor proteins family, which acts as negative feedback regulators that block cytokine signaling pathways in cells[26]. It specifically inhibits the JAK/STAT signaling pathway by targeting unphosphorylated JAK and blocking JAK phosphorylation through the kinase inhibitory region of SOCS1[27]. Moreover, the GSVA of SOCS1 confirmed the activation of the JAK/STAT signaling pathway in the low-expression group. SOCS1 is a crucial regulator of ferroptosis in cancer cells, indirectly increasing the sensitivity of cells to lipid oxidation and ferroptosis[28]. Notably, in our model, SOCS1 expression was downregulated, implying that it suppresses ferroptosis and plays a protector role in NAFLD. NR4A1, which controls the expression of adipogenesis-related genes and prevents adipocyte development, is essential for the cellular inflammatory response and glycolipid metabolism[29, 30]. In mice fed a high-fat chow diet, NR4A1 knockdown exacerbates insulin resistance and liver steatosis, whereas NR4A1 overexpression reduces hepatic triglyceride accumulation[31]. A possible mechanism for this could involve modulating the LKB1-AMPK signaling pathway and preventing downstream SREBP1c and FAS from altering the expression of fatty acid key enzyme and transporter protein CD36[32]. NR4A1 regulates tumor ferroptosis[33] and is consequently involved in both ferroptosis and NAFLD. The results of GSVA of ferroptosis gene NR4R1 revealed the activation of the "JAK-STAT signaling pathway" and "NOD-like signaling pathway" in the low-expression group. This finding indicates that NR4A1 is involved in NAFLD development by inhibiting these pathways. Recent studies have demonstrated the role of IL6 in ferroptosis. IL6 causes chondrocyte ferroptosis by inducing cellular oxidative stress and disrupting iron homeostasis[34]. This is because IL6 has isozyme-specific effects on GPX expression and can reduce its transcript concentration[35]. Increased IL6 expression in the presence of hepatic steatosis is associated with the severity of NAFLD as well as insulin resistance in the liver and adipose tissue[36]. However, our analysis revealed a decrease in IL6 expression in NAFLD. This finding suggests that the involvement of IL6 in NAFLD is complex and that the decrease in IL6 levels may be due to the excessive loss caused by ferroptosis in the liver.
The liver contains several innate and adaptive immune cells that play a crucial role in maintaining immune homeostasis[37], and immune dysregulation is implicated in NAFLD development. Thus, we examined the immune infiltration in the livers of NAFLD and healthy individuals using the CIBERSORT algorithm. Our findings showed considerably higher levels of M1 and M2 macrophages, resting dendritic cells, and resting mast cells in the livers of NAFLD patients than in those of normal individuals. Among these, M2 macrophages and γδ T cells were the most prevalent in the liver tissue, indicating their significance in the immunological microenvironment of NAFLD.
Simple steatosis leads to macrophage infiltration in the liver, and the number of macrophages increases with the severity of steatosis and inflammation in the liver[38]. Macrophage activation and polarization affect the progression of NAFLD[39, 40]. During the repair phase of NASH, hepatic macrophages polarize toward an inflammatory and pro-fibrotic M2 phenotype and release high levels of IL-12 and TGF-1 to promote fibrosis[41]. This observation indicates that although dysfunctional immune cells may exacerbate liver damage, an early inflammatory response could aid in the recovery and repair of liver tissues[42]. IL-4- and IL-13-stimulated macrophages are suggested to polarize toward the M2 type and promote arginase activity, leading to apoptosis of M1 macrophages and accelerating tissue damage repair and remodeling. However, in our study, hepatic M1 macrophages were highly abundant in NAFLD patients than in normal individuals. This may be due to the plasticity and flexibility of macrophages, allowing them to partially reverse their phenotype[43]. γδ T cells are a unique subset of T lymphocytes that play an essential role in host defense, immune surveillance, and immune system homeostasis. They comprise 15–25% of all hepatic T cells[44], making them a worthwhile target for research in the liver. IL-17A, a crucial pro-inflammatory cytokine in the liver, is primarily produced by γδ T cells[45]. The increased number of γδ T cells in the liver during NAFLD progression and exacerbation of steatohepatitis regulates CD4 T cells and increases IL-17 expression[46]. This observation is consistent with our study findings, which revealed a negative correlation between the expression levels of NAFLD ferroptosis-related genes and γδ T cells. KEGG enrichment analysis showed these genes were primarily enriched in the IL-17 signaling pathway, which promotes NAFLD development by recruiting neutrophils and inducing ROS production. Knockdown of the critical gene IL17RA in this pathway inhibits NAFLD development[47]. Therefore, γδ cells, which produce IL-17A, are the primary regulatory cells of NAFLD progression, and reducing the enrichment of γδ cells in the liver would be an effective way to stop NAFLD progression.
The expression of several ferroptosis-related genes showed a consistent correlation with different immune cells. In our study, IL6, SOCS1, and NR4A1 demonstrated a strong positive association with activated mast cells but a strong negative correlation with resting mast cells, whereas EGR1 had a substantial negative correlation with γδ T cells. Moreover, the expression of these genes was downregulated in NAFLD patients. We propose that these ferroptosis-related genes EGR1, IL6, SOCS1, and NR4A1 regulate immune cell aggregation via immunological or inflammatory pathways, ultimately affecting the NAFLD process. However, these conclusions are mostly based on the current data analysis, and further clinical trials or animal research are necessary to confirm and validate our findings.
Our analysis of the drugs targeting the marker and ceRNA networks identified 6 of the 20 drugs targeting IL6, namely, siltuximab, clazakizumab, sirukumab, elsilimomab, pf-04236921, olokizumab, as IL6 antagonists. These drugs have been used in rheumatoid arthritis, Crohn's bowel disease, and myeloma, but their effectiveness in treating NAFLD has not been reported. Genipin, which targets SREBP-1c and reduces hyperlipidemia and hepatic lipid buildup in mice[48], has beneficial functions, such as improving pancreatic β-cell function, in energy metabolism to treat metabolic disorders. Non-coding RNAs, such as miR-34a-3p, miR-23a-3p, miR-200b, and miR-200c, play essential roles in lipid metabolism in NAFLD[49–51]. However, the efficacy of these targeted drugs and non-coding RNAs in NAFLD treatment remains unclear and requires further research to determine their precise mechanisms. Hence, these selected gene-targeting drugs and non-coding RNAs warrant further investigation as potential treatments for NAFLD. There are some limitations to this study. First, the use of mouse models rather than human samples may limit the ability to validate differences in the expression of NAFLD marker genes; second, the sample size is relatively small and more samples should be obtained in animal studies and clinical cohort studies in the future.