Empirically, approximately half of ARDS instances are attributed to sepsis (2). Several studies have indicated that ferroptosis has been shown to exacerbate both lung injury and cellular damage (5, 8). Correlations between FAGs and immune cells in sepsis-induced ARDS were determined, for the first time, in this study. Thirty DEGs related to ferroptosis were identified from both the GSE32707 and FerrDb datasets, with 20 exhibiting upregulated expression and 10 exhibiting downregulated expression in lung injury serum samples compared to normal serum samples. Subsequently, pathway enrichment analysis was conducted on these DEGs. The top enriched pathways were mainly cellular response to chemical stimulus, oxygen-containing compound, and cytokine stimulus, etc. The major pathways identified through GO-MF analysis were oxidoreductase activity, CCR5 or CCR chemokine receptor binding. GO-CC analysis revealed that cytosol, extracellular exosome, and extracellular vesicle were the three most significant terms. The KEGG results primarily indicated the activation of TNF signaling pathway, NOD-like receptor signaling pathway, and metabolic pathways, etc. Ultimately, a panel of five pivotal genes (CTSB, LCN2, ZFP36, KLF2, and IRF1) was identified through analysis of PPI networks. Notably, CTSB and LCN2 demonstrated the most significant clinical diagnostic potential. The validation of these findings through external validation and experimentation with mice further substantiated the reliability of LCN2 as a diagnostic marker. From a bioinformatics perspective, these results offer valuable insights for investigating the pathological mechanisms underlying sepsis-induced ARDS.
Ferroptosis, a form of programmed cell death, is characterized by its reliance on lipid ROS and iron. Ferroptosis is known to exert significant influence on the development of pulmonary conditions, including chronic obstructive pulmonary disease, acute lung injury, lung cancer, and asthma (14–17). Prior research has indicated a significant correlation between ferroptosis and immune response in various cell types, including vascular endothelial and epithelial cells, smooth muscle cells, and macrophages (18). This study has revealed the significant role played by T cells, monocytes, and eosinophils in the development of sepsis-induced ARDS. These cell types are recognized as key contributors to the inflammatory response within the respiratory system (19). However, further in-depth investigations are required to elucidate the mechanism of ferroptosis in relation to the amelioration of sepsis-induced ARDS in these cells.
Ferroptosis is a process that induces intracellular oxidation and lipid ROS accumulation by regulating glutathione peroxidase (20). Our study revealed that certain oxidative stress pathways, including those related to oxygen-containing compounds and oxidoreductase activity, were enriched with differentially DEGs. This finding strongly suggests that oxidative stress is a significant factor in the development of sepsis-induced ARDS. Furthermore, He et al. demonstrated that ferroptosis is involved in sepsis-induced ARDS in mice through the Nrf2 pathway (6). Furthermore, experimental evidence has demonstrated the involvement of ferroptosis and stimulated autophagy in sepsis-induced ARDS through the inhibition of mTOR signaling (21). The KEGG pathway analysis revealed a significant enrichment of CCR5 or CCR chemokine receptor binding. Previous studies have reported that the activation of the CCR5 signaling pathway in endotoxin-induced lung injury leads to increased neutrophil infiltration (22). Research has demonstrated that CCR5 regulates ferroptosis activity, thereby facilitating the progression of epithelial to mesenchymal transition in individuals undergoing kidney transplantation (23). Nonetheless, there exists a paucity of research studies that have established correlations between CCR5 signaling and ferroptosis in the context of ARDS, thereby presenting a promising avenue for future investigations. The outcomes of the KEGG analysis primarily indicated the activation of various pathways, including the TNF signaling pathway, NOD-like receptor signaling pathway, and metabolic pathways. However, the precise role of the TNF signaling pathway in ferroptosis remains a subject of controversy. It has been suggested that heightened TNF levels may trigger the activation of the NF-κB pathway, leading to the biosynthesis of cellular glutathione and consequent protection of fibroblasts from ferroptosis (24). The TNF/TNFR1/NF-κB signaling pathway has been shown to promote the expression of NO, potentially contributing to the sensitization of ferroptosis (25, 26). Additionally, the NOD-like receptor signaling pathway within the innate immune response has been found to impact the disease process of sepsis-induced ARDS (27, 28). Previous research has indicated that a deficiency in the NLRP3 inflammasome in mice may reduce inflammation and ferroptosis by inhibiting the activation of the Keap1-Nrf2 pathway, ultimately alleviating cerebral ischemia/reperfusion injury (29). These findings suggest a potential interaction between the NOD-like receptor signaling pathway and ferroptosis in the context of sepsis.
The present study utilized gene microarray screening to identify key genes associated with ferroptosis-related DEGs, namely CTSB, LCN2, ZFP36, KLF2, and IRF1. Among these genes, LCN2 was found to hold the most potential for identifying and diagnosing sepsis-induced ARDS. LCN-2, a novel 198 amino acid secreted protein, belongs to a group of transporters responsible for circulating small molecules such as free fatty acids, LPS, and iron (30). Several studies have established the participation of LCN2 in diverse pathological states that impact multiple organs, including the liver, brain, renal system, lungs, and breast (31, 32). Furthermore, LCN2 has been linked to the initiation of both stress-induced behavioral responses and inflammatory pathways. The findings of An et al.'s experiments with mice demonstrated that the absence of LCN-2 led to the alleviation of acute lung inflammation triggered by LPS, as evidenced by the downregulation of genes associated with chemotaxis (33). Moreover, the upregulation of matrix metalloproteinase 9 in human neutrophil granulocytes can be facilitated by LCN2 (34), while LCN2 has been implicated in the exacerbation of acute lung inflammation and oxidative stress through the amplification of macrophage iron accumulation (35). However, the regulatory mechanism of the upstream transcription factor and promoter analysis of LCN2 remains poorly understood. Our investigation has identified miR-374b-3p as the most prominent miRNA involved in the targeting of LCN2. The vast majority of our discoveries are predicated upon a comprehensive analysis of bioinformatics, which in turn requires further experimentation to verify the molecules and pathways governed by LCN2 in the context of sepsis-induced ARDS. Although our study did not satisfy the criteria for expression validation and ROC curve analyses, previous research has indicated significant involvement of the CTSB, ZFP36, KLF2, and IRF1 genes in pulmonary disease.
Our study has certain limitations that must be acknowledged. Although we used the peripheral blood of sepsis patients to verify the value of the above key genes, there was a lack of signal pathway-related mechanisms for further verification.