Lung ischemia-reperfusion injury is a complex "aseptic" inflammation, which involves rapid oxidative stress and the follow-up response of all cells in the lung, which eventually leads to the destruction of endothelial and epithelial barriers, resulting in life-threatening edema and gas exchange defects. Some of the main participants are innate immune cells, which are rapidly activated during reperfusion and induce tissue injury or increase inflammation through the production of pro-inflammatory cytokines and DAMP. Although many experimental studies have described various methods to prevent or inhibit ischemia-reperfusion injury, these methods have not been translated into clinical application. Therefore, to better understand the mechanism of lung ischemia-reperfusion injury and determine effective therapeutic targets, it is helpful to better explore the complex intracellular and intercellular communication in the lung and to more effectively treat or prevent lung ischemia-reperfusion injury caused by lung transplantation. In this study, through the method of bioinformatics, based on GEO database, it is found that FOS,EGR1,ALB plays a key role in PPI network. Further study found that the expression of differential genes in IL-17 signal pathway and B cell receptor signal pathway was up-regulated, while the expression of fatty acid degradation, valine, leucine and isoleucine degradation, Fanconi anemia pathway was down-regulated.
4.1 FOS proto-oncogene
The FOS gene family consists of four members: FOS,FOSB,FOSL1 and FOSL2. These genes encode leucine zipper proteins, which can be dimerized with proteins of the JUN family to form a transcription factor complex AP-1. Therefore, FOS protein is considered to be a regulator of cell proliferation, differentiation and transformation. In some cases, the expression of FOS gene is also associated with apoptotic cell death, and nucleophosphorins that form tight but non-covalent complexes with JUN/AP-1 transcription factors. In heterodimers, both FOS and JUN/AP-1 bases seem to interact with symmetrical DNA half-sites. When TGF- β is activated, a polymer SMAD3/SMAD4/JUN/FOS complex is formed at the AP1/SMAD binding site to regulate the signal pathway mediated by TGF- β. It has the key function of regulating cell development to form and maintain bones. It is considered to play an important role in signal transduction, cell proliferation and differentiation. In growing cells, phospholipid synthesis may be activated by activating CDS1 and PI4K2A. This activity requires dephosphorylation of TYRL and binding to the endoplasmic reticulum [21]. The increase of FOS expression is enough to hinder the growth of cervical cancer[22]. In the adipose tissue around the abdominal aortic aneurysm, the characteristics of inflammatory infiltration may be indicated by the cellular network dominated by FOS, which is composed of activated mast cells, plasma cells and Tfh cells [23]. A large number of studies have shown that there is a close relationship between the activation of FOC and ischemic brain injury[24]. The study of Hua in 2022 showed that α-ketoglutarate attenuated cerebral ischemia-reperfusion injury through c-FOS/IL-10/Stat3 signal transduction pathway[25].
Through bioinformatics studies, we found that the expression of FOS was significantly up-regulated in the samples of pulmonary microvascular endothelial cells treated with cold ischemia for 6 hours and cold ischemia for 6 hours and reperfusion for 6 hours and 2 hours, and FOS was involved in myeloid cell differentiation, muscle tissue development, muscle organ development, MAPK signal pathway, endocrine resistance, RAS signal pathway and calcium signal pathway. As a key node in the PPI-protein interaction network, FOS interacts extensively with molecules encoded by multiple genes.
4.2 Human serum albumin (ALB)
The ALB gene encodes the most abundant protein in human blood. The protein has the function of regulating plasma colloidal osmotic pressure. it is a variety of endogenous molecules such as hormones, fatty acids, metabolites and carrier proteins of exogenous drugs. In addition, the protein showed a wide range of substrate-specific esterase-like activity. The encoded proprotein is hydrolyzed to form a mature protein. The peptide derived from this protein, EPI-X4, is an endogenous inhibitor of CXCR4 chemokine receptor. In 1998, Becker et al[26] showed that the protein osmotic reflection coefficient (sigmaALB) did not change after 45 minutes of ischemia in isolated ferret lungs, but decreased by 72% after 180 minutes of ischemia. A prospective, multicenter Cox regression analysis in 2021 showed that serum albumin and respiratory rate were independent prognostic variables for 30-day survival in patients with community-acquired pneumonia [27].
The results of this study showed that the expression of ALB in pulmonary microvascular endothelial cells after 2 h reperfusion was up-regulated, and it may have DNA-binding transcriptional activator activity, RNA polymerase II specificity, DNA-binding transcriptional activator molecular function, involved in calcium signal pathway, glycine, serine and threonine metabolism, regulation of pyruvate metabolic pathway, and may also be associated with benign tumors of organ system. Cellular benign tumor, musculoskeletal system tumor, high grade glioma, germ cell carcinoma, embryoma, myeloid tumor disease.
4.3 Early growth response 1 (EGR1)
The protein encoded by EGR1 belongs to the EGR family of C2H2 zinc finger proteins. It is a nuclear protein that plays a role in transcriptional regulation. The products of the target genes it activates are necessary for differentiation and mitosis. Studies have shown that this is a cancer suppressor gene. Overexpression of EGR1 can also promote cell proliferation, growth and migration, and inhibit apoptosis[28]. In 2022, Lehman et al[29] found that inflammatory genes CXCL3,CXCL8,CXCL10,TNF and PTGS2 were up-regulated in VEEV-infected cells, which were partly dependent on EGR1, transcription factors, including EGR1 itself, and ATF3,FOS,JUN,KLF4,EGR2 and EGR4 were found to be partially transcriptional dependent on EGR1. Genes identified as partially transcriptional dependent EGR1 in infected cells included ATF3 (EEEV,CHIKV,ZIKV), JUN (EEEV), KLF4 (SINV,ZIKV,RVFV), CXCL3 (EEEV,CHIKV,ZIKV). CXCL8 (EEEV,CHIKV,ZIKV,RVFV), CXCL10 (EEEV,RVFV), TNF- α (EEEV,ZIKV,RVFV) and PTGS2 (EEEV,CHIKV,ZIKV) indicate that EGR1 after virus infection induces stimulation to produce a variety of inflammatory mediators. The inhibition of EGR1 activity leads to the inhibition of inflammatory genes [30].
In our study, we found that the expression of EGR1 was up-regulated in pulmonary microvascular endothelial cells after ischemia-reperfusion. EGR1 exists in the nucleus and cytoskeleton, which may be involved in B cell receptor signal pathway, MAPK signal pathway, EGR1 may be involved in DNA binding transcriptional activator activity, RNA polymerase II specificity, DNA binding transcriptional activator activity and other cellular functions. However, its specific mechanism and potential need further experimental research.
It is worth mentioning that in order to determine in which pathways the expression of differential genes is up-regulated or down-regulated. We carried out gene set enrichment analysis and found that the expression of differential genes was up-regulated in B cell receptor signal, IL-17 signal pathway, osteoclast differentiation and choline metabolism pathway in cancer, but down-regulated in fatty acid degradation, valine, leucine and isoleucine degradation. Further experiments are needed to verify whether differential genes affect the repair of ischemia-reperfusion injury of pulmonary microvascular endothelial cells by regulating these pathways.
To sum up, based on bioinformatics methods and layer by layer analysis, the genes related to ischemia-reperfusion in pulmonary microvascular endothelial cells were screened by GSE172222 data set, their basic functions and related pathways were annotated and verified by GO,KEGG and DO analysis, and the regulatory role of differential genes in the pathway was further analyzed by GSEA enrichment analysis. In connection with the physiological and pathological manifestations and relatively complex pathogenesis of lung ischemia-reperfusion injury, this paper focuses on the possible mechanism of FOS,EGR1,ALB, in order to provide new insights into the pathogenesis, effective diagnosis and targeted treatment of lung ischemia-reperfusion injury.