CD is a long-term, chronic, recurring intestinal disease, collectively known with ulcerative colitis as inflammatory bowel disease(IBD)12.The incidence of CD was more pronounced in the Americas and Europe before the 20th century, however, the prevalence of CD has been steadily rising in Asian countries in recent years13.It mostly develops at the end of the ileum and can cause lesions from the mouth to the anus, as well as extraintestinal complications. CD were usually clinically insidious and are not easily detected and diagnosed, patients are often overlooked and missed when having a colonoscopy14. Patients' symptoms are dominated by diarrhea, abdominal pain, fever and anemia.Extraintestinal symptoms include inflammatory arthropathy, osteoporosis, scleritis, IgA nephropathy and others1516.Review of the Pubmed database revealed that the current findings on risk factors for CD include environmental factors, genetic factors, nutritional status,intestinal barrier disorders, immune response, microbial dysbiosis, gut viral and fungal317.However, the specific mechanisms or involving pathways are not clear yet.
Ferroptosis is a newly discovered iron-dependent, a non-apoptotic form of cell death, usually caused by intracellular lipid peroxidation and regulated by the oxidative and antioxidant systems in vivo, including redox homeostasis, mitochondrial activation, amino acid lipid and sugar metabolism, and other signaling pathways7. It has been observed to have a modulating impact in a number of benign and malignant disorders, including tumours, neurological disorders, kidney damage and Cardiovascular disease181920.
Ferroptosis has an important relationship with intestinal barrier and immune response, which are also the key mechanism causing CD.Liu et.al found that ferroptosis reverses intestinal mucosal barrier integrity maintained by Sestrin221.Ma et.al announced that CD36-directed CD8+ T cells ferroptosis suppresses the anti-tumour effects in vivo22.Xu et.al suggested that ferroptosis could exert anti-tumour effects by reversing the immune microenvironment23. For this reason we further speculate that there is some connection between ferroptosis and CD.
Based on the GEO databases and the ferrdb database, we obtained a total of 13 differential genes associated with ferroptosis and further explored the related functions of these genes by GO and KEGG enrichment analysis.As the figure shows in GO biological processes(BP) are most related to oxidative stress including response to oxidative stress,ROSmetabolic process and cellular response to oxidative stress.This result is more in line with our speculation, as oxidative stress processes are a key part of the occurrence of ferroptosis2425. In the course of the metabolic pathway of the cell due to the imbalance of the redox balance eventually leads to ferroptosis of the cell.Oxidative stress is also an important factor in inflammation, which activates a variety of proinflammatory factors such as NF-κB and p5326. The increased expression of P53 in Crohn promotes apoptosis of intestinal epithelial cells and aggravates inflammation2728. We hypothesized that oxidative stress-related ferroptosis may also be involved in the development of CD.
At the same time, we also obtained another BP enrichment result-vascular endothelial growth factor (VEGF) production.VEGF is a biomolecule that It is crucial for mitosis and preventing apoptosis in vascular endothelial cells29, including several members such as VEGF-A, VEGF-B, and VEGF-C.Inflammation and tumorigenesis are often accompanied by pathological vascular proliferation, which also implies an imbalance of oxygen homeostasis in the microenvironment.The concentration of ROS dependent on NADPH oxidase production is also significantly increased, which seems to form a circular pathway30. High production of ROS increases the likelihood of ferroptosis in cells31.This is also consistent with the enrichment results we obtained in Molecular function (MF) and cellular component (CC).
In other studies, the biomodulatory role of VEGF in IBD has been partially reported in other research.Scaldaferri et.al found that overexpression of VEGF-A in mice with DSS-induced IBD inflammation can exacerbate disease severity32. According to Eder et al,the degree of VEGF expression affected how severe CD was.33. However, the existing mechanism between VEGF and ferroptosis is still unclear. It is initially speculated that VEGF factors may lead to ferroptosis of intestinal epithelial cells and promote the occurrence of Crohn.
KEGG pathway analysis further revealed that the pertinent genes were primarily abundant in the TNF pathway, IL-17 pathway and Rheumatoid arthritis.Tumour necrosis factor (TNF) is an important cell regulatory molecule involved in the regulation of cell survival, apoptosis, as well as inflammation, and immune response34. Interleukin 17 (IL-7) is a inflammatory factor secreted by T helper 17 (Th17) cells35. Bishu et.al found elevated expression of both TNF and IL-17 in CD, which may be associated with a type CD 4+ T cell36.However, the regulatory relationship between ferroptosis with TNF factors and IL-17 in the currently available studies of CD remains unclear.
Next,We mapped the PPI network in Cytoscape and screened 5 hub genes using CytoHubba software IL6、PTGS2、HIF1A、DUOX2、NOS2. Three pairs of samples were performed qRC-PCR experiments to analyze the expression of the genes according to these five genes, in which the expression of IL6, DUOX2 and PTGS2 were different and p < 0.05.
Interleukin-6 (IL-6) is a pro-inflammatory factor that belongs to the interleukin family and crucial in the host's body for anti-inflammatory and emergency processes37.Shi et.al suggested that Toll-like receptor 4 ( TLR4) aggravates intestinal injury by regulating the expression of IL-638.In a study by V Gross et.al, who found significantly elevated IL-6 in CD in both active and inactive phases39.IL-6 was not only play a role in inflammation.Han et.al found that in asthma disease IL-6 disrupts iron homeostasis in BEAS-2B cells by promoting lipid peroxidation, which in turn promotes ferroptosis in bronchial epithelial cells40. Li et.al recently demonstrated that the expression of an amino acid countertransporter (xCT) in head and neck squamous cell carcinoma is associated with levels of IL-6, and xCT induces cellular ferroptosis41.Despite various results suggesting a link between IL-6 and inflammation and ferroptosis, there is no conclusive evidence for an association between IL-6 and ferroptosis in CD.
Prostaglandin endoperoxide synthase 2 (PTGS2), also called cyclooxygenase(COX-2), an enzyme that can be suppressed by nonsteroidal anti-inflammatory drugs (NSAID) such as aspirin and ibuprofen played a regulatory role in many types of inflammatory or neoplastic diseases42.Roberts et al. found through densitometric analysis that the expression of COX-2 in IBD tissues increased by 6–8 times43. Singer et al. Et al. found that COX-2 was predominantly present in apical epithelial cells and in lamina propria mononuclear cells in CD. Interestingly, both studies showed no expression of COX-2 in normal tissues44.This demonstrates that COX-2 has an important regulatory role in CD. However, no relevant studies have found whether the overexpression of COX-2 in Crohn is associated with ferroptosis.
DUOX2,Dual oxidase 2,is an enzyme first identified in the mammalian thyroid and a member of NADPH oxidase involved in ROS production.In a study of pediatric CD.Haberman et.al found that within the villous enterocytes of the CD ileum, DUOX2 was strongly expressed and may have exacerbated intestinal mucosal damage45.Lipinski et.al's study found that DUOX2 is involved in the production of ROS46, ROS is an important factor mediating the occurrence of ferroptosis.We, therefore, speculate that DUOX2 may promote ferroptosis in CD. However, the specific mechanism is not yet clear.
In the result of the 5 hub genes drug predicting targets,Kaempferol had found to inhibit the onset of cellular iron death, while other studies have shown that it can also be used to improve intestinal inflammation and intestinal barrier disorders4748.
Our research has some drawbacks.Firstly,the data set in this study was obtained from the GEO database and the same microarray was used for both data sets, and there was no further validation in tissue samples or cells or animals. Therefore, it is hoped that the association between iron death and CD will be further explored in future studies.