Identification of differentially expressed genes associated with ferroptosis in Crohn’s disease

Abstract

Objective:Ferroptosis-related genes may have a critical regulatory role in the pathogenetic process of Crohn’ disease(CD).The purpose of this study was to identify genes expressed in CD that are associated with ferroptosis and provide direction in the diagnosis and therapy of Crohn's disease.

Methods: The data for CD mRNA expression were first gathered from the Gene Expression Omnibus (GEO) database, and two gene sets were selected as major targets (GSE75214 and GSE102133) and analyzed differentially expressed genes.Next,R software (version 4.1.2) was used to analyze the common genes in CD differential expressed genes and ferroptosis-related genes.GO enrichment analysis,KEGG pathway analysis were used to identify differential related pathways and functions.Protein-protein interaction(PPI) analysis was performed to identify target genes.DSigDB website was used to predict potential target drugs for hub genes.Finally, qRT-PCR method were used to detect the expression of these ferroptosis related genes in clinical samples obtained from healthy control and CD patients.

Result: According to two GEO datasets, we finally identified 13 ferroptosis DEGs(10 upregulated genes and 2 downregulated genes) in crohn disease with the threshold of p-value < 0.05 and |log2 FC| > 1 and selected for continued analysis.Go enrichment analysis and KEGG pathways results were shown in the following figures.PPI analysis indicate the mutual effect between these genes and filtered out 5 hub genes.Top 10 potential targeted drugs were selected. Finally, the result of qRT-PCR shown that the expression of three genes IL-6,PTGS2 and DUOX2 were different between CD samples and healthy samples.This result was consistent with the results we obtained in the biological information analysis.

Conclusion: Bioinformatics analysis identified a total of 13 iron death-associated genes in CD. Three differential genes IL-6,PTG32 and DUOX2 were detected in tissue experiments.Our findings might provide new biomarkers and promising treatment targets in CD.

Introduction

Inflammatory bowel diseases(IBD) are currently known chronic inflammatory diseases of the gastrointestinal tract, including ulcerative colitis(UC) and Crohn's disease(CD)¹ .The incidence of CD has been increasing in recent years around the global scope²³.CD is usually found at the end of the ileum and can invade and spread to the surrounding intestinal tissues⁴ it also has a tendency to develop dysplasia and colorectal cancer(CRC)⁵.Patients with long-term CD have about 22% chance of developing cancer⁶. Ferroptosis is a newly discovered form of programmed cell death that is associated with many benign and malignant diseases of the digestive system⁷.However, the mechanisms involved in CD caused by ferroptosis-related genes are not fully understood.

Ferroptosis is a form of cell death that differs from apoptosis and pyroptosis, The usual causes are lipid peroxidation, iron accumulation, and cell membrane breakdown.⁸.Some other research had found that ferroptosis can be participant in the regulation of inflammatory bowel disease⁹. Huang et.al indicated that ferroptosis-related hub gene STAT3(signal transducer and activator of transcription 3) mediated the ferroptosis process and promoted ulcerative colitis occured¹⁰. Ferroptosis also has a significant part in the regulation of CRC.Glutathione peroxidase 4 (GPX4),a ferroptosis inhibitor,was an important central regulatory molecule that was involved in the progress of the CRC development¹¹.

Although ferroptosis is strongly associated with these digestive tract-related diseases, the mechanisms involved in ferroptosis in CD were not understood.So we search for the ferroptosis-related genes in CD to find out the correlation between CD and ferroptosis.GSE102133 and GSE75214 were found as target datasets to find different express genes in CD and such genes made cross-comparison with ferroptosis-related genes to finally determine the object of study. Continuous The result will provide new biomarkers for further study in CD research,to validate the potential effect of clinical diagnosis and treatment.

Materials And Methods

Source of data acquisition

We searched the CD-related microarray sequencing datasets on the GEO database (https://www.ncbi.nlm.nih.gov/geo/) to analysis in this study,the keyword is “crohn disease”.There are the following screening criteria:(a)the samples from human tissues.(b)the dataset was from Microarray expression data.(c)the dataset was required from CD ileal mucosa and healthy ileal mucosa.(d)the totally number of samples was more than 10.(e)the number of different expression genes(DEGs) was greater than 100.By this way, we finally determine two GEO datasets GSE75214 and GSE102133 to enroll in our study.Both of these datasets were present on GPL6244 (Affymetrix Human Gene 1.0 ST Array),GSE102133 included 65 CD patients and 12 controls.GSE75214 contained 67 CD patients and 11 controls,two GSE datasets specific informations was shown in table 1.Ferroptosis-related genes get from FerrDb website (http://www.zhounan.org/ferrdb/index.html)，there are 259 genes were found.

Identification of ferroptosis-related DEGs in CD 

  All microarray data were from the GEO database,the relative extracted data were normalized by log-2 transformation.Probes were converted to gene symbols according to the annotation information of the normalized data in the platform.The principal component analysis (PCA) plot was performed between two datasets.Data was standardized using the “Limma” toolkit in the R software (version 4.1.2). A | log2 (fold change)| > 1 and a P-value < 0.05 were used as the criteria for difffferential gene expression. The heatmap and volcano plot were drawn using the “Heatmap” and “ggplot2” packages in the R software (version 4.1.2). Then the DEGs made intersected with ferroptosis-related genes from FerrDb and generated a venn diagram by using online Bioinformatics tool  (http://bioinformatics.psb.ugent.be/webtools/Venn/).

GO functional enrichment and KEGG pathway and genes relationship for the common DEGs

To further investigate the biological function of the genes involved,We used the Gene Ontology (GO) enrichment analyses which included biological process (BP)、molecular function (MF) and cellular component (CC) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses for evaluating relating functions.R package clusterProfifiler toolkit in R software version(4.1.2) was used for such calculation.A p-value < 0.05 result was defined as significant. Gene Correlation Analysis Using Pearson correlation test to Assess Reciprocal Relationships at the mRNA Level in CD.
 Constructing Protein-protein interaction network and hub gene selecting

To further investigate the interactions between DEGs promoting proteins, PPI network analysis was performed by using the Search Tool for the Retrieval of Interacting Genes (STRING) database.The PPI network analysis map was visualized by Cytoscape software(version 3.9.1).Firstly, STRING database was used to establish the PPI network according to DEGs,after that the analysis results were then exported to Cytoscape software(version 3.9.1) for mapping.Nodes represent proteins, lines represent interactions between proteins, and different colors represent different functions.Hub gene was obtained from the cytoHubba plugin in Cytoscape (version 3.9.1) through maximal clique centrality (MCC) methods. The GeneMANIA website (https://genemania.org/) was used for co-expression, physical interaction, co-localization, pathways and other analyses of the 5 hub gene.

Potential target drug prediction

DSigDBv1.0 (http://dsigdb.tanlab.org/DSigDBv1.0/) was used to predict the five hub genes of potential drug targets, and select the top 10 scores as candidate drugs.

CD and controls tissues from patients

 From October 2021 to September 2022,we totally collected 3 pairs of tissues(3 CDs and 3 controls) from patients and healthy individuals at The Qingdao Municipal Hospital（Qingdao，China）. Written informed consent was obtained from all patients and approved by the medical ethics committee of the hospital（2022-066）.

Statistical analysis

The R software (version 4.1.2) is used to perform statistical analysis of biological information data. Results were analyzed by Student t test for comparison. In the qRT-PCR experiment, each sample was repeated three times.A p-value < 0.05 was considered statistically significant.

Result

Analysis of genes associated with ferroptosis in CD

   As shown in the flow chart (figure 1).We chose two CD dataset files (GSE102133 and GSE75214,table 1) and downloaded them using the same platform files. A total of 155 samples were included in the study (132 CD samples and 23 control samples).Principal component analysis (PCA) was performed between two datasets(Supplementary Figure 1A).The differential genes between the two groups of samples are shown in the volcano (Figure 2A). 13 differential genes in total(11 upregulated genes and 2 downregulated genes)were obtained after crossover with 259 ferroptosis-related genes showing in Venn diagram (Figure 2) and made heatmap plot between CD samples and normal samples.Expression of the 13 differential genes in CD's samples and healthy samples in box plots(Figure 3).The specific information with the differential genes is shown in table2.

GO enrichment and KEGG pathway and Gene interrelationships analysis of differential genes related to ferroptosis in CD's 

 According to the results of the GO enrichment analysis(Figure 4A,B), the biological process (BP) function was predominantly enriched in the response to oxidative stress, reactive oxygen metabolic processes, cellular response to oxidative stress, and vascular endothelial growth factor production.Changes in molecular function (MF) and cellular component (CC) were mainly in oxygen reductase activity and NADPH oxidase complex.KEGG pathway(Figure 4A) showed that genes were mainly enriched in TNF signaling pathway and IL-17 signaling pathway. Pearson correlation test showed that some of the 13 ferroptosis-related genes had significant positive and negative correlations in CD（Figure 4C）.

Protein-protein interaction network and hub genes associated with ferroptosis filtering

PPI analysis was used to show the interaction relationships between the genes of interest(Supplementary Fig 1B,Figure 5A).3 of the 13 genes were excluded which did not interact with the other. The other 10 genes were mapped for the PPI network.5 hub genes IL6,PTGS2,HIF1A,DUOX2, and NOS2 were discovered using the Cytoscape software's cytohubba plugin（Figure 5B）.Gene co-expression, functional analysis, and physical binding properties of the five hub genes were predicted using the GeneMANIA website in the figure 5C.

Targeted drug prediction for related genes

The DSigDB database was used to predict 5 hub gene-related target drugs that may treat CD by controlling the occurrence of ferroptosis. A total of 1378 possible drugs were predicted, and the top 10 target drugs were selected after ranking based on a composite score（Table 3）.

Expression of ferroptosis-associated genes in CD's clinical samples

  To further validate our results, we verified the expression of 5 hub genes in 3 pairs of CD's clinical samples(Figure 6). Among them, IL-6, PTGS2 and DUOX2 expressions were elevated and statistically significant in CD. Although the other two genes, HIF1A and NOS2, also showed some trends, the results were not statistically significant(Figure 7).The primer information were listed in Table 4.

Discussion

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.

Conclusion

Overall, 13 ferroptosis-associated genes were identified in CD by our bioinformatics study. Among them IL-6,DUOX2 and PTGS2 are most likely to regulate ferroptosis in CD involved in CD development and progression. But specific mechanistic studies need to be further explored. Our study provides theoretical support for CD and ferroptosis, increasing our understanding of CD and treatment strategies.

Declarations

Funding

The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. 

Competing Interests

The authors have no relevant financial or non-financial interests to disclose. 

Author Contributions

All authors contributed to the study conception and design.Material preparation, data collection and analysis were performed by WenquanZhang and Zhaoshui Li. The first draft of the manuscript was written by Wenquan Zhang and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. 

Hongbo Li conceived the concept, designed the manuscript, coordinated, and critically revised manuscript, and was responsible for its financial supports and the corresponding works. 

Ethics Approval

This study was performed in line with the principles of the Declaration of Helsinki. Approval was granted by the Ethics Committee of Qingdao municipal hospital.(2022-066) 

Consent to participate

Informed consent was obtained from all individual participants included in the study. 

Availability of data and materials 

The datasets presented in this study can be found in online repositories. Further inquiries can be directed to the corresponding authors.

References

1. Geremia, A., Biancheri, P., Allan, P., Corazza, G. R. & Di Sabatino, A. Innate and adaptive immunity in inflammatory bowel disease. Autoimmun. Rev. 13, 3–10 (2014).
2. Cosnes, J., Gower-Rousseau, C., Seksik, P. & Cortot, A. Epidemiology and natural history of inflammatory bowel diseases. Gastroenterology 140, 1785–1794 (2011).
3. Roda, G. et al. Crohn’s disease. Nat. Rev. Dis. Primer 6, 22 (2020).
4. Dulai, P. S. et al. Should We Divide Crohn’s Disease Into Ileum-Dominant and Isolated Colonic Diseases? Clin. Gastroenterol. Hepatol. Off. Clin. Pract. J. Am. Gastroenterol. Assoc. 17, 2634–2643 (2019).
5. Friedberg, S. & Rubin, D. T. Intestinal Cancer and Dysplasia in Crohn’s Disease. Gastroenterol. Clin. North Am. 51, 369–379 (2022).
6. Friedman, S. Cancer in Crohn’s disease. Gastroenterol. Clin. North Am. 35, 621–639 (2006).
7. Jiang, X., Stockwell, B. R. & Conrad, M. Ferroptosis: mechanisms, biology and role in disease. Nat. Rev. Mol. Cell Biol. 22, 266–282 (2021).
8. Li, J. et al. Ferroptosis: past, present and future. Cell Death Dis. 11, 88 (2020).
9. Xu, C., Liu, Z. & Xiao, J. Ferroptosis: A Double-Edged Sword in Gastrointestinal Disease. Int. J. Mol. Sci. 22, 12403 (2021).
10. Huang, F. et al. STAT3-mediated ferroptosis is involved in ulcerative colitis. Free Radic. Biol. Med. 188, 375–385 (2022).
11. Sui, X. et al. RSL3 Drives Ferroptosis Through GPX4 Inactivation and ROS Production in Colorectal Cancer. Front. Pharmacol. 9, 1371 (2018).
12. Veauthier, B. & Hornecker, J. R. Crohn’s Disease: Diagnosis and Management. Am. Fam. Physician 98, 661–669 (2018).
13. Chiba, M., Morita, N., Nakamura, A., Tsuji, K. & Harashima, E. Increased Incidence of Inflammatory Bowel Disease in Association with Dietary Transition (Westernization) in Japan. JMA J. 4, 347–357 (2021).
14. Chetcuti Zammit, S., Ellul, P. & Sidhu, R. The role of small bowel endoscopy for Crohn’s disease. Curr. Opin. Gastroenterol. 35, 223–234 (2019).
15. Wilkins, T., Jarvis, K. & Patel, J. Diagnosis and management of Crohn’s disease. Am. Fam. Physician 84, 1365–1375 (2011).
16. Ambruzs, J. M. & Larsen, C. P. Renal Manifestations of Inflammatory Bowel Disease. Rheum. Dis. Clin. North Am. 44, 699–714 (2018).
17. Nutritional Global Status and Its Impact in Crohn’s Disease - PubMed. https://pubmed.ncbi.nlm.nih.gov/34877468/.
18. Wu, X., Li, Y., Zhang, S. & Zhou, X. Ferroptosis as a novel therapeutic target for cardiovascular disease. Theranostics 11, 3052–3059 (2021).
19. L, M.-S. et al. Ferroptosis and its potential role in the physiopathology of Parkinson’s Disease. Prog. Neurobiol. 196, (2021).
20. Martin-Sanchez, D. et al. Ferroptosis and kidney disease. Nefrologia 40, 384–394 (2020).
21. Liu, W., Xu, C., Zou, Z., Weng, Q. & Xiao, Y. Sestrin2 suppresses ferroptosis to alleviate septic intestinal inflammation and barrier dysfunction. Immunopharmacol. Immunotoxicol. 1–10 (2022) doi:10.1080/08923973.2022.2121927.
22. Ma, X. et al. CD36-mediated ferroptosis dampens intratumoral CD8+ T cell effector function and impairs their antitumor ability. Cell Metab. 33, 1001-1012.e5 (2021).
23. Xu, H., Ye, D., Ren, M., Zhang, H. & Bi, F. Ferroptosis in the tumor microenvironment: perspectives for immunotherapy. Trends Mol. Med. 27, 856–867 (2021).
24. Yu, Y. et al. Ferroptosis: a cell death connecting oxidative stress, inflammation and cardiovascular diseases. Cell Death Discov. 7, 193 (2021).
25. Mancardi, D., Mezzanotte, M., Arrigo, E., Barinotti, A. & Roetto, A. Iron Overload, Oxidative Stress, and Ferroptosis in the Failing Heart and Liver. Antioxid. Basel Switz. 10, 1864 (2021).
26. Reuter, S., Gupta, S. C., Chaturvedi, M. M. & Aggarwal, B. B. Oxidative stress, inflammation, and cancer: how are they linked? Free Radic. Biol. Med. 49, 1603–1616 (2010).
27. Gu, L., Ge, Z., Wang, Y., Shen, M. & Zhao, P. Activating transcription factor 3 promotes intestinal epithelial cell apoptosis in Crohn’s disease. Pathol. Res. Pract. 214, 862–870 (2018).
28. Zhang, J. et al. Deficiency in the anti-apoptotic protein DJ-1 promotes intestinal epithelial cell apoptosis and aggravates inflammatory bowel disease via p53. J. Biol. Chem. 295, 4237–4251 (2020).
29. Melincovici, C. S. et al. Vascular endothelial growth factor (VEGF) - key factor in normal and pathological angiogenesis. Romanian J. Morphol. Embryol. Rev. Roum. Morphol. Embryol. 59, 455–467 (2018).
30. Fukai, T. & Ushio-Fukai, M. Cross-Talk between NADPH Oxidase and Mitochondria: Role in ROS Signaling and Angiogenesis. Cells 9, E1849 (2020).
31. Su, L.-J. et al. Reactive Oxygen Species-Induced Lipid Peroxidation in Apoptosis, Autophagy, and Ferroptosis. Oxid. Med. Cell. Longev. 2019, 5080843 (2019).
32. F, S. et al. VEGF-A links angiogenesis and inflammation in inflammatory bowel disease pathogenesis. Gastroenterology 136, (2009).
33. Eder, P. et al. Association of serum VEGF with clinical response to anti-TNFα therapy for Crohn’s disease. Cytokine 76, 288–293 (2015).
34. Bradley, J. R. TNF-mediated inflammatory disease. J. Pathol. 214, 149–160 (2008).
35. Iwakura, Y., Ishigame, H., Saijo, S. & Nakae, S. Functional specialization of interleukin-17 family members. Immunity 34, 149–162 (2011).
36. Bishu, S. et al. CD4+ Tissue-resident Memory T Cells Expand and Are a Major Source of Mucosal Tumour Necrosis Factor α in Active Crohn’s Disease. J. Crohns Colitis 13, 905–915 (2019).
37. Tanaka, T., Narazaki, M. & Kishimoto, T. IL-6 in inflammation, immunity, and disease. Cold Spring Harb. Perspect. Biol. 6, a016295 (2014).
38. Shi, Y.-J. et al. Toll-like receptor 4 (TLR4) deficiency aggravates dextran sulfate sodium (DSS)-induced intestinal injury by down-regulating IL6, CCL2 and CSF3. Ann. Transl. Med. 7, 713 (2019).
39. Gross, V., Andus, T., Caesar, I., Roth, M. & Schölmerich, J. Evidence for continuous stimulation of interleukin-6 production in Crohn’s disease. Gastroenterology 102, 514–519 (1992).
40. Han, F., Li, S., Yang, Y. & Bai, Z. Interleukin-6 promotes ferroptosis in bronchial epithelial cells by inducing reactive oxygen species-dependent lipid peroxidation and disrupting iron homeostasis. Bioengineered 12, 5279–5288 (2021).
41. Li, M., Jin, S., Zhang, Z., Ma, H. & Yang, X. Interleukin-6 facilitates tumor progression by inducing ferroptosis resistance in head and neck squamous cell carcinoma. Cancer Lett. 527, 28–40 (2022).
42. Kunzmann, A. T. et al. PTGS2 (Cyclooxygenase-2) expression and survival among colorectal cancer patients: a systematic review. Cancer Epidemiol. Biomark. Prev. Publ. Am. Assoc. Cancer Res. Cosponsored Am. Soc. Prev. Oncol. 22, 1490–1497 (2013).
43. Roberts, P. J., Morgan, K., Miller, R., Hunter, J. O. & Middleton, S. J. Neuronal COX-2 expression in human myenteric plexus in active inflammatory bowel disease. Gut 48, 468–472 (2001).
44. Singer, I. I. et al. Cyclooxygenase 2 is induced in colonic epithelial cells in inflammatory bowel disease. Gastroenterology 115, 297–306 (1998).
45. Haberman, Y. et al. Pediatric Crohn disease patients exhibit specific ileal transcriptome and microbiome signature. J. Clin. Invest. 124, 3617–3633 (2014).
46. Lipinski, S. et al. DUOX2-derived reactive oxygen species are effectors of NOD2-mediated antibacterial responses. J. Cell Sci. 122, 3522–3530 (2009).
47. Bian, Y. et al. Protective Effect of Kaempferol on LPS-Induced Inflammation and Barrier Dysfunction in a Coculture Model of Intestinal Epithelial Cells and Intestinal Microvascular Endothelial Cells. J. Agric. Food Chem. 68, 160–167 (2020).
48. Yuan, Y., Zhai, Y., Chen, J., Xu, X. & Wang, H. Kaempferol Ameliorates Oxygen-Glucose Deprivation/Reoxygenation-Induced Neuronal Ferroptosis by Activating Nrf2/SLC7A11/GPX4 Axis. Biomolecules 11, 923 (2021).

Tables

Table 1

GEO datasets information

Table 2

Comparison of 13 genes associated with ferroptosis between Crohn's samples and normal samples

Table 3

Top 10 predicted target drugs based on 5 hub genes

Table 4

Primer Sequence information