Effect And Mechanism Of Intestinal Epithelial Cell Ferroptosis In Necrotizing Enterocolitis

(1)Identifying of Ferroptosis in Intestinal Epithelial Cells of Necrotizing EnterocolitisBackground: Multiple RCDs exist in the intestinal epithelial cells of necrotizing enterocolitis(NEC),and intestinal barrier dysfunction resulting from RCDs is one of the causes of NEC.Ferroptosis,a new type of RCD,has not been investigated in NEC.Methods: Microarray data of intestinal epithelial tissues of 8 neonates were collected from GEO database(GSE46619 dataset),including 4 NEC and 4 normal controls.Differentially expressed genes(DEGs)in NEC tissues were identified based on the Limma package of R.Principle Component Analysis(PCA)and heat map analysis were used to explore differences in the expression of ferroptosis between the NEC group and the control group.Single-sample Gene Set Enrichment Analysis(ss GSEA)based on GSVA package of R was used to quantify the relative levels of ferroptosis in each intestinal tissue.In laboratory studies,iron concentration in intestinal tissues of NEC was detected by iron assay kit.Malondialdehyde(MDA)was evaluated for the levels of lipid peroxidation.The morphological changes of ferroptosis were observed by transmission electron microscopy.Finally,immunohistochemistry was used to measure the protein levels of key genes in the signaling pathway of ferroptosis.Results: After quality assessment,microarray data of 4 NEC and 4 normal intestinal tissues were statistically analyzed using bioinformatics analysis.We found that there were1571 DEGs between NEC intestinal tissues and normal intestinal tissues,among which 35 were shared genes between DEGs of NEC and ferroptosis;Ferroptosis related genes were enriched in NEC intestinal tissues,and the genes expression level of ferroptosis in NEC group was significantly higher than that of normal intestinal tissues(t test;P = 0.002921).Further laboratory studies showed iron concentration(t test,P = 0.0155)and the MDA levels(t test,P = 0.003)increased in the intestinal tissues of NEC.The protein levels of key genes in signaling pathway of ferroptosis including GPX4,ACSL4 and FTH1 changed and were consistent with the pattern when ferroptosis occurred.Typical ferroptosis subcellular morphological changes such as mitochondrial shrinkage,thickening of mitochondrial outer membrane and disappearance of mitochondrial crest were observed in intestinal epithelial cells in NEC intestinal tissues under transmission electron microscopy.Conclusion: There are iron accumulation,lipid peroxidation,typical ferroptosis morphology changes and key gene expression changes in signaling pathways of ferroptosis in the intestinal epithelial cells of NEC,which may link ferroptosis with NEC.Ferroptosis is a novel RCD mode in intestinal epithelial cells of NEC.This study will provide a theoretical basis for the pathogenesis of ferroptosis in NEC.(2)Validation of Multiple RCDs,Inflammation and Hypoxia in NEC PatientsBackground: Most of the current studies on RCDs,inflammation and hypoxia in the pathogenesis of NEC are conducted in animal or cell models,however research data from human specimens are indispensable.Methods: Microarray data of intestinal epithelial tissues of 8 neonates were collected from the GEO database(GSE46619 dataset),including 4 NEC and 4 normal controls.ss GSEA based on the GSVA package of R was used to quantify the relative levels of apoptosis,pyroptosis,autophagy,inflammation and hypoxia in each intestinal tissue.The limma package of R was used to identify DEGs in NEC tissues.The Database for Annotation,Visualization and Integrated Discovery(DAVID)was used for functional annotation of DEGs.The local software GSEA was used to analyze the signal pathways enriched in different groups based on the expression values of all genes.Results: Bioinformatics analysis was performed on 4 NEC and 4 normal controls in the GSE46619 dataset.It was found that the expression levels of related genes in autophagy(t test;P = 0.0002065),apoptosis(t test;P = 0.01583)and pyroptosis(t test;P = 0.06)were upregulated,the expression levels of inflammation-related genes(t test;P = 0.0009731)and hypoxia-related gene expression(t test;P = 0.003936)were also increased.ACSL4,a key gene in ferroptosis,was significantly associated with apoptosis(R = 0.95,P = 0.00028),autophagy(R = 0.97,P = 0.000079)and pyroptosis(R = 0.81,P = 0.015),hypoxia(R = 0.96,P = 0.000011)and inflammation(R = 0.96,P = 0.0004)in NEC.In addition,a variety of inflammatory related signaling pathways were enriched in the intestinal tissues of NEC,including nod-like receptor signaling pathway,cytokine-cytokine receptor interaction and toll-like receptor signaling pathway.Finally,a majority types of immune cells were significantly activated in NEC tissues,and ACSL4 was positively correlated with the abundance of multiple types of immune cellsConclusion: The data from human specimens showed that levels of apoptosis,pyroptosis,autophagy,inflammation and hypoxia were significantly increased in neonatal NEC intestinal tissues,as well as the expression of inflammatory molecules related to inflammatory injury.ACSL4 associated with multiple types of RCDs,hypoxia and inflammation may be a potential therapeutic target of NEC.(3)Validation of Ferroptosis in Animal Models of NECObjective: To determine whether ferroptosis exists in intestinal tissues of NEC animal models.Methods: The classical NEC animal model and transfusion-related NEC animal model were established using C57BL/6 newborn mice by "hypoxic-hypothermic-LPS " and "anemiatransfusion" methods.Iron concentration,levels of lipid peroxidation products,typical appearance of ferroptosis in intestinal epithelial cells under transmission electron microscopy and the protein levels of key genes in the signaling pathway of ferroptosis were detected to determine whether ferroptosis existed in intestinal tissues of NEC animal models.Results: We found that the iron concentration(t test,P =0.0003)and the levels of MDA(t test,P = 0.0010)were increased in intestinal tissues of NEC group.Similarly,the iron concentration(t test,P < 0.0001)and the levels of MDA(t test,P < 0.0001)were also increased in transfusion-related NEC animal models.Typical ferroptosis changes such as mitochondrial shrinkage and disappearance of mitochondrial cristae were observed in the intestinal epithelial cells of both mouse models.The expression levels of some key genes in the ferroptosis signaling pathway also altered in animal models.Conclusion: Ferroptosis was found in both classical NEC animal models and transfusion-related NEC animal models,which showed iron accumulation,lipid peroxidation damage and mitochondrial morphological changes,which were similar to the results from human specimens.Both NEC mouse models can be used as subjects for in vivo studies on the pathogenesis of ferroptosis in NEC.(4)Role of Heme Inducing Ferroptosis of Intestinal Epithelial Cells in Transfusionrelated NECBackground: Ferroptosis presents in the intestinal epithelial cells of patients with NEC.Our previous study found that red blood cells invaded the intestinal tissues of transfusion related NEC and may release heme under inflammation condition.Whether heme,carrying ferrous ions,is involved in iron accumulation inducing ferroptosis in transfusion related NEC intestinal epithelial cells has not been studied.Methods: Human epithelial cell line Caco-2 was cultured with heme and ferroptosis inhibitor liproxstatin-1 in different concentrations.Iron concentration,the levels of lipid peroxidation products(MDA),mitochondrial ROS,mitochondrial membrane potential(JC-1)and protein levels of key genes in the signaling pathways of ferroptosis in Caco-2 cells were detected.Transmission electron microscopy was used to observe the subcellular structure.Results: When heme and Caco-2 cells were cultured together,cell viability decreased with the increase of heme concentration(one-way ANOVA,P < 0.0001);iron concentration(one-way ANOVA,P < 0.0001)increased gradually,and the levels of MDA(one-way ANOVA,P =0.05)and mitochondrial ROS(one-way ANOVA,P < 0.0001)also increased.Transmission electron microscopy showed that the mitochondria of Caco-2 cells were significantly shrinked and the levels of mitochondrial membrane potential(JC-1)decreased after heme treatment.These changes could be alleviated by adding ferroptosis inhibitor liproxstatin-1 in the culture system.Conclusion: Heme can induce iron accumulation and lipid peroxidation damage in intestinal epithelial cells.Heme may cause ferroptosis of intestinal epithelial cells in transfusion related NEC.