Hyperoxia-activated Nrf2 regulates ferroptosis in intestinal epithelial cells and intervenes in inflammatory reaction through COX-2/PGE2/EP2 pathway

Hyperoxia-activated Nrf2 regulates ferroptosis in intestinal epithelial cells and intervenes in inflammatory reaction through COX-2/PGE2/EP2 pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Hyperoxia-activated Nrf2 regulates ferroptosis in intestinal epithelial cells and intervenes in inflammatory reaction through COX-2/PGE2/EP2 pathway Yanping Liu, Tianming Li, Changping Niu, Zhengwei Yuan, Siyu Sun, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4115151/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 03 Jan, 2025 Read the published version in Molecular Medicine → Version 1 posted 6 You are reading this latest preprint version Abstract The lack of knowledge about the mechanism of hyperoxia-induced intestinal injury has attracted considerable attention, due to the potential for this condition to cause neonatal complications. This study aimed to explore the relationship between hyperoxia-induced oxidative damage and ferroptosis in intestinal tissue and investigate the mechanism by which hyperoxia regulates inflammation through ferroptosis. The study systematically evaluated the effects of hyperoxia on oxidative stress, mitochondrial damage, ferroptosis, and inflammation of intestinal epithelial cells both in vitro and in vivo . The results showed that ferroptosis was involved in intestinal oxidative damage caused by hyperoxia and was regulated by Nrf2. Moreover, hyperoxia-induced oxidative damage regulated inflammation through ferroptosis by upregulating the COX-2/PGE2/EP2 signaling pathway. These findings have important implications for future clinical prevention and therapeutic approaches to neonatal organ injury caused by hyperoxia treatment. hyperoxia intestinal epithelial cells ferroptosis COX-2/PGE2/EP2 pathway inflammatory Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 1 Introduction Perinatal asphyxia leading to hypoxia is one of the main causes of neonatal morbidity and mortality, often requiring oxygen inhalation therapy (hyperoxia). However, hyperoxia for long time has been proven to be toxic[ 1 ], causing damage to neonatal lung, retina, and nerve development[ 2 – 4 ]. Researchers have also recently focused on the intestinal injury caused by hyperoxic toxicity. A previous study showed that after neonatal exposure to hyperoxia, the number of goblet cells in the intestines decreased, and the mucus layer was severely damaged, leading to higher intestinal permeability and bacterial translocation[ 5 ]. Moreover, under hyperoxia, there was a significant increase in the levels of interferon-gamma (IFN-γ) and interleukin 10 (IL-10), indicating intestinal injury[ 6 ]. Given that the development of the intestines plays a crucial role in early-stage nutrient absorption, it is important to investigate the effects and molecular mechanisms of hyperoxic toxicity on the newborn intestine. Under physiological conditions, the amount of reactive oxygen species (ROS) is balanced with antioxidant defense, precisely controlled by cells to avoid oxidative stress and cell damage[ 7 ]. However, under hyperoxia, ROS increases, and oxidative stress occurs in cells when ROS exceeds the antioxidant capacity[ 8 ]. In a previous study, we found that hyperoxia induced intestinal epithelial cells to release a large amount of ROS, leading to cell death, indicating that excessive ROS caused by hyperoxia is a major cause of oxidative damage[ 9 ]. Current evidence suggests that ferroptosis is a form of oxidative damage characterized by decreased antioxidant capacity and ROS accumulation[ 10 ]. Therefore, we speculate that ferroptosis is involved in hyperoxia-induced intestinal oxidative damage. Ferroptosis is a newly discovered form of cell death caused by the disorder of redox state in intracellular environment controlled by glutathione peroxidase 4(GPX4)[ 11 ]. When ferroptosis occurs, the antioxidant capacity of cells decreases, and ROS accumulates, resulting in lipid peroxidation of the cell membrane. Currently, most studies on ferroptosis focus on malignant tumors and degenerative diseases, with mechanistic studies primarily focusing on abnormalities in iron metabolism, lipid peroxidation, and the Kelch-like ECH-associated protein 1(Keap1)-Nuclear factor E2-related factor 2 (Nrf2) pathway[ 12 – 14 ]. Furthermore, investigations into ferroptosis in intestinal diseases mainly included intestinal ischemia/reperfusion injury and inflammatory bowel disease[ 15 – 16 ]. However, there have been no studies on ferroptosis in the process of hyperoxia-induced oxidative damage of intestinal tissue. Nrf2 is a transcription factor that plays a crucial role in the antioxidant process. It has been reported that Nrf2 can regulate ferroptosis in several ways, such as fighting ferroptosis through its targeting gene-ferritin heavy chain(FTH1)[ 17 ]. When cells are exposed to a ferroptosis inhibitor, the Keap1-Nrf2 pathway is activated, thereby reducing sensitivity to ferroptosis[ 18 ]. Additionally, Nrf2 can eliminate ROS through glutathione (GSH) metabolism and regulate the ROS level by controlling the steady flow of free ferrous ions[ 19 ]. Our previous research showed that in hyperoxia Nrf2 increased to neutralize much ROS in intestinal epithelial cells[ 20 ]. So Nrf2 might inhibite ferroptosis to protect intestinal epithelial cells in hyperoxia. Ferroptosis not only promotes cell death but also intensifies the inflammatory reaction by releasing damage-associated molecular patterns (DAMPs)[ 21 ]. This has been supported by the studies demonstrating the anti-inflammatory effect of ferroptosis inhibitors in animal models of many diseases[ 22 – 24 ]. COX-2 is a key inflammatory mediator that regulates inflammation, cell proliferation, and angiogenesis by synthesizing prostaglandins (PGs) and thromboxanes from arachidonic acid (AA)[ 25 ]. Prostaglandin E2 (PGE2) is synthesized by all human cells and plays a complex role in inflammation[ 26 ]. Yang et al. discovered that ferroptosis directly increases the expression of prostaglandin-endoperoxide synthase 2 (PTGS2) encoding cyclooxygenase-2(COX-2), accelerates the metabolism of AA, and promotes the secretion of inflammatory signal molecules[ 27 ]. Moreover, clinical studies have shown that dietary polyunsaturated fatty acids (PUFAs), especially AA, can cause intestinal inflammatory diseases such as Crohn's disease by promoting ferroptosis[ 28 – 29 ]. Hence, it is plausible that hyperoxia induces both ferroptosis and COX-2/PGE2-mediated inflammatory reactions in intestinal epithelial cells. Here, we hypothesize that Nrf2, activated by hyperoxia, participates in the regulation of ferroptosis and COX-2/PGE2-mediated inflammation in intestinal epithelial cells. To confirm this hypothesis, we systematically evaluate the effect of hyperoxia on oxidative damage and ferroptosis both in vitro and in vivo . We combine inhibitor intervention and protein expression analysis to determine the effect of Nrf2 on hyperoxia-induced ferroptosis and inflammation, as well as the underlying mechanism. The results of this study will provide a solid theoretical basis for preventing neonatal organ injury caused by hyperoxia treatment in clinical practice. 2 Materials and methods 2.1Animals The Animal Department of the Research and Development Center of Shengjing Hospital, China Medical University provided adult Sprague-Dawley (SD) rats, with a female to male mating ratio of 3 to 1. All animal experiments were conducted in accordance with the national animal protection regulations of China and the guidelines of the Animal Protection and Use Committee of China Medical University (Approval No.: 2018PS178K). And all animal experiments should be carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments or the National Research Council's Guide for the Care and Use of Laboratory Animals. Furthermore, reporting (not performance) of animal testing experiments should comply with the ARRIVE guidelines. 2.2Animal model and tissue harvest Within 12 h after birth, newborn SD rats were randomly divided into two groups: a control group (FiO 2 = 21%) and a hyperoxia group (FiO 2 = 85%), each consisting of 8 rats. To avoid differences between the groups caused by oxygen poisoning, the female rats were exchanged every 24 h. On the 3rd, 7th, 10th, and 14th day after birth, rats were randomly selected from both groups and euthanized for intestinal tissue harvesting. 2.3Cell lines and cell cultivation The NCM460 cells were cultured in RPMI 1640 incomplete medium (cat. no. Kgm31800-500 KeyGEN BioTECH, Jiangsu, China), supplemented with 10% fetal bovine serum and 1% penicillin/streptomycindoubleantibiotic solution.The cells in the logarithmic growth phase were digested and passaged. In the control group,the cells were culturedin a ordinaryincubator (FiO 2 21%, 37 ℃, 5% CO 2 ). In the hyperoxia group, after the cells were cultured in the ordinaryincubator for 24h, then were cultured in ahyperoxia incubator (FiO 2 85%, 37 ℃, 5% CO 2 )for 24h, 48h, and 72h, respectively. 2.4Immunohistochemical (IHC) staining 2.4.1. Small intestinal tissue samples A paraffin section of the intestinal tissue was taken, and dewaxing, antigen repair, blocking, and then were incubated with the primary antibodies: rabbit anti-recombinant divalent metal transporter 1(DMT1) (cat.no.20507-1-AP, Proteintech, Wuhan, China); rabbit anti-transferrin receptor(TFRC)( cat.no.A5865, Abclonal, Wuhan, China); rabbit anti-GPX4(cat.no.A13309, Abclonal, Wuhan, China); rabbit anti-FTH1(cat.no.A1144, Abclonal, Wuhan, China); rabbit anti-recombinant solute carrier family 7, member 11 (SLC7A11)(cat.no.A2413, Abclonal, Wuhan, China) were carried out at 4℃ overnight. Next the sections were in turn incubated with biotin-labeled goat anti-rabbit IgG and horseradish enzyme-labeled streptavidin working solution (cat. no. SP9001, Zhongshan Golden Bridge Biotech, Beijing, China) for 30 min. Images were taken using a light Microscopeanda Nikon image acquisition system (Eclipse NI, Nikon, Tokyo, Japan). The expressions of proteins were analyzed using Image J 1.48 (National Institutes of Health) software. 2.4.2. Intestinal epithelial cells NCM460cells were fixed on cover glass with 4% paraformaldehyde. Endogenous peroxidase of the cells was blocked with 3% H 2 O 2 and 10% goat serum. Then as described above, the cells were in turn incubated with the primary antibodies(rabbit anti-DMT1, TFRC, GPX4, FTH1, SLC7A11, respectively), biotin-labeled goat anti-rabbit IgG and horseradish enzyme-labeled streptavidin working solution. Finally, DAB and hematoxylin were used for staining. Image acquisition and analysis were carried out as described above. 2.5 Measurement of Lipid Peroxidation Levels The Lipid Peroxidation MDA Assay Kit (cat.no.BC0025,Solarbio,Beijing,China), the GSH Assay Kit (cat.no.BC1170,Solarbio,Beijing,China), and the Total SOD Assay Kit (cat.no.BC0175,Solarbio,Beijing,China) were used to analyse levels of malondialdehyde (MDA), GSH, and superoxide dismutase (SOD), respectively, following the kit instructions for all procedures. 2.6 Detection of ROS level NCM460 cells were fixed with 4% paraformaldehyde. Then the cells were incubated with PBS containing 0.1% Triton X-100 in an ice bath for 2 min. Next the cells were incubated with DCFH-DA probe in the dark at 37ºC and were observed by the fluorescence microscope. 2.7MMP assay The 5, 5', 6, 6'-tetrachloro-1, 1', 3, 3'-tetraethyl-benz imidazole carbon iodide (JC-1) fluorescent probe (cat. no. C2006, Beyotime, Shanghai, China) was used to detect MMP (mtΔΨ). NCM460 cells were fixed with 4% paraformaldehyde. Then the cells were incubated with PBS containing 0.1% Triton X-100 in an ice bath for 2 min. The cells were subsequently incubated with 0.5ml JC-1 working solution at 37℃ and were observed by the fluorescence microscope. 2.8Cell mortality rate Propidium iodide (PI) penetrates the membranes of dead cells, and can be inserted into double-stranded DNA, so the nuclei of dead cells are stained, but not living cells. And the cell mortality was measured by flow cytometry. 2.9Western blot analysis Protein was extracted from cells or intestinal tissueand quantified by BCA kit(cat.no.P0013C, Beyotime, Shanghai, China). The samples were transferred to 10%SDS-PAGE gel, then transferred to a polyvinylidene fluoride (PVDF) membranes and sealed using5% skim milk. The membranes were incubated with primary antibodies: rabbit anti - DMT1; TFRC; GPX4; FTH1; SLC7A11; Nrf2 (cat.no. ab31163, Abcam, Cambridge, USA); COX-2 (cat.no.ab179800, Abcam, Cambridge, USA); TNF alpha (cat. no. 17590-1 - AP, Proteintech, Wuhan, China); prostaglandin E receptor 4(EP4)(cat. no. 24895-1-AP, Proteintech, Wuhan, China); prostaglandin E receptor(EP2)(cat.no.ab167171, Abcam, Cambridge, USA) and mouse anti-IL-4 (cat.no.66142-1-Ig, Proteintech, Wuhan, China); IL-6 (cat.no.66146-1-Ig, Proteintech, Wuhan, China); β-actin (cat.no.66009-1-Ig, Proteintech, Wuhan, China) overnight at 4 ℃. Then the membranes were incubated with goat anti-rabbit or mouse IgG(cat.no.SA00001-2 or SA00001-1, Proteintech, Wuhan, China) and with an enhanced chemiluminescent substrate. Images were captured using Amersham Imager 680 (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Band density values were calculated using Image J 6.0 (National Institutes of Health) and normalized to β-actin. 2.10 Statistical analysis Experimental data are presented as the mean ± SD. SPSS25.0 software (IBM Corp, Armonk, NY, USA) was used for statistical analysis, and GraphPad Prism 8.0 software (GraphPad Software, San Diego, CA, USA) was used to prepare charts. The unpaired t -test was used for comparison between groups. Two-factor analysis of variance was used for comparisons between multiple groups, followed by Bonferroni post-hoc tests. 3 Results 3.1 Hyperoxia induced oxidative stress and mitochondrial injury in vivo and in vitro High concentrations of oxygen can disrupt the balance of ROS. Therefore, we first investigated hyperoxia-induced oxidative stress in the intestinal tissue of newborn rats and intestinal epithelial cells. Compared with the control group, after hyperoxia treatment, the GSH content and the activity of SOD decreased significantly, MDA content increased in rats, these differences were most significant on day 10 invivo ( P < 0.001; Fig. 1 A-C). We also evaluated hyperoxia-induced oxidative stress in vitro . The results showed that hyperoxia led to a decrease in GSH and SOD, and an increase in MDA, and after 72 h of hyperoxia treatment, the change in the oxidative stress index was the most significant ( P < 0.01 or P < 0.001; Fig. 1 D-F). Subsequently, we evaluated the ROS level in intestinal epithelial cells. Compared to the control group, the ROS level increased at 48h and peaked at 72h ( P < 0.01 or P < 0.001; Fig. 1 G-H). Finally, we explored hyperoxia-induced mitochondrial injury using the JC-1 kit. The results showed that the MMP level in intestinal epithelial cells decreased significantly at 48h and 72h ( P < 0.001; Fig. 1 I-J).In vitro and in vivo experiments indicated that hyperoxia increased the release of ROS, resulting in oxidative stress and mitochondrial injury in a time-dependent manner. 3.2 Hyperoxia induced ferroptosis in vivo and in vitro To investigate the impact of hyperoxia on ferroptosis, we examined the expression of ferroptosis-related proteins using IHC and western blot in both in vivo and in vitro settings. IHC results from rat intestinal tissue demonstrated that compared to the control group, the expressions of DMT1 and TFRC increased ( P < 0.01 or P < 0.001; Fig. 2 A-D), while GPX4 decreased from day 7 to day 14 ( P < 0.01 or P < 0.001; Fig. 2 G-H), but FTH1 increased on day 7 and decreased on day 10 and day 14( P < 0.01 or P < 0.001; Fig. 2 E-F), while SLC7A11 decreased only on day 14 ( P < 0.001; Fig. 2 I-J). However, western blot results showed compared to the control group, the expression of DMT1 increased, while GPX4, SLC7A11, and FTH1 decreased on day 10,and TFRC significantly increased on day 10 and day 14( P < 0.05, P < 0.01 or P < 0.001; Fig. 3 A-F).We also conducted protein expression analyses in vitro . The results of IHC indicated a significant increase in the expressions of DMT1 and TFRC from 24h to 72h, whereas FTH1, GPX4 and SLC7A11showed significant decreases compared to the control group ( P < 0.05 or P < 0.001; Fig. 4 A-J). Consistent with the IHC findings, western blot results showed that DMT1 and TFRC significantly increased at 72h, whereas FTH1, GPX4 and SLC7A11 showed a decrease in expression with increasing hyperoxia treatment time ( P < 0.05, P < 0.01, or P < 0.001; Fig. 5 A-F). Taken together, these findings suggest that hyperoxia-induced ferroptosis occurred in the intestinal tissue of neonatal rats and epithelial cells. 3.3Nrf2 mediated the regulation of hyperoxia-induced ferroptosis To investigate the effect of Nrf2 on hyperoxia-induced ferroptosis, we used the Nrf2 agonist tBHQ and inhibitor ML385 to up-regulate and down-regulate Nrf2, respectively. As hyperoxia time was prolonged, the levels of FTH1, GPX4, and SLC7A11 gradually decreased, while the expression of Nrf2 gradually increased ( P < 0.05, P < 0.01 or P < 0.001; Fig. 6 A-J). Compared to the hyperoxia group, the expression of FTH1, GPX4, SLC7A11 and Nrf2 significantly increased ( P < 0.05 or P < 0.01; Fig. 6 A-E) after treatment with tBHQ, but as expected, the levels of FTH1, GPX4, and SLC7A11 further decreased and the expression of Nrf2 was inhibited after treatment with ML385 ( P < 0.05 or P < 0.01; Fig. 6 F-J). These findings suggest that Nrf2 plays a role in mediating the regulation of hyperoxia-induced ferroptosis 3.4Hyperoxia activated COX-2/PGE2/EP signaling pathway and induced inflammation To investigate the effect of hyperoxia on the COX-2/PGE2/EP signaling pathway, we first assessed the expression of COX-2, EP2, and EP4 in the intestinal tissue of neonatal rats. The IHC results showed that compared to control group, in the hyperoxia group the expression of COX-2 increased on day 7, and EP4 increased on day 7 and day 10, and the expression of EP2 increased on day 3, day 10 and day 14, with the highest expression levels observed on day 10 ( P < 0.05 or P < 0.001; Fig. 7 A-F). The western blot results confirmed these findings, with the expression of COX-2, EP4, and EP2 increasing to varying degrees under hyperoxia ( P < 0.05, P < 0.01, or P < 0.001; Fig. 8 A-D), indicating the activation of the COX-2/PGE2/EP2 signaling pathway in hyperoxia. To further confirm this pathway, we used intestinal epithelial cells and observed that with an increase in hyperoxia exposure time, the expression of COX-2, EP4, and EP2 increased gradually ( P < 0.01 or P < 0.001; Fig. 8 E-F, H-I). Moreover, the level of TNF-α significantly increased at 48h and 72h ( P < 0.01; Fig. 8 E and G), suggesting that hyperoxia activates the COX-2/PGE2/EP signaling pathway and induces inflammation. 3.5 Hyperoxia-induced oxidative damage regulated inflammation through ferroptosis To elucidate the role of ferroptosis in hyperoxia-induced inflammation, we treated intestinal epithelial cells with the ferroptosis inhibitor Fer-1. Flow cytometry results showed that with increasing exposure time to hyperoxia, the cell death rate progressively increased, and after inhibiting ferroptosis, the cell death rate decreased significantly ( P < 0.05 or P < 0.01; Fig. 9 A-B). As cell damage was most pronounced at 72h, we chose this time point for further experiments. We observed that adding Fer-1 to hyperoxic cells increased the expression of GPX4 and SLC7A11, but decreased the expression of COX-2, TNF-α, EP4, and EP2 ( P < 0.05, P < 0.01or P < 0.001; Fig. 9 C-I), indicating that Fer-1 relieved hyperoxia-induced oxidative damage and suppressed hyperoxia-induced inflammation. These findings suggest that hyperoxia-induced oxidative damage regulates inflammation through ferroptosis. 3.6 Hyperoxia induced inflammation via COX-2/PGE2/EP signaling pathway To investigate the impact of COX-2 on inflammation in intestinal epithelial cells under hyperoxia, the cells were exposed to hyperoxia for 72 h and then treated with the COX-2 inhibitor Celecoxib. The results showed that the expression levels of COX-2, EP4, and EP2 were reduced in the Celecoxib-treated group compared to the hyperoxia group ( P < 0.05; Fig. 10 A-B, D-E), indicating that the COX-2 pathway was inhibited. Furthermore, the levels of the inflammatory factors TNF-α, IL-4, and IL-6 were significantly reduced ( P < 0.05 or P < 0.01; Fig. 10 A, C, F-G). These findings suggest that hyperoxia induces inflammation via the COX-2/PGE2/EP signaling pathway. 3.7COX-2 plays a pro-inflammatory role during hyperoxia via the EP2 receptor To determine the COX-2 receptor involved in hyperoxia-induced inflammation, we treated the cells with EP4 inhibitor CJ-42794 and EP2 inhibitor TG4-155 to down-regulate EP4 and EP2, respectively. The results demonstrated that compared to the hyperoxia group EP4 markedly decreased ( P 0.05; Fig. 11 A-C, E-G). However, after inhibiting EP2, the expression of COX, EP2, TNF-α, IL-4, and IL-6 markedly decreased ( P < 0.05 or P 0.05; Fig. 11 K).These results suggest that during hyperoxia, COX-2 plays a pro-inflammatory role through the EP2 receptor rather than the EP4 receptor. 4 Discussion The hyperoxic toxicity can damage the intestinal barrier of newborn rats, resulting in impaired intestinal development and function[ 5 ].As a result, long-term oxygen therapy can cause nutritional absorption disorders and even restrict the growth and development of children. In this study, we have confirmed that ferroptosis plays a significant role in hyperoxia-induced injury of the intestinal tissue, both in vitro and in vivo . Additionally, we have discovered that hyperoxia activates Nrf2 to regulate ferroptosis and mediate inflammatory reactions via the COX-2/PGE2/EP2 pathway. These findings can serve as a new experimental basis for preventing neonatal organ injury resulting from hyperoxia treatment in clinical practice. In this study, we developed a hyperoxia rat model by exposing newborn rats to hyperoxia on day 3, day 7, day 10, and day 14 after birth to investigate the mechanism of intestinal injury caused by hyperoxia. Excessive ROS can stimulate pathological redox signals leading to oxidative stress[ 30 ]. Cells develop their own antioxidant mechanisms, including several antioxidant enzymes such as the SOD enzyme family, glutathione peroxidase (GPX), and non-enzyme substances such as GSH to combat oxidative stress[ 31 ].Our results showed that compared to the control, SOD and GSH levels increased on day 3 and day 7, decreased significantly on day 10, and then increased again on the 14th day. Conversely, MDA, the end-product of lipid peroxidation, showed an opposite trend[ 32 ]. Therefore, we hypothesized that the early increase of GSH and SOD might be a response to oxidative stress induced by hyperoxia. As the exposure time to hyperoxia increased, the antioxidant capacity of the intestinal tissue of neonatal rats decreased, as evidenced by the reduction in GSH and SOD levels and the increase in MDA levels on the 10th day of hyperoxia. Additionally, hyperoxia led to the accumulation of ROS, causing oxidative stress. Mitochondria were identified as the primary source of intracellular ROS, and excessive ROS can lead to mitochondrial damage. Our findings indicate that hyperoxia causes a decrease in mitochondrial membrane potential, leading to oxidative stress and reduction of the cellular antioxidant capacity. This results in the accumulation of ROS in cells, leading to mitochondrial and cellular damage. These characteristics are also observed in ferroptosis, which leads us to speculate that ferroptosis may contribute to the intestinal oxidative damage induced by hyperoxia. Ferroptosis is an iron and oxidation-dependent form of cell death, and iron is essential for the accumulation of lipid peroxide and the onset of ferroptosis[ 33 ]. The balance of iron metabolism is maintained through the input, output, and storage of iron ions[ 34 ]. Iron input proteins include transferrin, TFRC, and DMT1, while iron output proteins include ferroportin 1 (Fpn1)[ 35 ]. Currently, it is believed that the occurrence of ferroptosis is related to abnormalities in iron metabolism, such as GPX4 inactivation, cystine/glutamate reverse transport system (System Xc-) inhibition, and lipid peroxidation, ultimately leading to an imbalance in ROS homeostasis and cell death[ 36 – 37 ]. In both in vivo and in vitro experiments, we found that hyperoxia increased DMT1 and TFRC, and decreased Nrf2 targeting gene FTH1 as well as the antioxidant protein GPX4 and SLC7A11, indicating the occurrence of ferroptosis. Nrf2 plays a crucial role in maintaining normal redox homeostasis and in mediating other metabolic pathways, including protease balance, iron/heme metabolism, lipid metabolism, and cell apoptosis[ 38 – 39 ]. So Nrf2 was involved in regulating the iron transporter and iron storage proteins[ 40 ]. And Nrf2 could directly or indirectly regulate the expression and function of GPX4[ 41 ]. In this study, we used the Nrf2 agonist tBHQ and its inhibitor ML385 to up/down-regulate its expression, respectively. The results showed that up-regulation of Nrf2 in intestinal epithelial cells inhibited the occurrence of ferroptosis under hyperoxia and played a protective role during cell injury, while down-regulation of Nrf2 had the opposite effect. These results suggest that a lot of Nrf2 protected cells from damage by inhibiting ferroptosis in hyperoxia. In ferroptosis, inflammatory mediators are produced by lipid peroxidation and AA metabolism, such as COX-2, which is also the key rate-limiting enzyme in the synthesis of PGs[ 42 – 43 ]. In this study, we detected COX-2 and its downstream molecules-EP2 and EP4, namely the two receptor subtypes of PGE2. We found that hyperoxia activated the COX-2/PGE2 pathway and up-regulated the expression of the pro-inflammatory factor TNF-α, suggesting that hyperoxia leads to ferroptosis in parallel with inflammatory damage in vivo and in vitro . In ferroptosis COX-2 is a key marker and increased significantly[ 44 – 45 ]. A recent study showed that anti-inflammatory treatment inhibited ferroptosis, and ferroptosis inhibitor Fer-1 inhibited COX-2, in other words, ferroptosis and inflammationmay also complement each other[ 46 ]. In this study Fer-1 reduced the expression of COX-2, EP4, and EP2, and reduced inflammation in intestinal epithelial cells in hyperoxia. Our findings were similar to a previous study in which Fer-1 decreased the level of ROS and alleviated inflammation[ 47 ]. So we suggest that in hyperoxia ferroptosis deteriorated oxidative damage and inflammation. To confirm the role of the COX-2/PGE2/EP pathway in hyperoxia-induced intestinal epithelial cell inflammation, we first added the COX-2 inhibitor Celecoxib to the intestinal epithelial cells. As expected, COX-2 inhibition reduced the expression of downstream receptors EP2 and EP4, and partially inhibited the inflammation caused by hyperoxia in intestinal epithelial cells. Previous studies have shown that PGE2 causes acute inflammation by relaxing vascular smooth muscle cells through the EP2/EP4 signal pathway[ 48 ]. PGE2 also promotes Th1 cell differentiation, Th17 cell proliferation, and IL-22 production of Th22 cell in vitro through EP2 and EP4 receptors[ 49 ]. This shows that EP2 and EP4 receptors play a significant role in inflammation. To explore the specific downstream receptors, EP2 and EP4 were inhibited, respectively. Interestingly, while the addition of EP2 inhibitor TG4-155 successfully inhibited the expression of COX-2 and EP2 in intestinal epithelial cells and reduced the levels of inflammation-related factors TNF-α, IL-4 and IL-6, the addition of EP4 inhibitor had no significant effect. These results indicated that the continuous up-regulation of COX-2 during hyperoxia increased the level of PGE2, which promoted inflammation through the EP2 receptor rather than the EP4 receptor. 5 Conclusions In conclusion, our study has revealed for the first time that ferroptosis is involved in the hyperoxia-induced intestinal oxidative damage both in vivo and in vitro . Furthermore, hyperoxia-activated Nrf2 regulates ferroptosis in intestinal epithelial cells and intervenes in inflammation through the COX-2/PGE2/EP2 pathway. These findings provide important experimental basis and theoretical basis for future clinical prevention and therapeutic approaches for neonatal intestinal injury caused by hyperoxia. Next, we will further study how COX-2/EP2 affects inflammation induced by hyperoxia. Declarations Competing interests The authors declare that they have no conflicts of interest. Ethical Approval and Consent to participate The Animal Department of the Research and Development Center of Shengjing Hospital, China Medical University provided adult Sprague-Dawley (SD) rats, with a female to male mating ratio of 3 to 1. All animal experiments were conducted in accordance with the national animal protection regulations of China and the guidelines of the Animal Protection and Use Committee of China Medical University (Approval No.: 2018PS178K). Consent for publication This work described has not been published in elsewhere. Its publication has been approved by all co-authors. Authors’ Contributions Liu Yanping finished the experiment and wrote the original manuscript draft. Li Tianming and Niu Changping performed the methodology and data analysis. Zhengwei Yuan revised final modifications. Sun Siyu drafted the work and revised it critically for important intellectual content drafted and revised the article critically for important intellectual content. Liu Dongyan conceptualised and designed the experiment, reviewed, and revised the manuscript, and gave the final approval of the submitted manuscript. All authors read and approved the submitted manuscript. Acknowledgements This work was supported by the National Natural Science Foundation of China [81170604];the Key Research and Development Joint Project of Liaoning Province [2020JH 2/10300136];the 345 Talent of Shengjing Hospital [M0738]; and the Free Researcher of Shengjing Hospital [MA66]. Availability of data and materials The authors confirm that the data supporting the findings of this study are available within the article [and/or its supplementary material. Funding This work was supported by the National Natural Science Foundation of China [81170604];the Key Research and Development Joint Project of Liaoning Province [2020JH 2/10300136];the 345 Talent of Shengjing Hospital [M0738]; and the Free Researcher of Shengjing Hospital [MA66]. References Ottolenghi S, Sabbatini G, Brizzolari A , et al. Hyperoxia and oxidative stress in anesthesia and critical care medicine [J]. Minerva Anestesiol, 2020, 86(1): 64-75. Zhang T, Day N J, Gaffrey M , et al. 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Cyclooxygenase Enzyme and Lipid Peroxidation Inhibitory Terpenoids and Steroidal compounds as Major Constituents in Cleome viscosa Leaves [J]. Planta Med, 2022, 88(14): 1287-92. Zhao Y, Yang Y, Liu M , et al. COX-2 is required to mediate crosstalk of ROS-dependent activation of MAPK/NF-kappaB signaling with pro-inflammatory response and defense-related NO enhancement during challenge of macrophage-like cell line with Giardia duodenalis [J]. PLoS Negl Trop Dis, 2022, 16(4): e0010402. Xu Y, Liu Y, Li K , et al. Correction to: COX-2/PGE2 Pathway Inhibits the Ferroptosis Induced by Cerebral Ischemia Reperfusion [J]. Mol Neurobiol, 2022, 59(12): 7542-3. Lin F, Chen W, Zhou J , et al. Mesenchymal stem cells protect against ferroptosis via exosome-mediated stabilization of SLC7A11 in acute liver injury [J]. Cell Death Dis, 2022, 13(3): 271. Zhang W, Lu J, Wang Y , et al. 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Cite Share Download PDF Status: Published Journal Publication published 03 Jan, 2025 Read the published version in Molecular Medicine → Version 1 posted Editorial decision: Major revision 12 Jun, 2024 Reviewers agreed at journal 22 Mar, 2024 Reviewers invited by journal 22 Mar, 2024 Editor invited by journal 21 Mar, 2024 Editor assigned by journal 20 Mar, 2024 First submitted to journal 18 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4115151","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":282697069,"identity":"79902c9f-7e38-463c-b1c8-917c91283893","order_by":0,"name":"Yanping Liu","email":"","orcid":"","institution":"Shengjing Hospital of China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yanping","middleName":"","lastName":"Liu","suffix":""},{"id":282697070,"identity":"03628317-e8ed-4769-9f54-e9519cf2b83d","order_by":1,"name":"Tianming Li","email":"","orcid":"","institution":"Shengjing Hospital of China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Tianming","middleName":"","lastName":"Li","suffix":""},{"id":282697071,"identity":"a17b6677-c5e7-48e2-8ccc-78572953c7d2","order_by":2,"name":"Changping Niu","email":"","orcid":"","institution":"Shengjing Hospital of China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Changping","middleName":"","lastName":"Niu","suffix":""},{"id":282697072,"identity":"b2b9a4b1-768d-4864-9992-4e7559b6c028","order_by":3,"name":"Zhengwei Yuan","email":"","orcid":"","institution":"Shengjing Hospital of China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhengwei","middleName":"","lastName":"Yuan","suffix":""},{"id":282697073,"identity":"cf3c5e20-98b8-45be-8695-4c2e6ae4d417","order_by":4,"name":"Siyu Sun","email":"","orcid":"","institution":"Shengjing Hospital of China Medical University","correspondingAuthor":false,"prefix":"","firstName":"Siyu","middleName":"","lastName":"Sun","suffix":""},{"id":282697074,"identity":"d9b2738b-c03d-48a7-b398-244720c69031","order_by":5,"name":"Dongyan Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7UlEQVRIiWNgGAWjYBACPmYGBgkGA5v6NmbGhgMJFQw8BLWwgbUUpDH2szMfPPDhDDFaGEBaPhxmnNnPlnxwZhsRDmNj5zG8zWNwmNngMJDknVcnY85+gPHDxxx8DuMxtuYxSGeDaNl2mMeyJ4FZcuY2vFrMpHkMgLogWg7wGBxIYGPmJayFWQKiZU4dj8H5B0RpcTaQbGZLODizgZnH4AZBW9iKLecYpCXwMzMfOPDhGNCuGw+b8fqFn//wxhtv/tgksPEfbP6QUFNnb3A++eCHj3i0gAATWvQxNuBXD1Lyg6CSUTAKRsEoGNEAAPMCSl6sV9j1AAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-3902-9316","institution":"Shengjing Hospital of China Medical University","correspondingAuthor":true,"prefix":"","firstName":"Dongyan","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2024-03-17 02:37:03","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4115151/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4115151/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s10020-024-00993-7","type":"published","date":"2025-01-03T15:57:36+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":53581251,"identity":"9deead30-686a-4b1d-b3e9-240223850b94","added_by":"auto","created_at":"2024-03-27 17:32:50","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":421603,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of hyperoxia on oxidative stress andmitochondrial injury. \u003c/strong\u003eThe levels of GSH (A, D), MDA (B, E), and SOD (C, F)\u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003einvitro\u003c/em\u003ewere detected by corresponding kit. ROS level in intestinal epithelial cells was evaluated by DCFH-DA(G). Quantification of ROS images (H). MMP level in intestinal epithelial cells was evaluated by JC-1(I). Quantification of MMP images(J).(The hyperoxia group was compared with the control group:*\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01，***\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001;Expression in the control group was compared between different time points, \u003csup\u003e△\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e△△\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01，\u003csup\u003e△△△\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001；Expression in the hyperoxia group was compared between different time points, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01，\u003csup\u003e###\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001,\u003cem\u003e n\u003c/em\u003e\u0026gt;8).\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4115151/v1/ed7b14784c73129f3492fe32.png"},{"id":53581249,"identity":"baa7da98-adb2-4a69-8103-d340bb3682da","added_by":"auto","created_at":"2024-03-27 17:32:50","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":658213,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe expressions of ferroptosis-related proteins in intestinal tissues under hyperoxia.\u003c/strong\u003e The expressions of DMT1 (A), TFRC (C), FTH1 (E), GPX4 (G), and SLC7A11 (I) wereevaluated by IHC. Quantification of IHC images of DMT1 (B), TFRC (D), FTH1 (F), GPX4 (H) and SLC7A11 (J).(The hyperoxia group was compared with the control group:*\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01，***\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001;Expression in the control group was compared between different time points, \u003csup\u003e△\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e△△\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01，\u003csup\u003e△△△\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001；Expression in the hyperoxia group was compared between different time points, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01，\u003csup\u003e###\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001,\u003cem\u003e n\u003c/em\u003e\u0026gt;8).\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4115151/v1/eda5ab9e8d93ac31da37137a.png"},{"id":53581253,"identity":"6e73c471-f3ff-408a-9cd0-9c36f5f1bdfb","added_by":"auto","created_at":"2024-03-27 17:32:50","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":280812,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of hyperoxia on ferroptosis-related proteins \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003eThe expressions of ferroptosis-related proteins wereevaluated by Western blot(A). Quantification of bands of DMT1 (B), TFRC (C), GPX4 (D), SLC7A11 (E) and FTH1 (F).(The hyperoxia group was compared with the control group:*\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01，***\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001;Expression in the hyperoxia group was compared between different time points, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01，\u003csup\u003e###\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001,\u003cem\u003e n\u003c/em\u003e\u0026gt;8).\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4115151/v1/4f64e0eb6feca8b9209b608a.png"},{"id":53581250,"identity":"b9124b63-7b42-49ca-972d-0ba3639e9b44","added_by":"auto","created_at":"2024-03-27 17:32:50","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":403940,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe ferroptosis-related proteins of intestinal epithelial cells in hyperoxia. \u003c/strong\u003eThe expressions of DMT1 (A), TFRC (C), FTH1 (E), GPX4 (G), and SLC7A11 (I) wereevaluated by IHC. Quantification of IHC images of DMT1 (B), TFRC (D), FTH1 (F), GPX4 (H), and SLC7A11 (J).(The hyperoxia group was compared with the control group:*\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01，***\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001; Expression in the hyperoxia group was compared between different time points, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01，\u003csup\u003e###\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001,\u003cem\u003e n\u003c/em\u003e\u0026gt;8).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4115151/v1/49f6f74d160c551079eb01d1.png"},{"id":53581252,"identity":"ca6dff2e-d2dc-483c-8ef7-1eeeb6d0a6d3","added_by":"auto","created_at":"2024-03-27 17:32:50","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":252957,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of hyperoxia on ferroptosis-related proteins \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003eThe expressions of ferroptosis-related proteins wereevaluated by western blot(A). Quantification of bands of DMT1 (B), TFRC (C), FTH1 (D), GPX4 (E), and SLC7A11 (F).(The hyperoxia group was compared with the control group:*\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01，***\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001; Expression in the hyperoxia group was compared between different time points, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01，\u003csup\u003e###\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001,\u003cem\u003e n\u003c/em\u003e\u0026gt;8).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4115151/v1/aad728c42cf1923a892c34a7.png"},{"id":53581257,"identity":"5d192075-cefc-46c8-9f74-9b7b75248e1f","added_by":"auto","created_at":"2024-03-27 17:32:50","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":323285,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of Nrf2 on hyperoxia-induced ferroptosis \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003eAfter up-regulating Nrf2, the expressions of ferroptosis-related proteins wereevaluated by western blot(A). Quantification of bands of FTH1 (B), GPX4 (C), SLC7A11 (D), and Nrf2 (E). After down-regulating Nrf2, the expressions of ferroptosis-related proteins wereevaluated by western blot(F). Quantification of bands of FTH1 (G), GPX4 (H), SLC7A11 (I), and Nrf2 (J).(The treatment group was compared with the hyperoxia group:*\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01，***\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001; Expression in the hyperoxia group was compared between different time points, \u003csup\u003e△\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e△△\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01，\u003csup\u003e△△△\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001；Expression in the treatment group was compared between different time points, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01，\u003csup\u003e###\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001,\u003cem\u003e n\u003c/em\u003e\u0026gt;8).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-4115151/v1/b8649d9784489499f451296e.png"},{"id":53582442,"identity":"a101100e-69e6-484a-b239-f31d1cabafb7","added_by":"auto","created_at":"2024-03-27 17:40:50","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":387450,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe COX-2/PGE2/EPof intestinal tissues in hyperoxia. \u003c/strong\u003eThe expressions ofCOX-2 (A), EP2 (C), and EP4 (E) proteins wereevaluated by IHC. Quantification of COX-2 (B), EP2 (D), and EP4 (F). (The hyperoxia group was compared with the control group:*\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01，***\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001;Expression in the control group was compared between different time points, \u003csup\u003e△\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e△△\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01，\u003csup\u003e△△△\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001；Expression in the hyperoxia group was compared between different time points, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01，\u003csup\u003e###\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001,\u003cem\u003e n\u003c/em\u003e\u0026gt;8).\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-4115151/v1/9ea5d1b3b2b2a1bb080bc79a.png"},{"id":53581255,"identity":"750352b8-8aeb-4f97-b866-b28b7075391e","added_by":"auto","created_at":"2024-03-27 17:32:50","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":383624,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of hyperoxia on COX-2/PGE2/EP \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vivo\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e and \u003c/strong\u003e\u003cem\u003e\u003cstrong\u003ein vitro\u003c/strong\u003e\u003c/em\u003e\u003cstrong\u003e. \u003c/strong\u003eThe expressions ofCOX-2, EP4, and EP2 wereevaluated by western blot\u003cem\u003ein vivo\u003c/em\u003e(A). Quantification of bands of COX-2 (B), EP2 (C), and EP4 (D). The expressions of COX-2, TNF-α, EP4and EP2proteins wereevaluated by western blot\u003cem\u003e in vitro \u003c/em\u003e(E)\u003cem\u003e.\u003c/em\u003e Quantification of bands of COX-2 (F), TNF-α (G), EP4 (H), and EP2 (I).(The hyperoxia group was compared with the control group:*\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01，***\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001;Expression in the control group was compared between different time points, \u003csup\u003e△\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e△△\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01，\u003csup\u003e△△△\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001；Expression in the hyperoxia group was compared between different time points, \u003csup\u003e#\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e##\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01，\u003csup\u003e###\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001,\u003cem\u003e n\u003c/em\u003e\u0026gt;8).\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-4115151/v1/7bc986ef3f56c6155a47b0b8.png"},{"id":53581260,"identity":"8977d9e3-440c-4b44-8fc0-c39903e911d1","added_by":"auto","created_at":"2024-03-27 17:32:51","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":496249,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of ferroptosis on COX-2/PGE2/EP in hyperoxia. \u003c/strong\u003eCell mortality rate was evaluated by flow cytometry(A). Quantification of cell mortality rate(B). The expressions ofCOX-2/PGE2/EP and ferroptosis-related proteins wereevaluated by western blot(C). Quantification of bands of COX-2 (D), TNF-α (E), GPX4 (F), SLC7A11 (G), EP4 (H), and EP2 (I).(The treatment group was compared with the hyperoxia group:\u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01，***\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001,\u003cem\u003e n\u003c/em\u003e\u0026gt;8).\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-4115151/v1/aa8f047eea69e607b3aa86e0.png"},{"id":53581254,"identity":"3f016c0d-1a9b-472c-acba-6a161a7e1f54","added_by":"auto","created_at":"2024-03-27 17:32:50","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":314964,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of COX-2 on COX-2/PGE2/EP and inflammation under hyperoxia. \u003c/strong\u003eThe expressions ofCOX-2/PGE2/EP and inflammation-related proteins wereevaluated by western blot(A). Quantification of bands of COX-2 (B), TNF-α (C), EP4 (D), EP2 (E), IL-4 (F), and IL-6 (G).(The treatment group was compared with the hyperoxia group:\u003csup\u003e*\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, \u003csup\u003e**\u003c/sup\u003e\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01，***\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001,\u003cem\u003e n\u003c/em\u003e\u0026gt;8).\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-4115151/v1/0e671218e9008d1554765cde.png"},{"id":53582443,"identity":"4dd2247f-43f5-46e9-b97e-59c86853ef69","added_by":"auto","created_at":"2024-03-27 17:40:50","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":428555,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of EP4/EP2 on COX-2/PGE2/EP and inflammation under hyperoxia. \u003c/strong\u003eAfter down-regulating EP4, the expressions of COX-2/PGE2/EP and inflammation-related proteins wereevaluated by western blot(A). Quantification of bands of COX-2 (B), TNF-α (C), EP4 (D), EP2 (E), IL-4 (F), and IL-6 (G). After down-regulating EP2, the expressions of related proteins wereevaluated by western blot(H). Quantification of bands of COX-2 (I), TNF-α (J), EP4 (K), EP2 (L), IL-4 (M) and IL-6 (N). (The treatment group was compared with the hyperoxia group:*\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.05, **\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.01，***\u003cem\u003eP\u003c/em\u003e\u0026lt; 0.001,\u003cem\u003e n\u003c/em\u003e\u0026gt;8).\u003c/p\u003e","description":"","filename":"floatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-4115151/v1/91a3534ea4ce23c18d6e1ef9.png"},{"id":73094765,"identity":"bfff2a4c-e975-4cc2-9c69-4cf5f37c36c3","added_by":"auto","created_at":"2025-01-06 16:24:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5259290,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4115151/v1/d014d5f6-59ac-4f51-917c-3752a4ed6c48.pdf"}],"financialInterests":"","formattedTitle":"Hyperoxia-activated Nrf2 regulates ferroptosis in intestinal epithelial cells and intervenes in inflammatory reaction through COX-2/PGE2/EP2 pathway","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003ePerinatal asphyxia leading to hypoxia is one of the main causes of neonatal morbidity and mortality, often requiring oxygen inhalation therapy (hyperoxia). However, hyperoxia for long time has been proven to be toxic[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e], causing damage to neonatal lung, retina, and nerve development[\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Researchers have also recently focused on the intestinal injury caused by hyperoxic toxicity. A previous study showed that after neonatal exposure to hyperoxia, the number of goblet cells in the intestines decreased, and the mucus layer was severely damaged, leading to higher intestinal permeability and bacterial translocation[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Moreover, under hyperoxia, there was a significant increase in the levels of interferon-gamma (IFN-γ) and interleukin 10 (IL-10), indicating intestinal injury[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Given that the development of the intestines plays a crucial role in early-stage nutrient absorption, it is important to investigate the effects and molecular mechanisms of hyperoxic toxicity on the newborn intestine.\u003c/p\u003e \u003cp\u003eUnder physiological conditions, the amount of reactive oxygen species (ROS) is balanced with antioxidant defense, precisely controlled by cells to avoid oxidative stress and cell damage[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. However, under hyperoxia, ROS increases, and oxidative stress occurs in cells when ROS exceeds the antioxidant capacity[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In a previous study, we found that hyperoxia induced intestinal epithelial cells to release a large amount of ROS, leading to cell death, indicating that excessive ROS caused by hyperoxia is a major cause of oxidative damage[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Current evidence suggests that ferroptosis is a form of oxidative damage characterized by decreased antioxidant capacity and ROS accumulation[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Therefore, we speculate that ferroptosis is involved in hyperoxia-induced intestinal oxidative damage.\u003c/p\u003e \u003cp\u003eFerroptosis is a newly discovered form of cell death caused by the disorder of redox state in intracellular environment controlled by glutathione peroxidase 4(GPX4)[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. When ferroptosis occurs, the antioxidant capacity of cells decreases, and ROS accumulates, resulting in lipid peroxidation of the cell membrane. Currently, most studies on ferroptosis focus on malignant tumors and degenerative diseases, with mechanistic studies primarily focusing on abnormalities in iron metabolism, lipid peroxidation, and the Kelch-like ECH-associated protein 1(Keap1)-Nuclear factor E2-related factor 2 (Nrf2) pathway[\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Furthermore, investigations into ferroptosis in intestinal diseases mainly included intestinal ischemia/reperfusion injury and inflammatory bowel disease[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. However, there have been no studies on ferroptosis in the process of hyperoxia-induced oxidative damage of intestinal tissue.\u003c/p\u003e \u003cp\u003eNrf2 is a transcription factor that plays a crucial role in the antioxidant process. It has been reported that Nrf2 can regulate ferroptosis in several ways, such as fighting ferroptosis through its targeting gene-ferritin heavy chain(FTH1)[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. When cells are exposed to a ferroptosis inhibitor, the Keap1-Nrf2 pathway is activated, thereby reducing sensitivity to ferroptosis[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Additionally, Nrf2 can eliminate ROS through glutathione (GSH) metabolism and regulate the ROS level by controlling the steady flow of free ferrous ions[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Our previous research showed that in hyperoxia Nrf2 increased to neutralize much ROS in intestinal epithelial cells[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. So Nrf2 might inhibite ferroptosis to protect intestinal epithelial cells in hyperoxia.\u003c/p\u003e \u003cp\u003eFerroptosis not only promotes cell death but also intensifies the inflammatory reaction by releasing damage-associated molecular patterns (DAMPs)[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. This has been supported by the studies demonstrating the anti-inflammatory effect of ferroptosis inhibitors in animal models of many diseases[\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. COX-2 is a key inflammatory mediator that regulates inflammation, cell proliferation, and angiogenesis by synthesizing prostaglandins (PGs) and thromboxanes from arachidonic acid (AA)[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Prostaglandin E2 (PGE2) is synthesized by all human cells and plays a complex role in inflammation[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Yang et al. discovered that ferroptosis directly increases the expression of prostaglandin-endoperoxide synthase 2 (PTGS2) encoding cyclooxygenase-2(COX-2), accelerates the metabolism of AA, and promotes the secretion of inflammatory signal molecules[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Moreover, clinical studies have shown that dietary polyunsaturated fatty acids (PUFAs), especially AA, can cause intestinal inflammatory diseases such as Crohn's disease by promoting ferroptosis[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Hence, it is plausible that hyperoxia induces both ferroptosis and COX-2/PGE2-mediated inflammatory reactions in intestinal epithelial cells.\u003c/p\u003e \u003cp\u003eHere, we hypothesize that Nrf2, activated by hyperoxia, participates in the regulation of ferroptosis and COX-2/PGE2-mediated inflammation in intestinal epithelial cells. To confirm this hypothesis, we systematically evaluate the effect of hyperoxia on oxidative damage and ferroptosis both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. We combine inhibitor intervention and protein expression analysis to determine the effect of Nrf2 on hyperoxia-induced ferroptosis and inflammation, as well as the underlying mechanism. The results of this study will provide a solid theoretical basis for preventing neonatal organ injury caused by hyperoxia treatment in clinical practice.\u003c/p\u003e"},{"header":"2 Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1Animals\u003c/h2\u003e \u003cp\u003eThe Animal Department of the Research and Development Center of Shengjing Hospital, China Medical University provided adult Sprague-Dawley (SD) rats, with a female to male mating ratio of 3 to 1. All animal experiments were conducted in accordance with the national animal protection regulations of China and the guidelines of the Animal Protection and Use Committee of China Medical University (Approval No.: 2018PS178K). And all animal experiments should be carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments or the National Research Council's Guide for the Care and Use of Laboratory Animals. Furthermore, reporting (not performance) of animal testing experiments should comply with the ARRIVE guidelines.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2Animal model and tissue harvest\u003c/h2\u003e \u003cp\u003eWithin 12 h after birth, newborn SD rats were randomly divided into two groups: a control group (FiO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;21%) and a hyperoxia group (FiO\u003csub\u003e2\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;85%), each consisting of 8 rats. To avoid differences between the groups caused by oxygen poisoning, the female rats were exchanged every 24 h. On the 3rd, 7th, 10th, and 14th day after birth, rats were randomly selected from both groups and euthanized for intestinal tissue harvesting.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3Cell lines and cell cultivation\u003c/h2\u003e \u003cp\u003eThe NCM460 cells were cultured in RPMI 1640 incomplete medium (cat. no. Kgm31800-500 KeyGEN BioTECH, Jiangsu, China), supplemented with 10% fetal bovine serum and 1% penicillin/streptomycindoubleantibiotic solution.The cells in the logarithmic growth phase were digested and passaged. In the control group,the cells were culturedin a ordinaryincubator (FiO\u003csub\u003e2\u003c/sub\u003e 21%, 37 ℃, 5% CO\u003csub\u003e2\u003c/sub\u003e). In the hyperoxia group, after the cells were cultured in the ordinaryincubator for 24h, then were cultured in ahyperoxia incubator (FiO\u003csub\u003e2\u003c/sub\u003e85%, 37 ℃, 5% CO\u003csub\u003e2\u003c/sub\u003e)for 24h, 48h, and 72h, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4Immunohistochemical (IHC) staining\u003c/h2\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1. Small intestinal tissue samples\u003c/h2\u003e \u003cp\u003eA paraffin section of the intestinal tissue was taken, and dewaxing, antigen repair, blocking, and then were incubated with the primary antibodies: rabbit anti-recombinant divalent metal transporter 1(DMT1) (cat.no.20507-1-AP, Proteintech, Wuhan, China); rabbit anti-transferrin receptor(TFRC)( cat.no.A5865, Abclonal, Wuhan, China); rabbit anti-GPX4(cat.no.A13309, Abclonal, Wuhan, China); rabbit anti-FTH1(cat.no.A1144, Abclonal, Wuhan, China); rabbit anti-recombinant solute carrier family 7, member 11 (SLC7A11)(cat.no.A2413, Abclonal, Wuhan, China) were carried out at 4℃ overnight. Next the sections were in turn incubated with biotin-labeled goat anti-rabbit IgG and horseradish enzyme-labeled streptavidin working solution (cat. no. SP9001, Zhongshan Golden Bridge Biotech, Beijing, China) for 30 min. Images were taken using a light Microscopeanda Nikon image acquisition system (Eclipse NI, Nikon, Tokyo, Japan). The expressions of proteins were analyzed using Image J 1.48 (National Institutes of Health) software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2. Intestinal epithelial cells\u003c/h2\u003e \u003cp\u003eNCM460cells were fixed on cover glass with 4% paraformaldehyde. Endogenous peroxidase of the cells was blocked with 3% H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e and 10% goat serum. Then as described above, the cells were in turn incubated with the primary antibodies(rabbit anti-DMT1, TFRC, GPX4, FTH1, SLC7A11, respectively), biotin-labeled goat anti-rabbit IgG and horseradish enzyme-labeled streptavidin working solution. Finally, DAB and hematoxylin were used for staining. Image acquisition and analysis were carried out as described above.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Measurement of Lipid Peroxidation Levels\u003c/h2\u003e \u003cp\u003eThe Lipid Peroxidation MDA Assay Kit (cat.no.BC0025,Solarbio,Beijing,China), the GSH Assay Kit (cat.no.BC1170,Solarbio,Beijing,China), and the Total SOD Assay Kit (cat.no.BC0175,Solarbio,Beijing,China) were used to analyse levels of malondialdehyde (MDA), GSH, and superoxide dismutase (SOD), respectively, following the kit instructions for all procedures.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Detection of ROS level\u003c/h2\u003e \u003cp\u003eNCM460 cells were fixed with 4% paraformaldehyde. Then the cells were incubated with PBS containing 0.1% Triton X-100 in an ice bath for 2 min. Next the cells were incubated with DCFH-DA probe in the dark at 37\u0026ordm;C and were observed by the fluorescence microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.7MMP assay\u003c/h2\u003e \u003cp\u003eThe 5, 5', 6, 6'-tetrachloro-1, 1', 3, 3'-tetraethyl-benz imidazole carbon iodide (JC-1) fluorescent probe (cat. no. C2006, Beyotime, Shanghai, China) was used to detect MMP (mtΔΨ). NCM460 cells were fixed with 4% paraformaldehyde. Then the cells were incubated with PBS containing 0.1% Triton X-100 in an ice bath for 2 min. The cells were subsequently incubated with 0.5ml JC-1 working solution at 37℃ and were observed by the fluorescence microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.8Cell mortality rate\u003c/h2\u003e \u003cp\u003ePropidium iodide (PI) penetrates the membranes of dead cells, and can be inserted into double-stranded DNA, so the nuclei of dead cells are stained, but not living cells. And the cell mortality was measured by flow cytometry.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.9Western blot analysis\u003c/h2\u003e \u003cp\u003eProtein was extracted from cells or intestinal tissueand quantified by BCA kit(cat.no.P0013C, Beyotime, Shanghai, China). The samples were transferred to 10%SDS-PAGE gel, then transferred to a polyvinylidene fluoride (PVDF) membranes and sealed using5% skim milk. The membranes were incubated with primary antibodies: rabbit anti - DMT1; TFRC; GPX4; FTH1; SLC7A11; Nrf2 (cat.no. ab31163, Abcam, Cambridge, USA); COX-2 (cat.no.ab179800, Abcam, Cambridge, USA); TNF alpha (cat. no. 17590-1 - AP, Proteintech, Wuhan, China); prostaglandin E receptor 4(EP4)(cat. no. 24895-1-AP, Proteintech, Wuhan, China); prostaglandin E receptor(EP2)(cat.no.ab167171, Abcam, Cambridge, USA) and mouse anti-IL-4 (cat.no.66142-1-Ig, Proteintech, Wuhan, China); IL-6 (cat.no.66146-1-Ig, Proteintech, Wuhan, China); β-actin (cat.no.66009-1-Ig, Proteintech, Wuhan, China) overnight at 4 ℃. Then the membranes were incubated with goat anti-rabbit or mouse IgG(cat.no.SA00001-2 or SA00001-1, Proteintech, Wuhan, China) and with an enhanced chemiluminescent substrate. Images were captured using Amersham Imager 680 (GE Healthcare Life Sciences, Pittsburgh, PA, USA). Band density values were calculated using Image J 6.0 (National Institutes of Health) and normalized to β-actin.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Statistical analysis\u003c/h2\u003e \u003cp\u003eExperimental data are presented as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. SPSS25.0 software (IBM Corp, Armonk, NY, USA) was used for statistical analysis, and GraphPad Prism 8.0 software (GraphPad Software, San Diego, CA, USA) was used to prepare charts. The unpaired \u003cem\u003et\u003c/em\u003e-test was used for comparison between groups. Two-factor analysis of variance was used for comparisons between multiple groups, followed by Bonferroni post-hoc tests.\u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Hyperoxia induced oxidative stress and mitochondrial injury \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eHigh concentrations of oxygen can disrupt the balance of ROS. Therefore, we first investigated hyperoxia-induced oxidative stress in the intestinal tissue of newborn rats and intestinal epithelial cells. Compared with the control group, after hyperoxia treatment, the GSH content and the activity of SOD decreased significantly, MDA content increased in rats, these differences were most significant on day 10\u003cem\u003einvivo\u003c/em\u003e(\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-C). We also evaluated hyperoxia-induced oxidative stress \u003cem\u003ein vitro\u003c/em\u003e. The results showed that hyperoxia led to a decrease in GSH and SOD, and an increase in MDA, and after 72 h of hyperoxia treatment, the change in the oxidative stress index was the most significant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 or \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-F). Subsequently, we evaluated the ROS level in intestinal epithelial cells. Compared to the control group, the ROS level increased at 48h and peaked at 72h (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 or \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eG-H). Finally, we explored hyperoxia-induced mitochondrial injury using the JC-1 kit. The results showed that the MMP level in intestinal epithelial cells decreased significantly at 48h and 72h (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eI-J).In vitro and in vivo experiments indicated that hyperoxia increased the release of ROS, resulting in oxidative stress and mitochondrial injury in a time-dependent manner.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Hyperoxia induced ferroptosis \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e\u003c/h2\u003e \u003cp\u003eTo investigate the impact of hyperoxia on ferroptosis, we examined the expression of ferroptosis-related proteins using IHC and western blot in both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e settings. IHC results from rat intestinal tissue demonstrated that compared to the control group, the expressions of DMT1 and TFRC increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 or \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-D), while GPX4 decreased from day 7 to day 14 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 or \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG-H), but FTH1 increased on day 7 and decreased on day 10 and day 14(\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 or \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE-F), while SLC7A11 decreased only on day 14 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eI-J). However, western blot results showed compared to the control group, the expression of DMT1 increased, while GPX4, SLC7A11, and FTH1 decreased on day 10,and TFRC significantly increased on day 10 and day 14(\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 or \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-F).We also conducted protein expression analyses \u003cem\u003ein vitro\u003c/em\u003e. The results of IHC indicated a significant increase in the expressions of DMT1 and TFRC from 24h to 72h, whereas FTH1, GPX4 and SLC7A11showed significant decreases compared to the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-J). Consistent with the IHC findings, western blot results showed that DMT1 and TFRC significantly increased at 72h, whereas FTH1, GPX4 and SLC7A11 showed a decrease in expression with increasing hyperoxia treatment time (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, or \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-F). Taken together, these findings suggest that hyperoxia-induced ferroptosis occurred in the intestinal tissue of neonatal rats and epithelial cells.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.3Nrf2 mediated the regulation of hyperoxia-induced ferroptosis\u003c/h2\u003e \u003cp\u003eTo investigate the effect of Nrf2 on hyperoxia-induced ferroptosis, we used the Nrf2 agonist tBHQ and inhibitor ML385 to up-regulate and down-regulate Nrf2, respectively. As hyperoxia time was prolonged, the levels of FTH1, GPX4, and SLC7A11 gradually decreased, while the expression of Nrf2 gradually increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 or \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-J). Compared to the hyperoxia group, the expression of FTH1, GPX4, SLC7A11 and Nrf2 significantly increased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eA-E) after treatment with tBHQ, but as expected, the levels of FTH1, GPX4, and SLC7A11 further decreased and the expression of Nrf2 was inhibited after treatment with ML385 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eF-J). These findings suggest that Nrf2 plays a role in mediating the regulation of hyperoxia-induced ferroptosis\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.4Hyperoxia activated COX-2/PGE2/EP signaling pathway and induced inflammation\u003c/h2\u003e \u003cp\u003eTo investigate the effect of hyperoxia on the COX-2/PGE2/EP signaling pathway, we first assessed the expression of COX-2, EP2, and EP4 in the intestinal tissue of neonatal rats. The IHC results showed that compared to control group, in the hyperoxia group the expression of COX-2 increased on day 7, and EP4 increased on day 7 and day 10, and the expression of EP2 increased on day 3, day 10 and day 14, with the highest expression levels observed on day 10 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA-F). The western blot results confirmed these findings, with the expression of COX-2, EP4, and EP2 increasing to varying degrees under hyperoxia (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01, or \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA-D), indicating the activation of the COX-2/PGE2/EP2 signaling pathway in hyperoxia. To further confirm this pathway, we used intestinal epithelial cells and observed that with an increase in hyperoxia exposure time, the expression of COX-2, EP4, and EP2 increased gradually (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01 or \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE-F, H-I). Moreover, the level of TNF-α significantly increased at 48h and 72h (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eE and G), suggesting that hyperoxia activates the COX-2/PGE2/EP signaling pathway and induces inflammation.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.5 Hyperoxia-induced oxidative damage regulated inflammation through ferroptosis\u003c/h2\u003e \u003cp\u003eTo elucidate the role of ferroptosis in hyperoxia-induced inflammation, we treated intestinal epithelial cells with the ferroptosis inhibitor Fer-1. Flow cytometry results showed that with increasing exposure time to hyperoxia, the cell death rate progressively increased, and after inhibiting ferroptosis, the cell death rate decreased significantly (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA-B). As cell damage was most pronounced at 72h, we chose this time point for further experiments. We observed that adding Fer-1 to hyperoxic cells increased the expression of GPX4 and SLC7A11, but decreased the expression of COX-2, TNF-α, EP4, and EP2 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01or \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eC-I), indicating that Fer-1 relieved hyperoxia-induced oxidative damage and suppressed hyperoxia-induced inflammation. These findings suggest that hyperoxia-induced oxidative damage regulates inflammation through ferroptosis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.6 Hyperoxia induced inflammation via COX-2/PGE2/EP signaling pathway\u003c/h2\u003e \u003cp\u003eTo investigate the impact of COX-2 on inflammation in intestinal epithelial cells under hyperoxia, the cells were exposed to hyperoxia for 72 h and then treated with the COX-2 inhibitor Celecoxib. The results showed that the expression levels of COX-2, EP4, and EP2 were reduced in the Celecoxib-treated group compared to the hyperoxia group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eA-B, D-E), indicating that the COX-2 pathway was inhibited. Furthermore, the levels of the inflammatory factors TNF-α, IL-4, and IL-6 were significantly reduced (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eA, C, F-G). These findings suggest that hyperoxia induces inflammation via the COX-2/PGE2/EP signaling pathway.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e3.7COX-2 plays a pro-inflammatory role during hyperoxia via the EP2 receptor\u003c/h2\u003e \u003cp\u003eTo determine the COX-2 receptor involved in hyperoxia-induced inflammation, we treated the cells with EP4 inhibitor CJ-42794 and EP2 inhibitor TG4-155 to down-regulate EP4 and EP2, respectively. The results demonstrated that compared to the hyperoxia group EP4 markedly decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eD), but there was no statistical difference in the expression of COX, EP2, TNF-α, IL-4 and IL-6 after adding CJ-42794 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eA-C, E-G). However, after inhibiting EP2, the expression of COX, EP2, TNF-α, IL-4, and IL-6 markedly decreased (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 or \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01; Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eH-N),except for EP4 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05; Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eK).These results suggest that during hyperoxia, COX-2 plays a pro-inflammatory role through the EP2 receptor rather than the EP4 receptor.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eThe hyperoxic toxicity can damage the intestinal barrier of newborn rats, resulting in impaired intestinal development and function[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].As a result, long-term oxygen therapy can cause nutritional absorption disorders and even restrict the growth and development of children. In this study, we have confirmed that ferroptosis plays a significant role in hyperoxia-induced injury of the intestinal tissue, both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. Additionally, we have discovered that hyperoxia activates Nrf2 to regulate ferroptosis and mediate inflammatory reactions via the COX-2/PGE2/EP2 pathway. These findings can serve as a new experimental basis for preventing neonatal organ injury resulting from hyperoxia treatment in clinical practice.\u003c/p\u003e \u003cp\u003eIn this study, we developed a hyperoxia rat model by exposing newborn rats to hyperoxia on day 3, day 7, day 10, and day 14 after birth to investigate the mechanism of intestinal injury caused by hyperoxia. Excessive ROS can stimulate pathological redox signals leading to oxidative stress[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. Cells develop their own antioxidant mechanisms, including several antioxidant enzymes such as the SOD enzyme family, glutathione peroxidase (GPX), and non-enzyme substances such as GSH to combat oxidative stress[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].Our results showed that compared to the control, SOD and GSH levels increased on day 3 and day 7, decreased significantly on day 10, and then increased again on the 14th day. Conversely, MDA, the end-product of lipid peroxidation, showed an opposite trend[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Therefore, we hypothesized that the early increase of GSH and SOD might be a response to oxidative stress induced by hyperoxia. As the exposure time to hyperoxia increased, the antioxidant capacity of the intestinal tissue of neonatal rats decreased, as evidenced by the reduction in GSH and SOD levels and the increase in MDA levels on the 10th day of hyperoxia. Additionally, hyperoxia led to the accumulation of ROS, causing oxidative stress. Mitochondria were identified as the primary source of intracellular ROS, and excessive ROS can lead to mitochondrial damage. Our findings indicate that hyperoxia causes a decrease in mitochondrial membrane potential, leading to oxidative stress and reduction of the cellular antioxidant capacity. This results in the accumulation of ROS in cells, leading to mitochondrial and cellular damage. These characteristics are also observed in ferroptosis, which leads us to speculate that ferroptosis may contribute to the intestinal oxidative damage induced by hyperoxia.\u003c/p\u003e \u003cp\u003eFerroptosis is an iron and oxidation-dependent form of cell death, and iron is essential for the accumulation of lipid peroxide and the onset of ferroptosis[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The balance of iron metabolism is maintained through the input, output, and storage of iron ions[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Iron input proteins include transferrin, TFRC, and DMT1, while iron output proteins include ferroportin 1 (Fpn1)[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Currently, it is believed that the occurrence of ferroptosis is related to abnormalities in iron metabolism, such as GPX4 inactivation, cystine/glutamate reverse transport system (System Xc-) inhibition, and lipid peroxidation, ultimately leading to an imbalance in ROS homeostasis and cell death[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. In both \u003cem\u003ein vivo\u003c/em\u003e and in \u003cem\u003evitro\u003c/em\u003e experiments, we found that hyperoxia increased DMT1 and TFRC, and decreased Nrf2 targeting gene FTH1 as well as the antioxidant protein GPX4 and SLC7A11, indicating the occurrence of ferroptosis. Nrf2 plays a crucial role in maintaining normal redox homeostasis and in mediating other metabolic pathways, including protease balance, iron/heme metabolism, lipid metabolism, and cell apoptosis[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. So Nrf2 was involved in regulating the iron transporter and iron storage proteins[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. And Nrf2 could directly or indirectly regulate the expression and function of GPX4[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. In this study, we used the Nrf2 agonist tBHQ and its inhibitor ML385 to up/down-regulate its expression, respectively. The results showed that up-regulation of Nrf2 in intestinal epithelial cells inhibited the occurrence of ferroptosis under hyperoxia and played a protective role during cell injury, while down-regulation of Nrf2 had the opposite effect. These results suggest that a lot of Nrf2 protected cells from damage by inhibiting ferroptosis in hyperoxia.\u003c/p\u003e \u003cp\u003eIn ferroptosis, inflammatory mediators are produced by lipid peroxidation and AA metabolism, such as COX-2, which is also the key rate-limiting enzyme in the synthesis of PGs[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. In this study, we detected COX-2 and its downstream molecules-EP2 and EP4, namely the two receptor subtypes of PGE2. We found that hyperoxia activated the COX-2/PGE2 pathway and up-regulated the expression of the pro-inflammatory factor TNF-α, suggesting that hyperoxia leads to ferroptosis in parallel with inflammatory damage \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e. In ferroptosis COX-2 is a key marker and increased significantly[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. A recent study showed that anti-inflammatory treatment inhibited ferroptosis, and ferroptosis inhibitor Fer-1 inhibited COX-2, in other words, ferroptosis and inflammationmay also complement each other[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. In this study Fer-1 reduced the expression of COX-2, EP4, and EP2, and reduced inflammation in intestinal epithelial cells in hyperoxia. Our findings were similar to a previous study in which Fer-1 decreased the level of ROS and alleviated inflammation[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. So we suggest that in hyperoxia ferroptosis deteriorated oxidative damage and inflammation.\u003c/p\u003e \u003cp\u003eTo confirm the role of the COX-2/PGE2/EP pathway in hyperoxia-induced intestinal epithelial cell inflammation, we first added the COX-2 inhibitor Celecoxib to the intestinal epithelial cells. As expected, COX-2 inhibition reduced the expression of downstream receptors EP2 and EP4, and partially inhibited the inflammation caused by hyperoxia in intestinal epithelial cells. Previous studies have shown that PGE2 causes acute inflammation by relaxing vascular smooth muscle cells through the EP2/EP4 signal pathway[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. PGE2 also promotes Th1 cell differentiation, Th17 cell proliferation, and IL-22 production of Th22 cell \u003cem\u003ein vitro\u003c/em\u003e through EP2 and EP4 receptors[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. This shows that EP2 and EP4 receptors play a significant role in inflammation. To explore the specific downstream receptors, EP2 and EP4 were inhibited, respectively. Interestingly, while the addition of EP2 inhibitor TG4-155 successfully inhibited the expression of COX-2 and EP2 in intestinal epithelial cells and reduced the levels of inflammation-related factors TNF-α, IL-4 and IL-6, the addition of EP4 inhibitor had no significant effect. These results indicated that the continuous up-regulation of COX-2 during hyperoxia increased the level of PGE2, which promoted inflammation through the EP2 receptor rather than the EP4 receptor.\u003c/p\u003e"},{"header":"5 Conclusions","content":"\u003cp\u003eIn conclusion, our study has revealed for the first time that ferroptosis is involved in the hyperoxia-induced intestinal oxidative damage both \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e. Furthermore, hyperoxia-activated Nrf2 regulates ferroptosis in intestinal epithelial cells and intervenes in inflammation through the COX-2/PGE2/EP2 pathway. These findings provide important experimental basis and theoretical basis for future clinical prevention and therapeutic approaches for neonatal intestinal injury caused by hyperoxia. Next, we will further study how COX-2/EP2 affects inflammation induced by hyperoxia.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Approval and Consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe Animal Department of the Research and Development Center of Shengjing Hospital, China Medical University provided adult Sprague-Dawley (SD) rats, with a female to male mating ratio of 3 to 1. All animal experiments were conducted in accordance with the national animal protection regulations of China and the guidelines of the Animal Protection and Use Committee of China Medical University (Approval No.: 2018PS178K).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work described has not been published in\u0026nbsp;elsewhere. Its\u0026nbsp;publication\u0026nbsp;has been approved by all co-authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLiu Yanping finished the experiment and wrote the original manuscript draft. Li Tianming and Niu Changping performed the methodology and data analysis. Zhengwei Yuan revised final modifications. Sun Siyu drafted the work and revised it critically for important intellectual content drafted and revised the article critically for important intellectual content. Liu Dongyan conceptualised and designed the experiment, reviewed, and revised the manuscript, and gave the final approval of the submitted manuscript. All authors read and approved the submitted manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China [81170604];the Key Research and Development Joint Project of Liaoning Province [2020JH 2/10300136];the 345 Talent of Shengjing Hospital [M0738]; and the Free Researcher of Shengjing Hospital [MA66].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors confirm that the data supporting the findings of this study are available within the article [and/or its supplementary material.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China [81170604];the Key Research and Development Joint Project of Liaoning Province [2020JH 2/10300136];the 345 Talent of Shengjing Hospital [M0738]; and the Free Researcher of Shengjing Hospital [MA66].\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eOttolenghi S, Sabbatini G, Brizzolari A\u003cem\u003e, et al.\u003c/em\u003e Hyperoxia and oxidative stress in anesthesia and critical care medicine [J]. 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The Journal of allergy and clinical immunology, 2019, 143(2): 631-43.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mome","sideBox":"Learn more about [Molecular Medicine](https://molmed.biomedcentral.com)","snPcode":"10020","submissionUrl":"https://submission.springernature.com/new-submission/10020/3","title":"Molecular Medicine","twitterHandle":"@MolecularMedic1","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"hyperoxia, intestinal epithelial cells, ferroptosis, COX-2/PGE2/EP2 pathway, inflammatory","lastPublishedDoi":"10.21203/rs.3.rs-4115151/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4115151/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe lack of knowledge about the mechanism of hyperoxia-induced intestinal injury has attracted considerable attention, due to the potential for this condition to cause neonatal complications. This study aimed to explore the relationship between hyperoxia-induced oxidative damage and ferroptosis in intestinal tissue and investigate the mechanism by which hyperoxia regulates inflammation through ferroptosis. The study systematically evaluated the effects of hyperoxia on oxidative stress, mitochondrial damage, ferroptosis, and inflammation of intestinal epithelial cells both \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e. The results showed that ferroptosis was involved in intestinal oxidative damage caused by hyperoxia and was regulated by Nrf2. Moreover, hyperoxia-induced oxidative damage regulated inflammation through ferroptosis by upregulating the COX-2/PGE2/EP2 signaling pathway. These findings have important implications for future clinical prevention and therapeutic approaches to neonatal organ injury caused by hyperoxia treatment.\u003c/p\u003e","manuscriptTitle":"Hyperoxia-activated Nrf2 regulates ferroptosis in intestinal epithelial cells and intervenes in inflammatory reaction through COX-2/PGE2/EP2 pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-27 17:32:45","doi":"10.21203/rs.3.rs-4115151/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2024-06-12T10:02:02+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2024-03-22T13:19:34+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-03-22T12:45:38+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Molecular Medicine","date":"2024-03-21T14:56:31+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-21T00:20:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Medicine","date":"2024-03-18T22:23:53+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mome","sideBox":"Learn more about [Molecular Medicine](https://molmed.biomedcentral.com)","snPcode":"10020","submissionUrl":"https://submission.springernature.com/new-submission/10020/3","title":"Molecular Medicine","twitterHandle":"@MolecularMedic1","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a6b89362-b300-46fe-a926-8353ce8173c0","owner":[],"postedDate":"March 27th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-01-06T16:22:12+00:00","versionOfRecord":{"articleIdentity":"rs-4115151","link":"https://doi.org/10.1186/s10020-024-00993-7","journal":{"identity":"molecular-medicine","isVorOnly":false,"title":"Molecular Medicine"},"publishedOn":"2025-01-03 15:57:36","publishedOnDateReadable":"January 3rd, 2025"},"versionCreatedAt":"2024-03-27 17:32:45","video":"","vorDoi":"10.1186/s10020-024-00993-7","vorDoiUrl":"https://doi.org/10.1186/s10020-024-00993-7","workflowStages":[]},"version":"v1","identity":"rs-4115151","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4115151","identity":"rs-4115151","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}