Sodium butyrate inhibits ferroptosis and ameliorates intestinal ischemia-reperfusion injury by modulating the NRF2/SLC7A11/GPX4 pathway

Sodium butyrate inhibits ferroptosis and ameliorates intestinal ischemia-reperfusion injury by modulating the NRF2/SLC7A11/GPX4 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 Sodium butyrate inhibits ferroptosis and ameliorates intestinal ischemia-reperfusion injury by modulating the NRF2/SLC7A11/GPX4 pathway Zicen Zhao, Yufang Leng, Liya Chang, Yu Wang, Dongbin Li, Yang Xing, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3988128/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose Sodium butyrate, a short-chain fatty acid produced by the metabolism of intestinal flora, has been shown to have a protective effect against intestinal ischemia reperfusion injury (IRI), but its effect on intestinal IRI-associated ferroptosis has not yet been demonstrated. Methods In this experiment, we used a model of superior mesenteric artery occlusion in mice to examine the effects and principles of sodium butyrate on ferroptosis related to intestinal IRI. We assessed the role of sodium butyrate by constructing mice intestinal IRI models and determining the extent of intestinal tissue damage and changes in ferroptosis-related factors. Results Results show that Intestinal IRI mice showed increased tissue damage, massive infiltration of inflammatory cells, severe destruction of villus structure, and elevated MDA. In addition, intestinal IRI led to downregulation of GSH, GPX4, FTH1 and SLC7A11, regulators of ferroptosis, and a rise in Fe 2+ , as well as downregulation of NRF2 by intestinal IRI. Sodium butyrate had an attenuating effect on intestinal IRI, whereas mice exogenously supplemented with sodium butyrate showed less tissue damage, some restoration of villus structure and decreased MDA, up-regulation of GSH, GSH/GSSG, GPX4, FTH1, SLC7A11, and NRF2, and decreased Fe 2+ . Conclusion This experiment demonstrates that sodium butyrate regulates the SLC7A11/GPX4 pathway by up-regulating NRF2, thereby inhibiting ferroptosis and attenuating intestinal IRI. gut intestinal ischemia reperfusion injury sodium butyrate ferroptosis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The gut plays a crucial role in maintaining physiological homeostasis and performing various functions such as nutrient absorption, toxin excretion, immune homeostasis, and hormone release[ 1 ]. However, critical illnesses such as traumatic shock, severe infections, and severe burns can result in intestinal IRI. This injury occurs when intestinal flora translocates and there is a massive release of endotoxins and inflammatory factors during blood reperfusion. These events not only harm the intestinal mucosa but also lead to extra-intestinal multi-organ dysfunction or failure, which can worsen the patient's condition and even contribute to their death[ 2 ]. Current research shows that targeting intestinal IRI is an Important factor in addressing the risk of death in high-risk patients, but no systematic treatment approach has been developed to date. Therefore, the finding of new therapeutic strategies to mitigate intestinal IRI is an essential direction of its research. Numerous experiments have demonstrated the significance of programmed cell death (PCD) in intestinal IRI [ 3 – 6 ]. One type of PCD is ferroptosis, which is an iron-dependent form of cell death. Ferroptosis is characterized by a decrease in mitochondrial volume, an increase in membrane density, and the occurrence of iron overaccumulation along with lipid peroxidation during the process of cell death[ 7 ]. Factors that induce ferroptosis can directly or indirectly affect the levels of glutathione (GSH) through various pathways, resulting in reduced antioxidant capacity, accumulation of reactive oxygen species (ROS), and ultimately leading to cell death[ 8 ]. Many studies have found that modulation of ferroptosis attenuates intestinal IRI[ 9 – 11 ]. Nuclear factor erythroid 2-related factor 2 (NRF2) is an essential regulator of cellular antioxidant responses. It coordinates the expression of many cytoprotective and metabolic genes as well as playing a vital role in controlling the expression of genes in response to antioxidant and pro-electrophilic stress[ 12 ]. The target genes regulated by NRF2 help prevent lipid peroxidation and the accumulation of free iron[ 13 ]. Both the light and heavy chains of ferritin (FTL/FTH1) and ferritin SLC40A1 are regulated by NRF2[ 14 ]. Additionally, NRF2 also controls the activity of various enzymes involved in glutathione synthesis and metabolism, such as glutamate-cysteine ligase (GCLC/GCLM), glutathione synthetase (GSS), and a subunit of cystine/glutamate transporter (SLC7A11) [ 13 ]. NRF2 also regulates the expression of glutathione peroxidase 4 (GPX4), which plays a crucial role in reducing lipid peroxidation and ferroptosis[ 15 ]. Short-chain fatty acids (SCFAs) are substances produced by microbial fermentation of carbohydrates in the gut, which can act as substrates or signaling molecules at the organ level to regulate certain cellular processes and systemic effects[ 16 ]. Among them, sodium butyrate has received widespread attention due to its beneficial effects on cellular energy metabolism and intestinal homeostasis. Many in vitro and in vivo studies have shown that butyrate plays an important role in regulating immune and inflammatory responses as well as intestinal barrier function. Studie has found that oral butyrate supplementation attenuates myocardial IRI [ 17 ]. It has also been demonstrated that butyrate ameliorates ferroptosis in ulcerative colitis by modulating the Nrf2/GPX4 signaling pathway and improving the intestinal barrier[ 18 ]. However, the role of sodium butyrate on intestinal ischemia-reperfusion-induced ferroptosis remains to be investigated. Therefore, we propose to envision that sodium butyrate could attenuate intestinal IRI by inhibiting ferroptosis through modulation of NRF2. we applied the method of clamping the superior mesenteric artery of mice to establish an intestinal IRI model in mice to probe the outcome of the action of sodium butyrate on ferroptosis correlated with intestinal IRI. Materials and methods Animals thirty-six (36) specific-pathogen-free (SPF)– grade healthy male C57BL mice with weight of 20–23 g were purchased at Laboratory Animal Centre of Lanzhou University (Animal certificate number SCXK (Gan) 2023-0003). Animals were reared in an SPF animal laboratory at an ambient temperature of 23–25°C, with a daily light period of 12 h and no restrictions on food, water, or movement, for one week of acclimatization. All experiments were executed in conformity with the Provision and General Recommendation of Chinese Experimental Animals Administration Legislation. And this study was approval by LZU No.1 Hospital Ethics Committee (Approval No.: LDYYLL-2023-498). Reagents Sodium butyrate (5 g) was purchased from Sigma-Aldrich (USA); Lysate, buffer and skimmed milk powder for Western Blot were purchased from Solarbio Science Technology (Beijing) Co., Ltd (China); ECL luminescent solution and protein electrophoresis Marker were purchased from Yeasen Biotechnology (Shanghai) Co., Ltd (China); PVDF membrane purchased from Millipore (USA); Monoclonal and secondary antibodies for NRF2/GPX4/SLC7A11/FTH1 were purchased from ImmunoWay (USA); GSH kit, GSH/GSSG kit, tissue iron (Fe 2+ ) kit and MDA kit were purchased from Nanjing Jiancheng Bioengineering Institute. Intestinal Ischemia–Reperfusion Model Construction Mice were fasted for 12 h and 2 h preoperatively. 2% sodium pentobarbital (40 mg/kg) was injected intraperitoneally at the time of surgery, and the mice were immobilized on the operating table and incubated. After performing routine abdominal skin preparation, the abdomen was sterilized and toweled, followed by an incision in the middle of the abdomen to open the abdomen. A portion of the small intestinal was pivoted to the side to expose and bluntly isolate the superior mesenteric artery (SMA). The SMA was clamped with a microvascular clip, and was released after 45 min. The intestine is gently placed back into the abdominal cavity and closed layer by layer with 30 minutes of reperfusion. Experimental Protocol The animals were randomly divided into six groups, each consisting of 6 subjects, using a random number table method. The groups were as follows: sham surgery group (SHAM group), intestinal ischemia reperfusion group (IR group), intestinal ischemia reperfusion + sodium butyrate group (IN group), intestinal ischemia reperfusion + sodium butyrate + FER-1(INF group), intestinal ischemia reperfusion + sodium butyrate + erastin group (INE group), and intestinal ischemia reperfusion + sodium butyrate + ML385 group (INM group). In the SHAM group, only the SMA was isolated, while the remaining five groups underwent intestinal ischemia-reperfusion modeling. The IN, INF, INE and INM groups were gavage with sodium butyrate 500 mg/kg daily for 7 days prior to modeling. On the other hand, the SHAM and IR groups were injected with equal amounts of saline 7 days prior to modeling. Additionally, FER-1, erastin, and ML385 were intraperitoneally injected into the INF, INE, and INM groups at doses of 5 mg/kg, 10 mg/kg, and 30 mg/kg in the three groups, respectively, 1 h before modeling. Detection of Fe 2+ , MDA, GSH in Intestinal Tissue After 30 minutes of reperfusion, the tissue was taken 5 cm from the end of the mouse cecum and snap-frozen in liquid nitrogen. Subsequently, the frozen tissue was ground to a white powder using a pre-cooled grinding bowl with pre-cooled saline and centrifuged in a centrifuge at 3500 r/min for 10 minutes. The supernatant was taken as per the concentration of the sample was calculated according to the instructions of the kit. Western Blot Detection of Nrf2, GPX4, SLC7A11, and FTH1 Expression Levels in Intestinal Tissue After reperfusion for 30 min, the tissue was taken 5 cm from the end of the mouse cecum and stored in a -80% refrigerator. One portion of frozen small intestinal tissue was taken, cut into pieces with tissue scissors, added with pre-cooled saline, rinsed twice, and lysate was added and placed in a low-temperature homogenizer for thorough homogenization, and then centrifuge it at 12000rpm at 4℃ for 15min, and then take the supernatant as a backup. After electrophoresis, membrane transfer and closure, the membranes were incubated overnight at 4°C with primary antibodies (Nrf2 [1: 2000], FTH1 [1: 2000], GPX4 [1: 2000], SLC7A11 [1: 2000] and β-actin [1: 2000] antibodies). Following 5 washes of the membrane with TBST, the secondary antibody (1:10,000) was allowed to incubate with the membrane on a shaker at room temperature for 1 h. The membranes were then exposed with a fully automated chemiluminescence imaging system (WD-9423C), and finally the gray values were measured with Image-J and graphs were created with GraphPad Prism 9. Pathological Observations Small intestine tissue was fixed in 4% paraformaldehyde solution for 48 hours and then went through the steps of ethanol dehydration, xylene immersion and paraffin embedding. Finally, the intestinal histopathological results were observed under light microscope after hematoxylin-eosin staining. Chiu score Chiu score was used to assess the degree of injury. 0 point, normal intestinal mucosal villi. 1 point, formation of subepithelial gap at the tip of the intestinal villi with capillary dilatation. 2 points, enlargement of the subepithelial gap, moderate edema of the lamina propria. 3 points, degeneration, and necrosis of the cells of the epithelial layer of the intestinal mucosa, significant edema of the lamina propria. 4 points, necrosis and detachment of the villi and the lamina propria, dilatation of the capillaries and increase of cells in the lamina propria. 5 points, decomposition of the lamina propria, hemorrhage of blood vessels, and ulcer formation. Bioinformatic Analysis Microarray data originating (GSE37013) from the GEO database were analyzed for differences and normalized with the limma package, followed by the ggplot2 package to analyze the expression levels of the NRF2 gene in the Control group versus the ischemia-reperfusion group[ 19 ]. We obtained the genes related to sodium butyrate, intestinal IRI, and ferroptosis from Gene Card website, used microbiology letter making website to draw the Wayne map and Enrichment Bar Graph, and finally visualized and analyzed the intersecting genes with Cytoscape and made the PPI maps. Statistical Analysis Statistical analysis of the data was carried out using GraphPad Prism 9 software. Comparisons of means between multiple groups that conformed to normal distribution were analyzed by one-way ANOVA, with pairwise comparisons involving equal variances being conducted through the least significant difference (LSD) method. In cases of unequal variances, the Dunnett T3 test was applied. If the data did not adhere to a normal distribution, a rank sum test was utilized. Results were considered statistically significant at p < 0.05. Results Sodium butyrate alleviates ischemia-reperfusion-induced intestinal injury In the study of sodium butyrate action on intestinal epithelial cells, this experiment that the intestinal mucosal epithelial cells in the SHAM group were well aligned and structurally intact with normal villi morphology. The IR group showed necrosis of the small intestinal epithelial cells, massive infiltration of the inflammatory cells, and severe disruption of the villi structure as compared to the SHAM group, whereas in the IN group there was less damage, better cellular alignment, and some restoration of the intestinal villi structure (Fig. 1 a.b). HE staining results showed an increase in tissue damage scores in the IR group compared to the SHAM group and a decrease in scores after the addition of sodium butyrate (Fig. 1 d). In addition, MDA levels were significantly higher in the IR group compared to the SHAM group and decreased in the IN group compared to the IR group (Fig. 1 c). Sodium butyrate alleviates ferroptosis induced by intestinal ischemia-reperfusion injury This experiment was followed by a study of the performance of sodium butyrate in ferroptosis associated with intestinal ischemia-reperfusion injury, and results showed that GSH (Fig. 2 b) and GSH/GSSG (Fig. 2 c) were decreased and Fe 2+ (Fig. 2 a) was increased in the IR group compared to the SHAM group. At the same time, the protein levels of the ferroptosis negative regulators GPX4 (Fig. 2 e), FTH1(Fig. 2 f) and SLC7A11(Fig. 2 g) were downregulated, whereas sodium butyrate reversed the changes in the above factors, suggesting that sodium butyrate inhibited ferroptosis by modulating the levels of SLC7A11/ GPX4. Fe 2+ declined in the INF group, and GSH and SLC7A11 did not change significantly compared with the IN group, although the changes in the IN group were reversed by the addition of erastin, an inducer of ferroptosis. (Fig. 2 a-g). NRF2 is a pathway by which sodium butyrate affects intestinal IR-induced ferroptosis We determined the signaling pathways associated with sodium butyrate when it acts on ferroptosis by bioinformatics analysis, and the levels of NRF2 in the intestinal IRI patients showed a decreasing trend compared to the control group (Fig. 3 a.b), and the intersecting genes were enriched on ferroptosis in the Wein plot with the enrichment analysis bar graphs (Fig. 3 c.d), and the role of NRF2(NFE2L2) was at the forefront (Fig. 3 e). Sodium butyrate affects ferroptosis by regulating NRF2 NRF2 was found to play a role in effect of sodium butyrate on ferroptosis, followed by addition of an NRF2 inhibitor, which was found to reverse the mitigating effect of sodium butyrate on ferroptosis by ML385. Compared with the IN group, GSH, GSH/GSSG, GPX4, FTH1 and SLC7A11 decreased in the INM group (Fig. 4 b-h), and Fe 2+ (Fig. 4 a) increased in the INM group. The results of HE staining and HE score also showed that tissue damage was more severe in the INM group than in the IN group (Fig. 4 c). Although there was a decrease in MDA (Fig. 4 d) in the INM group compared to the IN group, it was not statistically significant. In addition, NRF2 rose in the IN group compared to the IR group and decreased after the addition of an NRF2 inhibitor (Fig. 4 i). These results suggested that sodium butyrate attenuated ferroptosis by upregulating NRF2 pathway. Discussion In this study, we investigated the role of sodium butyrate on ferroptosis associated with intestinal ischemia-reperfusion injury, and we found that sodium butyrate, as a metabolite of intestinal flora, attenuated intestinal IRI, and we also found that sodium butyrate had a role in ferroptosis induced by intestinal IRI, which means that sodium butyrate inhibited ferroptosis and thus attenuated intestinal IRI by affecting the level of SLC7A11/GPX4 through upregulation of NRF2. The extent and progression of tissue injury after ischemia-reperfusion is irreversible damage and is directly related to the duration of ischemia. Among them, the tight junctions between epithelial cells in the mucosal layer are disrupted after intestinal IR, leading to bacterial or enterotoxin infiltration of the intestinal mesenchymal space, and in severe cases of ischemia, even sepsis and multi-organ failure may occur. Therefore, restoration of blood supply and reduction of cell death are the main goals of treatment[ 20 ]. Many studies have demonstrated the involvement of intestinal flora and metabolites in intestinal IRI[ 21 , 22 ]. Butyrate is a metabolite produced in the gut flora, synthesized by the intestinal flora through a variety of pathways, and is one of the most abundant short-chain fatty acids in the gut[ 23 ]. Sodium butyrate has been found to have various beneficial effects on the human body. A study reveals that butyrate improves skeletal muscle atrophy in patients with diabetic nephropathy by enhancing FFA2-mediated PI3K/Akt/mTOR signaling[ 24 ], Sodium butyrate has also been found to inhibit cerebral IRI in mice by reducing MDA[ 25 ]. In this study. In this study, mice with the addition of sodium butyrate significantly lowered the rise in MDA caused by intestinal IRI, and the intestinal structures damaged by ischemia-reperfusion injury were partially restored, confirming that sodium butyrate inhibits oxidative stress to attenuate intestinal ischemia-reperfusion-induced epithelial cell injury. Ferroptosis is an important PCD that Fig.s prominently in intestinal IRI. The results of the present study showed that intestinal ischemia-reperfusion injury decreased the ferroptosis-related factors GSH and FTH1, suggesting that ischemia-reperfusion injury induced the occurrence of ferroptosis in mice. It has been found that butyrate inhibits ferroptosis in m2-like macrophages in experimental colitis[ 26 ], but the effect of butyrate on intestinal IRI-induced ferroptosis has not been reported. To determine whether the beneficial effects of sodium butyrate are related to ferroptosis, in this experiment we exogenously added sodium butyrate and found that it works by augmenting the levels of the negative regulators of ferroptosis, GPX4 and SLC7A11. And this effect was reversed by the ferroptosis agonist, thus proving that sodium butyrate attenuates intestinal IRI by inhibiting ferroptosis. In contrast, sodium butyrate has been found to enhance ferroptosis in tumor cells, thereby promoting tumor cell death [ 27 – 29 ]. This suggests that sodium butyrate shows different results of action on ferroptosis in intestinal cells versus tumor cells. NRF2, a key factor regulating ferroptosis[ 30 , 31 ], interacts with Keap1 at low levels under general conditions. In contrast, upon controlled activation, cysteine residues (Cys) in Keap1 are induced by electrophilic species through covalent modification to induce a conformational change in Keap1, which hinders the ubiquitination of NRF2, thereby increasing the level of the NRF2 protein, which translocate to the nucleus to activate target genes to function[ 32 ]. NRF2 and heme oxygenase 1 (HO-1) were found to be involved in GPX4 synthesis[ 33 ],and sodium butyrate, acting as an activator of NRF2, promotes the expression of the renal NRF2 gene[ 34 ]. Whether sodium butyrate affects intestinal IRI-associated ferroptosis through modulation of NRF2. Our bioinformatic analysis identified genes associated with sodium butyrate and intestinal IRI that can be enriched for ferroptosis, demonstrating that sodium butyrate can act on ferroptosis and that NRF2 occupies a prostate position, suggesting the importance of NRF2 in sodium butyrate's influence on ferroptosis. Then in our experiments we showed a significant increase in NRF2, SLC7A11, and GPX4 in mice compared to the IR group after administration with sodium butyrate. And reversed these changes with the addition of an NRF2 inhibitor, which inhibited the attenuating effect of sodium butyrate on intestinal IRI. These results suggest that sodium butyrate exerts its protective effects against intestinal ischemia-reperfusion injury by activating NRF2/ SLC7A11/GPX4 axis. What is interesting is that the different results of sodium butyrate's action on ferroptosis in tumor versus non-tumor cells are more than just what has been stated above. Exploring the role of NRF2 on iron homeostasis and ferroptosis, Anandhan et al. found that NRF2-WT-type tumor cells undergo ferroptosis with decreased levels of FTH1, whereas NRF2-KO tumor cells inhibit Fe 2+ binding to ferritin because of elevated levels of NCOA4, which hinders the process of ferritinophagy. Tumor cells were enriched in Fe 2+ , which resulted in a positive correlation between ferroptosis and FTH1 levels (aggravation of ferroptosis was accompanied by an increase in FTH1 levels), demonstrating a strong association between ferroptosis and ferritinophagy. [ 35 ]. In contrast to this result, in our study, sodium butyrate ameliorated intestinal IRI-induced ferroptosis, and GSH, GPX4, and FTH1 levels were decreased by the addition of the NRF2 inhibitor, which means that ferroptosis rose along with a decrease in FTH1, obviously inconsistent with the outcome in the tumor cells, and it remains to be investigated whether ferritinophagy plays a role in this. In summary, the results of this article prove that sodium butyrate can regulate SLC7A11/GPX4 levels by upregulating NRF2, inhibiting ferroptosis and reducing intestinal IRI. Declarations Funding This study was supported by National Natural Science Foundation of China (Grant No. 82260381), Medical Innovation and Development Project of Lanzhou University (Grant No. lzuyxcx-2022-108). Competing interests All authors declare that they have no competing interests related to this study. Data Availability Data will be made available on request. Author Contribution Zizen Zhao: the main writer of the article, the main operator of the experiment, produced Figures 1 and 2.Yufang Leng: Corresponding author, ensured all authors approved the manuscript and were responsible for the article.Liya Chang: assisted with experiments and revised the manuscript.Yu Wang: Assist with experiments, suggest experimental directions, and manage data.Dongbin Li: Assist with experiments, management and supervision.Yang Xing: Assisted with experiments and presented methodology.Yuxuan Wu: Assist with experiments to produce Figures 3-5. 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Ferroptosis: regulation by competition between NRF2 and BACH1 and propagation of the death signal. FEBS Journal. 2023;290(7):1688-1704. https://doi.org/10.1111/febs.16382 Harder B, Jiang T, Wu T, Tao S, Rojo de la Vega M et al. Molecular mechanisms of Nrf2 regulation and how these influence chemical modulation for disease intervention. Biochemical Society Transactions. 2015;43(4):680-686. https://doi.org/10.1042/bst20150020 Song X, Long D. Nrf2 and Ferroptosis: A New Research Direction for Neurodegenerative Diseases. Frontiers in Neuroscience. 2020;14:267. https://doi.org/10.3389/fnins.2020.00267 Cheng X, Zhou T, He Y, Xie Y, Xu Y et al. The role and mechanism of butyrate in the prevention and treatment of diabetic kidney disease. Frontiers in Microbiology. 2022;13:961536. https://doi.org/10.3389/fmicb.2022.961536 Anandhan A, Dodson M, Shakya A, Chen J, Liu P et al. NRF2 controls iron homeostasis and ferroptosis through HERC2 and VAMP8. Science Advances. 2023;9(5):eade9585. https://doi.org/10.1126/sciadv.ade9585 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted 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-3988128","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":275362340,"identity":"79aa8ed0-1734-4f4d-87bb-392fe23c9b2c","order_by":0,"name":"Zicen Zhao","email":"","orcid":"","institution":"Lanzhou University","correspondingAuthor":false,"prefix":"","firstName":"Zicen","middleName":"","lastName":"Zhao","suffix":""},{"id":275362341,"identity":"61c64860-fca2-43c7-ba3b-c04bd9e9f50d","order_by":1,"name":"Yufang Leng","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAy0lEQVRIiWNgGAWjYBACPjBpYyMDpnmI0cIGJtPSeEjWcpgULey9h1/zJJznkZ+RwPjgbRuDvDlBLTzn0qx5Em7zMM5IYDac28ZguLOBkBaJHDNj3h+3eZglEtikedsYEgwOENIi/8bMmCfhHA+bRAL7b+K0SPAYP+ZJOMDDA7SFmTgtPDlmjHMSknkkeB42S845J2G4gZAWfvYzxh/eJNjJybcnH/zwpsxGnqAtIIukINHB2AAkJAirBwLmjz+IUjcKRsEoGAUjFgAAzEYz0MdfEPIAAAAASUVORK5CYII=","orcid":"","institution":"Lanzhou University","correspondingAuthor":true,"prefix":"","firstName":"Yufang","middleName":"","lastName":"Leng","suffix":""},{"id":275362342,"identity":"520709c1-ce34-4f88-8ced-d37ae1d74a6d","order_by":2,"name":"Liya Chang","email":"","orcid":"","institution":"Lanzhou University","correspondingAuthor":false,"prefix":"","firstName":"Liya","middleName":"","lastName":"Chang","suffix":""},{"id":275362343,"identity":"af7e18d5-e3e4-45af-8843-4bdaa474d58e","order_by":3,"name":"Yu Wang","email":"","orcid":"","institution":"Lanzhou University","correspondingAuthor":false,"prefix":"","firstName":"Yu","middleName":"","lastName":"Wang","suffix":""},{"id":275362344,"identity":"068cafc0-b652-4c60-87e8-ddfcc8d00922","order_by":4,"name":"Dongbin Li","email":"","orcid":"","institution":"Lanzhou University","correspondingAuthor":false,"prefix":"","firstName":"Dongbin","middleName":"","lastName":"Li","suffix":""},{"id":275362345,"identity":"d096f95c-3a6a-4b65-b416-6245c733b3d8","order_by":5,"name":"Yang Xing","email":"","orcid":"","institution":"Lanzhou University","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Xing","suffix":""},{"id":275362346,"identity":"ff89fc76-16c2-4c45-8f98-630c754608b0","order_by":6,"name":"Yuxuan Wu","email":"","orcid":"","institution":"Fenyang College of Shanxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Yuxuan","middleName":"","lastName":"Wu","suffix":""}],"badges":[],"createdAt":"2024-02-25 13:46:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3988128/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3988128/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":51832421,"identity":"4ddcd954-8a0f-44d1-a131-44d2fde897cf","added_by":"auto","created_at":"2024-02-29 19:02:35","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":11709869,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of sodium butyrate on intestinal ischemia-reperfusion injury in mice. (a) The state of the intestine before and during ischemia in mice. (b) Hematoxylin-eosin staining. (c) Determination of MDA by ELISA. Data are expressed as mean ± standard deviation, n= 6.\u003csup\u003e ****\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001 vs SHAM group, \u003csup\u003e####\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001 vs IR group, \u003csup\u003e^^\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e=0.0076 vs IN group. (d) HE Score,\u003csup\u003e **\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e= 0.0058 vs SHAM group.\u003c/p\u003e","description":"","filename":"figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-3988128/v1/47a20baf9f0c4fa56acf956a.png"},{"id":51832418,"identity":"06674016-c46d-46e4-a2f9-d710b343acd4","added_by":"auto","created_at":"2024-02-29 19:02:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":943445,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in ferroptosis-related factors after the action of sodium butyrate on intestinal IRI mice. (a) Determination of Fe\u003csup\u003e2+\u003c/sup\u003e by ELISA. Data are expressed as mean ± standard deviation, n= 6. \u003csup\u003e****\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001 vs SHAM group, \u003csup\u003e####\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001 vs IR group, \u003csup\u003e$ $ $ $\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001 vs IN group, \u003csup\u003e^^^^\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001 vs IN group. (b) Determination of GSH by ELISA. Data are expressed as mean ± standard deviation, n= 6. \u003csup\u003e****\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001 vs SHAM group, \u003csup\u003e####\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001 vs IR group,\u003csup\u003e ^^^\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e=0.0003 vs IN group. (c) Determination of Fe\u003csup\u003e2+\u003c/sup\u003e by Biochemical Tests. Data are expressed as mean ± standard deviation, n= 6. \u003csup\u003e****\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001 vs SHAM group, \u003csup\u003e####\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001 vs IR group,\u003csup\u003e $$$\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e= 0.0008 vs IN group,\u003csup\u003e ^^^^\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001 vs IN group. (d) Western\u0026nbsp;blot. (e) Determination of GPX4 by WB. Data are expressed as mean ± standard deviation, n= 6.\u003csup\u003e ****\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001 vs SHAM group, \u003csup\u003e####\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001 vs IR group, \u003csup\u003e$\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e=0.0313 vs IN group, \u003csup\u003e^^^\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e=0.0007 vs IN group. (f) Determination of FTH1 by WB. Data are expressed as mean ± standard deviation, n= 6. \u003csup\u003e****\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001 vs SHAM group, \u003csup\u003e####\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001 vs IR group, \u003csup\u003e^^\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e=0.0029 vs IN group. (g) Determination of SLC7A11 by WB. Data are expressed as mean ± standard deviation, n= 6. \u003csup\u003e****\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001 vs SHAM group, \u003csup\u003e####\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001 vs IR group, \u003csup\u003e$$\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e=0.0019 vs IN group, \u003csup\u003e^^^^\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001 vs IN group.\u003c/p\u003e","description":"","filename":"figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-3988128/v1/26b6982a63eada3e6a21de25.png"},{"id":51832417,"identity":"8e820400-5d09-48de-b13b-4401b6386a10","added_by":"auto","created_at":"2024-02-29 19:02:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4467252,"visible":true,"origin":"","legend":"\u003cp\u003eGenes associated with intestinal IRI, ferroptosis and sodium butyrate. (a) Data standardization. (b) Levels of NRF2 change before and after intestinal IRI. (c) Wayne plot of genes associated with sodium butyrate, intestinal IRI, and ferroptosis. (d) KEGG pathway diagram. (e) PPI chart.\u003c/p\u003e","description":"","filename":"figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-3988128/v1/f10749d87e0523216beca1ab.png"},{"id":51832420,"identity":"23408ecf-a162-4af9-adf6-df0cb0fcd60b","added_by":"auto","created_at":"2024-02-29 19:02:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":5863566,"visible":true,"origin":"","legend":"\u003cp\u003eChanges in NRF2 pathway-associated protein expression after sodium butyrate action on intestinal IRI mice. (a) Determination of Fe\u003csup\u003e2+\u003c/sup\u003e by ELISA. Data are expressed as mean ± standard deviation, n= 6. \u003csup\u003e****\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001 vs SHAM group, \u003csup\u003e####\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001 vs IR group, \u003csup\u003e$ $ $ $\u003c/sup\u003e \u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001 vs IN group, \u003csup\u003e^^^^\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001 vs IN group. (b) Determination of GSH and GSH/GSSG. Data are expressed as mean ± standard deviation, n= 6. GSH, \u003csup\u003e****\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001 vs SHAM group, \u003csup\u003e####\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001 vs IR group,\u003csup\u003e ^^\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e=0.0046 vs IN group. GSH/GSSG, \u003csup\u003e****\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001 vs SHAM group, \u003csup\u003e####\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001 vs IR group,\u003csup\u003e ^\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e=0.0214 vs IN group. (c) Hematoxylin-eosin staining, and HE Score.\u003csup\u003e **\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e= 0.0058 vs SHAM group, \u003csup\u003e^\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e=0.0474 vs IN group. (d) Determination of MDA by ELISA. Data are expressed as mean ± standard deviation, n= 6.\u003csup\u003e ****\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001 vs SHAM group, \u003csup\u003e####\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001 vs IR group. (e) Western\u0026nbsp;blot. (f) Determination of GPX4 by WB. Data are expressed as mean ± standard deviation, n= 6.\u003csup\u003e ****\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001 vs SHAM group, \u003csup\u003e####\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001 vs IR group, \u003csup\u003e^^\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e=0.0026 vs IN group. (g) Determination of FTH1 by WB. Data are expressed as mean ± standard deviation, n= 6. \u003csup\u003e****\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001 vs SHAM group, \u003csup\u003e####\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001 vs IR group, \u003csup\u003e^\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e=0.0160 vs IN group. (h) Determination of SLC7A11 by WB. Data are expressed as mean ± standard deviation, n= 6. \u003csup\u003e****\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001 vs SHAM group, \u003csup\u003e####\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001 vs IR group, \u003csup\u003e^^\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0010 vs IN group. (i) Determination of NRF2 by WB. Data are expressed as mean ± standard deviation, n= 6. \u003csup\u003e****\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001 vs SHAM group, \u003csup\u003e####\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001 vs IR group, \u003csup\u003e^^^^\u003c/sup\u003e\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.0001 vs IN group.\u003c/p\u003e","description":"","filename":"figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-3988128/v1/f806dac891ac096836b72282.png"},{"id":51832419,"identity":"a02166f1-b181-42a7-97de-1f52aacda15d","added_by":"auto","created_at":"2024-02-29 19:02:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1768306,"visible":true,"origin":"","legend":"\u003cp\u003eMechanistic map of the effect of sodium butyrate on ferroptosis induced by intestinal ischemia-reperfusion injury.\u003c/p\u003e","description":"","filename":"figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-3988128/v1/bdabd8c2ac090cc9df6e0f23.png"},{"id":52750663,"identity":"1f769757-fe78-4de2-a22f-81325ce7cc12","added_by":"auto","created_at":"2024-03-15 10:17:05","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2056138,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3988128/v1/671844da-e1ba-42dd-82fb-0a157b7d1b69.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Sodium butyrate inhibits ferroptosis and ameliorates intestinal ischemia-reperfusion injury by modulating the NRF2/SLC7A11/GPX4 pathway","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe gut plays a crucial role in maintaining physiological homeostasis and performing various functions such as nutrient absorption, toxin excretion, immune homeostasis, and hormone release[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. However, critical illnesses such as traumatic shock, severe infections, and severe burns can result in intestinal IRI. This injury occurs when intestinal flora translocates and there is a massive release of endotoxins and inflammatory factors during blood reperfusion. These events not only harm the intestinal mucosa but also lead to extra-intestinal multi-organ dysfunction or failure, which can worsen the patient's condition and even contribute to their death[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Current research shows that targeting intestinal IRI is an Important factor in addressing the risk of death in high-risk patients, but no systematic treatment approach has been developed to date. Therefore, the finding of new therapeutic strategies to mitigate intestinal IRI is an essential direction of its research.\u003c/p\u003e \u003cp\u003eNumerous experiments have demonstrated the significance of programmed cell death (PCD) in intestinal IRI [\u003cspan additionalcitationids=\"CR4 CR5\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. One type of PCD is ferroptosis, which is an iron-dependent form of cell death. Ferroptosis is characterized by a decrease in mitochondrial volume, an increase in membrane density, and the occurrence of iron overaccumulation along with lipid peroxidation during the process of cell death[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Factors that induce ferroptosis can directly or indirectly affect the levels of glutathione (GSH) through various pathways, resulting in reduced antioxidant capacity, accumulation of reactive oxygen species (ROS), and ultimately leading to cell death[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Many studies have found that modulation of ferroptosis attenuates intestinal IRI[\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNuclear factor erythroid 2-related factor 2 (NRF2) is an essential regulator of cellular antioxidant responses. It coordinates the expression of many cytoprotective and metabolic genes as well as playing a vital role in controlling the expression of genes in response to antioxidant and pro-electrophilic stress[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The target genes regulated by NRF2 help prevent lipid peroxidation and the accumulation of free iron[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBoth the light and heavy chains of ferritin (FTL/FTH1) and ferritin SLC40A1 are regulated by NRF2[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Additionally, NRF2 also controls the activity of various enzymes involved in glutathione synthesis and metabolism, such as glutamate-cysteine ligase (GCLC/GCLM), glutathione synthetase (GSS), and a subunit of cystine/glutamate transporter (SLC7A11) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. NRF2 also regulates the expression of glutathione peroxidase 4 (GPX4), which plays a crucial role in reducing lipid peroxidation and ferroptosis[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eShort-chain fatty acids (SCFAs) are substances produced by microbial fermentation of carbohydrates in the gut, which can act as substrates or signaling molecules at the organ level to regulate certain cellular processes and systemic effects[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Among them, sodium butyrate has received widespread attention due to its beneficial effects on cellular energy metabolism and intestinal homeostasis. Many in vitro and in vivo studies have shown that butyrate plays an important role in regulating immune and inflammatory responses as well as intestinal barrier function. Studie has found that oral butyrate supplementation attenuates myocardial IRI [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. It has also been demonstrated that butyrate ameliorates ferroptosis in ulcerative colitis by modulating the Nrf2/GPX4 signaling pathway and improving the intestinal barrier[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. However, the role of sodium butyrate on intestinal ischemia-reperfusion-induced ferroptosis remains to be investigated.\u003c/p\u003e \u003cp\u003eTherefore, we propose to envision that sodium butyrate could attenuate intestinal IRI by inhibiting ferroptosis through modulation of NRF2. we applied the method of clamping the superior mesenteric artery of mice to establish an intestinal IRI model in mice to probe the outcome of the action of sodium butyrate on ferroptosis correlated with intestinal IRI.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals\u003c/h2\u003e \u003cp\u003ethirty-six (36) specific-pathogen-free (SPF)\u0026ndash; grade healthy male C57BL mice with weight of 20\u0026ndash;23 g were purchased at Laboratory Animal Centre of Lanzhou University (Animal certificate number SCXK (Gan) 2023-0003). Animals were reared in an SPF animal laboratory at an ambient temperature of 23\u0026ndash;25\u0026deg;C, with a daily light period of 12 h and no restrictions on food, water, or movement, for one week of acclimatization. All experiments were executed in conformity with the Provision and General Recommendation of Chinese Experimental Animals Administration Legislation. And this study was approval by LZU No.1 Hospital Ethics Committee (Approval No.: LDYYLL-2023-498).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eReagents\u003c/h2\u003e \u003cp\u003eSodium butyrate (5 g) was purchased from Sigma-Aldrich (USA); Lysate, buffer and skimmed milk powder for Western Blot were purchased from Solarbio Science Technology (Beijing) Co., Ltd (China); ECL luminescent solution and protein electrophoresis Marker were purchased from Yeasen Biotechnology (Shanghai) Co., Ltd (China); PVDF membrane purchased from Millipore (USA); Monoclonal and secondary antibodies for NRF2/GPX4/SLC7A11/FTH1 were purchased from ImmunoWay (USA); GSH kit, GSH/GSSG kit, tissue iron (Fe\u003csup\u003e2+\u003c/sup\u003e) kit and MDA kit were purchased from Nanjing Jiancheng Bioengineering Institute.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eIntestinal Ischemia\u0026ndash;Reperfusion Model Construction\u003c/h2\u003e \u003cp\u003eMice were fasted for 12 h and 2 h preoperatively. 2% sodium pentobarbital (40 mg/kg) was injected intraperitoneally at the time of surgery, and the mice were immobilized on the operating table and incubated. After performing routine abdominal skin preparation, the abdomen was sterilized and toweled, followed by an incision in the middle of the abdomen to open the abdomen. A portion of the small intestinal was pivoted to the side to expose and bluntly isolate the superior mesenteric artery (SMA). The SMA was clamped with a microvascular clip, and was released after 45 min. The intestine is gently placed back into the abdominal cavity and closed layer by layer with 30 minutes of reperfusion.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eExperimental Protocol\u003c/h2\u003e \u003cp\u003eThe animals were randomly divided into six groups, each consisting of 6 subjects, using a random number table method. The groups were as follows: sham surgery group (SHAM group), intestinal ischemia reperfusion group (IR group), intestinal ischemia reperfusion\u0026thinsp;+\u0026thinsp;sodium butyrate group (IN group), intestinal ischemia reperfusion\u0026thinsp;+\u0026thinsp;sodium butyrate\u0026thinsp;+\u0026thinsp;FER-1(INF group), intestinal ischemia reperfusion\u0026thinsp;+\u0026thinsp;sodium butyrate\u0026thinsp;+\u0026thinsp;erastin group (INE group), and intestinal ischemia reperfusion\u0026thinsp;+\u0026thinsp;sodium butyrate\u0026thinsp;+\u0026thinsp;ML385 group (INM group). In the SHAM group, only the SMA was isolated, while the remaining five groups underwent intestinal ischemia-reperfusion modeling. The IN, INF, INE and INM groups were gavage with sodium butyrate 500 mg/kg daily for 7 days prior to modeling. On the other hand, the SHAM and IR groups were injected with equal amounts of saline 7 days prior to modeling. Additionally, FER-1, erastin, and ML385 were intraperitoneally injected into the INF, INE, and INM groups at doses of 5 mg/kg, 10 mg/kg, and 30 mg/kg in the three groups, respectively, 1 h before modeling.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eDetection of Fe\u003csup\u003e2+\u003c/sup\u003e, MDA, GSH in Intestinal Tissue\u003c/h2\u003e \u003cp\u003eAfter 30 minutes of reperfusion, the tissue was taken 5 cm from the end of the mouse cecum and snap-frozen in liquid nitrogen. Subsequently, the frozen tissue was ground to a white powder using a pre-cooled grinding bowl with pre-cooled saline and centrifuged in a centrifuge at 3500 r/min for 10 minutes. The supernatant was taken as per the concentration of the sample was calculated according to the instructions of the kit.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eWestern Blot Detection of Nrf2, GPX4, SLC7A11, and FTH1 Expression Levels in Intestinal Tissue\u003c/h2\u003e \u003cp\u003eAfter reperfusion for 30 min, the tissue was taken 5 cm from the end of the mouse cecum and stored in a -80% refrigerator. One portion of frozen small intestinal tissue was taken, cut into pieces with tissue scissors, added with pre-cooled saline, rinsed twice, and lysate was added and placed in a low-temperature homogenizer for thorough homogenization, and then centrifuge it at 12000rpm at 4℃ for 15min, and then take the supernatant as a backup. After electrophoresis, membrane transfer and closure, the membranes were incubated overnight at 4\u0026deg;C with primary antibodies (Nrf2 [1: 2000], FTH1 [1: 2000], GPX4 [1: 2000], SLC7A11 [1: 2000] and β-actin [1: 2000] antibodies). Following 5 washes of the membrane with TBST, the secondary antibody (1:10,000) was allowed to incubate with the membrane on a shaker at room temperature for 1 h. The membranes were then exposed with a fully automated chemiluminescence imaging system (WD-9423C), and finally the gray values were measured with Image-J and graphs were created with GraphPad Prism 9.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003ePathological Observations\u003c/h2\u003e \u003cp\u003eSmall intestine tissue was fixed in 4% paraformaldehyde solution for 48 hours and then went through the steps of ethanol dehydration, xylene immersion and paraffin embedding. Finally, the intestinal histopathological results were observed under light microscope after hematoxylin-eosin staining.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eChiu score\u003c/h2\u003e \u003cp\u003eChiu score was used to assess the degree of injury. 0 point, normal intestinal mucosal villi. 1 point, formation of subepithelial gap at the tip of the intestinal villi with capillary dilatation. 2 points, enlargement of the subepithelial gap, moderate edema of the lamina propria. 3 points, degeneration, and necrosis of the cells of the epithelial layer of the intestinal mucosa, significant edema of the lamina propria. 4 points, necrosis and detachment of the villi and the lamina propria, dilatation of the capillaries and increase of cells in the lamina propria. 5 points, decomposition of the lamina propria, hemorrhage of blood vessels, and ulcer formation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eBioinformatic Analysis\u003c/h2\u003e \u003cp\u003eMicroarray data originating (GSE37013) from the GEO database were analyzed for differences and normalized with the limma package, followed by the ggplot2 package to analyze the expression levels of the NRF2 gene in the Control group versus the ischemia-reperfusion group[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. We obtained the genes related to sodium butyrate, intestinal IRI, and ferroptosis from Gene Card website, used microbiology letter making website to draw the Wayne map and Enrichment Bar Graph, and finally visualized and analyzed the intersecting genes with Cytoscape and made the PPI maps.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analysis of the data was carried out using GraphPad Prism 9 software. Comparisons of means between multiple groups that conformed to normal distribution were analyzed by one-way ANOVA, with pairwise comparisons involving equal variances being conducted through the least significant difference (LSD) method. In cases of unequal variances, the Dunnett T3 test was applied. If the data did not adhere to a normal distribution, a rank sum test was utilized. Results were considered statistically significant at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\n\u003ch2\u003eSodium butyrate alleviates ischemia-reperfusion-induced intestinal injury\u003c/h2\u003e\n\u003cp\u003eIn the study of sodium butyrate action on intestinal epithelial cells, this experiment that the intestinal mucosal epithelial cells in the SHAM group were well aligned and structurally intact with normal villi morphology. The IR group showed necrosis of the small intestinal epithelial cells, massive infiltration of the inflammatory cells, and severe disruption of the villi structure as compared to the SHAM group, whereas in the IN group there was less damage, better cellular alignment, and some restoration of the intestinal villi structure (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea.b). HE staining results showed an increase in tissue damage scores in the IR group compared to the SHAM group and a decrease in scores after the addition of sodium butyrate (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ed). In addition, MDA levels were significantly higher in the IR group compared to the SHAM group and decreased in the IN group compared to the IR group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\n\u003ch2\u003eSodium butyrate alleviates ferroptosis induced by intestinal ischemia-reperfusion injury\u003c/h2\u003e\n\u003cp\u003eThis experiment was followed by a study of the performance of sodium butyrate in ferroptosis associated with intestinal ischemia-reperfusion injury, and results showed that GSH (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb) and GSH/GSSG (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec) were decreased and Fe\u003csup\u003e2+\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea) was increased in the IR group compared to the SHAM group. At the same time, the protein levels of the ferroptosis negative regulators GPX4 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ee), FTH1(Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ef) and SLC7A11(Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eg) were downregulated, whereas sodium butyrate reversed the changes in the above factors, suggesting that sodium butyrate inhibited ferroptosis by modulating the levels of SLC7A11/ GPX4. Fe\u003csup\u003e2+\u003c/sup\u003e declined in the INF group, and GSH and SLC7A11 did not change significantly compared with the IN group, although the changes in the IN group were reversed by the addition of erastin, an inducer of ferroptosis. (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea-g).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n\u003ch2\u003eNRF2 is a pathway by which sodium butyrate affects intestinal IR-induced ferroptosis\u003c/h2\u003e\n\u003cp\u003eWe determined the signaling pathways associated with sodium butyrate when it acts on ferroptosis by bioinformatics analysis, and the levels of NRF2 in the intestinal IRI patients showed a decreasing trend compared to the control group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea.b), and the intersecting genes were enriched on ferroptosis in the Wein plot with the enrichment analysis bar graphs (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec.d), and the role of NRF2(NFE2L2) was at the forefront (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ee).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\n\u003ch2\u003eSodium butyrate affects ferroptosis by regulating NRF2\u003c/h2\u003e\n\u003cp\u003eNRF2 was found to play a role in effect of sodium butyrate on ferroptosis, followed by addition of an NRF2 inhibitor, which was found to reverse the mitigating effect of sodium butyrate on ferroptosis by ML385. Compared with the IN group, GSH, GSH/GSSG, GPX4, FTH1 and SLC7A11 decreased in the INM group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb-h), and Fe\u003csup\u003e2+\u003c/sup\u003e (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea) increased in the INM group. The results of HE staining and HE score also showed that tissue damage was more severe in the INM group than in the IN group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ec). Although there was a decrease in MDA (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ed) in the INM group compared to the IN group, it was not statistically significant. In addition, NRF2 rose in the IN group compared to the IR group and decreased after the addition of an NRF2 inhibitor (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ei). These results suggested that sodium butyrate attenuated ferroptosis by upregulating NRF2 pathway.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we investigated the role of sodium butyrate on ferroptosis associated with intestinal ischemia-reperfusion injury, and we found that sodium butyrate, as a metabolite of intestinal flora, attenuated intestinal IRI, and we also found that sodium butyrate had a role in ferroptosis induced by intestinal IRI, which means that sodium butyrate inhibited ferroptosis and thus attenuated intestinal IRI by affecting the level of SLC7A11/GPX4 through upregulation of NRF2.\u003c/p\u003e \u003cp\u003eThe extent and progression of tissue injury after ischemia-reperfusion is irreversible damage and is directly related to the duration of ischemia. Among them, the tight junctions between epithelial cells in the mucosal layer are disrupted after intestinal IR, leading to bacterial or enterotoxin infiltration of the intestinal mesenchymal space, and in severe cases of ischemia, even sepsis and multi-organ failure may occur. Therefore, restoration of blood supply and reduction of cell death are the main goals of treatment[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Many studies have demonstrated the involvement of intestinal flora and metabolites in intestinal IRI[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eButyrate is a metabolite produced in the gut flora, synthesized by the intestinal flora through a variety of pathways, and is one of the most abundant short-chain fatty acids in the gut[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Sodium butyrate has been found to have various beneficial effects on the human body. A study reveals that butyrate improves skeletal muscle atrophy in patients with diabetic nephropathy by enhancing FFA2-mediated PI3K/Akt/mTOR signaling[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], Sodium butyrate has also been found to inhibit cerebral IRI in mice by reducing MDA[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. In this study. In this study, mice with the addition of sodium butyrate significantly lowered the rise in MDA caused by intestinal IRI, and the intestinal structures damaged by ischemia-reperfusion injury were partially restored, confirming that sodium butyrate inhibits oxidative stress to attenuate intestinal ischemia-reperfusion-induced epithelial cell injury.\u003c/p\u003e \u003cp\u003eFerroptosis is an important PCD that Fig.s prominently in intestinal IRI. The results of the present study showed that intestinal ischemia-reperfusion injury decreased the ferroptosis-related factors GSH and FTH1, suggesting that ischemia-reperfusion injury induced the occurrence of ferroptosis in mice. It has been found that butyrate inhibits ferroptosis in m2-like macrophages in experimental colitis[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], but the effect of butyrate on intestinal IRI-induced ferroptosis has not been reported. To determine whether the beneficial effects of sodium butyrate are related to ferroptosis, in this experiment we exogenously added sodium butyrate and found that it works by augmenting the levels of the negative regulators of ferroptosis, GPX4 and SLC7A11. And this effect was reversed by the ferroptosis agonist, thus proving that sodium butyrate attenuates intestinal IRI by inhibiting ferroptosis. In contrast, sodium butyrate has been found to enhance ferroptosis in tumor cells, thereby promoting tumor cell death [\u003cspan additionalcitationids=\"CR28\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. This suggests that sodium butyrate shows different results of action on ferroptosis in intestinal cells versus tumor cells.\u003c/p\u003e \u003cp\u003eNRF2, a key factor regulating ferroptosis[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], interacts with Keap1 at low levels under general conditions. In contrast, upon controlled activation, cysteine residues (Cys) in Keap1 are induced by electrophilic species through covalent modification to induce a conformational change in Keap1, which hinders the ubiquitination of NRF2, thereby increasing the level of the NRF2 protein, which translocate to the nucleus to activate target genes to function[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. NRF2 and heme oxygenase 1 (HO-1) were found to be involved in GPX4 synthesis[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e],and sodium butyrate, acting as an activator of NRF2, promotes the expression of the renal NRF2 gene[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Whether sodium butyrate affects intestinal IRI-associated ferroptosis through modulation of NRF2. Our bioinformatic analysis identified genes associated with sodium butyrate and intestinal IRI that can be enriched for ferroptosis, demonstrating that sodium butyrate can act on ferroptosis and that NRF2 occupies a prostate position, suggesting the importance of NRF2 in sodium butyrate's influence on ferroptosis. Then in our experiments we showed a significant increase in NRF2, SLC7A11, and GPX4 in mice compared to the IR group after administration with sodium butyrate. And reversed these changes with the addition of an NRF2 inhibitor, which inhibited the attenuating effect of sodium butyrate on intestinal IRI. These results suggest that sodium butyrate exerts its protective effects against intestinal ischemia-reperfusion injury by activating NRF2/ SLC7A11/GPX4 axis.\u003c/p\u003e \u003cp\u003eWhat is interesting is that the different results of sodium butyrate's action on ferroptosis in tumor versus non-tumor cells are more than just what has been stated above. Exploring the role of NRF2 on iron homeostasis and ferroptosis, Anandhan et al. found that NRF2-WT-type tumor cells undergo ferroptosis with decreased levels of FTH1, whereas NRF2-KO tumor cells inhibit Fe\u003csup\u003e2+\u003c/sup\u003e binding to ferritin because of elevated levels of NCOA4, which hinders the process of ferritinophagy. Tumor cells were enriched in Fe\u003csup\u003e2+\u003c/sup\u003e, which resulted in a positive correlation between ferroptosis and FTH1 levels (aggravation of ferroptosis was accompanied by an increase in FTH1 levels), demonstrating a strong association between ferroptosis and ferritinophagy. [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. In contrast to this result, in our study, sodium butyrate ameliorated intestinal IRI-induced ferroptosis, and GSH, GPX4, and FTH1 levels were decreased by the addition of the NRF2 inhibitor, which means that ferroptosis rose along with a decrease in FTH1, obviously inconsistent with the outcome in the tumor cells, and it remains to be investigated whether ferritinophagy plays a role in this.\u003c/p\u003e \u003cp\u003eIn summary, the results of this article prove that sodium butyrate can regulate SLC7A11/GPX4 levels by upregulating NRF2, inhibiting ferroptosis and reducing intestinal IRI.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis study was supported by National Natural Science Foundation of China (Grant No. 82260381), Medical Innovation and Development Project of Lanzhou University (Grant No. lzuyxcx-2022-108).\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eAll authors declare that they have no competing interests related to this study.\u003c/p\u003e\n\u003ch2\u003eData Availability\u003c/h2\u003e\n\u003cp\u003eData will be made available on request.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eZizen Zhao: the main writer of the article, the main operator of the experiment, produced Figures 1 and 2.Yufang Leng: Corresponding author, ensured all authors approved the manuscript and were responsible for the article.Liya Chang: assisted with experiments and revised the manuscript.Yu Wang: Assist with experiments, suggest experimental directions, and manage data.Dongbin Li: Assist with experiments, management and supervision.Yang Xing: Assisted with experiments and presented methodology.Yuxuan Wu: Assist with experiments to produce Figures 3-5.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eDeng F, Lin ZB, Sun QS, Min Y, Zhang Y et al. 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Science Advances. 2023;9(5):eade9585. https://doi.org/10.1126/sciadv.ade9585\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"gut, intestinal ischemia reperfusion injury, sodium butyrate, ferroptosis","lastPublishedDoi":"10.21203/rs.3.rs-3988128/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3988128/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eSodium butyrate, a short-chain fatty acid produced by the metabolism of intestinal flora, has been shown to have a protective effect against intestinal ischemia reperfusion injury (IRI), but its effect on intestinal IRI-associated ferroptosis has not yet been demonstrated.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eIn this experiment, we used a model of superior mesenteric artery occlusion in mice to examine the effects and principles of sodium butyrate on ferroptosis related to intestinal IRI. We assessed the role of sodium butyrate by constructing mice intestinal IRI models and determining the extent of intestinal tissue damage and changes in ferroptosis-related factors.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eResults show that Intestinal IRI mice showed increased tissue damage, massive infiltration of inflammatory cells, severe destruction of villus structure, and elevated MDA. In addition, intestinal IRI led to downregulation of GSH, GPX4, FTH1 and SLC7A11, regulators of ferroptosis, and a rise in Fe\u003csup\u003e2+\u003c/sup\u003e, as well as downregulation of NRF2 by intestinal IRI. Sodium butyrate had an attenuating effect on intestinal IRI, whereas mice exogenously supplemented with sodium butyrate showed less tissue damage, some restoration of villus structure and decreased MDA, up-regulation of GSH, GSH/GSSG, GPX4, FTH1, SLC7A11, and NRF2, and decreased Fe\u003csup\u003e2+\u003c/sup\u003e.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThis experiment demonstrates that sodium butyrate regulates the SLC7A11/GPX4 pathway by up-regulating NRF2, thereby inhibiting ferroptosis and attenuating intestinal IRI.\u003c/p\u003e","manuscriptTitle":"Sodium butyrate inhibits ferroptosis and ameliorates intestinal ischemia-reperfusion injury by modulating the NRF2/SLC7A11/GPX4 pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-29 19:02:29","doi":"10.21203/rs.3.rs-3988128/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d830777d-b94d-4d7f-a126-c23c51847983","owner":[],"postedDate":"February 29th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-05-02T15:01:24+00:00","versionOfRecord":[],"versionCreatedAt":"2024-02-29 19:02:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3988128","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3988128","identity":"rs-3988128","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}