FGF21 inhibited NETs formation induced by myocardial I/R injury via AMPK

FGF21 inhibited NETs formation induced by myocardial I/R injury via AMPK | 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 FGF21 inhibited NETs formation induced by myocardial I/R injury via AMPK Lingyun Gu, Chengao Jia, Zuozhen Sheng, Wenlong Jiang, Zhuowen Xu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4111215/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 Background Previous investigations have established the anti-inflammatory properties of fibroblast growth factor 21 (FGF21). However, the specific mechanism through which FGF21 mitigates myocardial ischemia/reperfusion (I/R) injury by inhibiting neutrophil extracellular traps(NETs) formation remains unclear. Methods A mice model of myocardial I/R injury was induced, and myocardial tissue was stained with immunofluorescence to assess the formation of NETs. Serum NETs levels were quantified using a PicoGreen kit. In addition, the expression levels of AMP-activated protein kinase (AMPK) and FGF21 were evaluated by Wes fully automated protein blotting quantitative analysis system. Moreover, an hypoxia/reoxygenation (H/R) model was established using AMPK inhibitor and agonist pretreated H9c2 cells to further explore the relationship between FGF21 and AMPK. Results Compared with the control group, serum NETs levels were significantly higher in I/R mice, and a large number of NETs were formed in myocardial tissues (97.63±11.45 vs 69.65±3.33, P<0.05). However, NETs levels were reversed in FGF21 pretreated mice (P<0.05). Further studies showed that FGF21 enhanced AMPK expression, which was significantly increased after inhibition of AMPK and decreased after promotion of AMPK (P<0.05). Conclusions FGF21 may exert cardioprotective effects by inhibiting I/R injury-induced NETs formation via AMPK. FGF21 Myocardial I/R injury NETs AMPK Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Background Acute myocardial infarction (AMI) is a common clinical cardiovascular emergency. Emergency percutaneous coronary intervention (PCI), which significantly reduces mortality, is the main strategy for reperfusion therapy in patients with a AMI [ 1 ]. However, myocardial reperfusion therapy, while essential for restoring coronary blood flow, may also cause additional myocardial damage called myocardial ischemia/reperfusion (I/R) injury, which accounts for approximately 50% of the final myocardial area of injury [ 2 – 4 ]. Despite technological advances in myocardial reperfusion therapy, including continuous improvements in PCI techniques and drugs, an effective approach to preventing I/R injury remains elusive [ 5 ]. Myocardial I/R injury encompasses various pathophysiological mechanisms, prominently featuring inflammatory response, oxidative stress, and mitochondrial dysfunction. Among these, inflammatory response and oxidative stress emerge as pivotal factors [ 6 ]. In the initial hours of AMI, inflammatory cells, notably neutrophils, extensively infiltrate the infarcted area [ 7 ]. Neutrophils become activated in response to various factors, including pro-inflammatory cytokines, activated platelets, damaged vascular endothelium, and oxygen free radicals. Activated neutrophils release chromatin and granulocyte enzymes outside the cell, forming a net-like structure termed neutrophil extracellular traps (NETs) [ 8 ]. While NETs play a critical role in the inflammatory response and offer benefits in various infectious diseases, in sterile diseases, they promote inflammation by releasing active molecules in substantial quantities [ 9 ]. NETs levels in the peripheral blood of AMI patients correlate with disease severity and prognosis, and elevated NETs levels in intracoronary thrombi correlate with reduced left ventricular ejection fraction and infarct size [ 10 – 13 ]. NET levels independently predict major adverse cardiac events (MACE) occurrence within a year following AMI [ 14 ]. NETs promote thrombosis by causing damage to vascular endothelial cells, inducing an inflammatory response, serving as a scaffold for platelet aggregation and activation, and activating the coagulation system while inhibiting the fibrinolytic process [ 15 ]. In a mice AMI model, the anti-inflammatory effects of colchicine significantly inhibited NETs formation, inflammatory responses, improved myocardial tissue remodeling, and increased survival rates [ 16 ]. Fibroblast growth factor 21 (FGF21), which has anti-inflammatory properties and attenuates myocardial I/R injury, is significantly increased in the serum of patients with AMI and correlates with MACE [ 17 , 18 ]. However, whether FGF21 can mitigate myocardial I/R injury by inhibiting NETs remains unexplored. In this study, we explored the mechanism of FGF21 inhibition of NETs during I/R injury by using a mice I/R injury model and a myocardial hypoxia/reoxygenation (H/R) model. Materials and methods Experimental study design Six- to eight-week-old male C57BL/6WT mice were purchased from Spivey (Beijing) Biotechnology Co. All mice were maintained in SPF chambers with temperatures between 20–26°C, humidity between 40–75%, and a light/dark cycle of 12 hours. Mice had free access to standard laboratory chow and water. Following one week of adaptive feeding, the mice were randomly allocated to one of three groups: control group (n = 6), I/R group (n = 6), I/R + FGF21 group (n = 3) and I/R + Compound C group (n = 3). Before establishing the myocardial I/R injury model, mice in the I/R + FGF21 group received subcutaneous injections of recombinant FGF21 protein (0.1 mg/kg/day, Novoprotein, China) for fourteen consecutive days. Mice in the I/R + Compound C group were administered intraperitoneal injections of 5 mg/kg of Compound C (MCE, USA) one hour before inducing I/R injury. H9c2 cells were pretreated with Compound C (10 µmol/L, MCE, USA) and AICAR (1 mmol/L, MCE, USA) for 24 hours before modeling. Then, H9c2 cells were randomly divided into control group (n = 3), H/R group (n = 3), H/R + Compound C group (n = 3), and H/R + AICAR group (n = 3). Establishment of an animal model of myocardial I/R injury After 10% chloral hydrate (3.5 ml/kg) intraperitoneal anaesthesia of mice, the 4th rib at the left margin of the sternum was clipped. After bluntly separating and clipping the pericardium, the anterior descending branch was ligated with sutures under a body mirror to establish a model of myocardial I/R injury. The I/R injury model was induced by occluding the left anterior descending coronary artery for 30 minutes, followed by 24 hours of reperfusion. There were no extra surviving mice in this experiment due to strict control of the use of experimental animals. Mice were executed by cervical dislocation at the end of the experiment. Hearts were promptly immersed in liquid nitrogen for preservation. Peripheral blood samples were collected, and after centrifugation at 3000 rpm for 10 minutes, the supernatant was stored in tubes at -80 degrees Celsius. Cell culture and establishment of H/R models H9c2 cardiomyocytes obtained from ATCC were cultured in a medium comprising 90% DMEM and 10% FBS, maintained in an incubator at 37°C with 5% CO2. For establishing anaerobic conditions, H9c2 cardiomyocytes were placed in a 2.5 L sealed culture jar (MGC, Japan) with a 2.5 L anaerobic gas-producing bag (MGC, Japan). The sealed jars were incubated at 37°C for 12 hours. Subsequently, H9c2 cells were removed from the sealed jars and incubated for an additional 24 hours at 37°C to establish the H/R model. TTC staining The myocardial tissue underwent rapid cooling to -20°C for approximately 15 minutes. Subsequently, it was meticulously sliced into five 2 mm thick sections in the coronal direction. These sections were then immersed in a 2% TTC solution at 37°C until normal myocardial tissue displayed a red hue, while infarcted tissue exhibited a white appearance. Once the color change was achieved, the sections were carefully retrieved. The treated sections were immersed in a 4% paraformaldehyde solution for one hour and subsequently examined at a macro level. Utilizing Image-Pro Plus 5.0 image analysis software, we quantified the area of myocardial infarction. To calculate the volume of myocardial infarction for each section, the mean number of infarction areas was multiplied by the section's thickness. The total myocardial infarct volume for the mice was then determined by summing the infarct volumes of all slices. To express the magnitude of myocardial infarction as a percentage, the infarct volume was divided by the total volume, and the resulting value was multiplied by 100%. HE staining The myocardial tissue was fixed using a 4% formaldehyde solution and underwent dehydration through a series of graded ethanol solutions (75%, 85%, 95%, 95%, 100%, 100%). After washing with xylene, the myocardial tissues were embedded in paraffin and sectioned to a thickness of 2 µm. The resulting sections were subjected to a sequential treatment involving immersion in xylene, graded ethanol, pure water, and hematoxylin staining solution. Following this, sections were rinsed with tap water and reblued using Scott's Bluing Solution. To facilitate staining, the previously rinsed sections were immersed in eosin staining solution, followed by swift dehydration in a graded ethanol series and ultimate sealing with a sealer. Sections were meticulously examined using an Olympus microscope from various angles and magnifications, and photographs were captured for subsequent analysis. The obtained results were subjected to detailed analysis. Immunofluorescence staining The dewaxed sections underwent heat treatment in an autoclave using an antigen repair solution until boiling. Subsequent to natural cooling, the sections were subjected to treatment with 5% bovine serum albumin (Solarbio, China) for 30 minutes at 37°C. Primary antibodies, specifically myeloperoxidase (MPO) (Proteintech, USA) and Histone H3 antibodies (Abcam, UK), were employed to incubate the myocardial tissue sections overnight at 4°C. Following this incubation, sections were rinsed with PBS and then treated with either cy3 goat anti-rabbit IgG antibody (ABdonal, China) or goat-anti-rabbit IgG/488 (ZSGB, China) for 30 minutes at 37°C, with protection from light. Subsequently, nuclei were stained with DAPI (KeyGEN, China), and the sections were subjected to blocking with a solution containing an anti-fluorescence quencher. The treated sections were then observed and analyzed using a fluorescence microscope (BX53, OLYMPUS). Picogreen Quantification of dsDNA The working solution was prepared following the instructions provided with the PicoGreen kit (Yesen, China). Subsequently, 100 µL of the sample and 100 µL of the prepared working solution were added to a 96-well plate and incubated at room temperature for 5 minutes while avoiding exposure to light. The fluorescence intensity of the samples was measured using an enzyme marker with excitation at 480 nm and emission at 520 nm. Cell Activity Assay The medium from H9c2 cardiomyocytes inoculated into 96-well plates was aspirated. Subsequently, 80 µL of CellCounting-Lite 2.0 (Vazyme, China) was added to each well, shaken and mixed, and left at room temperature for 5 minutes. Following this incubation, the luminescence signal was detected. Wes fully automated protein blotting quantitative analysis system to detect protein expression Proteins extracted from mice myocardial tissue and H9C2 cells underwent lysis and centrifugation, with their concentrations determined using the BCA method (MA0082, China). Samples, primary antibodies, including FGF21 antibody (Abcam, UK), AMP-activated protein kinase (AMPK) antibody (Affinity, USA), GAPDH antibody (TransGen, China) and luminescent solutions were prepared according to the guidance instructions of the Wes Fully Automated Protein Blotting and Quantitative Analysis System (Bio-Techne, USA), after which they were added sequentially to the plates and run on the machine. Assay results were analysed by using Compass software. Statistical analysis Statistical analyses were conducted using the SPSS 22.0 statistical package, and graphical representations were generated with GraphPad Prism 8.0.0. Quantitative variables were presented as mean ± standard deviation and compared using the Student t-test. For comparisons involving multiple groups, one-way ANOVA was employed, and statistical significance was considered at a P value of < 0.05 (two-tailed). Results FGF21 reduced the volume of myocardial I/R injury Myocardial infarction volume post I/R was assessed using TTC staining. The results revealed that mice subjected to I/R injury developed larger myocardial infarcts, constituting approximately (11.15 ± 2.97) % of the infarct area in the left ventricle compared to the control group (P < 0.05). In contrast, the percentage of myocardial infarct area in mice pretreated with FGF21 before I/R injury was approximately (7.70 ± 1.75) %, significantly lower than the I/R group, with a statistically significant difference (P < 0.05) (Fig. 1 ). FGF21 attenuated myocardial tissue damage caused by I/R injury HE staining illustrated that myocardial cells in the control group displayed a neat and tightly arranged structure, regular morphology, and no apparent edema or inflammatory cell infiltration. Conversely, myocardial cells in the I/R injury group exhibited abnormal morphology, disorganized arrangement, and a substantial influx of inflammatory cells. Notably, the pathological changes in the myocardium were markedly improved in mice with I/R injury pretreated with FGF21 (Fig. 2 ). These findings demonstrated the ability of FGF21 to attenuate myocardial I/R injury. FGF21 inhibited I/R injury-induced NETs formation To further investigate the mechanism by which FGF21 attenuates myocardial I/R injury, we first examined the formation of NETs after myocardial I/R injury by immunofluorescence. Immunofluorescence revealed almost no NETs formation in the myocardial tissue of the control group. However, histone H3 and MPO expression significantly increased in the myocardial tissue of the I/R group, indicating substantial NETs formation due to I/R. In contrast, NETs formation was significantly reduced in I/R-injured mice pretreated with FGF21 compared to the I/R group (Fig. 3 ). Detection of dsDNA, the main component of NETs, using PicoGreen revealed that serum levels of NETs were significantly higher in the I/R group of mice (97.63 ± 11.45 vs 69.65 ± 3.33) compared to the control group (P < 0.05). In contrast, NETs levels were significantly lower in the I/R + FGF21 group compared to the I/R group (69.60 ± 5.66 vs 97.63 ± 11.45, P < 0.05) (Fig. 4 ). These studies highlighted that FGF21 could inhibit the formation of NETs induced by myocardial I/R injury. Inhibition of AMPK exacerbates myocardial tissue I/R injury To investigate the mechanism by which FGF21 inhibited the formation of NETs induced by myocardial I/R injury, we first observed myocardial tissues after inhibition of AMPK during I/R injury by HE staining. In the I/R injury group, myocardial cells exhibited abnormal morphology, disorganization, and infiltration by a large number of inflammatory cells. Conversely, these pathological changes were significantly exacerbated in I/R-injured mice pretreated with Compound C, an inhibitor of AMPK (Fig. 5 ). The study demonstrated that inhibition of AMPK exacerbated myocardial I/R injury. Increased formation of NETs after AMPK inhibition The formation of NETs in the myocardium of mice pretreated with AMPK inhibitor was detected by immunofluorescence. Immunofluorescence demonstrated that there was almost no histone H3 and MPO expression in the myocardial tissue of the control group, indicating no NETs formation. In contrast, NETs formation increased further in I/R-injured mice pretreated with Compound C, compared to the I/R group (Fig. 6 ). Next, NETs levels were quantified by PicoGreen and found to be significantly higher in the serum of mice in the I/R group compared to the control group (103.03 ± 21.79 vs 75.08 ± 8.03), with a statistically significant difference (P < 0.05). In contrast, NETs levels were significantly higher in mice in the I/R + Compound C group compared to the I/R group (153.00 ± 20.11 vs 103.03 ± 21.79, P < 0.05) (Fig. 7 ). AMPK Mediated the Inhibition of Myocardial I/R Injury by FGF21 To further explore the relationship between FGF21 and AMPK, AMPK protein expression levels were measured by establishing an I/R injury model in mice pre-injected with FGF21. The study showed that AMPK protein expression levels were increased in the I/R group compared with the control group, and further increased in the I/R + FGF21 group (P < 0.05) (Fig. 8 A). Subsequently, FGF21 protein expression levels were measured by establishing an I/R injury model in mice pre-injected with Compound C. The study showed that FGF21 protein expression levels were significantly higher in the I/R group compared with the control group and further increased in the I/R + Compound C group (all P < 0.05) (Fig. 8 B). These studies identified the involvement of AMPK in the cardioprotective effects of FGF21. The H/R model was then established after pre-treatment of H9c2 cells with Compound C, an inhibitor of AMPK, and AICAR, an agonist. Different hypoxia groups (2h, 6h, 12h) were set up with a reoxygenation time of 12h to detect cellular activity. The study demonstrated that AMPK was able to inhibit H/R-induced cardiomyocyte injury and had a cardioprotective effect. It was found that with the prolongation of hypoxia time, the activity of each group of cells gradually decreased and reached a trough at 12 hours of hypoxia, so the time point of 12 hours of hypoxia was chosen for the follow-up study. Finally, FGF21 protein expression levels were examined after pre-treatment of H9c2 cells with the AMPK inhibitor Compound C and the agonist AICAR. The FGF21 protein expression level in the H/R + Compound C group was further increased compared to the H/R group (P < 0.05), while the FGF21 protein expression level in the H/R + AICAR group was significantly decreased compared to the H/R + Compound C group (P < 0.05) (Fig. 8 C). The study suggested that AMPK played an important role in the mechanism of FGF21 inhibition of myocardial H/R injury. Discussion The results of the present study suggested that FGF21 has the ability to attenuate myocardial I/R injury by inhibiting NET formation. Furthermore, the cardioprotective effect of FGF21 was mediated through the involvement of AMPK. A large number of previous studies have found that FGF21 attenuates myocardial I/R injury, increases cardiomyocyte activity, and decreases the rate of cardiomyocyte apoptosis, which is consistent with our study [ 19 – 21 ]. In a rat model of myocardial I/R injury, a notable upregulation in the expression of NETs was observed within the ischemic zone. Interestingly, facilitating the degradation of NETs through deoxy ribonuclease I led to a reduction in myocardial infarct size and a significant improvement in left ventricular remodeling induced by I/R injury in rats [ 22 ]. In a mice model of chronic pancreatitis, mice treated with FGF21 significantly reduced levels of MPO, a biomarker of NETs, and improved the inflammatory state of serum, pancreatic, and peritoneal macrophages [ 23 ]. Significantly, our current investigation, within a myocardial I/R injury model, has unveiled for the first time that FGF21 exhibits the capacity to inhibit the formation of NETs induced by I/R injury, thereby attenuating myocardial I/R injury. Previous studies have shown that FGF21 is an endocrine factor that can regulate energy metabolism and that AMPK, as an energy sensor, is a target for FGF21 signaling [ 24 ]. FGF21 activates AMPK both directly through the FGFR1/klotho-β complex and indirectly by inducing the expression of lipofuscin and corticosterone [ 25 ]. Once activated, AMPK plays a crucial role in regulating mitochondrial activity by stimulating SIRT1 and PGC-1α [ 26 ]. Furthermore, AMPK contributes to the maintenance of lipid homeostasis, protein synthesis regulation, and promotion of glucose metabolism, all aimed at sustaining optimal intracellular adenosine triphosphate levels [ 27 ]. Beyond its role in stimulating AMPK to regulate energy metabolism, FGF21 also exhibits anti-inflammatory properties. Studies have shown that a lack of FGF21 increases susceptibility to inflammation, leading to local and systemic inflammatory responses, and supplementation with exogenous FGF21 has been shown to reverse elevated levels of these inflammatory markers [ 28 – 30 ]. In hypertensive and diabetic mice models, the absence of FGF21 resulted in a pronounced exacerbation of inflammatory response and oxidative stress. Conversely, administration of FGF21 significantly alleviated these conditions by promoting the phosphorylation of AMPK, highlighting the crucial role of FGF21 in mitigating inflammatory and oxidative stress pathways [ 31 , 32 ]. Subsequent investigations have unveiled that FGF21 exerts its anti-inflammatory, antioxidant stress, and inhibitory effects on myocardial remodeling and apoptosis through two distinct pathways. The first pathway involves the AMPK-AKT2-Nrf2 antioxidant pathway, while the second pathway operates through the AMPK-ACC-CPT-1 lipid-lowering pathway, both of which are mediated by AMPK [ 33 ]. Studies have shown that imbalances in the mechanisms that produce or eliminate NETs can lead to tissue damage, which is an important mechanism in autoimmune and inflammatory diseases that are closely linked to cardiovascular disease, and that inhibiting NETs formation has been shown to be beneficial [ 34 , 35 ]. Several studies have consistently demonstrated that AMPK, known for its anti-inflammatory effects, plays a pivotal role in the formation of NETs [ 36 – 39 ]. Activation of AMPK both inhibits NETs formation and promotes NETs clearance to reduce NETs levels[ 40 , 41 ]. However, inhibition of AMPK significantly increased the levels of NETs in the peripheral circulation and in ischemic tissues [ 42 ]. The present study has some limitations. Due to the limitation of funds and experimental equipment, cardiac ultrasonography was not performed in I/R-injured mice in this study, so it was not possible to judge the improvement of cardiac function in mice after the application of FGF21. Although the present study suggests a possible mechanism by which FGF21 inhibits the formation of NETs through activation of AMPK, the actual biological mechanism is likely to be more complex, and other unconsidered signaling pathways or cell types may also be involved. Conclusion In the present study, we observed a significant elevation in NETs levels in both serum and myocardial tissue during myocardial I/R injury. Notably, the administration of FGF21 demonstrated a remarkable reversal of this increase. Subsequent studies delved into the mechanisms, suggesting that FGF21 may possess the ability to inhibit I/R injury-induced NETs formation through the activation of AMPK. These findings highlight a potential therapeutic role for FGF21 in mitigating NETs-mediated responses in the context of myocardial I/R injury. Declarations Ethics approval and consent to participate All protocols in this study were approved by the Nantong University Laboratory Animal Center (Approval No: S20210820-002) in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85 − 23, revised 1996). All methods in our manuscript are reported in accordance with the ARRIVE guidelines ( https://arriveguidelines.org ) for reporting animal experiments. Consent for publication Not applicable. Competing interests The authors have no conflicts of interest to disclose. Funding Sources This research was supported by the development fund of Affiliated Hospital of Xuzhou Medical University (grant no. XYFY2021029), Medical Research Program of Jiangsu Provincial Health and Wellness Commission (grant no. Z2021042) and Jiangyin Young and Middle-aged Reserve Excellent Talents Program (grant no. JYROYT202309). Author Contribution Lingyun Gu contributed to the conception, methodology and writing-original draft. Chengao Jia and Zuozhen Sheng carried out the methodology. Zhuowen Xu and Wenlong Jiang carried out the interpretation of data. Weizhang Li and Hua Zhang carried out the conception, resource and supervision. Availability of data and materials The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. References Bhatt DL, Lopes RD, Harrington RA. Diagnosis and Treatment of Acute Coronary Syndromes: A Review. JAMA. 2022;327(7):662–75. Ibanez B, Heusch G, Ovize M, Van de Werf F. Evolving therapies for myocardial ischemia/reperfusion injury. J Am Coll Cardiol. 2015;65(14):1454–71. Frank A, Bonney M, Bonney S, Weitzel L, Koeppen M, Eckle T. Myocardial ischemia reperfusion injury: from basic science to clinical bedside. Semin Cardiothorac Vasc Anesth. 2012;16(3):123–32. 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Hexavalent chromium inhibits the formation of neutrophil extracellular traps and promotes the apoptosis of neutrophils via AMPK signaling pathway. Ecotoxicol Environ Saf. 2021;223:112614. Ong CW, Elkington PT, Brilha S, Ugarte-Gil C, Tome-Esteban MT, Tezera LB, et al. Neutrophil-Derived MMP-8 Drives AMPK-Dependent Matrix Destruction in Human Pulmonary Tuberculosis. PLoS Pathog. 2015;11(5):e1004917. Rodriguez C, Munoz M, Contreras C, Prieto D. AMPK, metabolism, and vascular function. FEBS J. 2021;288(12):3746–71. Gregoire M, Uhel F, Lesouhaitier M, Gacouin A, Guirriec M, Mourcin F et al. Impaired efferocytosis and neutrophil extracellular trap clearance by macrophages in ARDS. Eur Respir J. 2018; 52(2). Chiang N, Sakuma M, Rodriguez AR, Spur BW, Irimia D, Serhan CN. Resolvin T-series reduce neutrophil extracellular traps. Blood. 2022;139(8):1222–33. Wu ML, Zou X, Chen XY, Ma KT, Chen C, Yu NW, et al. Phthalide derivative CD21 regulates the platelet- neutrophil extracellular trap-thrombin axis and protects against ischemic brain injury in rodents. Int Immunopharmacol. 2023;114:109547. Additional Declarations No competing interests reported. Supplementary Files rawdata.zip Figurelegend.docx Figurelegend.docx rawdata.zip 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-4111215","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":283487662,"identity":"60d06d87-a026-48d8-a48b-3ce8dfca79a6","order_by":0,"name":"Lingyun Gu","email":"","orcid":"","institution":"The Jiangyin Clinical College of Xuzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Lingyun","middleName":"","lastName":"Gu","suffix":""},{"id":283487663,"identity":"824d60be-57f0-41a7-9569-08d221d903b0","order_by":1,"name":"Chengao Jia","email":"","orcid":"","institution":"The Jiangyin Clinical College of Xuzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Chengao","middleName":"","lastName":"Jia","suffix":""},{"id":283487664,"identity":"102181d4-4ad3-4692-b067-613a1edacd05","order_by":2,"name":"Zuozhen Sheng","email":"","orcid":"","institution":"Jiangyin Hospital Affiliated to Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Zuozhen","middleName":"","lastName":"Sheng","suffix":""},{"id":283487665,"identity":"6cc69676-dbbf-4a75-b646-45aaa60efc98","order_by":3,"name":"Wenlong Jiang","email":"","orcid":"","institution":"The Jiangyin Clinical College of Xuzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Wenlong","middleName":"","lastName":"Jiang","suffix":""},{"id":283487666,"identity":"666aaf09-bdee-417b-891e-194be3b974a5","order_by":4,"name":"Zhuowen Xu","email":"","orcid":"","institution":"The Jiangyin Clinical College of Xuzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Zhuowen","middleName":"","lastName":"Xu","suffix":""},{"id":283487667,"identity":"309747b7-f4fa-4cc0-aa69-b5bb22d2423a","order_by":5,"name":"Weizhang Li","email":"","orcid":"","institution":"The Jiangyin Clinical College of Xuzhou Medical University","correspondingAuthor":false,"prefix":"","firstName":"Weizhang","middleName":"","lastName":"Li","suffix":""},{"id":283487668,"identity":"dddb923a-bbd2-4581-b6d6-42f4af9b8c7e","order_by":6,"name":"Hua Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA00lEQVRIiWNgGAWjYHACNhCRwMDAfODAhx+kaWFLPDizhzQtPMaHOdiIUG8+I/3Zg587avMMjp/5cJiBh0GeX+wAfi0yNxLSDXvPHC82OJO74XCBBYPhzNkJ+LVISCQck+BtO5a44QBQywwehgSD2wS1JLZJ/gVpOf/mwWEeNqK0JLNJ87bVJG64kcNApBaeZ2zSsm0HEmfeeGYADGQJIvzCnv5M8m1bXWLf+eTHHz78sJHnlyaghUEArOAw3AgCykGA/wCIrCNC5SgYBaNgFIxYAABIHEtNBFzfaAAAAABJRU5ErkJggg==","orcid":"","institution":"The Jiangyin Clinical College of Xuzhou Medical University","correspondingAuthor":true,"prefix":"","firstName":"Hua","middleName":"","lastName":"Zhang","suffix":""}],"badges":[],"createdAt":"2024-03-16 04:29:23","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4111215/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4111215/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53450541,"identity":"e8c3fb8c-2b7d-440b-8c48-dba578d271a9","added_by":"auto","created_at":"2024-03-26 06:34:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":637004,"visible":true,"origin":"","legend":"\u003cp\u003eFGF21 inhibited I/R injury in myocardial tissue (TTC staining). (A) TTC staining of myocardial tissue. (B) Myocardial Infarction Volume Calculated Based on TTC Staining. *P\u0026lt;0.05, compared to control group; #P\u0026lt;0.05, compared to I/R group.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4111215/v1/664263d4a031878da861e915.png"},{"id":53450543,"identity":"e7cced1f-5172-4ef3-8bfc-95a2e7a50f4f","added_by":"auto","created_at":"2024-03-26 06:34:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1957883,"visible":true,"origin":"","legend":"\u003cp\u003eMyocardial tissue after myocardial I/R injury in mice pretreated with FGF21 (HE staining, ×400).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4111215/v1/9c07701558a7d89bb60b7dd7.png"},{"id":53451219,"identity":"8491eac7-7150-448b-a79a-a2d88df129ab","added_by":"auto","created_at":"2024-03-26 06:42:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1242729,"visible":true,"origin":"","legend":"\u003cp\u003eNETs formation in myocardial necrotic tissues was detected after the establishment of a myocardial I/R injury model in FGF21 pretreated mice (immunofluorescence, ×400).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4111215/v1/2a56d750b7c045b03901cbe1.png"},{"id":53450549,"identity":"b69ebe5f-0c26-4c44-b181-7b0ebb81c966","added_by":"auto","created_at":"2024-03-26 06:34:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":38993,"visible":true,"origin":"","legend":"\u003cp\u003eMeasurement of peripheral serum dsDNA levels in FGF21 pretreated mice using the PicoGreen assay. *P\u0026lt;0.05, compared to control group; #P\u0026lt;0.05, compared to I/R group.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4111215/v1/6accc2d5ff83ae541a20905e.png"},{"id":53450546,"identity":"aac7cf3c-1806-4956-978a-6aead84bf4fd","added_by":"auto","created_at":"2024-03-26 06:34:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1865363,"visible":true,"origin":"","legend":"\u003cp\u003eMyocardial tissue after myocardial I/R injury in mice pretreated with AMPK inhibitor (HE staining, ×400).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4111215/v1/7ea591bac5cc9710e7421998.png"},{"id":53450550,"identity":"bfc126cd-2c10-47b2-8684-109d191fc479","added_by":"auto","created_at":"2024-03-26 06:34:01","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2005766,"visible":true,"origin":"","legend":"\u003cp\u003eNETs formation in myocardial necrotic tissues was detected after the establishment of a myocardial I/R injury model in Compound C pretreated mice (immunofluorescence, ×400).\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4111215/v1/ec84a024152a18386b97eebe.png"},{"id":53450544,"identity":"9e5ac9b9-d898-4e19-8f9d-b431c4c173a9","added_by":"auto","created_at":"2024-03-26 06:34:01","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":41709,"visible":true,"origin":"","legend":"\u003cp\u003eMeasurement of peripheral serum dsDNA levels in Compound C pretreated mice using the PicoGreen assay. *P\u0026lt;0.05, compared to control group; #P\u0026lt;0.05, compared to I/R group.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-4111215/v1/2c4d5eff20c31893dc40c255.png"},{"id":53450551,"identity":"180e985f-0eb5-469d-9505-cbfb37c95f5c","added_by":"auto","created_at":"2024-03-26 06:34:01","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":220244,"visible":true,"origin":"","legend":"\u003cp\u003eThe interaction of FGF21 and AMPK was detected in a mouse model of myocardial I/R injury or in an H/R model of H9c2 cells. (A) AMPK protein expression levels were measured in mice pre-injected with FGF21. (B) FGF21 protein expression levels were measured in mice pre-injected with AMPK inhibitor Compound C. (C) FGF21 protein expression levels were measured in H9c2 cells pretreated with Compound C, an inhibitor of AMPK, or with AICAR, an agonist. *P\u0026lt;0.05, compared to control; #P\u0026lt;0.05, compared to I/R group; \u0026amp;P\u0026lt;0.05, compared to H/R+Compound C group.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-4111215/v1/798e6e89f04cf94a5d64db40.png"},{"id":60652117,"identity":"bc1a960d-77c4-4059-8d7f-30e463df1b69","added_by":"auto","created_at":"2024-07-19 06:44:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11610931,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4111215/v1/f40a30c3-4b2a-4a05-8c93-f126aedf89bd.pdf"},{"id":53450553,"identity":"59b2bba6-2449-4d3a-9e11-a82800a3fb7b","added_by":"auto","created_at":"2024-03-26 06:34:05","extension":"zip","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":63284991,"visible":true,"origin":"","legend":"","description":"","filename":"rawdata.zip","url":"https://assets-eu.researchsquare.com/files/rs-4111215/v1/e207ecd34e21782dd5f48d23.zip"},{"id":53450542,"identity":"866d09d1-7aec-405f-8c68-10be0fc6d750","added_by":"auto","created_at":"2024-03-26 06:34:01","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":35146,"visible":true,"origin":"","legend":"","description":"","filename":"Figurelegend.docx","url":"https://assets-eu.researchsquare.com/files/rs-4111215/v1/a32030ec6fbe328593e8c15e.docx"},{"id":53450548,"identity":"fef99572-efcd-4321-894e-92c528cfaf61","added_by":"auto","created_at":"2024-03-26 06:34:01","extension":"docx","order_by":10,"title":"","display":"","copyAsset":false,"role":"supplement","size":35146,"visible":true,"origin":"","legend":"","description":"","filename":"Figurelegend.docx","url":"https://assets-eu.researchsquare.com/files/rs-4111215/v1/86fcd2d209ef08a55e511a68.docx"},{"id":53450552,"identity":"68f20214-44e4-4b60-b705-1520509fde4b","added_by":"auto","created_at":"2024-03-26 06:34:04","extension":"zip","order_by":11,"title":"","display":"","copyAsset":false,"role":"supplement","size":63284991,"visible":true,"origin":"","legend":"","description":"","filename":"rawdata.zip","url":"https://assets-eu.researchsquare.com/files/rs-4111215/v1/b6a59893b0cb327f92c52aca.zip"}],"financialInterests":"No competing interests reported.","formattedTitle":"FGF21 inhibited NETs formation induced by myocardial I/R injury via AMPK","fulltext":[{"header":"Background","content":"\u003cp\u003eAcute myocardial infarction (AMI) is a common clinical cardiovascular emergency. Emergency percutaneous coronary intervention (PCI), which significantly reduces mortality, is the main strategy for reperfusion therapy in patients with a AMI [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. However, myocardial reperfusion therapy, while essential for restoring coronary blood flow, may also cause additional myocardial damage called myocardial ischemia/reperfusion (I/R) injury, which accounts for approximately 50% of the final myocardial area of injury [\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Despite technological advances in myocardial reperfusion therapy, including continuous improvements in PCI techniques and drugs, an effective approach to preventing I/R injury remains elusive [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMyocardial I/R injury encompasses various pathophysiological mechanisms, prominently featuring inflammatory response, oxidative stress, and mitochondrial dysfunction. Among these, inflammatory response and oxidative stress emerge as pivotal factors [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. In the initial hours of AMI, inflammatory cells, notably neutrophils, extensively infiltrate the infarcted area [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Neutrophils become activated in response to various factors, including pro-inflammatory cytokines, activated platelets, damaged vascular endothelium, and oxygen free radicals. Activated neutrophils release chromatin and granulocyte enzymes outside the cell, forming a net-like structure termed neutrophil extracellular traps (NETs) [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. While NETs play a critical role in the inflammatory response and offer benefits in various infectious diseases, in sterile diseases, they promote inflammation by releasing active molecules in substantial quantities [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eNETs levels in the peripheral blood of AMI patients correlate with disease severity and prognosis, and elevated NETs levels in intracoronary thrombi correlate with reduced left ventricular ejection fraction and infarct size [\u003cspan additionalcitationids=\"CR11 CR12\" citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. NET levels independently predict major adverse cardiac events (MACE) occurrence within a year following AMI [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. NETs promote thrombosis by causing damage to vascular endothelial cells, inducing an inflammatory response, serving as a scaffold for platelet aggregation and activation, and activating the coagulation system while inhibiting the fibrinolytic process [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In a mice AMI model, the anti-inflammatory effects of colchicine significantly inhibited NETs formation, inflammatory responses, improved myocardial tissue remodeling, and increased survival rates [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFibroblast growth factor 21 (FGF21), which has anti-inflammatory properties and attenuates myocardial I/R injury, is significantly increased in the serum of patients with AMI and correlates with MACE [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. However, whether FGF21 can mitigate myocardial I/R injury by inhibiting NETs remains unexplored. In this study, we explored the mechanism of FGF21 inhibition of NETs during I/R injury by using a mice I/R injury model and a myocardial hypoxia/reoxygenation (H/R) model.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExperimental study design\u003c/h2\u003e \u003cp\u003eSix- to eight-week-old male C57BL/6WT mice were purchased from Spivey (Beijing) Biotechnology Co. All mice were maintained in SPF chambers with temperatures between 20\u0026ndash;26\u0026deg;C, humidity between 40\u0026ndash;75%, and a light/dark cycle of 12 hours. Mice had free access to standard laboratory chow and water. Following one week of adaptive feeding, the mice were randomly allocated to one of three groups: control group (n\u0026thinsp;=\u0026thinsp;6), I/R group (n\u0026thinsp;=\u0026thinsp;6), I/R\u0026thinsp;+\u0026thinsp;FGF21 group (n\u0026thinsp;=\u0026thinsp;3) and I/R\u0026thinsp;+\u0026thinsp;Compound C group (n\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e \u003cp\u003eBefore establishing the myocardial I/R injury model, mice in the I/R\u0026thinsp;+\u0026thinsp;FGF21 group received subcutaneous injections of recombinant FGF21 protein (0.1 mg/kg/day, Novoprotein, China) for fourteen consecutive days. Mice in the I/R\u0026thinsp;+\u0026thinsp;Compound C group were administered intraperitoneal injections of 5 mg/kg of Compound C (MCE, USA) one hour before inducing I/R injury.\u003c/p\u003e \u003cp\u003eH9c2 cells were pretreated with Compound C (10 \u0026micro;mol/L, MCE, USA) and AICAR (1 mmol/L, MCE, USA) for 24 hours before modeling. Then, H9c2 cells were randomly divided into control group (n\u0026thinsp;=\u0026thinsp;3), H/R group (n\u0026thinsp;=\u0026thinsp;3), H/R\u0026thinsp;+\u0026thinsp;Compound C group (n\u0026thinsp;=\u0026thinsp;3), and H/R\u0026thinsp;+\u0026thinsp;AICAR group (n\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eEstablishment of an animal model of myocardial I/R injury\u003c/h2\u003e \u003cp\u003eAfter 10% chloral hydrate (3.5 ml/kg) intraperitoneal anaesthesia of mice, the 4th rib at the left margin of the sternum was clipped. After bluntly separating and clipping the pericardium, the anterior descending branch was ligated with sutures under a body mirror to establish a model of myocardial I/R injury. The I/R injury model was induced by occluding the left anterior descending coronary artery for 30 minutes, followed by 24 hours of reperfusion. There were no extra surviving mice in this experiment due to strict control of the use of experimental animals. Mice were executed by cervical dislocation at the end of the experiment. Hearts were promptly immersed in liquid nitrogen for preservation. Peripheral blood samples were collected, and after centrifugation at 3000 rpm for 10 minutes, the supernatant was stored in tubes at -80 degrees Celsius.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eCell culture and establishment of H/R models\u003c/h2\u003e \u003cp\u003eH9c2 cardiomyocytes obtained from ATCC were cultured in a medium comprising 90% DMEM and 10% FBS, maintained in an incubator at 37\u0026deg;C with 5% CO2. For establishing anaerobic conditions, H9c2 cardiomyocytes were placed in a 2.5 L sealed culture jar (MGC, Japan) with a 2.5 L anaerobic gas-producing bag (MGC, Japan). The sealed jars were incubated at 37\u0026deg;C for 12 hours. Subsequently, H9c2 cells were removed from the sealed jars and incubated for an additional 24 hours at 37\u0026deg;C to establish the H/R model.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eTTC staining\u003c/h2\u003e \u003cp\u003eThe myocardial tissue underwent rapid cooling to -20\u0026deg;C for approximately 15 minutes. Subsequently, it was meticulously sliced into five 2 mm thick sections in the coronal direction. These sections were then immersed in a 2% TTC solution at 37\u0026deg;C until normal myocardial tissue displayed a red hue, while infarcted tissue exhibited a white appearance. Once the color change was achieved, the sections were carefully retrieved. The treated sections were immersed in a 4% paraformaldehyde solution for one hour and subsequently examined at a macro level. Utilizing Image-Pro Plus 5.0 image analysis software, we quantified the area of myocardial infarction. To calculate the volume of myocardial infarction for each section, the mean number of infarction areas was multiplied by the section's thickness. The total myocardial infarct volume for the mice was then determined by summing the infarct volumes of all slices. To express the magnitude of myocardial infarction as a percentage, the infarct volume was divided by the total volume, and the resulting value was multiplied by 100%.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eHE staining\u003c/h2\u003e \u003cp\u003eThe myocardial tissue was fixed using a 4% formaldehyde solution and underwent dehydration through a series of graded ethanol solutions (75%, 85%, 95%, 95%, 100%, 100%). After washing with xylene, the myocardial tissues were embedded in paraffin and sectioned to a thickness of 2 \u0026micro;m. The resulting sections were subjected to a sequential treatment involving immersion in xylene, graded ethanol, pure water, and hematoxylin staining solution. Following this, sections were rinsed with tap water and reblued using Scott's Bluing Solution. To facilitate staining, the previously rinsed sections were immersed in eosin staining solution, followed by swift dehydration in a graded ethanol series and ultimate sealing with a sealer. Sections were meticulously examined using an Olympus microscope from various angles and magnifications, and photographs were captured for subsequent analysis. The obtained results were subjected to detailed analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eImmunofluorescence staining\u003c/h2\u003e \u003cp\u003eThe dewaxed sections underwent heat treatment in an autoclave using an antigen repair solution until boiling. Subsequent to natural cooling, the sections were subjected to treatment with 5% bovine serum albumin (Solarbio, China) for 30 minutes at 37\u0026deg;C. Primary antibodies, specifically myeloperoxidase (MPO) (Proteintech, USA) and Histone H3 antibodies (Abcam, UK), were employed to incubate the myocardial tissue sections overnight at 4\u0026deg;C. Following this incubation, sections were rinsed with PBS and then treated with either cy3 goat anti-rabbit IgG antibody (ABdonal, China) or goat-anti-rabbit IgG/488 (ZSGB, China) for 30 minutes at 37\u0026deg;C, with protection from light. Subsequently, nuclei were stained with DAPI (KeyGEN, China), and the sections were subjected to blocking with a solution containing an anti-fluorescence quencher. The treated sections were then observed and analyzed using a fluorescence microscope (BX53, OLYMPUS).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003ePicogreen Quantification of dsDNA\u003c/h2\u003e \u003cp\u003eThe working solution was prepared following the instructions provided with the PicoGreen kit (Yesen, China). Subsequently, 100 \u0026micro;L of the sample and 100 \u0026micro;L of the prepared working solution were added to a 96-well plate and incubated at room temperature for 5 minutes while avoiding exposure to light. The fluorescence intensity of the samples was measured using an enzyme marker with excitation at 480 nm and emission at 520 nm.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCell Activity Assay\u003c/h2\u003e \u003cp\u003eThe medium from H9c2 cardiomyocytes inoculated into 96-well plates was aspirated. Subsequently, 80 \u0026micro;L of CellCounting-Lite 2.0 (Vazyme, China) was added to each well, shaken and mixed, and left at room temperature for 5 minutes. Following this incubation, the luminescence signal was detected.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eWes fully automated protein blotting quantitative analysis system to detect protein expression\u003c/h2\u003e \u003cp\u003eProteins extracted from mice myocardial tissue and H9C2 cells underwent lysis and centrifugation, with their concentrations determined using the BCA method (MA0082, China). Samples, primary antibodies, including FGF21 antibody (Abcam, UK), AMP-activated protein kinase (AMPK) antibody (Affinity, USA), GAPDH antibody (TransGen, China) and luminescent solutions were prepared according to the guidance instructions of the Wes Fully Automated Protein Blotting and Quantitative Analysis System (Bio-Techne, USA), after which they were added sequentially to the plates and run on the machine. Assay results were analysed by using Compass software.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were conducted using the SPSS 22.0 statistical package, and graphical representations were generated with GraphPad Prism 8.0.0. Quantitative variables were presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation and compared using the Student t-test. For comparisons involving multiple groups, one-way ANOVA was employed, and statistical significance was considered at a P value of \u0026lt;\u0026thinsp;0.05 (two-tailed).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eFGF21 reduced the volume of myocardial I/R injury\u003c/h2\u003e \u003cp\u003eMyocardial infarction volume post I/R was assessed using TTC staining. The results revealed that mice subjected to I/R injury developed larger myocardial infarcts, constituting approximately (11.15\u0026thinsp;\u0026plusmn;\u0026thinsp;2.97) % of the infarct area in the left ventricle compared to the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In contrast, the percentage of myocardial infarct area in mice pretreated with FGF21 before I/R injury was approximately (7.70\u0026thinsp;\u0026plusmn;\u0026thinsp;1.75) %, significantly lower than the I/R group, with a statistically significant difference (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eFGF21 attenuated myocardial tissue damage caused by I/R injury\u003c/h2\u003e \u003cp\u003eHE staining illustrated that myocardial cells in the control group displayed a neat and tightly arranged structure, regular morphology, and no apparent edema or inflammatory cell infiltration. Conversely, myocardial cells in the I/R injury group exhibited abnormal morphology, disorganized arrangement, and a substantial influx of inflammatory cells. Notably, the pathological changes in the myocardium were markedly improved in mice with I/R injury pretreated with FGF21 (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). These findings demonstrated the ability of FGF21 to attenuate myocardial I/R injury.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eFGF21 inhibited I/R injury-induced NETs formation\u003c/h2\u003e \u003cp\u003eTo further investigate the mechanism by which FGF21 attenuates myocardial I/R injury, we first examined the formation of NETs after myocardial I/R injury by immunofluorescence. Immunofluorescence revealed almost no NETs formation in the myocardial tissue of the control group. However, histone H3 and MPO expression significantly increased in the myocardial tissue of the I/R group, indicating substantial NETs formation due to I/R. In contrast, NETs formation was significantly reduced in I/R-injured mice pretreated with FGF21 compared to the I/R group (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eDetection of dsDNA, the main component of NETs, using PicoGreen revealed that serum levels of NETs were significantly higher in the I/R group of mice (97.63\u0026thinsp;\u0026plusmn;\u0026thinsp;11.45 vs 69.65\u0026thinsp;\u0026plusmn;\u0026thinsp;3.33) compared to the control group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In contrast, NETs levels were significantly lower in the I/R\u0026thinsp;+\u0026thinsp;FGF21 group compared to the I/R group (69.60\u0026thinsp;\u0026plusmn;\u0026thinsp;5.66 vs 97.63\u0026thinsp;\u0026plusmn;\u0026thinsp;11.45, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). These studies highlighted that FGF21 could inhibit the formation of NETs induced by myocardial I/R injury.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eInhibition of AMPK exacerbates myocardial tissue I/R injury\u003c/h2\u003e \u003cp\u003eTo investigate the mechanism by which FGF21 inhibited the formation of NETs induced by myocardial I/R injury, we first observed myocardial tissues after inhibition of AMPK during I/R injury by HE staining. In the I/R injury group, myocardial cells exhibited abnormal morphology, disorganization, and infiltration by a large number of inflammatory cells. Conversely, these pathological changes were significantly exacerbated in I/R-injured mice pretreated with Compound C, an inhibitor of AMPK (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). The study demonstrated that inhibition of AMPK exacerbated myocardial I/R injury.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eIncreased formation of NETs after AMPK inhibition\u003c/h2\u003e \u003cp\u003eThe formation of NETs in the myocardium of mice pretreated with AMPK inhibitor was detected by immunofluorescence. Immunofluorescence demonstrated that there was almost no histone H3 and MPO expression in the myocardial tissue of the control group, indicating no NETs formation. In contrast, NETs formation increased further in I/R-injured mice pretreated with Compound C, compared to the I/R group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eNext, NETs levels were quantified by PicoGreen and found to be significantly higher in the serum of mice in the I/R group compared to the control group (103.03\u0026thinsp;\u0026plusmn;\u0026thinsp;21.79 vs 75.08\u0026thinsp;\u0026plusmn;\u0026thinsp;8.03), with a statistically significant difference (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In contrast, NETs levels were significantly higher in mice in the I/R\u0026thinsp;+\u0026thinsp;Compound C group compared to the I/R group (153.00\u0026thinsp;\u0026plusmn;\u0026thinsp;20.11 vs 103.03\u0026thinsp;\u0026plusmn;\u0026thinsp;21.79, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eAMPK Mediated the Inhibition of Myocardial I/R Injury by FGF21\u003c/h2\u003e \u003cp\u003eTo further explore the relationship between FGF21 and AMPK, AMPK protein expression levels were measured by establishing an I/R injury model in mice pre-injected with FGF21. The study showed that AMPK protein expression levels were increased in the I/R group compared with the control group, and further increased in the I/R\u0026thinsp;+\u0026thinsp;FGF21 group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eSubsequently, FGF21 protein expression levels were measured by establishing an I/R injury model in mice pre-injected with Compound C. The study showed that FGF21 protein expression levels were significantly higher in the I/R group compared with the control group and further increased in the I/R\u0026thinsp;+\u0026thinsp;Compound C group (all P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). These studies identified the involvement of AMPK in the cardioprotective effects of FGF21.\u003c/p\u003e \u003cp\u003eThe H/R model was then established after pre-treatment of H9c2 cells with Compound C, an inhibitor of AMPK, and AICAR, an agonist. Different hypoxia groups (2h, 6h, 12h) were set up with a reoxygenation time of 12h to detect cellular activity. The study demonstrated that AMPK was able to inhibit H/R-induced cardiomyocyte injury and had a cardioprotective effect. It was found that with the prolongation of hypoxia time, the activity of each group of cells gradually decreased and reached a trough at 12 hours of hypoxia, so the time point of 12 hours of hypoxia was chosen for the follow-up study.\u003c/p\u003e \u003cp\u003eFinally, FGF21 protein expression levels were examined after pre-treatment of H9c2 cells with the AMPK inhibitor Compound C and the agonist AICAR. The FGF21 protein expression level in the H/R\u0026thinsp;+\u0026thinsp;Compound C group was further increased compared to the H/R group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), while the FGF21 protein expression level in the H/R\u0026thinsp;+\u0026thinsp;AICAR group was significantly decreased compared to the H/R\u0026thinsp;+\u0026thinsp;Compound C group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). The study suggested that AMPK played an important role in the mechanism of FGF21 inhibition of myocardial H/R injury.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe results of the present study suggested that FGF21 has the ability to attenuate myocardial I/R injury by inhibiting NET formation. Furthermore, the cardioprotective effect of FGF21 was mediated through the involvement of AMPK.\u003c/p\u003e \u003cp\u003eA large number of previous studies have found that FGF21 attenuates myocardial I/R injury, increases cardiomyocyte activity, and decreases the rate of cardiomyocyte apoptosis, which is consistent with our study [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In a rat model of myocardial I/R injury, a notable upregulation in the expression of NETs was observed within the ischemic zone. Interestingly, facilitating the degradation of NETs through deoxy ribonuclease I led to a reduction in myocardial infarct size and a significant improvement in left ventricular remodeling induced by I/R injury in rats [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. In a mice model of chronic pancreatitis, mice treated with FGF21 significantly reduced levels of MPO, a biomarker of NETs, and improved the inflammatory state of serum, pancreatic, and peritoneal macrophages [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Significantly, our current investigation, within a myocardial I/R injury model, has unveiled for the first time that FGF21 exhibits the capacity to inhibit the formation of NETs induced by I/R injury, thereby attenuating myocardial I/R injury.\u003c/p\u003e \u003cp\u003ePrevious studies have shown that FGF21 is an endocrine factor that can regulate energy metabolism and that AMPK, as an energy sensor, is a target for FGF21 signaling [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. FGF21 activates AMPK both directly through the FGFR1/klotho-β complex and indirectly by inducing the expression of lipofuscin and corticosterone [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Once activated, AMPK plays a crucial role in regulating mitochondrial activity by stimulating SIRT1 and PGC-1α [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Furthermore, AMPK contributes to the maintenance of lipid homeostasis, protein synthesis regulation, and promotion of glucose metabolism, all aimed at sustaining optimal intracellular adenosine triphosphate levels [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBeyond its role in stimulating AMPK to regulate energy metabolism, FGF21 also exhibits anti-inflammatory properties. Studies have shown that a lack of FGF21 increases susceptibility to inflammation, leading to local and systemic inflammatory responses, and supplementation with exogenous FGF21 has been shown to reverse elevated levels of these inflammatory markers [\u003cspan additionalcitationids=\"CR29\" citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In hypertensive and diabetic mice models, the absence of FGF21 resulted in a pronounced exacerbation of inflammatory response and oxidative stress. Conversely, administration of FGF21 significantly alleviated these conditions by promoting the phosphorylation of AMPK, highlighting the crucial role of FGF21 in mitigating inflammatory and oxidative stress pathways [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Subsequent investigations have unveiled that FGF21 exerts its anti-inflammatory, antioxidant stress, and inhibitory effects on myocardial remodeling and apoptosis through two distinct pathways. The first pathway involves the AMPK-AKT2-Nrf2 antioxidant pathway, while the second pathway operates through the AMPK-ACC-CPT-1 lipid-lowering pathway, both of which are mediated by AMPK [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eStudies have shown that imbalances in the mechanisms that produce or eliminate NETs can lead to tissue damage, which is an important mechanism in autoimmune and inflammatory diseases that are closely linked to cardiovascular disease, and that inhibiting NETs formation has been shown to be beneficial [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Several studies have consistently demonstrated that AMPK, known for its anti-inflammatory effects, plays a pivotal role in the formation of NETs [\u003cspan additionalcitationids=\"CR37 CR38\" citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Activation of AMPK both inhibits NETs formation and promotes NETs clearance to reduce NETs levels[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. However, inhibition of AMPK significantly increased the levels of NETs in the peripheral circulation and in ischemic tissues [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe present study has some limitations. Due to the limitation of funds and experimental equipment, cardiac ultrasonography was not performed in I/R-injured mice in this study, so it was not possible to judge the improvement of cardiac function in mice after the application of FGF21. Although the present study suggests a possible mechanism by which FGF21 inhibits the formation of NETs through activation of AMPK, the actual biological mechanism is likely to be more complex, and other unconsidered signaling pathways or cell types may also be involved.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn the present study, we observed a significant elevation in NETs levels in both serum and myocardial tissue during myocardial I/R injury. Notably, the administration of FGF21 demonstrated a remarkable reversal of this increase. Subsequent studies delved into the mechanisms, suggesting that FGF21 may possess the ability to inhibit I/R injury-induced NETs formation through the activation of AMPK. These findings highlight a potential therapeutic role for FGF21 in mitigating NETs-mediated responses in the context of myocardial I/R injury.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e \u003cp\u003e All protocols in this study were approved by the Nantong University Laboratory Animal Center (Approval No: S20210820-002) in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85\u0026thinsp;\u0026minus;\u0026thinsp;23, revised 1996). All methods in our manuscript are reported in accordance with the ARRIVE guidelines (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://arriveguidelines.org\u003c/span\u003e\u003cspan address=\"https://arriveguidelines.org\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) for reporting animal experiments.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication\u003c/strong\u003e \u003cp\u003eNot applicable.\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors have no conflicts of interest to disclose.\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding Sources\u003c/h2\u003e \u003cp\u003eThis research was supported by the development fund of Affiliated Hospital of Xuzhou Medical University (grant no. XYFY2021029), Medical Research Program of Jiangsu Provincial Health and Wellness Commission (grant no. Z2021042) and Jiangyin Young and Middle-aged Reserve Excellent Talents Program (grant no. JYROYT202309).\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eLingyun Gu contributed to the conception, methodology and writing-original draft. Chengao Jia and Zuozhen Sheng carried out the methodology. Zhuowen Xu and Wenlong Jiang carried out the interpretation of data. Weizhang Li and Hua Zhang carried out the conception, resource and supervision.\u003c/p\u003e\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e \u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBhatt DL, Lopes RD, Harrington RA. Diagnosis and Treatment of Acute Coronary Syndromes: A Review. 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Int Immunopharmacol. 2023;114:109547.\u003c/span\u003e\u003c/li\u003e\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":"FGF21, Myocardial I/R injury, NETs, AMPK","lastPublishedDoi":"10.21203/rs.3.rs-4111215/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4111215/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground \u003c/strong\u003ePrevious investigations have established the anti-inflammatory properties of fibroblast growth factor 21 (FGF21). However, the specific mechanism through which FGF21 mitigates myocardial ischemia/reperfusion (I/R) injury by inhibiting neutrophil extracellular traps(NETs) formation remains unclear.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods \u003c/strong\u003eA mice model of myocardial I/R injury was induced, and myocardial tissue was stained with immunofluorescence to assess the formation of NETs. Serum NETs levels were quantified using a PicoGreen kit. In addition, the expression levels of AMP-activated protein kinase (AMPK) and FGF21 were evaluated by Wes fully automated protein blotting quantitative analysis system. Moreover, an hypoxia/reoxygenation (H/R) model was established using AMPK inhibitor and agonist pretreated H9c2 cells to further explore the relationship between FGF21 and AMPK.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e Compared with the control group, serum NETs levels were significantly higher in I/R mice, and a large number of NETs were formed in myocardial tissues (97.63±11.45 vs 69.65±3.33, P\u0026lt;0.05). However, NETs levels were reversed in FGF21 pretreated mice (P\u0026lt;0.05). Further studies showed that FGF21 enhanced AMPK expression, which was significantly increased after inhibition of AMPK and decreased after promotion of AMPK (P\u0026lt;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e FGF21 may exert cardioprotective effects by inhibiting I/R injury-induced NETs formation via AMPK.\u003c/p\u003e","manuscriptTitle":"FGF21 inhibited NETs formation induced by myocardial I/R injury via AMPK","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-26 06:33:56","doi":"10.21203/rs.3.rs-4111215/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":"dd80ba9b-1aa2-4935-8181-0a6383cff62f","owner":[],"postedDate":"March 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-07-19T06:36:05+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-26 06:33:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4111215","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4111215","identity":"rs-4111215","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}