Therapeutic potential of ANXA1 in hepatic encephalopathy via the inhibition of NLPR3 inflammasome activation in microglia

Therapeutic potential of ANXA1 in hepatic encephalopathy via the inhibition of NLPR3 inflammasome activation in microglia | 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 Article Therapeutic potential of ANXA1 in hepatic encephalopathy via the inhibition of NLPR3 inflammasome activation in microglia Weiliang Hu, Xurui Lu, Haoming Yan, Qian Gu, Xiaoyun Zhou, Xiaoling Ding, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7205070/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 Hepatic encephalopathy (HE) is a severe syndrome characterized by profound cognitive and neurological deficits and an increased risk of mortality. Unfortunately, targeted therapeutic interventions for this condition are lacking. ANXA1 is a pleiotropic cytokine that exhibits either proinflammatory or anti-inflammatory properties depending on the context; however, its involvement in the pathogenesis of HE has yet to be elucidated. Methods RNA sequencing data from both murine and human samples were used to investigate ANXA1 expression in HE. HE cell models were developed using the mouse microglial cell line BV2 and the endothelial cell line bEnd.3, and the model cells were cultured in the presence of ammonium chloride. A murine model of HE was established via intraperitoneal injection of thioacetamide (TAA), and the success of modeling was confirmed through behavioral, histological, and enzymatic assessments. The expression levels of ANXA1, along with those of cytokines and proteins associated with NLRP3 inflammasome activation, were analyzed via Western blotting and enzyme-linked immunosorbent assay (ELISA). The biologically active domain of ANXA1, AC2-26, was employed to investigate the effects of ANXA1 in HE in vitro and in vivo. Results We detected increased ANXA1 expression in microglia from HE model mice and brain tissues from cirrhosis patients. Microglia cultured under hyperammonemic conditions in vitro exhibited increased aNLRP3 inflammasome activation and increased secretion of inflammatory mediators, including ANXA1, TNF-α, IL-1β, and IL-6. Importantly, AC2-26 treatment effectively inhibited NLRP3 inflammasome activation and cytokine production by microglia, and this effect was diminished by the ANXA1 receptor antagonist Boc2, suggesting that ANXA1 plays a role in limiting microglia-mediated neuroinflammation by modulating NLRP3 inflammasome activation. In addition, microglia-derived cytokines reduced tight junction protein expression by endothelial cells under hyperammonemic conditions, indicating impaired blood‒brain barrier (BBB) function, which was ameliorated by AC2-26 treatment, suggesting that ANXA1 can protect BBB integrity in HE. Furthermore, we demonstrated that AC2-26 administration alleviated HE symptoms in a mouse model. Conclusion In conclusion, our findings indicate that ANXA1 counteracts NLRP3 inflammasome activation and mitigates neuroinflammation in HE, suggesting a potential therapeutic strategy for managing HE in the clinic. Health sciences/Diseases Biological sciences/Immunology Health sciences/Neurology Biological sciences/Neuroscience Hepatic encephalopathy ANXA1 NLRP3 inflammasome Blood–brain barrier Neuroinflammation Full Text 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-7205070","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":512823112,"identity":"c7af185d-a768-4f9f-a311-cfab7ccd2be9","order_by":0,"name":"Weiliang Hu","email":"","orcid":"","institution":"Affiliated Hospital 2 of Nantong University","correspondingAuthor":false,"prefix":"","firstName":"Weiliang","middleName":"","lastName":"Hu","suffix":""},{"id":512823113,"identity":"f921ca6e-cc05-4a91-b7a1-1103c2910b6d","order_by":1,"name":"Xurui Lu","email":"","orcid":"","institution":"Affiliated Hospital 2 of Nantong 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11:23:30","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7205070/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7205070/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":106216592,"identity":"0741adcf-b02f-4342-b01f-fc7d7c05e762","added_by":"auto","created_at":"2026-04-06 08:42:42","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4508491,"visible":true,"origin":"","legend":"","description":"","filename":"Manuscript0814.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7205070/v1_covered_84a690b0-7af4-42ae-882a-c2061170d4b6.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Therapeutic potential of ANXA1 in hepatic encephalopathy via the inhibition of NLPR3 inflammasome activation in microglia","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"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":"Hepatic encephalopathy, ANXA1, NLRP3 inflammasome, Blood–brain barrier, Neuroinflammation","lastPublishedDoi":"10.21203/rs.3.rs-7205070/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7205070/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground Hepatic encephalopathy (HE) is a severe syndrome characterized by profound cognitive and neurological deficits and an increased risk of mortality. Unfortunately, targeted therapeutic interventions for this condition are lacking. ANXA1 is a pleiotropic cytokine that exhibits either proinflammatory or anti-inflammatory properties depending on the context; however, its involvement in the pathogenesis of HE has yet to be elucidated.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMethods RNA sequencing data from both murine and human samples were used to investigate ANXA1 expression in HE. HE cell models were developed using the mouse microglial cell line BV2 and the endothelial cell line bEnd.3, and the model cells were cultured in the presence of ammonium chloride. A murine model of HE was established via intraperitoneal injection of thioacetamide (TAA), and the success of modeling was confirmed through behavioral, histological, and enzymatic assessments. The expression levels of ANXA1, along with those of cytokines and proteins associated with NLRP3 inflammasome activation, were analyzed via Western blotting and enzyme-linked immunosorbent assay (ELISA). The biologically active domain of ANXA1, AC2-26, was employed to investigate the effects of ANXA1 in HE in vitro and in vivo.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eResults We detected increased ANXA1 expression in microglia from HE model mice and brain tissues from cirrhosis patients. Microglia cultured under hyperammonemic conditions in vitro exhibited increased aNLRP3 inflammasome activation and increased secretion of inflammatory mediators, including ANXA1, TNF-α, IL-1β, and IL-6. Importantly, AC2-26 treatment effectively inhibited NLRP3 inflammasome activation and cytokine production by microglia, and this effect was diminished by the ANXA1 receptor antagonist Boc2, suggesting that ANXA1 plays a role in limiting microglia-mediated neuroinflammation by modulating NLRP3 inflammasome activation. In addition, microglia-derived cytokines reduced tight junction protein expression by endothelial cells under hyperammonemic conditions, indicating impaired blood‒brain barrier (BBB) function, which was ameliorated by AC2-26 treatment, suggesting that ANXA1 can protect BBB integrity in HE. Furthermore, we demonstrated that AC2-26 administration alleviated HE symptoms in a mouse model.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConclusion In conclusion, our findings indicate that ANXA1 counteracts NLRP3 inflammasome activation and mitigates neuroinflammation in HE, suggesting a potential therapeutic strategy for managing HE in the clinic.\u003c/p\u003e","manuscriptTitle":"Therapeutic potential of ANXA1 in hepatic encephalopathy via the inhibition of NLPR3 inflammasome activation in microglia","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-11 14:47:34","doi":"10.21203/rs.3.rs-7205070/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":"0ab352a1-b651-4a22-a13e-2f0353e25da3","owner":[],"postedDate":"September 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":54472911,"name":"Health sciences/Diseases"},{"id":54472912,"name":"Biological sciences/Immunology"},{"id":54472913,"name":"Health sciences/Neurology"},{"id":54472914,"name":"Biological sciences/Neuroscience"}],"tags":[],"updatedAt":"2026-04-06T08:42:05+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-11 14:47:34","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7205070","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7205070","identity":"rs-7205070","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}