Ergosterol Ameliorates Hypoxic-Ischemic Encephalopathy in Mice via Akt/GSK-3β Pathway Activation

Ergosterol Ameliorates Hypoxic-Ischemic Encephalopathy in Mice via Akt/GSK-3β Pathway Activation | 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 Ergosterol Ameliorates Hypoxic-Ischemic Encephalopathy in Mice via Akt/GSK-3β Pathway Activation Lingyu Fang, Baozhou Hong, Xiaomei Tong, He Wang, Dongliang Chen This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8371288/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 9 You are reading this latest preprint version Abstract Hypoxic-ischemic encephalopathy (HIE) is a leading cause of neonatal mortality and long-term neurological disability, with limited therapeutic options. Ergosterol (ERG), a natural sterol with antioxidant, anti-inflammatory, and anti-apoptotic properties that crosses the blood-brain barrier, exhibits neuroprotective potential. Here, we demonstrate that ERG exerts comprehensive neuroprotection in HIE by activating the Akt/GSK-3β pathway. In vitro, ERG significantly improved cell viability, reduced apoptosis, suppressed reactive oxygen species (ROS) accumulation, and inhibited pro-inflammatory cytokine expression in oxygen-glucose deprivation/reoxygenation (OGD/R)-injured HT22 neurons. In vivo, ERG treatment reduced cerebral infarction volume, alleviated histopathological damage and neuronal apoptosis, upregulated neurotrophic proteins (BDNF, PSD95, synaptophysin), and cognitive function in mouse HIE models. Mechanistically, ERG activated Akt phosphorylation and subsequent GSK-3β inhibition, while the Akt inhibitor MK-2206 completely abolished ERG-mediated protection. These findings establish ERG as a promising multi-target therapeutic candidate for HIE through Akt/GSK-3β pathway modulation. Biological sciences/Cell biology Health sciences/Diseases Biological sciences/Drug discovery Health sciences/Neurology Biological sciences/Neuroscience Hypoxic-ischemic encephalopathy ergosterol Akt/GSK-3β signaling pathway neuroprotection apoptosis oxidative stress Full Text Additional Declarations No competing interests reported. Supplementary Files Supplementarymaterials.pdf Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 02 May, 2026 Reviews received at journal 19 Apr, 2026 Reviewers agreed at journal 11 Apr, 2026 Reviewers agreed at journal 29 Mar, 2026 Reviewers invited by journal 16 Jan, 2026 Editor assigned by journal 16 Jan, 2026 Editor invited by journal 01 Jan, 2026 Submission checks completed at journal 31 Dec, 2025 First submitted to journal 31 Dec, 2025 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-8371288","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":576090634,"identity":"8a3851f3-5d26-4fa2-b1a2-cb4cd85ba6be","order_by":0,"name":"Lingyu Fang","email":"","orcid":"","institution":"QuanZhou Women's and Children's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Lingyu","middleName":"","lastName":"Fang","suffix":""},{"id":576090635,"identity":"6b26fb79-1ef3-4ea2-ac45-6b1441222245","order_by":1,"name":"Baozhou Hong","email":"","orcid":"","institution":"Mindong Hospital, Fujian Medical University","correspondingAuthor":false,"prefix":"","firstName":"Baozhou","middleName":"","lastName":"Hong","suffix":""},{"id":576090638,"identity":"2fb17fe8-a492-4262-9a56-f0014c5c0bfd","order_by":2,"name":"Xiaomei Tong","email":"","orcid":"","institution":"QuanZhou Women's and Children's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xiaomei","middleName":"","lastName":"Tong","suffix":""},{"id":576090640,"identity":"92c929c1-c93b-4552-9102-a7fcfd1e607c","order_by":3,"name":"He Wang","email":"","orcid":"","institution":"QuanZhou Women's and Children's Hospital","correspondingAuthor":false,"prefix":"","firstName":"He","middleName":"","lastName":"Wang","suffix":""},{"id":576090642,"identity":"7558b3bd-df61-4bea-a7f1-9072a65cf9bd","order_by":4,"name":"Dongliang Chen","email":"data:image/png;base64,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","orcid":"","institution":"Mindong Hospital, Fujian Medical University","correspondingAuthor":true,"prefix":"","firstName":"Dongliang","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2025-12-16 03:23:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8371288/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8371288/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":100736297,"identity":"8db1c606-4e52-4331-81ec-8b3caa17596f","added_by":"auto","created_at":"2026-01-20 22:37:34","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":7604764,"visible":true,"origin":"","legend":"","description":"","filename":"Manuscript1.docx","url":"https://assets-eu.researchsquare.com/files/rs-8371288/v1/2d2a18408d88c5a75ab953e8.docx"},{"id":100736461,"identity":"045b9798-9965-4aee-85b8-d85643c18194","added_by":"auto","created_at":"2026-01-20 22:40:25","extension":"json","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":6397,"visible":true,"origin":"","legend":"","description":"","filename":"186bbdc7234c462c9a5ed273c56aba8b.json","url":"https://assets-eu.researchsquare.com/files/rs-8371288/v1/0ec727a28dca89aacda48090.json"},{"id":100736672,"identity":"8d2fe85a-9c0c-4d4d-a639-029573001dea","added_by":"auto","created_at":"2026-01-20 22:44:28","extension":"xml","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":85153,"visible":true,"origin":"","legend":"","description":"","filename":"186bbdc7234c462c9a5ed273c56aba8b1enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-8371288/v1/776552a9a617345f0fdb5540.xml"},{"id":100736446,"identity":"4cab3707-4588-494f-b907-795876d8b927","added_by":"auto","created_at":"2026-01-20 22:39:55","extension":"jpeg","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":5152374,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8371288/v1/63ba93fa3e48e1f32f8a9562.jpeg"},{"id":100736429,"identity":"6b3b8321-d0aa-4f3a-a6f6-95d9536c004e","added_by":"auto","created_at":"2026-01-20 22:39:25","extension":"jpeg","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":5129588,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8371288/v1/d4ac3fc0ab01d209b7c2a44b.jpeg"},{"id":100736317,"identity":"f7555269-595f-4d92-b92c-8fb903c77107","added_by":"auto","created_at":"2026-01-20 22:38:07","extension":"jpeg","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":1411588,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8371288/v1/47c85295f61fada09001c187.jpeg"},{"id":100736451,"identity":"0f5eafab-2396-410c-9d50-b9346d6b0a5a","added_by":"auto","created_at":"2026-01-20 22:40:09","extension":"jpeg","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":4306362,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8371288/v1/ff6f6946b98486084669b0ae.jpeg"},{"id":100736437,"identity":"3d641d62-fbc6-4161-92a7-eec22eae6dcb","added_by":"auto","created_at":"2026-01-20 22:39:45","extension":"jpeg","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":3324770,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8371288/v1/b20fa7427db5d67522cf1828.jpeg"},{"id":100736571,"identity":"2021bb68-c74f-4318-ab89-8fa0f8262677","added_by":"auto","created_at":"2026-01-20 22:42:48","extension":"jpeg","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2206232,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8371288/v1/af2177bd154a0e4d984d2fbd.jpeg"},{"id":100736463,"identity":"a99ddcd0-9255-4019-a1c4-a548e8aa8b60","added_by":"auto","created_at":"2026-01-20 22:40:27","extension":"png","order_by":10,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":100039,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-8371288/v1/84146e74a4c61663c5b892b8.png"},{"id":100736663,"identity":"9659f373-53b7-46b6-ba40-5dfae4437e05","added_by":"auto","created_at":"2026-01-20 22:43:56","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2220030,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-8371288/v1/8c545c9c65ef6b4d3fd190f6.png"},{"id":100736368,"identity":"d357c5e2-0b61-4262-979e-4b11eb071a6d","added_by":"auto","created_at":"2026-01-20 22:38:59","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":455690,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-8371288/v1/ac99b836c2c08c62086863d3.png"},{"id":100736499,"identity":"bd626fba-9e20-45b3-b238-c3d6c218e91f","added_by":"auto","created_at":"2026-01-20 22:41:43","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":97166,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-8371288/v1/3c5086c3daa2d6bcb61aff1d.png"},{"id":100736442,"identity":"23fe5bdd-a956-45bf-ba2d-010bccda941b","added_by":"auto","created_at":"2026-01-20 22:39:50","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":50987,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-8371288/v1/21ef5d9f48dbff9afc1aa461.png"},{"id":100736580,"identity":"47aecd7b-f785-447b-9117-6e6b4e731f05","added_by":"auto","created_at":"2026-01-20 22:43:04","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":759225,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-8371288/v1/2d6f990fcbf9679a7cc1b0b2.png"},{"id":100736291,"identity":"5719b3a2-5589-4142-a6b0-d7ef96812f91","added_by":"auto","created_at":"2026-01-20 22:37:29","extension":"xml","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":82976,"visible":true,"origin":"","legend":"","description":"","filename":"186bbdc7234c462c9a5ed273c56aba8b1structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-8371288/v1/d709a2223387b51226100b5b.xml"},{"id":100736512,"identity":"02cce0bf-30ba-49da-9ade-cbe52249fe33","added_by":"auto","created_at":"2026-01-20 22:42:45","extension":"html","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":93576,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8371288/v1/31d3c451f6447012b687bc23.html"},{"id":100736977,"identity":"04ae2751-7f2d-4690-8e5f-00c18dcacd82","added_by":"auto","created_at":"2026-01-20 22:48:10","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":987891,"visible":true,"origin":"","legend":"","description":"","filename":"Manuscript1.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8371288/v1_covered_b41b7bc9-d83d-4be6-bfa2-fa9338a1aff8.pdf"},{"id":100736496,"identity":"53b546c4-be9b-4ff2-af45-8c91138a8acd","added_by":"auto","created_at":"2026-01-20 22:41:32","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":336266,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterials.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8371288/v1/8cc4c08ecb65d627d54993c0.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Ergosterol Ameliorates Hypoxic-Ischemic Encephalopathy in Mice via Akt/GSK-3β Pathway Activation","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Hypoxic-ischemic encephalopathy, ergosterol, Akt/GSK-3β signaling pathway, neuroprotection, apoptosis, oxidative stress","lastPublishedDoi":"10.21203/rs.3.rs-8371288/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8371288/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eHypoxic-ischemic encephalopathy (HIE) is a leading cause of neonatal mortality and long-term neurological disability, with limited therapeutic options. Ergosterol (ERG), a natural sterol with antioxidant, anti-inflammatory, and anti-apoptotic properties that crosses the blood-brain barrier, exhibits neuroprotective potential. Here, we demonstrate that ERG exerts comprehensive neuroprotection in HIE by activating the Akt/GSK-3β pathway. In vitro, ERG significantly improved cell viability, reduced apoptosis, suppressed reactive oxygen species (ROS) accumulation, and inhibited pro-inflammatory cytokine expression in oxygen-glucose deprivation/reoxygenation (OGD/R)-injured HT22 neurons. In vivo, ERG treatment reduced cerebral infarction volume, alleviated histopathological damage and neuronal apoptosis, upregulated neurotrophic proteins (BDNF, PSD95, synaptophysin), and cognitive function in mouse HIE models. Mechanistically, ERG activated Akt phosphorylation and subsequent GSK-3β inhibition, while the Akt inhibitor MK-2206 completely abolished ERG-mediated protection. These findings establish ERG as a promising multi-target therapeutic candidate for HIE through Akt/GSK-3β pathway modulation.\u003c/p\u003e","manuscriptTitle":"Ergosterol Ameliorates Hypoxic-Ischemic Encephalopathy in Mice via Akt/GSK-3β Pathway Activation","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-20 22:13:49","doi":"10.21203/rs.3.rs-8371288/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-05-02T06:05:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-04-19T13:15:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"242134614989977727429265440603605923567","date":"2026-04-11T16:46:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"148674893896705406888085141975097856686","date":"2026-03-29T23:38:10+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-01-16T14:28:50+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-01-16T14:14:33+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-01-01T15:18:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-12-31T15:03:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-12-31T14:55:01+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"a484757e-ab8c-4f59-accd-804c194cce14","owner":[],"postedDate":"January 20th, 2026","published":true,"recentEditorialEvents":[{"type":"editorInvitedReview","content":"","date":"2026-05-02T06:05:57+00:00","index":111,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[{"id":61299739,"name":"Biological sciences/Cell biology"},{"id":61299740,"name":"Health sciences/Diseases"},{"id":61299741,"name":"Biological sciences/Drug discovery"},{"id":61299742,"name":"Health sciences/Neurology"},{"id":61299743,"name":"Biological sciences/Neuroscience"}],"tags":[],"updatedAt":"2026-01-20T22:13:49+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-20 22:13:49","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8371288","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8371288","identity":"rs-8371288","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}