Suppressive effect of chrysin on macrophage LC3B autophagy through miR-204-5p in diabetic atherosclerosis

Suppressive effect of chrysin on macrophage LC3B autophagy through miR-204-5p in diabetic atherosclerosis | 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 Suppressive effect of chrysin on macrophage LC3B autophagy through miR-204-5p in diabetic atherosclerosis Chiu-Mei Lin, Bao-Wei Wang, Wei-Jen Fang, Chun-Ming Pan, Kou-Gi Shyu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8343027/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 12 You are reading this latest preprint version Abstract Flavonoid chrysin possesses many biological properties. Autophagy and inflammation interact to cause diabetic cardiovascular diseases. However, whether chrysin affects macrophage autophagy and microRNA 204-5p regulation under hyperglycemic conditions remain unclear. In this study, we examined that the effects of chrysin on the molecular regulatory mechanisms of miR-204-5p in LC3B macrophage autophagy under hyperglycemic stimulation. Macrophages were cultured with 25 mM glucose for hyperglycemia stimulation. A diabetic rat model of carotid artery balloon injury was established. Reverse transcription and real-time quantitative polymerase chain reaction, Western blotting, immunohistochemical staining, and luciferase activity assay were performed. The results indicated that chrysin augmented the macrophage autophagy marker LC3B presentation under high-glucose (HG)-stimulation significantly, however, chrysin attenuated the miR-204-5p level in macrophage cultures. Treatment with chrysin, rapamycin or mutant miR-204-5p increased LC3B presentation through the suppression of miR-204-5p level in macrophage cultures under HG-stimulated condition. By luciferase activity assay, the combination of chrysin with mutant miR-204-5p reduced LC3B binding activity under hyperglycemic stress. Treatment with chrysin (100 mg/kg/day) and wild-type miR-204-5p considerably reduced the neointimal sizes by 46.3% and 36.3%, respectively, at 14 days after carotid artery balloon injury in STZ-induced diabetic rats; ameliorated M1 polarization simultaneously. However, by treating with chrysin suppressed LC3B presentation in diabetic rats. Our findings indicate, under hyperglycemic stress, chrysin regulates the presentation of LC3B autophagy in hyperglycemia-stimulated macrophage cultures and neointimal hyperplasia by mediating the miR-204-5p expression. The results imply that chrysin is potentially effective for preventing hyperglycemia related cardiovascular diseases. Biological sciences/Biochemistry Health sciences/Cardiology Biological sciences/Cell biology Health sciences/Diseases Health sciences/Endocrinology Biological sciences/Molecular biology Biological sciences/Physiology chrysin macrophage autophagy miR-204-5p diabetes neointima Full Text Additional Declarations No competing interests reported. Supplementary Files westernblotraw2B1.jpg westernblotraw2B2.jpg westernblotraw4E1.jpg westernblotraw4E2.jpg westernblotraw4E3.jpg westernblotraw4E4.jpg studydesignsupp1.tif Supplementary data 1: Animal experimental design. Representative of timeline of the study, treatment with STZ or balloon injury, or chrysin, or wild miR-204-5p or mutant miR-204-5p received by the male Wistar rats for each group. The group were named after the treatment they received in treatment: 0 day = sham, STZ = streptozotocin, balloon injury = balloon injury of rat carotid artery, CH = chrysin, miR = wild miR-204-5p, miR-MU = mutant miR-204-5p. These groups originally numbered 5 rats. MTTRSupp2.tif Supplementary data 2: Cell viability assays are used to measure the treatment of chrysin in macrophage cultures under hyperglycemia stress. TOCchrysinmiR2045p.tif Graphical Abstract: Proposed pathway for macrophage LC3B autophagy induced by chrysin to regulate microRNA (miR)-204-5p expression and counteract the inhibitory effect of miR-204-5p on chrysin expression in macrophages. This provides an outline of the key role of the herbal chrysin, which may serve as a valuable therapeutic supplement that provides diabetic atheroprotection. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 23 Mar, 2026 Reviews received at journal 21 Mar, 2026 Reviews received at journal 21 Mar, 2026 Reviewers agreed at journal 12 Mar, 2026 Reviewers agreed at journal 12 Mar, 2026 Reviews received at journal 02 Mar, 2026 Reviewers agreed at journal 25 Feb, 2026 Reviewers invited by journal 27 Jan, 2026 Editor assigned by journal 27 Jan, 2026 Editor invited by journal 02 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. 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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-8343027","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":581065372,"identity":"fafb8a22-333d-4088-8f2d-c1ced1922253","order_by":0,"name":"Chiu-Mei Lin","email":"","orcid":"","institution":"Shin Kong Wu Ho-Su Memorial Hospital","correspondingAuthor":false,"prefix":"","firstName":"Chiu-Mei","middleName":"","lastName":"Lin","suffix":""},{"id":581065373,"identity":"21eb48fb-043e-4174-8252-1f2c74f89040","order_by":1,"name":"Bao-Wei Wang","email":"","orcid":"","institution":"Shin Kong Wu Ho-Su Memorial 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Representative of timeline of the study, treatment with STZ or balloon injury, or chrysin, or wild miR-204-5p or mutant miR-204-5p received by the male Wistar rats for each group. The group were named after the treatment they received in treatment: 0 day = sham, STZ = streptozotocin, balloon injury = balloon injury of rat carotid artery, CH = chrysin, miR = wild miR-204-5p, miR-MU = mutant miR-204-5p. 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Autophagy and inflammation interact to cause diabetic cardiovascular diseases. However, whether chrysin affects macrophage autophagy and microRNA 204-5p regulation under hyperglycemic conditions remain unclear. In this study, we examined that the effects of chrysin on the molecular regulatory mechanisms of miR-204-5p in LC3B macrophage autophagy under hyperglycemic stimulation. Macrophages were cultured with 25 mM glucose for hyperglycemia stimulation. A diabetic rat model of carotid artery balloon injury was established. Reverse transcription and real-time quantitative polymerase chain reaction, Western blotting, immunohistochemical staining, and luciferase activity assay were performed. The results indicated that chrysin augmented the macrophage autophagy marker LC3B presentation under high-glucose (HG)-stimulation significantly, however, chrysin attenuated the miR-204-5p level in macrophage cultures. 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