Thonningianin A from Penthorum chinense Pursh alleviates apoptosis and pyroptosis in cerebral ischemia-reperfusion injury through the activation of mitophagy by the PINK1/Parkin signaling pathway

Thonningianin A from Penthorum chinense Pursh alleviates apoptosis and pyroptosis in cerebral ischemia-reperfusion injury through the activation of mitophagy by the PINK1/Parkin signaling pathway | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Thonningianin A from Penthorum chinense Pursh alleviates apoptosis and pyroptosis in cerebral ischemia-reperfusion injury through the activation of mitophagy by the PINK1/Parkin signaling pathway Qianfang Yao, Guishan Hu, Can Yin, Anguo Wu, Guangqiang Hu, Dalian Qin, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6273676/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Jan, 2026 Read the published version in Chinese Medicine → Version 1 posted 5 You are reading this latest preprint version Abstract Background Cerebral ischemia-reperfusion injury (CI/RI) poses a significant challenge in the treatment of ischemic stroke (IS). Activation of mitophagy can effectively inhibit cell apoptosis and necroptosis, thereby improving CI/RI. Penthorum chinense Pursh (PCP) has been used for many years as a hepatoprotective agent, and Thonningianin A (TA), a key bioactive compound in PCP, has shown autophagic activity. However, studies on the effect and mechanism of TA on CI/RI treatment are currently lacking. Methods A rat model of middle cerebral artery occlusion/reperfusion (MCAO/R) was used as a vivo model. The preliminary therapeutic effects of TA on CI/RI were assessed through TTC staining, Longa neurological scoring, and immunofluorescence staining. And an vitro model of oxygen-glucose deprivation/reoxygenation (OGD/R) was employed to examine the impact of TA on cell viability using MTT and Hoechst/PI staining in HT22 and BV2 cells. Furtherly, the effects of TA on mitochondrial oxidative damage, apoptosis in HT22 and pyroptosis in BV2 were evaluated by using Dihydroethidium (DHE) staining, flow cytometry, JC-1 and Tetramethylrhodamine methyl ester perchlorate (TMRM) staining, Mito-Tracker staining, plasmid transfection, EthD-2/YO-PRO-1 staining and Western blot. The effects of TA on autophagy was assessed by using the positive control drug rapamycin (Rap) and the inhibitor 3-MA, and mitophagy by using the positive control drug Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and the inhibitor AC220 in EGFP-LC3-U87 and mCherry-GFP-FIS1-293T cells, Western blot and immunofluorescence co-localization were applied to determine whether TA inhibits apoptosis and pyroptosis through mitophagy. Finally, the molecular mechanism of TA on CI/RI were validated in the MCAO/R model through immunofluorescence co- localization and Western blot analysis. Results TA significantly improved neurological function, reduced infarct volume, and mitigated neuronal damage in the MCAO/R model. TA decreased OGD/Rinduced mitochondrial oxidative damage and apoptosis in HT22 cells and improved pyroptosis in BV2 cells, these effects were mediated via the PINK1/Parkin mitophagy signaling pathway, which was corroborated in the MCAO/R model. Conclusion This study is the first to confirm that TA is a potential natural active component for the treatment of CI/RI. Its therapeutic effect and mechanism are associated with the inhibition of apoptosis and pyroptosis through the activation of the PINK1/Parkin mitophagy signaling pathway. Cerebral ischemia-reperfusion injury Apoptosis Pyroptosis Autophagy Mitophagy PINK1/Parkin signaling pathway Penthorum chinense Pursh Thonningianin A Full Text Supplementary Files Supplementarymaterials20250321215458.pdf Cite Share Download PDF Status: Published Journal Publication published 16 Jan, 2026 Read the published version in Chinese Medicine → Version 1 posted Editorial decision: Major revision 15 Apr, 2025 Reviewers agreed at journal 29 Mar, 2025 Reviewers invited by journal 29 Mar, 2025 Editor assigned by journal 25 Mar, 2025 First submitted to journal 21 Mar, 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-6273676","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":435673334,"identity":"e6b21d4a-671c-4129-a770-7139c5f81a63","order_by":0,"name":"Qianfang Yao","email":"","orcid":"","institution":"Southwest Medical University","correspondingAuthor":false,"prefix":"","firstName":"Qianfang","middleName":"","lastName":"Yao","suffix":""},{"id":435673335,"identity":"a406e2c2-08c8-454e-8f36-3942a2843976","order_by":1,"name":"Guishan Hu","email":"","orcid":"","institution":"Southwest Medical 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pathway","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"chinese-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cmed","sideBox":"Learn more about [Chinese Medicine](http://cmjournal.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/cmed/default.aspx","title":"Chinese Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Cerebral ischemia-reperfusion injury, Apoptosis, Pyroptosis, Autophagy, Mitophagy, PINK1/Parkin signaling pathway, Penthorum chinense Pursh, Thonningianin A","lastPublishedDoi":"10.21203/rs.3.rs-6273676/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6273676/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground \u003c/strong\u003eCerebral ischemia-reperfusion injury (CI/RI) poses a significant challenge in the treatment of ischemic stroke (IS). Activation of mitophagy can effectively inhibit cell apoptosis and necroptosis, thereby improving CI/RI.\u003c/p\u003e\n\u003cp\u003ePenthorum chinense Pursh (PCP) has been used for many years as a hepatoprotective agent, and Thonningianin A (TA), a key bioactive compound in PCP, has shown autophagic activity. However, studies on the effect and mechanism of TA on CI/RI treatment are currently lacking.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods \u003c/strong\u003eA rat model of middle cerebral artery occlusion/reperfusion (MCAO/R) was used as a vivo model. The preliminary therapeutic effects of TA on CI/RI were assessed through TTC staining, Longa neurological scoring, and immunofluorescence staining. And an vitro model of oxygen-glucose deprivation/reoxygenation (OGD/R) was employed to examine the impact of TA on cell viability using MTT and Hoechst/PI staining in HT22 and BV2 cells. Furtherly, the effects of TA on mitochondrial oxidative damage, apoptosis in HT22 and pyroptosis in BV2 were evaluated by using Dihydroethidium (DHE) staining, flow cytometry, JC-1 and Tetramethylrhodamine methyl ester perchlorate (TMRM) staining, Mito-Tracker staining, plasmid transfection, EthD-2/YO-PRO-1 staining and Western blot. The effects of TA on autophagy was assessed by using the positive control drug rapamycin (Rap) and the inhibitor 3-MA, and mitophagy by using the positive control drug Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) and the inhibitor AC220 in EGFP-LC3-U87 and mCherry-GFP-FIS1-293T cells, Western blot and immunofluorescence co-localization were applied to determine whether TA inhibits apoptosis and pyroptosis through mitophagy. Finally, the molecular mechanism of TA on CI/RI were validated in the MCAO/R model through immunofluorescence co- localization and Western blot analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults \u003c/strong\u003eTA significantly improved neurological function, reduced infarct volume, and mitigated neuronal damage in the MCAO/R model. TA decreased OGD/Rinduced mitochondrial oxidative damage and apoptosis in HT22 cells and improved pyroptosis in BV2 cells, these effects were mediated via the PINK1/Parkin mitophagy signaling pathway, which was corroborated in the MCAO/R model.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion \u003c/strong\u003eThis study is the first to confirm that TA is a potential natural active component for the treatment of CI/RI. Its therapeutic effect and mechanism are associated with the inhibition of apoptosis and pyroptosis through the activation of the PINK1/Parkin mitophagy signaling pathway.\u003c/p\u003e","manuscriptTitle":"Thonningianin A from Penthorum chinense Pursh alleviates apoptosis and pyroptosis in cerebral ischemia-reperfusion injury through the activation of mitophagy by the PINK1/Parkin signaling pathway","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-18 04:49:31","doi":"10.21203/rs.3.rs-6273676/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2025-04-15T23:59:06+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"","date":"2025-03-29T12:43:19+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-29T09:59:14+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-25T12:08:26+00:00","index":"","fulltext":""},{"type":"submitted","content":"Chinese Medicine","date":"2025-03-22T03:58:42+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"chinese-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"cmed","sideBox":"Learn more about [Chinese Medicine](http://cmjournal.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/cmed/default.aspx","title":"Chinese Medicine","twitterHandle":"@BioMedCentral","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bf23ffa4-f7e6-429c-837d-9e92c57affbc","owner":[],"postedDate":"April 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-19T17:05:10+00:00","versionOfRecord":{"articleIdentity":"rs-6273676","link":"https://doi.org/10.1186/s13020-025-01247-2","journal":{"identity":"chinese-medicine","isVorOnly":false,"title":"Chinese Medicine"},"publishedOn":"2026-01-16 16:29:09","publishedOnDateReadable":"January 16th, 2026"},"versionCreatedAt":"2025-04-18 04:49:31","video":"","vorDoi":"10.1186/s13020-025-01247-2","vorDoiUrl":"https://doi.org/10.1186/s13020-025-01247-2","workflowStages":[]},"version":"v1","identity":"rs-6273676","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6273676","identity":"rs-6273676","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}