{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,30]],"date-time":"2026-07-30T14:27:03Z","timestamp":1785421623508,"version":"3.56.0"},"reference-count":35,"publisher":"MDPI AG","issue":"12","license":[{"start":{"date-parts":[[2026,6,12]],"date-time":"2026-06-12T00:00:00Z","timestamp":1781222400000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["82401669"],"award-info":[{"award-number":["82401669"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"award":["82401669"],"award-info":[{"award-number":["82401669"]}],"id":[{"id":"https:\/\/ror.org\/01h0zpd94","id-type":"ROR","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100004731","name":"Zhejiang Provincial Natural Science Foundation","doi-asserted-by":"publisher","award":["LMS26H090008"],"award-info":[{"award-number":["LMS26H090008"]}],"id":[{"id":"10.13039\/501100004731","id-type":"DOI","asserted-by":"publisher"}]},{"award":["LMS26H090008"],"award-info":[{"award-number":["LMS26H090008"]}],"id":[{"id":"https:\/\/ror.org\/04aqat463","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cells"],"abstract":"<jats:p>Background: Mitochondrial dysfunction is a central event in the pathogenesis of ischemic stroke. The roles of specific mitochondrial complex subunits, such as NDUFA4 and NDUFB3, in cerebral ischemia\u2013reperfusion injury remain poorly defined. This study aims to investigate the dynamic expressions and functional impact of NDUFA4 and NDUFB3 in ischemic stroke. Methods: A transient middle cerebral artery occlusion (MCAO) model was established in male C57BL\/6J mice. Label-free quantitative proteomics and Western blotting were employed to analyze protein expression in the ischemic penumbra. Highly differentiated PC12 cells were subjected to oxygen-glucose deprivation\/reperfusion (OGD\/R) or glutamate excitotoxicity to mimic ischemic injury in vitro. The functional consequences of NDUFB3 knockdown and overexpression were assessed by measuring ATP levels, reactive oxygen species (ROS), mitochondrial membrane potential (\u0394\u03a8m), and apoptosis. The involvement of the JNK-mediated mitochondrial apoptotic pathway was also examined. Results: Proteomic analysis revealed a significant upregulation of NDUFA4 and NDUFB3 in the ischemic penumbra of MCAO mice, as verified by western blot. In highly differentiated PC12 cells, both OGD\/R and glutamate exposure induced a time-dependent increase in these proteins in mitochondrial fractions. Functional studies demonstrated that NDUFB3 knockdown significantly rescued OGD\/R-induced mitochondrial dysfunction, as indicated by restored ATP production, reduced ROS generation, and stabilized \u0394\u03a8m. Furthermore, NDUFB3 silencing attenuated apoptosis by inhibiting JNK phosphorylation and decreasing BAX levels. Conversely, overexpression of NDUFB3 alone was sufficient to induce mitochondrial abnormalities, including loss of \u0394\u03a8m and elevated oxidative stress in highly differentiated PC12 cells. Conclusions: Ischemic injury triggers the upregulation of mitochondrial complex subunits NDUFA4 and NDUFB3. While this may initially act as a compensatory response, our findings identify NDUFB3 as a critical mediator of ischemic stroke pathology, whose overexpression drives mitochondrial dysfunction and apoptosis. In contrast, the suppression of NDUFB3 provides protection against ischemic injury. Therefore, NDUFB3 may be a potential candidate therapeutic target for reducing mitochondrial damage in ischemic stroke, but this role requires further validation in additional experimental and translational models.<\/jats:p>","DOI":"10.3390\/cells15121071","type":"journal-article","created":{"date-parts":[[2026,6,15]],"date-time":"2026-06-15T00:44:58Z","timestamp":1781484298000},"page":"1071","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["The Upregulation of NDUFB3 Is Implicated in Mitochondrial Dysfunction and Neuronal Apoptosis in Ischemic Stroke"],"prefix":"10.3390","volume":"15","author":[{"given":"Shuyue","family":"Cheng","sequence":"first","affiliation":[{"name":"Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health), Institute of Mental Health and Drug Discovery, School of Psychiatry, Wenzhou Medical University, Wenzhou 325000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zeyue","family":"Mu","sequence":"additional","affiliation":[{"name":"Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health), Institute of Mental Health and Drug Discovery, School of Psychiatry, Wenzhou Medical University, Wenzhou 325000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Feng","family":"Zhang","sequence":"additional","affiliation":[{"name":"Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health), Institute of Mental Health and Drug Discovery, School of Psychiatry, Wenzhou Medical University, Wenzhou 325000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jianyou","family":"Song","sequence":"additional","affiliation":[{"name":"Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health), Institute of Mental Health and Drug Discovery, School of Psychiatry, Wenzhou Medical University, Wenzhou 325000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiapeng","family":"Shao","sequence":"additional","affiliation":[{"name":"School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou 325000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yunqi","family":"Yan","sequence":"additional","affiliation":[{"name":"Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health), Institute of Mental Health and Drug Discovery, School of Psychiatry, Wenzhou Medical University, Wenzhou 325000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3302-6429","authenticated-orcid":false,"given":"Anastasios A.","family":"Daskalakis","sequence":"additional","affiliation":[{"name":"Campbell Family Mental Health Research Institute, Centre for Addiction and Mental Health, Toronto, ON M5T1R8, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yunjie","family":"Wang","sequence":"additional","affiliation":[{"name":"Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health), Institute of Mental Health and Drug Discovery, School of Psychiatry, Wenzhou Medical University, Wenzhou 325000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bin","family":"Zhang","sequence":"additional","affiliation":[{"name":"School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou 325000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yashuang","family":"Jiang","sequence":"additional","affiliation":[{"name":"School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou 325000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0002-8539-6018","authenticated-orcid":false,"given":"Le","family":"Wang","sequence":"additional","affiliation":[{"name":"Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health), Institute of Mental Health and Drug Discovery, School of Psychiatry, Wenzhou Medical University, Wenzhou 325000, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fang","family":"Liu","sequence":"additional","affiliation":[{"name":"Oujiang Laboratory (Zhejiang Lab for Regenerative Medicine, Vision and Brain Health), Institute of Mental Health and Drug Discovery, School of Psychiatry, Wenzhou Medical University, Wenzhou 325000, China"},{"name":"School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou 325000, China"},{"name":"Campbell Family Mental Health Research Institute, Centre for Addiction and Mental Health, Toronto, ON M5T1R8, Canada"},{"name":"Department of Pharmacology & Toxicology, Institute of Medical Science, University of Toronto, Toronto, ON M5T1R8, Canada"},{"name":"Department of Physiology, Institute of Medical Science, University of Toronto, Toronto, ON M5T1R8, Canada"},{"name":"Department of Psychiatry, Institute of Medical Science, University of Toronto, Toronto, ON M5T1R8, Canada"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,6,12]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"152","DOI":"10.1007\/s00415-026-13702-7","article-title":"Early-phase fluid diagnostic biomarkers in acute ischemic stroke: A hybrid umbrella review","volume":"273","author":"Bombaci","year":"2026","journal-title":"J. Neurol."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"815","DOI":"10.1055\/s-0040-1708878","article-title":"Plasma Levels of Protein Z in Ischemic Stroke: A Systematic Review and Meta-Analysis","volume":"120","author":"Kowalewski","year":"2020","journal-title":"Thromb. Haemost."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"70","DOI":"10.1038\/s41572-019-0118-8","article-title":"Ischaemic stroke","volume":"5","author":"Campbell","year":"2019","journal-title":"Nat. Rev. Dis. Prim."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Zhou, X., Chen, H., Wang, L., Lenahan, C., Lian, L., Ou, Y., and He, Y. (2021). Mitochondrial Dynamics: A Potential Therapeutic Target for Ischemic Stroke. Front. Aging Neurosci., 13.","DOI":"10.3389\/fnagi.2021.721428"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"101883","DOI":"10.1016\/j.mito.2024.101883","article-title":"Mitochondrial mechanisms in Cerebral Ischemia-Reperfusion Injury: Unravelling the intricacies","volume":"77","author":"Singh","year":"2024","journal-title":"Mitochondrion"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"924","DOI":"10.14336\/AD.2017.1126","article-title":"Mitochondria in Ischemic Stroke: New Insight and Implications","volume":"9","author":"Liu","year":"2018","journal-title":"Aging Dis."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"103426","DOI":"10.1016\/j.redox.2024.103426","article-title":"Regulation of mitochondrial oxidative phosphorylation through tight control of cytochrome c oxidase in health and disease-Implications for ischemia\/reperfusion injury, inflammatory diseases, diabetes, and cancer","volume":"78","author":"Pham","year":"2024","journal-title":"Redox Biol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"946","DOI":"10.1016\/j.bbabio.2015.12.013","article-title":"Ischemic A\/D transition of mitochondrial complex I and its role in ROS generation","volume":"1857","author":"Stepanova","year":"2016","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"eabc4209","DOI":"10.1126\/science.abc4209","article-title":"The coupling mechanism of mammalian respiratory complex I","volume":"370","author":"Kampjut","year":"2020","journal-title":"Science"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"431","DOI":"10.1038\/nature13909","article-title":"Ischaemic accumulation of succinate controls reperfusion injury through mitochondrial ROS","volume":"515","author":"Chouchani","year":"2014","journal-title":"Nature"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"3649","DOI":"10.1177\/0271678X17730242","article-title":"Reverse electron transfer results in a loss of flavin from mitochondrial complex I: Potential mechanism for brain ischemia reperfusion injury","volume":"37","author":"Stepanova","year":"2017","journal-title":"J. Cereb. Blood Flow. Metab."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"103047","DOI":"10.1016\/j.redox.2024.103047","article-title":"Reactive oxygen species generation by reverse electron transfer at mitochondrial complex I under simulated early reperfusion conditions","volume":"70","author":"Dancil","year":"2024","journal-title":"Redox Biol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"123","DOI":"10.1038\/nature19754","article-title":"Accessory subunits are integral for assembly and function of human mitochondrial complex I","volume":"538","author":"Stroud","year":"2016","journal-title":"Nature"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"20241050","DOI":"10.1515\/med-2024-1050","article-title":"Role of COX6C and NDUFB3 in septic shock and stroke","volume":"19","author":"Tian","year":"2024","journal-title":"Open Med."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"452","DOI":"10.1016\/j.tem.2018.03.009","article-title":"NDUFA4 (Renamed COXFA4) Is a Cytochrome-c Oxidase Subunit","volume":"29","author":"Pitceathly","year":"2018","journal-title":"Trends Endocrinol. Metab."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"1026","DOI":"10.1038\/s41422-018-0071-1","article-title":"Structure of the intact 14-subunit human cytochrome c oxidase","volume":"28","author":"Zong","year":"2018","journal-title":"Cell Res."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"1795","DOI":"10.1016\/j.celrep.2013.05.005","article-title":"NDUFA4 mutations underlie dysfunction of a cytochrome c oxidase subunit linked to human neurological disease","volume":"3","author":"Pitceathly","year":"2013","journal-title":"Cell Rep."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"15","DOI":"10.1186\/s13041-018-0357-8","article-title":"Targeting NMDA receptors in stroke: New hope in neuroprotection","volume":"11","author":"Wu","year":"2018","journal-title":"Mol. Brain"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"79","DOI":"10.1016\/j.neulet.2017.08.048","article-title":"Beyond the critical point: An overview of excitotoxicity, calcium overload and the downstream consequences","volume":"663","author":"Bano","year":"2018","journal-title":"Neurosci. Lett."},{"key":"ref_20","doi-asserted-by":"crossref","unstructured":"Morotti, A., Barale, C., Melchionda, E., and Russo, I. (2022). Platelet Redox Imbalance in Hypercholesterolemia: A Big Problem for a Small Cell. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms231911446"},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"50","DOI":"10.1016\/j.ab.2017.07.009","article-title":"Mitochondrial membrane potential","volume":"552","author":"Zorova","year":"2018","journal-title":"Anal. Biochem."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"44","DOI":"10.1186\/s12974-024-03033-7","article-title":"Mitochondrial stress: A key role of neuroinflammation in stroke","volume":"21","author":"Gao","year":"2024","journal-title":"J. Neuroinflamm."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"45","DOI":"10.1186\/s13619-025-00268-4","article-title":"Modulating mitochondrial metabolism: A neuroprotective mechanism for hypoxic-ischemic preconditioning","volume":"14","author":"Li","year":"2025","journal-title":"Cell Regen."},{"key":"ref_24","doi-asserted-by":"crossref","unstructured":"Wu, X., Zhou, X., Xiong, L., Pei, J., Yao, X., Liang, C., Bao, P., Chu, M., Guo, X., and Yan, P. (2020). Transcriptome Analysis Reveals the Potential Role of Long Non-coding RNAs in Mammary Gland of Yak During Lactation and Dry Period. Front. Cell Dev. Biol., 8.","DOI":"10.3389\/fcell.2020.579708"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"1321","DOI":"10.4161\/auto.25132","article-title":"Cerebral ischemia-reperfusion-induced autophagy protects against neuronal injury by mitochondrial clearance","volume":"9","author":"Zhang","year":"2013","journal-title":"Autophagy"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"682","DOI":"10.1038\/nrn2911","article-title":"Synaptic versus extrasynaptic NMDA receptor signalling: Implications for neurodegenerative disorders","volume":"11","author":"Hardingham","year":"2010","journal-title":"Nat. Rev. Neurosci."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"6125","DOI":"10.1523\/JNEUROSCI.16-19-06125.1996","article-title":"Mitochondrial dysfunction is a primary event in glutamate neurotoxicity","volume":"16","author":"Schinder","year":"1996","journal-title":"J. Neurosci."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"366","DOI":"10.1038\/1577","article-title":"Glutamate-induced neuron death requires mitochondrial calcium uptake","volume":"1","author":"Stout","year":"1998","journal-title":"Nat. Neurosci."},{"key":"ref_29","first-page":"1452696","article-title":"PGC-1\u03b1, Inflammation, and Oxidative Stress: An Integrative View in Metabolism","volume":"2020","author":"Ortega","year":"2020","journal-title":"Oxidative Med. Cell. Longev."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1186\/s13578-025-01519-2","article-title":"PGC-1\u03b1: Key regulator of mitochondrial biogenesis and cellular differentiation in metabolic and regenerative tissues","volume":"16","author":"Cao","year":"2025","journal-title":"Cell Biosci."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"792639","DOI":"10.1155\/2011\/792639","article-title":"Mitogen-Activated Protein Kinases and Reactive Oxygen Species: How Can ROS Activate MAPK Pathways?","volume":"2011","author":"Son","year":"2011","journal-title":"J. Signal Transduct."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"4350965","DOI":"10.1155\/2016\/4350965","article-title":"ROS and ROS-Mediated Cellular Signaling","volume":"2016","author":"Zhang","year":"2016","journal-title":"Oxidative Med. Cell. Longev."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"118749","DOI":"10.1016\/j.biopha.2025.118749","article-title":"Role of JNK3 signaling in acute ischemic stroke cerebroprotection: A systematic review","volume":"193","author":"Ferrari","year":"2025","journal-title":"Biomed. Pharmacother."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"1889","DOI":"10.1038\/sj.emboj.7600194","article-title":"JNK promotes Bax translocation to mitochondria through phosphorylation of 14-3-3 proteins","volume":"23","author":"Tsuruta","year":"2004","journal-title":"EMBO J."},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"91","DOI":"10.1038\/s41392-025-02176-0","article-title":"The BCL2 family: From apoptosis mechanisms to new advances in targeted therapy","volume":"10","author":"Vogler","year":"2025","journal-title":"Signal Transduct. Target. Ther."}],"container-title":["Cells"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2073-4409\/15\/12\/1071\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,24]],"date-time":"2026-06-24T06:05:50Z","timestamp":1782281150000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2073-4409\/15\/12\/1071"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,6,12]]},"references-count":35,"journal-issue":{"issue":"12","published-online":{"date-parts":[[2026,6]]}},"alternative-id":["cells15121071"],"URL":"https:\/\/doi.org\/10.3390\/cells15121071","relation":{},"ISSN":["2073-4409"],"issn-type":[{"value":"2073-4409","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,6,12]]}}}