Search Results (361)

Neonatal hypoxic–ischaemic encephalopathy (HIE) is a major cause of neonatal mortality and long-term neurological disability, affecting 1–3 per 1000 live births in developed countries and occurring at substantially higher rates in developing countries. Neuroinflammation is a key contributor to disease progression, with growing evidence implicating the activation of the NLR family pyrin domain containing 3 (NLRP3) inflammasome following hypoxic–ischaemic (HI) injury. In this study, we investigated the role and regulation of the NLRP3 inflammasome in neonatal HIE using in vitro and in vivo models. Primary microglial cultures subjected to oxygen–glucose deprivation and the Vannucci neonatal rat model of HI were used to characterize NLRP3 activation and its contribution to injury. We demonstrated that HI induces NLRP3 inflammasome activation, whereas pharmacological inhibition of NLRP3 enhances cell viability and attenuates brain damage. Our findings identify microglia as a central mediator of NLRP3-driven neuroinflammation and highlight microglial NLRP3 signaling as a promising therapeutic target for neuroinflammatory diseases. Collectively, this study provides an integrated view of NLRP3 regulation in neonatal HIE and supports inflammasome-directed strategies for neuroprotection following neonatal HI injury. Full article

Hydrogen (H₂) is a promising renewable energy carrier because of its high energy density and environmentally friendly characteristics. The halotolerant cyanobacterium Aphanothece halophytica is a potential biological H₂ producer because of its ability to generate H₂ via oxygen-sensitive hydrogenase activity under nitrogen-deprived dark anaerobic conditions. This study investigated the effects of various chemical supplements on H₂ production by A. halophytica. Initial screening identified glucose, sodium dithionite, and dithiothreitol (DTT) as effective stimulants for enhancing H₂ production. Optimization experiments established 10 mM glucose, 5 mM DTT, and 1 mM sodium dithionite as the optimal concentrations for H₂ production. Combined supplementation under these optimized conditions resulted in the highest H₂ production rate of 0.588 ± 0.028 mL·L⁻¹·h⁻¹ and maximum cumulative H₂ yield of 8.445 ± 0.750 mL·L⁻¹ after incubation for 72 h, representing increases of approximately 170–180%, compared with the untreated control. Simultaneously, maximum cumulative O₂ production decreased from 20.176 ± 0.850 to 10.405 ± 0.480 mL·L⁻¹, corresponding to approximately a 50% reduction. These findings demonstrate that simultaneous optimization of electron supply, oxygen minimization, and intracellular redox balance using selected stimulants effectively enhances H₂ production by A. halophytica, providing a simple and practical strategy for improving biological H₂ production. Full article

Background/Objectives: Ischemic stroke (IS) induces substantial phenotypic remodeling within the neurovascular unit, and pericytes have been implicated in the cellular responses to ischemic injury. However, the molecular characteristics and regulatory mechanisms underlying pericyte phenotypic remodeling after ischemic stroke remain incompletely understood. Methods: In this study, we used PDGFR-β-CreERT2; ZsGreen lineage-tracing mice and a middle cerebral artery occlusion (MCAO) model to characterize the temporal changes in PDGFR-β-lineage cells following ischemic injury. In vitro, human brain vascular pericytes (HBVPs) exposed to oxygen–glucose deprivation/reperfusion (OGD/R) and subsequently maintained in neurobasal medium to examine ischemia-related phenotypic changes. The involvement of KLF2-associated signaling was further investigated using KLF2 knockdown approaches. Results: We observed increased expression of Nestin in PDGFR-β-lineage cells during the early post-ischemic period, followed by the emergence of subsets co-expressing GFAP or DCX during the subacute stage. These findings indicate the acquisition of neuroglial- and neuronal-associated molecular features, rather than providing definitive evidence of lineage conversion or functional differentiation. The expression of these markers declined at later stages, whereas PDGFR-β-lineage cells were also associated with vascular remodeling during the recovery phase. In vitro, OGD/R-treated HBVPs exhibited increased expression of DCX, MAP2, and NeuN. KLF2 knockdown attenuated these molecular changes. Consistently, OGD/R was associated with increased KLF2, phosphorylated ERK1/2, and MAP2 expression, whereas KLF2 knockdown reduced ERK1/2 phosphorylation and MAP2 expression. These findings suggest that KLF2 is associated with ERK1/2 activation and MAP2 expression during ischemia-related phenotypic remodeling of PDGFR-β-lineage cells. Conclusions: Overall, our results identify transient neuroglial- and neuronal-associated molecular changes in pericytes after ischemic injury and provide evidence for an association between KLF2 and ERK1/2–MAP2 signaling in this process, while further studies are required to determine whether these changes represent stable lineage conversion or functional neuronal differentiation. Full article

The neurovascular unit (NVU) is composed of a diverse array of cells and an extracellular matrix (ECM). Neural cells and blood vessels are intricately interconnected, forming a cohesive whole. Specific cellular components and structures within the NVU play an indispensable role in maintaining homeostasis of the central nervous system (CNS). With the advancement and maturation of cell co-culture technology, various three-dimensional (3D) NVU models continue to emerge, offering a more objective and comprehensive perspective for in vitro studies of CNS diseases. Specifically, these 3D NVU models include Transwell Chamber models, gel-PDNS-based 3D models, self-assembled NVU models and microfluidic NVU models, which reconstruct the complex NVU architecture to varying degrees. This review systematically summarizes multiple 3D construction strategies for in vitro NVU to overcome the limitations of conventional cellular tests or animal experiments, highlights the critical roles of biomimetic gel in recapitulating native cell-gel crosstalk, comparatively analyzes four major 3D NVU technical routes in terms of cellular composition, vascular morphology, barrier performance, and reproducibility, categorizes application scenarios of 3D NVU platforms oriented to practical research demands, including oxygen-glucose deprivation/reoxygenation (OGD/R) injury modeling, blood-brain barrier (BBB) permeability assay, CNS drug penetration screening, neuroinflammation and neurotoxicity evaluation, proposes practical principles for model selection under different experimental purposes, and concludes with current bottlenecks, including imperfect vascular network maturation and lack of unified evaluation criteria, together with future perspectives for standardized 3D NVU in vitro. By comparing the advantages and limitations of these approaches, we aim to clarify their optimal applicability for investigating specific pathological mechanisms and screening potential therapeutics. Full article

Background/Objectives: Cerebral ischemia–reperfusion injury (CIRI) poses therapeutic challenges because of oxidative stress, blood–brain barrier disruption, and neuronal apoptosis, limiting current treatments. Paeoniflorin (PF) from Paeonia lactiflora has neuroprotective potential, but its multi-target mechanisms remain unclear. This study investigated the role and mechanisms of PF in CIRI, focusing on neuron–endothelial crosstalk. Methods: Oxygen–glucose deprivation/reoxygenation (OGD/R) models were established using SH-SY5Y (human neuroblastoma) and HCMEC/D3 cells (human cerebral microvascular endothelial). Network pharmacology was used to predict potential PF targets and pathways. RNA sequencing, molecular docking, and molecular dynamics simulation were performed to screen and evaluate PF binding characteristics with key targets, and MTT, flow cytometry, Western blotting, and co-cultures were employed to detect paracrine interactions. Results: Network pharmacology and transcriptomics identified VEGF/PI3K-AKT/mTOR pathway enrichment. Molecular docking confirmed stable PF binding to VEGF-A (−8.4 kcal/mol), AKT (−5.5 kcal/mol), and mTOR (−9.6 kcal/mol). PF (10–80 μM) showed no cytotoxicity and reduced OGD/R injury in a concentration-dependent manner (maximal at 40 μM). PF activated VEGF/PI3K-AKT/mTOR signaling, reducing apoptosis by 57% (SH-SY5Y) and 33% (HCMEC/D3); PI3K inhibitor LY294002 abolished these effects. PF-treated HCMEC/D3-conditioned media enhanced OGD/R neuronal viability, verifying paracrine crosstalk. Conclusions: PF alleviated CIRI by directly protecting neurons and indirectly modulating neuron–endothelial crosstalk through VEGF/PI3K-AKT/mTOR activation, supporting its multi-target therapeutic potential. Full article

Background/Objectives: Ischemia-reperfusion injury (IRI) contributes significantly to cardiomyocyte death following myocardial infarction, largely through sustained inflammatory signaling. This study aimed to identify stress-responsive deubiquitinating enzymes involved in cardiomyocyte injury under ischemic stress. Methods: HL-1 cardiomyocytes were subjected to oxygen-glucose deprivation and reoxygenation (OGD/R), followed by transcriptomic analysis, genetic manipulation of Usp31, biochemical assessment of p65 ubiquitination, NF-κB reporter assays, and in vitro deubiquitinase activity assays using sodium tanshinone IIA sulfonate (STS). Results: Usp31 was persistently upregulated in HL-1 cardiomyocytes after OGD/R. Depletion of Usp31 improved cell viability and reduced lactate dehydrogenase (LDH) release under OGD/R conditions. Mechanistically, Usp31 stabilized p65 and supported NF-κB activation, whereas Usp31 deficiency increased p65 ubiquitination and limited NF-κB transcriptional activity. STS inhibited recombinant Usp31 enzymatic activity under the experimental conditions tested and reduced Usp31-associated NF-κB activation and cardiomyocyte injury in cellular assays. Conclusions: These findings identify Usp31 as a candidate regulator of NF-κB-associated inflammatory responses in cardiomyocytes and support STS as a pharmacological modulator of Usp31-associated signaling with potential relevance to myocardial ischemic injury. Full article

Cerebral ischemia–reperfusion injury is a key determinant of a poor outcome after stroke. The mitochondrial uncoupling protein 2 (UCP2) has been implicated in cerebral ischemia-reperfusion injury and in the outcome of ischemic stroke, although its role remains controversial. In C57BL/6J and B6.129S4-Ucp2^tm1Lowl/J (UCP2^−/−) mice, we analyzed cognitive function and lifespan. In an MCAO model induced for one hour, infarct volumes, neurological deficits, and gene expression patterns were determined after 24 h. The UCP2 inhibitor genipin was used in an oxygen-glucose deprivation (OGD) model to investigate synaptic transmission in the hippocampus. Compared to controls, UCP2^−/− mice exhibited a reduced lifespan and displayed impaired cognition. However, in 6-month-old UCP2^−/− mice, the infarct volume was reduced, primarily due to a smaller core size, but not in 18-month-old animals. In both strains, ischemia induced upregulation of antioxidant defense genes, including catalase and SOD1. In the ex vivo ODG model, synaptic transmission was depressed, but pretreatment with genipin prevented the tissue from this impairment. Our findings indicate an infarct-reducing effect of UCP2 deficiency, especially in young-adult mice, and, mechanistically, a neuroprotective effect by genipin in hippocampal slices. Full article

Background: Danshen–Gegen is a classic herb pair in traditional Chinese medicine, which has been used to treat cardiovascular and cerebrovascular diseases. Ischemic stroke (IS) is a prevalent cerebrovascular condition; ferroptosis is one of the contributing factors driving the progression of IS. This study aims to determine the underlying mechanism and examine if Danshen–Gegen (DG) extract may prevent cerebral ischemia–reperfusion injury by preventing ferroptosis. Methods: The comprehensive compositional characterization of DG was analyzed by ultra-high-performance liquid chromatography coupled with hybrid quadrupole-orbitrap high-resolution mass spectrometry (UPLC-Q-orbitrap MS). The experiments were conducted in middle cerebral artery occlusion/reperfusion (MCAO/R) rats and oxygen-glucose deprivation/re-oxygenation (OGD/R) cells. The neuroprotective effects of DG on IS were assessed by examining rat survival rates, infarct volume, behavioral scores, and cerebral water content. Then, we tested the accumulation of Fe²⁺ and lipid peroxidation products such as reactive oxygen species (ROS), glutathione (GSH), malondialdehyde (MDA), myeloperoxidase (MPO), and 4-hydroxynonenal (4-HNE) in rats and cells. The expression of nuclear factor erythroid-derived 2-like 2 (Nrf2), Solute Carrier Family 7 Member 11 (-xCT), Glutathione peroxidase 4 (GPX4), Cyclooxygenase-2 (COX-2), Transferrin Receptor 1 (TFR1), and Long-chain-fatty-acid–CoA ligase 4 (ACSL4) was also assessed in vivo and in vitro. Results: UPLC-Q-orbitrap MS analysis was performed to characterize the chemical profile of DG, and a total of 33 chemical constituents were successfully identified. DG significantly alleviated the ischemic damage to brain tissue, reduced infarct volume, and improved neurological dysfunction. The content of Fe²⁺ and lipid peroxidation products was markedly decreased. Furthermore, DG could restore the expression of Nrf2, -xCT, and GPX4 with the inhibition of COX-2, TFR1, and ACSL4, thus achieving a suppressive effect on ferroptosis. Conclusions: The regulatory influence of DG via the Nrf2/System xc-/GPX4 axis may play a crucial role in alleviating ferroptosis and enhancing recovery from cerebral ischemia injury. Full article

Reperfusion after acute ischemic stroke (AIS) triggers a series of ferroptosis-related molecular events, including iron dyshomeostasis, oxidative/nitrative stress, antioxidant depletion, and membrane lipid peroxidation. Conventional ferroptosis assays mainly rely on ex vivo or endpoint measurements, limiting their ability to dynamically monitor the spatiotemporal evolution of these events during ischemia–reperfusion. Recent advances in chemical sensing and optical imaging have enabled in situ detection of key ferroptosis-related nodes, such as Fe²⁺/labile iron pool, ROS/ONOO⁻, GSH/Cys/GPX4, H₂S/Cys–Met metabolism, and lipid peroxidation. In this review, we summarize sensing targets, reaction-based probe design, near-infrared and two-photon imaging, photoacoustic imaging, and multimodal validation strategies for AIS-related ferroptosis. Representative probes for H₂O₂, ONOO⁻, H₂S, Fe²⁺, and lipid peroxidation are discussed in the context of cellular models, oxygen-glucose deprivation/reoxygenation, middle cerebral artery occlusion/reperfusion, and in vivo brain imaging. We emphasize that a single probe signal cannot independently confirm ferroptosis and should be interpreted together with GPX4/ACSL4 alterations, MDA/4-HNE levels, tissue injury, neurological outcomes, and Fer-1/Lip-1 rescue experiments. Finally, we discuss current challenges, including limited tissue penetration, blood–brain barrier delivery, quantitative stability, probe safety, and clinical translation, and highlight future directions involving ratiometric, NIR/NIR-II, two-photon, multitarget, and imaging-guided validation strategies. Full article

Brain injury after hypoxia–ischemia (HI) is the leading cause of morbidity and mortality in term and near-term neonates worldwide. The ferret is a promising translational model to study HI due to its gyrified brain and white-to-gray matter ratio that more closely resembles humans compared to rodents. Caffeine, an adenosine A2A receptor (A2AR) antagonist, shows neuroprotective potential after HI, but its effects have not been fully characterized. We sought to evaluate caffeine’s effect on neuronal cell death, cytotoxicity, and inflammatory and oxidative stress markers in a term-equivalent ferret organotypic brain slice model of HI. Slices were cultured for 72 h, exposed to two hours of oxygen–glucose deprivation (OGD), and randomized to OGD alone, OGD with caffeine (20 or 50 mg/L), or OGD with caffeine and an A2AR agonist. Healthy slices served as controls. Outcomes included global cell death, regional cell death, microglial morphology, and expression of inflammatory and oxidative stress genes (46–48 slices/group for cell death assays and 18 slices/group for imaging, balanced by sex). Caffeine 50 mg/L significantly reduced global cell death compared to OGD (p = 0.02), and this effect persisted despite co-administration of an A2AR agonist (p = 0.01), suggesting that protection was not primarily mediated through A2AR signaling. Caffeine also did not change regional pyknotic nuclei counts (p > 0.05). Caffeine altered microglial morphology, increasing the proportion of microglia with features characteristic of control conditions. OGD significantly increased expression of inflammatory and oxidative stress-related genes (p < 0.05) compared with control slices, whereas caffeine did not significantly alter gene expression. In summary, caffeine partially reversed global cell death after OGD and altered microglial morphology. Larger, higher-powered studies are needed to further investigate caffeine’s effects on neonatal HI. Full article

Ischemia plays a critical role in the pathogenesis of sensorineural hearing loss through the induction of severe cochlear apoptosis and mitochondrial dysfunction. Exosomes derived from adipose-derived mesenchymal stem cells (ADMSC-Exo) have robust protective effects under non-otologic ischemic conditions. However, their otoprotective effects remain unclear. This study aimed to investigate the protective effects of human ADMSC-Exo against cochlear damage and mitochondrial dysfunction under oxygen–glucose deprivation (OGD), an in vitro and ex vivo model of cochlear ischemia. ADMSC-Exo attenuated OGD-induced cytotoxicity and apoptosis in HEI-OC1 cells and reduced the OGD-induced loss of cochlear hair cells in the organ of Corti explants. OGD caused a decrease in mitochondrial mass and mitochondrial membrane potential depolarization and impaired mitochondrial respiration in auditory cells. ADMSC-Exo preserved mitochondrial integrity and improved mitochondrial bioenergetic function following OGD exposure. These effects were accompanied by increased LC3-II conversion and formation of autolysosome-like structures and elevated expression of PINK1 and Parkin, indicating the activation of autophagy and mitophagy-related protective mechanisms. Importantly, 3-methyladenine, an autophagy inhibitor, attenuated the cytoprotective effect of ADMSC-Exo, supporting the involvement of autophagy in ADMSC-Exo-mediated protection. Collectively, these findings suggest that ADMSC-Exo protect against OGD-induced cochlear injury by promoting autophagy-associated mitochondrial protection. Full article

Background: Hypoxic–ischaemic brain injury (HI) is a major contributor to neurological deficits following stroke. Understanding what happens to the smallest functional and structural unit of the central nervous system in the face of oxygen and nutrient deprivation is essential to fully comprehend the pathogenesis of diseases and disorders associated with HI, such as ischaemic stroke. Aim: The aim of this study was to develop a robust in vitro tool for initial screening of potential therapeutics and identification of diagnostic markers of brain hypoxic injury. Methods: This study details and validates a comprehensive protocol for modelling HI using differentiated SH-SY5Y neuroblastoma cells (Neuron-like Cells, NLCs). First, we optimized the differentiation process and confirmed the maturity and purity of NLCs via standard molecular markers. The NLCs exhibited functional excitotoxicity, demonstrating a graded cell death response to N-methyl-D-aspartate (NMDA), thus validating their functional application. To simulate HI, we initially optimized the oxygen-glucose deprivation (OGD) treatment using graded concentrations of CoCl₂ (0.125 mM to 2 mM) in glucose-free media. The validated NLCs were then subjected to the refined OGD protocol (1 mM CoCl₂ in glucose-free media) for 3 h, followed by various periods of reoxygenation (1 h, 3 h, 6 h, 12 h, 18 h, and 24 h). Result: Bulk RNA-sequencing revealed a distinct temporal transcriptional response to HI. Injury-associated genes, including heat shock proteins and stress markers, were significantly (p < 0.05) upregulated at 3 h of reoxygenation, peaked at 6 h, and declined thereafter, remaining above baseline at 24 h. Upstream regulator analysis identified IL-1β, TNF-α, and HIF-1α as key drivers during OGD, with additional regulators emerging during reoxygenation. TNF-α and β-oestradiol were consistently identified across time points, while TGF-β1 and NTRK1 became prominent during peak injury and later phases. Analysis of secreted factors showed increased release of inflammatory (TNF-α) and neurotrophic (β-NGF, BDNF, VEGF) mediators with reoxygenation, while maximal cell death occurred at 24 h. Conclusions: This study identifies a transient, time-dependent transcriptional cascade following hypoxic–ischaemic injury, highlighting a critical window for early neuronal response. The model provides a reproducible platform for studying neuronal injury and recovery, and identifies known (TNF-α, IL-β, and HIF-1α), context-specific (NTRK1 and TGF-β) and novel (β-oestradiol) regulators of the injury response with potential relevance for therapeutic targeting. Full article

Background/Objectives: Ischemic stroke remains a major global health challenge, yet therapeutic options are severely restricted by narrow treatment windows and the risk of hemorrhagic transformation. Natural small molecules represent a valuable reservoir for discovering novel neuroprotective leads with favorable safety profiles. Cinchonidine, a natural quinoline alkaloid, has shown anti-inflammatory and cytoprotective properties, but its potential in treating ischemic stroke is largely unexplored. This study aimed to evaluate the neurovascular protective effects and hemostatic safety of cinchonidine in preclinical stroke models. Methods: We evaluated cinchonidine using a mouse model of middle cerebral artery occlusion (MCAO) and in vitro oxygen–glucose deprivation (OGD) models in cerebral endothelial cells (CECs) and Neuro2A cells. Infarct volume, brain edema, and neurological recovery were assessed. Blood–brain barrier (BBB) integrity was measured via Evans blue extravasation. Mechanistic markers, including microglial activation, pro-inflammatory mediators (iNOS, COX-2), and apoptosis-related signaling, were examined. Additionally, cinchonidine’s effect on platelet aggregation was also tested. Results: Cinchonidine significantly reduced infarct volume and brain edema while improving neurological functional recovery. It effectively preserved BBB integrity and enhanced cell viability under OGD conditions. Furthermore, cinchonidine suppressed microglial activation and decreased the expression of pro-inflammatory mediators. These protective effects were associated with the modulation of apoptotic signaling pathways. These protective effects were accompanied by reduced p53-associated stress signaling in endothelial cells and ischemic brain tissue. Importantly, cinchonidine did not significantly interfere with platelet aggregation, suggesting a potentially favorable hemostatic profile. Conclusions: Cinchonidine attenuates ischemic brain injury and is associated with endothelial protection, preservation of BBB integrity, and modulation of inflammatory and apoptotic responses. As a natural lead compound that does not compromise hemostasis, cinchonidine represents a promising lead compound for further development as a neurovascular protective strategy in ischemic stroke. Full article

Background: Scutellaria baicalensis (Scu) extract has been traditionally used in the treatment of stroke-related syndromes, yet its underlying molecular mechanisms, particularly those involving ferroptosis, remain to be fully elucidated. Purpose: This study aims to validate the hypothesis that Scu extract improves cerebral ischemia-reperfusion injury (CIRI) by inhibiting ferroptosis through the PI3K/AKT signaling pathway. Methods: This study employed middle cerebral artery occlusion (MCAO) in male Sprague-Dawley (SD) rats and oxygen–glucose deprivation/reoxygenation (OGD/R) models to evaluate the protective effects of Scu extract against CIRI. Multiple approaches were integrated to elucidate the underlying mechanisms. Furthermore, a range of experimental techniques, including neurological function assessment, TTC staining, histopathological analysis, biochemical assays, qPCR, transmission electron microscopy (TEM), reactive oxygen species (ROS) detection, Western blotting, and immunofluorescence, were used to comprehensively validate its neuroprotective effects. Results: Scu extract significantly improved neurological outcomes and attenuated brain injury in MCAO rats. Proteomic analysis revealed significant enrichment of ferroptosis-related pathways, which was supported by reduced mitochondrial damage, decreased iron accumulation, and restoration of the SLC7A11/GPX4 axis. Subsequently, UPLC/Q-TOF-MS analysis revealed that four major bioactive components were absorbed in MCAO rats. KEGG pathway analysis based on network pharmacology further indicated that the PI3K/AKT signaling pathway is a key regulatory target. Notably, pharmacological inhibition of PI3K with LY294002 markedly abolished the anti-ferroptotic effects of Scu extract, which was further confirmed in vitro. Conclusions: This study demonstrates that Scu extract confers neuroprotection against CIRI in MCAO rats potentially through inhibiting ferroptosis via activation of the PI3K/AKT pathway. Full article

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–reperfusion 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 (ΔΨm), 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 ΔΨm. 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 ΔΨm 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. Full article