Search Results (77)

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

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

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

The link between neutrophil extracellular traps (NETs) and hepatocyte ferroptosis in liver ischemia–reperfusion injury (LIRI) is unclear. Adipose-derived mesenchymal stem cell exosomes (ADSCs-Exo) hold therapeutic potential for LIRI. This study employed miniature pigs to investigate the NETs’ role and ADSCs-Exo’s protection in LIRI. In vitro, established hepatocyte oxygen-glucose deprivation/reoxygenation (OGD/R) model and Transwell co-culture system with polymorphonuclear neutrophils (PMNs). In vivo, a laparoscopic minimally invasive LIRI model was constructed in miniature pigs, followed by ADSCs-Exo intervention. Results demonstrated that NETs exacerbate OGD/R-induced hepatocyte ferroptosis via myeloperoxidase. ADSCs-Exo inhibited NET formation via the NADPH/MAPK pathway, thereby mitigating ferroptosis, and ultimately improved liver histopathology and function. This study is the first to demonstrate in a large animal model that ADSCs-Exo alleviate LIRI by inhibiting NET formation via the NADPH/MAPK pathway, consequently attenuating hepatocyte ferroptosis. These findings provide novel insights into LIRI pathogenesis, support the translational potential of ADSCs-Exo as a cell-free therapeutic strategy, and highlight the value of the miniature pig model in liver research. Full article

Background: Hydroxysafflor Yellow A (HSYA), the major bioactive component from Carthamus tinctorius L., exerts significant protective effects against myocardial ischemia-reperfusion injury (MIRI). Mitophagy is pivotal in the pathological process of MIRI, yet the specific molecular mechanism underlying HSYA-mediated mitophagy regulation remains unclear. Objective: This study aimed to investigate the association between HSYA treatment and mitochondrial autophagy in murine MIRI and to explore the potential mechanistic role of the SIRT1-FOXO3-BNIP3 signaling pathway using functional loss-of-function and rescue experiments. These findings may provide preliminary evidence supporting the clinical translational potential in MIRI therapy. Methods: Mouse myocardial ischemia-reperfusion injury (MIRI) model and oxygen-glucose deprivation/reoxygenation (OGD/R)-induced AC16 cardiomyocyte injury models were established. Metabolomics, molecular docking, and surface plasmon resonance (SPR) techniques were combined to screen the potential targets of HSYA. The SIRT1 inhibitor EX527 and SIRT1 siRNA were used to verify the underlying mechanism. Cardiac function, myocardial infarct size, mitochondrial function, the expression of autophagy-related proteins, and protein–protein interaction were detected and analyzed. Results: Compared with the MIRI group, HSYA significantly improved cardiac function in mice, as evidenced by increased left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) (p < 0.01), attenuated ST-segment elevation, and improved myocardial perfusion. HSYA also markedly reduced myocardial infarct size (p < 0.01) and serum levels of CK-MB, LDH, and cTnI (all p < 0.01) and ameliorated myocardial histopathological damage and mitochondrial ultrastructural integrity. Mechanistic studies revealed that HSYA significantly upregulated the expression of SIRT1, FOXO3, BNIP3, Beclin-1, and the LC3II/I ratio while downregulating p62 expression (p < 0.01), consistent with enhanced mitophagy-related activity. Furthermore, these protective effects were markedly attenuated upon SIRT1 inhibition or siRNA-mediated silencing, whereas HSYA intervention partially reversed these alterations. Additionally, co-immunoprecipitation (Co-IP) and pull-down assays demonstrated that HSYA promoted protein–protein interactions between SIRT1-FOXO3, FOXO3-BNIP3, and BNIP3-LC3B. Conclusions: These findings highlight that HSYA is associated with improved cardiac function, enhanced mitophagy-related activity, and upregulated SIRT1-FOXO3-BNIP3 signaling, providing robust experimental evidence for its clinical translational application in MIRI treatment. Full article

We previously demonstrated that fatty acid-binding protein 3 (FABP3) is significantly upregulated in ischemic neurons, and its inhibition mitigates ischemic brain injury in mice and attenuates mitochondrial damage under rotenone-induced oxidative stress. These findings suggest a potential role for FABP3 in mitochondrial dysfunction in ischemic neurons, although the underlying mechanism remains unclear. In this study, we further investigated the role of FABP3 in mitochondrial injury and apoptosis in ischemic neurons. Our findings indicated that FABP3 deficiency significantly decreased infarct volume following middle cerebral artery occlusion/reperfusion (MCAO/R) in mice, improved cognitive and spontaneous activity deficits, and suppressed BAX activation and mitochondrial translocation, caspase-3 activation, and cytochrome c release. In HT22 cells subjected to oxygen-glucose deprivation/reoxygenation (OGD/R), FABP3 deficiency increased cell viability, reduced apoptosis, and alleviated the loss of mitochondrial membrane potential. Conversely, FABP3 overexpression further exacerbated mitochondrial dysfunction and apoptosis, effects that were partially reversed by the BAX inhibitor BAI1. Furthermore, FABP3 overexpression promoted abnormal mitochondrial lipid accumulation and increased lipid peroxidation. Both the mitochondria-targeted antioxidant MitoQ and the ferroptosis inhibitor Ferrostatin-1 alleviated FABP3 overexpression-induced mitochondrial damage and apoptotic signaling. Collectively, our findings suggest that FABP3 is an important promoter of cerebral ischemia–reperfusion injury. FABP3 may aggravate ischemic neuronal injury by promoting abnormal mitochondrial lipid accumulation and lipid peroxidation, thereby enhancing BAX-dependent mitochondrial apoptotic signaling. Targeting FABP3 may provide a potential therapeutic strategy for neuroprotection in ischemic stroke. Full article

The marine benthic invertebrate Urechis unicinctus exhibits extraordinary tolerance to hypoxic environments, making its coelomic fluid a unique and promising biological source for discovering novel stress-adapting macromolecules. Polysaccharides derived from the coelomic fluid of U. unicinctus were systematically extracted, fractionated, and characterized to investigate their structural features and associated biological activities. Gradient ethanol precipitation (30–80%) combined with DEAE-52 ion exchange chromatography yielded twelve fractions with distinct physicochemical properties. Significant variations were observed in molecular weight (10³–10⁵ Da), sulfate content (3.77–24.26%), and monosaccharide composition. High-ethanol fractions, particularly U68P and U18P (extracted at 60 °C and 100 °C, respectively, and both precipitated with 80% ethanol), were enriched in low-molecular-weight, highly sulfated heteropolysaccharides composed of galactose, fucose, glucosamine, and ribose. These fractions exhibited superior antioxidant activities, including strong scavenging effects against DPPH, ABTS, and hydroxyl radicals. Moreover, they demonstrated pronounced neuroprotective effects in the oxygen–glucose deprivation/reoxygenation (OGD/R) model using SH-SY5Y cells, significantly improving cell viability. Structure–activity relationship analysis revealed that reduced molecular weight, increased sulfation degree, and more diverse monosaccharide composition (e.g., more diverse monosaccharide composition) synergistically contribute to improved bioactivity by facilitating cellular uptake and exposing functional groups. In contrast, high-molecular-weight homoglucan fractions showed relatively weak effects. Overall, this study identifies U. unicinctus coelomic fluid as a promising source of bioactive polysaccharides and provides a theoretical basis for the development of marine-derived anti-hypoxic and antioxidant agents. Full article

Neuronal ferroptosis is a key contributor to secondary brain injury following cerebral ischemia, yet the metabolic mechanisms governing this process remain poorly understood. Enriched environment (EE) is a housing paradigm that provides enhanced sensory, cognitive, and social stimulation through complex physical surroundings and increased opportunities for voluntary activity. Our preliminary data indicate that EE confers cerebroprotection against ischemia-induced ferroptosis; however, whether this effect is associated with glycogen metabolic regulation and the underlying molecular pathways has not been elucidated. This study aimed to determine whether EE may influence ferroptosis-associated pathways, potentially via Sirtuin 1 (SIRT1)/protein kinase B (AKT)/glycogen synthase kinase-3β (GSK3β)-related mechanisms of glycogen metabolism. Using a mouse model of middle cerebral artery occlusion (MCAO) and an oxygen–glucose deprivation/reoxygenation (OGD/R) cellular model, we performed behavioral assessments, molecular and biochemical analyses, and pharmacological interventions to elucidate mechanistic pathways. EE was associated with improved neurological outcomes and reduced infarct volume after ischemia. Mechanistically, EE appeared to activate the SIRT1/AKT pathway and increase the inhibitory phosphorylation of GSK3β and relieving its suppressive effect on glycogen synthase, which may underlie the observed increase in glycogen levels within ischemic brain tissue. Pharmacological inhibition of SIRT1 largely diminished these metabolic and neuroprotective benefits. Consistently, at the cellular level, SIRT1 overexpression contributed to the restoration of glycogen metabolism and robustly attenuated ferroptosis under ischemic conditions. Collectively, these findings suggest that EE may attenuate ferroptosis-related pathways possibly involving SIRT1/AKT/GSK3β-dependent glycogen metabolic remodeling, providing a novel metabolic perspective on EE-induced cerebroprotection and highlighting SIRT1-centered regulation of glycogen metabolism as a potential therapeutic target for ischemic stroke. Full article

Cytochrome c (Cytc) tyrosine 48 (Y48) has been previously shown to be phosphorylated in bovine liver, and phosphomimetic substitution (Y48E) inhibits key functions of Cytc in vitro, including respiration and apoptosis. In this study, we investigated the effect of Y48 modification in a double-knockout cell culture model that stably expressed either unphosphorylated wild-type (WT) Cytc, control Y48F Cytc, or phosphomimetic Y48E Cytc. Our findings revealed that Y48E Cytc caused partial inhibition of mitochondrial respiration in intact cells, which corresponded with lower mitochondrial membrane potentials (ΔΨ_m) and reduced reactive oxygen species (ROS) production. When subjected to an oxygen–glucose deprivation/reoxygenation (OGD/R) model, which simulates ischemia/reperfusion injury, the Y48E phosphomimetic cell line showed lower ROS production compared to the unphosphorylated WT and Y48F Cytc cell lines, the latter of which generated higher levels of ROS upon reoxygenation. As a result, the Y48E Cytc cell line had significantly lower cell death rates when exposed to OGD/R, confirming the cytoprotective role of Y48 phosphorylation of Cytc. In summary, our research indicates that the loss of Y48 phosphorylation in Cytc during ischemia leads to reperfusion injury by driving maximum electron transport chain flow, hyperpolarization of ΔΨ_m, bursts of ROS, and death of cells through apoptosis. Full article

Retinal ischemia–reperfusion (I/R) injury results in irreversible vision loss largely through retinal ganglion cell (RGC) death, with ferroptosis being a key mechanism. This study evaluated the therapeutic potential of the ferroptosis inhibitor Liproxstatin-1 (Lip-1) and deciphered its underlying mechanism. Using a mouse retinal I/R model and primary RGC cultures subjected to oxygen–glucose deprivation/reoxygenation (OGD/R), we demonstrated that Lip-1 effectively inhibits ferroptosis. Lip-1 treatment preserved retinal architecture (as assessed by H&E staining and SD-OCT) and partially restored visual function (as measured by electroretinography). Integrated molecular analyses—including immunofluorescence, Western blotting, and RNA sequencing—showed that Lip-1 downregulates early growth response 1 (EGR1), thereby inhibiting p53 and consequently restoring solute carrier family 7 member 11 (xCT) expression. Crucially, lentivirus-mediated EGR1 knockdown attenuated OGD/R-induced ferroptosis, confirming its pivotal role. Our work defines a coherent EGR1–p53–xCT signaling axis driving ferroptosis in retinal I/R injury and identifies Lip-1 as a neuroprotective agent targeting this pathway. These findings establish a druggable ferroptotic cascade and provide a mechanistic rationale for targeting EGR1 in the treatment of ischemic retinopathies. Full article

Background: Mitophagy is a critical mitochondrial quality control mechanism that limits neuronal injury following cerebral ischemia/reperfusion injury (CI/RI). Tetramethylpyrazine (TMP), a bioactive alkaloid from Ligusticum chuanxiong Hort., exhibits neuroprotective effects in cerebrovascular disorders. However, whether these effects involve mitophagy regulation remains unclear. Methods: CI/RI was induced using a middle cerebral artery occlusion/reperfusion (MCAO/R) model in mice and an oxygen–glucose deprivation/reoxygenation (OGD/R) model in HT22 cells. Neurological function, infarct volume, mitochondrial function, and mitophagy-related markers were assessed. Pharmacological inhibitors and genetic manipulation of YAP and Parkin were used to investigate underlying mechanisms. Results: TMP treatment significantly reduced infarct volume and improved neurological deficits in MCAO/R mice, accompanied by enhanced mitophagy, as indicated by increased mitochondrial LC3 recruitment and Parkin expression. In OGD/R-injured HT22 cells, TMP promoted mitophagosome and mitolysosome formation, reduced mitochondrial reactive oxygen species, and restored mitochondrial membrane potential. Inhibition of mitophagy with Mdivi-1 attenuated TMP-mediated neuroprotection. Mechanistically, TMP promoted YAP nuclear localization, and inhibition of YAP or silencing of Parkin abolished TMP-induced mitophagy, while Parkin overexpression restored mitophagy under YAP inhibition. Conclusions: TMP alleviates CI/RI by promoting mitophagy through the YAP/Parkin signaling pathway, suggesting mitophagy modulation as a potential therapeutic strategy for ischemic brain injury. Full article

Myocardial ischemia–reperfusion injury (MIRI) is a pathological process in which reperfusion-induced oxidative stress and metabolic derangement further aggravate myocardial damage and blunt the benefit of reperfusion. Ferroptosis is increasingly implicated in MIRI, with the glutathione (GSH)–glutathione peroxidase 4 (GPX4) axis constituting a key antioxidant barrier. Although GSH depletion is recognized as a critical event, its upstream regulation in MIRI remains unclear. Against this background, we investigate the BACH1–CHAC1–GSH pathway as a putative upstream regulatory axis of ferroptosis in MIRI and a potential molecular target. Here, using an oxygen–glucose deprivation/reoxygenation (OGD/R) model in AC16 and the reversibility conferred by the ferrostatin-1, RNA sequencing identified the GSH-degrading enzyme CHAC1 as a modulator that is induced by stress and promotes ferroptosis. Experiments showed that CHAC1 overexpression aggravated OGD/R-induced injury, depleted GSH, suppressed GPX4 and enhanced lipid peroxidation, whereas CHAC1 knockdown was partially protective. N-acetylcysteine (NAC) replenished GSH, restored GPX4 activity and partially rescued CHAC1-driven injury. In a mouse myocardial I/R model, cardiotropic adeno-associated virus-mediated CHAC1 overexpression worsened cardiac dysfunction, enlarged infarct and fibrosis areas, and increased myocardial iron deposition. Dual-luciferase assays revealed that the transcription factor BACH1 activates the CHAC1 promoter, and BACH1 silencing attenuated ferroptosis by suppressing CHAC1 and restoring the GSH–GPX4 axis. Collectively, our data identify the BACH1–CHAC1–GSH axis as an upstream amplifier of ferroptosis in MIRI through glutathione depletion and impairment of GPX4-dependent antioxidant defense. These findings refine the mechanistic link between reperfusion-phase redox imbalance and ferroptosis and highlight BACH1/CHAC1 inhibition or augmentation of GSH precursors as potential cardioprotective strategies in ischemic heart disease. Full article

As a natural flavonoid, the flavonoid luteolin is characterized by its powerful antioxidant and anti-inflammatory effects. While its precise mechanisms require further elucidation, existing evidence confirms its efficacy in ameliorating myocardial ischemia–reperfusion injury (MIRI). This research was designed to investigate the mechanism through which luteolin protects against MIRI. We established MIRI rat models through the ligation of left anterior descending coronary artery (LAD). To evaluate the cardioprotective effects of luteolin, echocardiographic analysis was performed, Hematoxylin and Eosin (HE) staining, and serum cardiac injury markers creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH). Cardiac vascular permeability was determined using Evans blue staining. To mimic ischemia–reperfusion injury, endothelial cells (ECs) were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) in vitro. Endothelial cell barrier function was evaluated through F-actin phalloidin staining and FITC-Dextran fluorescence leakage experiments. To elucidate the molecular mechanism, FOXP1 small interfering RNA (siRNA) and NLRP3 inhibitor MCC950 were administered. In MIRI rats, luteolin significantly improved cardiac function and preserved endothelial barrier integrity. These effects were associated with upregulation of FOXP1 and suppression of NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome. In OGD/R-treated endothelial cells, luteolin restored barrier function and cell viability. The protective effects of luteolin were abolished after FOXP1 silencing. Pharmacological NLRP3 inhibition (MCC950) mirrored luteolin’s protection. Our study indicates that luteolin enhances endothelial barrier function and attenuates MIRI via the FOXP1-NLRP3 pathway. The current study provides a potential drug for MIRI treatment. Full article