Search Results (503)

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

Background: Chronic exposure to artificial light at night (light pollution) causes circadian desynchronosis and melatonin deficiency, which is considered an independent driver of metabolic disorders and accelerated aging. However, the long-term effects of chronic desynchronosis on systemic metabolism and liver structure throughout the life cycle, as well as the potential of preventive melatonin administration, remain poorly understood. Objective: To evaluate the effects of chronic dark deprivation and prevention of metabolic disorders by exogenous melatonin on plasma melatonin levels, metabolic profile, liver function, and morphological changes in rats over a 24-month experiment. Methods: A 24-month experiment was conducted on 360 male Wistar rats divided into three groups: control (standard 10:14 h light/dark photoperiod), dark deprivation (DD, constant illumination), and correction (DD+Mel, constant illumination + melatonin 10 mg/kg five times per week). Animals were sacrificed at 6, 12, 18, and 24 months. Plasma melatonin was assessed by ELISA. Biochemical parameters (ALT, AST, LDH, total protein, albumin, bilirubin, glucose, triglycerides, and cholesterol), body weight, liver weight, relative liver weight, and histological parameters (steatosis, fibrosis, nuclear area, nuclear/cytoplasmic ratio, and binucleated hepatocytes) were analyzed. Results: In the DD group, a persistent progressive melatonin deficiency was detected (5.1-fold decrease by 6 months, p < 0.0005), accompanied by hypertriglyceridemia (Cohen’s d = 6.40), hypercholesterolemia (d = 4.59), biphasic dysglycemia (hypoglycemia followed by hyperglycemia), elevated ALT and AST activity (d = 2.60 and 2.46, respectively), hypoproteinemia (d = 5.33), hypoalbuminemia (d = 3.34), and hyperbilirubinemia (d = 3.22–4.37), as well as progressive steatosis (2.8 ± 0.3 points, d = 7.20) and pericellular fibrosis (1.8 ± 0.4 points, d = 4.50). In the DD group, a decrease in relative liver weight during the first 12 months was observed (metabolic disproportion, d = 2.31), reflecting disproportionate body weight gain. In the DD+Mel group, exogenous melatonin restored the biochemical parameters to values that did not differ statistically from the control values (Cohen’s d < 0.2 for most parameters), prevented steatosis (0.8 ± 0.3 points, d = 0.80) and fibrosis (0 points), increased relative liver weight by 24 months (3.83 ± 0.49 vs. 3.27 ± 0.029 in the control, d = 1.60), and increased the hepatocyte nuclear area (58.4 ± 4.1 vs. 48.6 ± 3.8 μm², d = 2.32). Conclusions: Chronic desynchronosis induced by constant illumination leads to persistent melatonin deficiency and complex metabolic and structural liver disturbances modeling accelerated aging. Exogenous melatonin (10 mg/kg five times per week) exhibits pronounced geroprotective, hepatoprotective, and antifibrotic effects, normalizing all biochemical parameters and preventing age-related liver involution. 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

Peptidylarginine deiminases (PADs) are a family of five isozymes (PAD1–4, PAD6) in humans, with PAD2, 3 and 4 associated with the central nervous system. PAD-mediated post-translational citrullination/deimination of target proteins contributes to pathobiological processes, including in the central nervous system (CNS), where the potential of PAD inhibitor treatment has been reported. This study aimed to identify PAD-dependent pro-regenerative responses in neuronal and astrocytic cells, respectively, using human cellular in vitro models to assess the therapeutic effects of pan-PAD, PAD2- and PAD4 isozyme-specific inhibitors in an oxygen–glucose deprivation/reperfusion model of ischaemia (OGD/R) at different time windows (30 min, 1 h and 4 h) in conjunction with scratch injury and LPS stimulation. Key findings suggest that pan-PAD inhibitor Cl-amidine promotes CNS regeneration through enhancing wound-healing of both neuronal and astrocytic cells, indicating roles for several PAD isozymes in acute CNS injury. Astrocyte cells showed the most prominent PAD4 detection, with significantly lower levels of PAD1, PAD2, PAD3 and PAD6, while differentiated SH-SY5Y neuronal cells showed the highest detection of PAD3, followed by PAD2 and PAD1, as well as strong PAD6 positivity, but negligible PAD4 detection. Histone H3 citrullination was significantly reduced in response to Cl-amidine treatment in both cell types, indicating changes in histone H3-dependent events in CNS injury. Cl-amidine treatment modulated key neuronal (beta-3 tubulin) and astrocytic (GFAP) markers and also reduced inflammatory cytokine IL-6 levels in astrocytes following 4 h OGD/R in conjunction with LPS stimulation. This study indicates roles for several PAD isozymes, with differing prominence in neurons and astrocytes, and emphasises the potential for pharmacological PAD inhibitor treatment in CNS injury. Full article

Kelch-like ECH-associated protein 1 (Keap1) acts as a repressor of nuclear factor-erythroid 2-related factor 2 (Nrf2), a major transcription factor regulating cellular antioxidant response. Keap1 is the substrate adaptor subunit of the cullin 3-RING E3 ubiquitin ligase complex that specifically facilitates Nrf2 ubiquitination and its proteasomal degradation. Keap1 is rich in cysteine residues, and several of them undergo various modifications, such as sulphydration, nitrosylation and glutathionylation under cellular stress conditions. Some of these modifications alter the conformation of Keap1, preventing Nrf2 from ubiquitination and subsequent proteasome-mediated degradation. As a result, newly synthesised Nrf2 translocates to the nucleus to induce the expression of diverse genes involved in protecting cells against oxidative stress. Protein CoAlation is a reversible redox-dependent post-translational modification (PTM) in which coenzyme A (CoA) forms disulphide bonds with oxidised cysteine residues under oxidative or metabolic stress. In this study, we demonstrate for the first time that disulphide Keap1 dimer undergoes CoAlation in cellular response to oxidative stress induced by various oxidising compounds. Furthermore, glucose deprivation also induces CoAlation of the disulphide Keap1 dimer in HEK293/Pank1β cells. We also demonstrate that the Keap1^{11 Cys-less} mutant is not CoAlated in response to diamide treatment or glucose deprivation. In summary, this study uncovers a novel PTM of Keap1 by the key metabolic integrator CoA, which provides new insights into the regulation of the Keap1-Nrf2 antioxidant pathway under oxidative and metabolic stress. 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

N-acetylneuraminate pyruvate lyase (NPL) is a key enzyme in sialic acid catabolism that links sialylation to cellular metabolism, but its role in cancer cell metabolic adaptation is poorly defined. In particular, it remains unclear whether NPL contributes to ATP maintenance in hepatocellular carcinoma (HCC) under nutrient stress through its role in sialic acid catabolism. HCC is a highly lethal malignancy characterized by extensive metabolic reprogramming. Here, we investigated whether NPL links sialic acid catabolism to ATP maintenance in HCC cells under glucose deprivation. Glucose deprivation induced NPL expression in Huh7 and PLC/PRF/5 cells, which is consistent with an adaptive response to energetic stress. Stable NPL knockdown reduced intracellular ATP levels. Consistently, cell growth was significantly reduced, as assessed by Cell Counting Kit-8 (CCK-8) and colony formation assays. These effects were more pronounced in the absence of glucose. Exogenous pyruvate partially restored ATP levels and the growth inhibition caused by NPL knockdown, particularly in the absence of glucose. This rescue further suggests that NPL may support ATP maintenance under glucose deprivation partly through pyruvate metabolism. Together, these findings indicate that NPL contributes to maintaining intracellular ATP levels during glucose deprivation, thereby supporting HCC cell adaptation to metabolic stress. To extend these biological findings to potential therapeutic exploration, we performed virtual screening and molecular docking using a U.S. Food and Drug Administration (FDA)-approved drug library. Candidate compounds predicted to bind NPL were identified, providing a basis for further validation and optimization. Full article

Impairment in blood supply to the brain deprives its cells of the much-needed nutrients and molecules such as oxygen and glucose necessary for its development, growth and survival. This will set up a host of pathological processes such as impaired homeostasis, energy failure, excitotoxicity, oxidative stress, impaired protein synthesis, inflammation, cytokine-mediated toxicity and impairment of blood–brain barrier. These pathological processes will result in the damage or death of the cells depending on the extent of the deprivation. Similarly, they will impair synthesis of acetylcholine (Ach) and norepinephrine (NE), which are important neurotransmitters in the cholinergic and adrenergic systems responsible for cellular communication and functions. Thus, interventions to help arrest and/or modulate the initial and subsequent pathological states and help recover the functions of the brain are needed. One of such interventions is vagus nerve stimulation, which helps activate the cholinergic and the adrenergic systems via projections of the afferent fibers of the vagus nerve to the nucleus of the solitary tract (NTS). Activation of the cholinergic and the adrenergic systems results in reduction in pro-inflammatory factors such as tumor necrosis α, increase in pro-angiogenic factors and increase in firing of adrenergic neurons in the central nervous system (CNS). Full article

Background: Prostate cancer (PCa) is the most commonly diagnosed malignancy in men and a leading cause of cancer-related mortality worldwide. Growing evidence indicates that metabolic syndrome components, including obesity, insulin resistance, and hyperglycemia, contribute to PCa development, and progression to more aggressive form. At the same time, standard treatments such as androgen deprivation therapy (ADT) and androgen receptor pathway inhibitors (ARPIs) significantly improve oncologic outcomes but are associated with adverse metabolic effects, including increased fat mass, insulin resistance, and sarcopenia, potentially worsening patients’ overall metabolic profile and quality of life. Tumor progression in PCa is strongly driven by androgen receptor (AR) signaling, which is closely linked to cellular metabolic reprogramming, highlighting metabolism as a potential therapeutic target. Aim: The aim of this study was to evaluate and synthesize current evidence on the role of the ketogenic diet (KD) in PCa, with particular emphasis on its interaction with hormonal therapies, underlying metabolic and endocrine mechanisms, and its potential application as an adjunctive strategy in integrated oncologic care. Results: The KD, characterized by high fat and very low carbohydrate intake, induces a metabolic state of ketosis that reduces circulating glucose, insulin, and insulin-like growth factor 1 (IGF-1), potentially counteracting metabolic alterations associated with PCa and its treatments. Preclinical studies consistently demonstrate that carbohydrate restriction and KD can slow tumor growth, modulate key oncogenic pathways such as PI3K/AKT/mTOR, reduce systemic insulin signaling, and enhance survival in prostate cancer models. Additionally, emerging evidence suggests possible synergistic effects when KD is combined with standard therapies, including ADT and immunotherapy. Clinical data, although limited, indicate that low-carbohydrate dietary interventions may improve metabolic parameters and could delay biochemical progression, as suggested by increased prostate-specific antigen (PSA) doubling time. However, results across studies remain heterogeneous, and robust evidence on long-term oncologic outcomes is lacking. Conclusions: Overall, the KD represents a promising but still experimental strategy in PCa management, requiring careful nutritional supervision to avoid adverse effects such as unintended weight loss or sarcopenia. Further well-designed randomized clinical trials are needed to clarify its safety, efficacy, and role in routine clinical practice. Full article

Pterostilbene (Pts), a small molecule stilbenoid and a dimethyl analogue of the star molecule resveratrol, exerts significant blood–brain barrier protection on cerebral ischemia-reperfusion injury and has received extensive attention. This study performed structural optimizations on Pts to obtain a series of derivatives and investigated their anti-ischemic activities both in vitro and in vivo, aiming to identify candidates with high safety and improved efficacy compared with Pts. The ADMET method was used to predict the drug-likeness of a series of Pts derivatives, and in vitro MTT cell viability analysis was conducted on neuroblastoma cells (SH-SY5Y) and brain microvascular endothelial cells (BMECs) after oxygen-glucose deprivation/reperfusion (OGD/R) injury. On the basis of the cytotoxicity results, four derivatives (NO. 1, NO. 3, NO. 5, and NO. 7) were selected for subsequent in vitro and in vivo biological activities evaluation. These compounds exhibited significantly higher TI values (18.29–30.61) in OGD/R-injured hBMECs compared with Pts (7.63) and effectively suppressed apoptosis, promoted cell migration, and enhanced tube formation capacity. In vivo, NO. 3 (5 mg/kg, ip., 7 d) demonstrated superior efficacy compared to Pts in improving cerebral blood flow, reducing infarction volume, enhancing neurological function, and modulating serum biomarker levels in middle cerebral artery occlusion/reperfusion (MCAO/R) rats, whereas NO. 1 and NO. 7 showed comparable efficacy to Pts. The acute intraperitoneal toxicity of NO. 3 was conducted and showed that the LD₅₀ of NO. 3 was estimated to be more than 300 mg/kg. In this study, the rational design and comprehensive evaluation of Pts derivatives were reported. Compound NO. 3 demonstrated superior pharmacological efficacy to Pts both in vitro and in vivo, and it may be a promising therapeutic candidate for ischemic stroke intervention. 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

Microfluidic systems are increasingly used in neuroprotection research, but their clearest value may be to show why candidate compounds fail before costly downstream models. This critical framework review examines CNS-relevant microfluidic studies through a within-program triage logic linking chemistry-aware prescreening, blood-brain barrier/neurovascular unit (BBB/NVU) filtering, and timed validation in neuronal ischemia/reperfusion models, and treats non-CNS organ-on-a-chip and analytical microfluidic studies as engineering analogies only. The available evidence most strongly supports BBB/NVU chips as exposure- and safety-aware filters and compartmentalized neuronal oxygen-glucose deprivation platforms as timing-sensitive validation tools; droplet microfluidics contributes mainly upstream through dense dose mapping, aggregation assays and counterscreens for assay interference. A compound-centered reading also suggests that apparent activity often fails for distinct reasons, including timing mismatch, poor solubility, surface adsorption, optical artifact, inadequate multicellular context, or loss of efficacy under transport-aware testing. Taken together, the literature supports a cautious, within-program triage logic in which microfluidics is used not as a universal disease model, but as an operational framework for exposing transport, barrier, timing and assay liabilities early in neuroprotective discovery. 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