Search

Mitochondrial dysfunction is a key event in many pathological conditions, including neurodegenerative processes. When mitochondria are damaged, they release damage‐associated molecular patterns (DAMPs) that activate mito‐inflammation. The present study assessed mito‐inflammation after in vitro oxygen–glucose deprivation as a representation of ischaemia, followed by reoxygenation (OGD/R) of HT22 cells and modulation of the inflammatory response by melatonin. We observed that melatonin prevented mitochondrial structural damage and dysfunction caused by OGD/R. Melatonin reduced oxidative damage and preserved the enzymatic activity for complexes I, III and IV, encoded by mitochondrial DNA, which were reduced by OGD/R. No effect was observed on complex II activity encoded by nuclear DNA. The release of mtDNA into the cytosol was also prevented with a consequent reduction of the cGAS–STING pathway and IFNβ and IL‐6 production. Interestingly, melatonin also increased the early release of the fibroblast growth factor‐21 (FGF‐21), a mitokine secreted in response to mitochondrial stress. These data indicate that melatonin reduces mito‐inflammation and modulates FGF‐21 release, further highlighting the key role of this molecule in preserving mitochondrial integrity in OGD/R deprivation‐type ischaemic brain injury. [ABSTRACT FROM AUTHOR]

Cofilactin rods (CARs), which are 1:1 aggregates of cofilin-1 and actin, lead to neurite loss in ischemic stroke and other disorders. The biochemical pathways driving CAR formation are well-established, but how these pathways are engaged under ischemic conditions is less clear. Brain ischemia produces both ATP depletion and glutamate excitotoxicity, both of which have been shown to drive CAR formation in other settings. Here, we show that CARs are formed in cultured neurons exposed to ischemia-like conditions: oxygen-glucose deprivation (OGD), glutamate, or oxidative stress. Of these conditions, only OGD produced significant ATP depletion, showing that ATP depletion is not required for CAR formation. Moreover, the OGD-induced CAR formation was blocked by the glutamate receptor antagonists MK-801 and kynurenic acid; the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitors GSK2795039 and apocynin; as well as an ROS scavenger. The findings identify a biochemical pathway leading from OGD to CAR formation in which the glutamate release induced by energy failure leads to activation of neuronal glutamate receptors, which in turn activates NADPH oxidase to generate oxidative stress and CARs.

Naringin (NAR), a flavanone glycoside, occurs widely in citrus fruits, vegetables, and alcoholic beverages. Despite evidence of the neuroprotective effects of NAR on animal models of ischemic stroke, brain cell-type-specific data about the antioxidant efficacy of NAR and possible protein targets of such beneficial effects are limited. Here, we demonstrate the brain cell type-specific prophylactic role of NAR, an FDA-listed food additive, in an in vitro oxygen-glucose deprivation (OGD) model of cerebral ischemia using MTT and DCFDA assays. Using Bayes' theorem-based predictive model, we first ranked the top-10 protein targets (ALDH2, ACAT1, CTSB, FASN, LDHA, PTGS1, CTSD, LGALS1, TARDBP, and CDK1) from a curated list of 289 NAR-interacting proteins in neurons that might be mediating its antioxidant effect in the OGD model. When preincubated with NAR for 2 days, N2a and CTX-TNA2 cells could withstand up to 8 h of OGD without a noticeable decrease in cell viability. This cerebroprotective effect is partly mediated by reducing intracellular ROS production in the above two brain cell types. The antioxidant effect of NAR was comparable with the equimolar (50 µM) concentration of clinically used ROS-scavenger and neuroprotective edaravone. Molecular docking of NAR with the top-10 protein targets from Bayes' analysis showed the lowest binding energy for CDK1 (− 8.8 kcal/M). Molecular dynamics simulation analysis showed that NAR acts by inhibiting CDK1 by stably occupying its ATP-binding cavity. Considering diet has been listed as a risk factor for stroke, NAR may be explored as a component of functional food for stroke or related neurological disorders. [ABSTRACT FROM AUTHOR]

Abstract Background Neurovascular deficits and blood-brain barrier (BBB) dysfunction are major hallmarks of brain trauma and neurodegenerative diseases. Oxidative stress is a prominent contributor to neurovascular unit (NVU) dysfunction and can propagate BBB disruption. Oxidative damage results in an imbalance of mitochondrial homeostasis, which can further drive functional impairment of brain capillaries. To this end, we developed a method to track mitochondrial-related changes after oxidative stress in the context of neurovascular pathophysiology as a critical endophenotype of neurodegenerative diseases. Methods To study brain capillary-specific mitochondrial function and dynamics in response to oxidative stress, we developed an ex vivo model in which we used isolated brain capillaries from transgenic mice that express dendra2 green specifically in mitochondria (mtD2g). Isolated brain capillaries were incubated with 2,2’-azobis-2-methyl-propanimidamide dihydrochloride (AAPH) or hydrogen peroxide (H2O2) to induce oxidative stress through lipid peroxidation. Following the oxidative insult, mitochondrial bioenergetics were measured using the Seahorse XFe96 flux analyzer, and mitochondrial dynamics were measured using confocal microscopy with Imaris software. Results We optimized brain capillary isolation with intact endothelial cell tight-junction and pericyte integrity. Further, we demonstrate consistency of the capillary isolation process and cellular enrichment of the isolated capillaries. Mitochondrial bioenergetics and morphology assessments were optimized in isolated brain capillaries. Finally, we found that oxidative stress significantly decreased mitochondrial respiration and altered mitochondrial morphology in brain capillaries, including mitochondrial volume and count. Conclusions Following ex vivo isolation of brain capillaries, we confirmed the stability of mitochondrial parameters, demonstrating the feasibility of this newly developed platform. We also demonstrated that oxidative stress has profound effects on mitochondrial homeostasis in isolated brain capillaries. This novel method can be used to evaluate pharmacological interventions to target oxidative stress or mitochondrial dysfunction in cerebral small vessel disease and neurovascular pathophysiology as major players in neurodegenerative disease.

Abstract Investigating blood-brain barrier (BBB) dysfunction has become a pre-clinical and clinical research focus as it accompanies many neurological disorders. Nevertheless, knowledge of how diagnostic BBB tracers cross the endothelium from blood-to-brain or vice versa often remains incomplete. In particular, brain-to-blood transport (efflux) may reduce tracer extravasation of intravascularly (i.v.) applied tracers. Conversely, impaired efflux could mimic phenotypic extravasation. Both processes would affect conclusions on BBB properties primarily attributed to blood-to-brain leakage. Here, we specifically investigated efflux of fluorescent BBB tracers, focusing on the most common non-toxic marker, sodium fluorescein, which is applicable in patients. We used acute neocortical slices from mice and applied fluorescein, sulforhodamine-B, rhodamine-123, FITC dextran to the artificial cerebrospinal fluid. Anionic low molecular weight (MW) fluorescein and sulforhodamine-B, but not ~ 10-fold larger FITC-dextran and cationic low MW rhodamine-123, showed efflux into the lumen of blood vessels. Our data suggest that fluorescein efflux depends on organic anion transporter polypeptides (Oatp) rather than P-glycoprotein. Furthermore, sodium-potassium ATPase inhibition and incomplete oxygen-glucose deprivation (OGD, 20% O2) reduced fluorescein efflux, while complete OGD (0% O2) abolished efflux. We provide evidence for active efflux of fluorescein in vitro. Impaired efflux of fluorescein could thus contribute to the frequently observed BBB dysfunction in neuropathologies in addition to blood-to-brain leakage.

[Display omitted] • HIF-1α eases OGD/R −induced ferroptosis. • HIF-1α enhances the transcriptional activity of transferrin FPN1. • FPN1 knockdown annuls HIF-1α-induced alleviation on OGD/R-induced ferroptosis. • HIF-1α boosts FPN1 to activate Nrf2/HO-1 and ease OGD/R-induced ferroptosis. • HIF-1α boosts FPN1 to activate Nrf2/HO-1 and ease cerebral I/R injury in rats. Cerebral ischemic/reperfusion (I/R) injury has high disability and morbidity. Hypoxia-inducible factor-1α (HIF-1α) may enhance the transcriptional activity of transferrin ferroportin 1 (FPN1) in regulating ferroptosis after cerebral ischemia injury (CII). In this study, cerebral I/R injury rat models were established and treated with pcDNA3.1-HIF-1α, pcDNA3.1-NC lentiviral plasmid, or ML385 (a specific Nrf2 inhibitor). Additionally, oxygen-glucose deprivation/reoxygenation (OGD/R) exposed PC12 cells were used as an in vitro model of cerebral ischemia and treated with pcDNA3.1-HIF-1α, si-FPN1, or ML385. The results elicited that cerebral I/R injury rats exhibited increased Longa scores, TUNEL and NeuN co-positive cells, Fe2+ concentration, ROS and HIF-1α levels, and MDA content, while reduced cell density and number, GSH content, and GPX4 protein level. Morphologically abnormal and disordered hippocampal neurons were also observed in CII rats. HIF-1α inhibited brain neuron ferroptosis and ameliorated I/R injury. HIF-1α alleviated OGD-induced PC12 cell ferroptosis. OGD/R decreased FPN1 protein level in PC12 cells, and HIF-1α enhanced FPN1 transcriptional activity. FPN1 knockdown reversed HIF-1α-mediated alleviation of OGD/R-induced ferroptosis. HIF-1α activated the Nrf2/HO-1 pathway by enhancing FPN1 expression and alleviating OGD/R-induced ferroptosis. Conjointly, HIF-1α enhanced the transcriptional activity of FPN1, activated the Nrf2/HO-1 pathway, and inhibited ferroptosis of brain neurons, thereby improving I/R injury in CII rats. [ABSTRACT FROM AUTHOR]

Objective: Previously, we have successfully purified and synthesized viscolin, an agent derived from Viscum coloratum extract, which has shown significant potential in the treatment of stroke. Our study aimed to evaluate the neuroprotective effects of viscolin. Methods: We first assessed the cytotoxicity of viscolin on primary neuronal cultures and determined its antioxidant and radical scavenging properties. Subsequently, we identified the optimal dose-response of viscolin in protecting against glutamate-induced neurotoxicity. Results: Our results demonstrated that viscolin at a concentration of 10 μM effectively reduced neuronal cell death up to 6 hours after glutamate-induced neurotoxicity. Additionally, we investigated the therapeutic window of opportunity and the potential of viscolin in preventing necrotic and apoptotic damage in cultured neurons exposed to oxygen glucose deprivation-induced neurotoxicity. Our findings showed that viscolin treatment significantly reduced DNA breakage, prevented the release of cytochrome c from mitochondria to cytosol, increased the expression of anti-apoptotic protein Bcl-2, decreased the expression of pro-apoptotic protein Bax, and reduced the number of TUNEL-positive cells. Additionally, our in vivo investigation demonstrated a reduction in brain infarction following middle cerebral artery occlusion. Conclusion: Viscolin has potential utility as a therapeutic agent in the treatment of stroke. [ABSTRACT FROM AUTHOR]

Background: Neurovascular deficits and blood-brain barrier (BBB) dysfunction are major hallmarks of brain trauma and neurodegenerative diseases. Oxidative stress is a prominent contributor to neurovascular unit (NVU) dysfunction and can propagate BBB disruption. Oxidative damage results in an imbalance of mitochondrial homeostasis, which can further drive functional impairment of brain capillaries. To this end, we developed a method to track mitochondrial-related changes after oxidative stress in the context of neurovascular pathophysiology as a critical endophenotype of neurodegenerative diseases. Methods: To study brain capillary-specific mitochondrial function and dynamics in response to oxidative stress, we developed an ex vivo model in which we used isolated brain capillaries from transgenic mice that express dendra2 green specifically in mitochondria (mtD2g). Isolated brain capillaries were incubated with 2,2'-azobis-2-methyl-propanimidamide dihydrochloride (AAPH) or hydrogen peroxide (H2O2) to induce oxidative stress through lipid peroxidation. Following the oxidative insult, mitochondrial bioenergetics were measured using the Seahorse XFe96 flux analyzer, and mitochondrial dynamics were measured using confocal microscopy with Imaris software. Results: We optimized brain capillary isolation with intact endothelial cell tight-junction and pericyte integrity. Further, we demonstrate consistency of the capillary isolation process and cellular enrichment of the isolated capillaries. Mitochondrial bioenergetics and morphology assessments were optimized in isolated brain capillaries. Finally, we found that oxidative stress significantly decreased mitochondrial respiration and altered mitochondrial morphology in brain capillaries, including mitochondrial volume and count. Conclusions: Following ex vivo isolation of brain capillaries, we confirmed the stability of mitochondrial parameters, demonstrating the feasibility of this newly developed platform. We also demonstrated that oxidative stress has profound effects on mitochondrial homeostasis in isolated brain capillaries. This novel method can be used to evaluate pharmacological interventions to target oxidative stress or mitochondrial dysfunction in cerebral small vessel disease and neurovascular pathophysiology as major players in neurodegenerative disease. [ABSTRACT FROM AUTHOR]

The non-coding RNA LINC00894 modulates tumor proliferation and drug resistance. However, its role in brain is still unclear. Using RNA-pull down combined with mass spectrometry and RNA binding protein immunoprecipitation, EIF5 was identified to interact with LINC00894. Furthermore, LINC00894 knockdown decreased EIF5 protein expression, whereas LINC00894 overexpression increased EIF5 protein expression in SH-SY5Y and BE(2)-M17 (M17) neuroblastoma cells. Additionally, LINC00894 affected the ubiquitination modification of EIF5. Adeno-associated virus (AAV) mediated LINC00894 overexpression in the brain inhibited the expression of activated Caspase-3, while increased EIF5 protein level in rats and mice subjected to transient middle cerebral artery occlusion reperfusion (MCAO/R). Meanwhile, LINC00894 knockdown increased the number of apoptotic cells and expression of activated Caspase-3, and its overexpression decreased them in the oxygen–glucose deprivation and reoxygenation (OGD/R) in vitro models. Further, LINC00894 was revealed to regulated ATF4 protein expression in condition of OGD/R and normoxia. LINC00894 knockdown also decreased the expression of glutamate-cysteine ligase catalytic subunit (GCLC) and ATF4, downregulated glutathione (GSH), and the ratio of GSH to oxidized GSH (GSH: GSSG) in vitro. By using RNA-seq combined with qRT-PCR and immunoblot, we identified that fibroblast growth factor 21 (FGF21) and aconitate decarboxylase 1 (ACOD1), as the ATF4 target genes were regulated by LINC00894 in the MCAO/R model. Finally, we revealed that ATF4 transcriptionally regulated FGF21 and ACOD1 expression; ectopic overexpression of FGF21 or ACOD1 in LINC00894 knockdown cells decreased activated Caspase-3 expression in the OGD/R model. Our results demonstrated that LINC00894 regulated cerebral ischemia injury by stabilizing EIF5 and facilitating EIF5-ATF4-dependent induction of FGF21 and ACOD1. [ABSTRACT FROM AUTHOR]

Background: Ischaemic stroke can lead to many complications, but treatment options are limited. Icariin is a traditional Chinese medicine with reported neuroprotective effects against ischaemic cerebral injury; however, the underlying mechanisms by which icariin ameliorates cell apoptosis require further study. Purpose: This study aimed to investigate the therapeutic potential of icariin after ischaemic stroke and the underlying molecular mechanisms. Methods: N2a neuronal cells were used to create an in vitro oxygen-glucose deprivation (OGD) model. The effects of icariin on OGD cells were assessed using the CCK-8 kit to detect the survival of cells and based on the concentration, apoptosis markers, inflammation markers, and M2 pyruvate kinase isoenzyme (PKM2) expression were detected using western blotting, RT-qPCR, and flow cytometry. To investigate the underlying molecular mechanisms, we used the PKM2 agonist TEPP-46 and detected apoptosis-related proteins. Results: We demonstrated that icariin alleviated OGD-induced apoptosis in vitro. The expression levels of the apoptosis marker proteins caspase-3 and Bax were upregulated and Bcl-2 was downregulated. Furthermore, icariin reduced inflammation and downregulated the expression of PKM2. Moreover, activation of the PKM2 by pretreatment with the PKM2 agonist TEPP-46 enhanced the effects on OGD induced cell apoptosis in vitro. Conclusion: This study elucidated the underlying mechanism of PKM2 in OGD-induced cell apoptosis and highlighted the potential of icariin in the treatment of ischaemic stroke.

Perinatal asphyxia (PA) and hypoxic-ischemic encephalopathy can result in severe, long-lasting neurological deficits. In vitro models, such as oxygen–glucose deprivation (OGD), are used experimentally to investigate neuronal response to metabolic stress. However, multiple variables can affect the severity level of OGD/PA and may confound any measured treatment effect. Oxytocin (OXT) has emerged as a potential neuroprotective agent against the deleterious effects of PA. Previous studies have demonstrated OXT’s potential to enhance neuronal survival in immature hippocampal cultures exposed to OGD, possibly by modulating gamma-aminobutyric acid-A receptor activity. Moreover, OXT’s precise impact on developing hippocampal neurons under different severities of OGD/PA remains uncertain. In this study, we investigated the effects of OXT (0.1 µM and 1 µM) on 7-day-old primary rat hippocampal cultures subjected to 2 h OGD/sham normoxic conditions. Cell culture viability was determined using the resazurin assay. Our results indicate that the efficacy of 1 µM OXT treatment varied according to the severity of the OGD-induced lesion, exhibiting a protective effect (p = 0.022) only when cellular viability dropped below 49.41% in non-treated OGD cultures compared to normoxic ones. Furthermore, administration of 0.1 µM OXT did not yield significant effects, irrespective of lesion severity (p > 0.05). These findings suggest that 1 µM OXT treatment during OGD confers neuroprotection exclusively in severe lesions in hippocampal neurons after 7 days in vitro. Further research is warranted to elucidate the mechanisms involved in OXT-mediated neuroprotection.

Blood–brain barrier (BBB) disruption is a major pathophysiological event of ischemic stroke. Brain microvascular endothelial cells are critical to maintain homeostasis between central nervous system and periphery. Resveratrol protects against ischemic stroke. 3,3′,4,5′‐tetramethoxy‐trans‐stilbene (3,3′,4,5′‐TMS) and 3,4′,5‐trimethoxy‐trans‐stilbene (3,4′,5‐TMS) are resveratrol derivatives with addition of methoxy groups, showing better pharmacokinetic performance. We aimed to explore their protective effects and underlying mechanisms. Oxygen–glucose deprivation (OGD) model was applied in bEnd.3 cell line, mouse brain microvascular endothelium to mimic ischemia. The cells were pre‐treated with 3,3′,4,5′‐TMS or 3,4′,5‐TMS (1 and 5 μM, 24 h) and then subjected to 2‐h OGD injury. Cell viability, levels of proinflammatory cytokines and reactive oxygen species (ROS), and protein expressions were measured by molecular assays and fluorescence staining. OGD injury triggered cell death, inflammatory responses, ROS production and nuclear factor‐kappa B (NF‐κB) signalling pathway. These impairments were remarkably attenuated by the two stilbenes, 3,3′,4,5′‐TMS and 3,4′,5‐TMS. They also alleviated endothelial barrier injuries through upregulating the expression of tight junction proteins. Moreover, 3,3′,4,5′‐TMS and 3,4′,5‐TMS activated 5′ adenosine monophosphate‐activated protein kinase (AMPK) and endothelial nitric oxide synthase (eNOS). Overall, 3,3′,4,5′‐TMS and 3,4′,5‐TMS exert protective effects against OGD damage through suppressing cell death, inflammatory responses, oxidative stress, as well as BBB disruption on bEnd.3 cells. [ABSTRACT FROM AUTHOR]

Background: Exogenous neural cell transplantation may be therapeutic for stroke, cerebral ischemic injury. Among other mechanisms, increasing findings indicated circular RNAs (circRNAs) regulate the pathogenesis progression of cerebral ischemia. Mmu_circ_0015034 (circEfnb2) was upregulated in focal cortical infarction established by middle cerebral artery occlusion (MCAO) in mice. Our study was designed to probe the molecular mechanism of circEfnb2 in the oxygen-glucose deprivation/reperfusion (OGD/R)-induced neuronal damage in cerebral ischemia. Methods: We established an in vitro OGD/R cell model. CircEfnb2 and microRNA-202-5p (miR-202-5p) levels were detected using real-time quantitative polymerase chain reaction (RT-qPCR). Lactate dehydrogenase (LDH), malondialdehyde (MDA), and reactive oxygen species (ROS) levels were assessed using specific kits. Tumor necrosis factor-« (TNF-a) and Interleukin-lß (IL-lß) levels were examined using an Enzyme-linked immunosorbent assay (ELISA). Flow cytometry analysis evaluated cell apoptosis. Protein levels of B-cell lymphoma-2 (Bcl-2), Bcl-2 related X protein (Bax), cleaved caspase 3, and Tumor necrosis factor receptor-associated factor 3 (TRAF3) were determined using Western blot assay. Results: Overall, circEfnb2 was highly expressed whereas miR-202-5p was decreased in OGD/R-treated mouse hippocampal neuronal HT22 cells compared to normal controls (both p > 0.05). From an in vitro functional perspective, circEfnb2 knockdown attenuated an OGD/R-triggered neuronal injury compared to controls (p > 0.05). Mechanically, circEfnb2 acted as a sponge of miR-202-5p; downregulation of miR-202-5p annulled the inhibitory roles of circEfnb2 silencing in an OGD/R-caused neuronal injury model. Our analysis showed that miR-202-5p directly targeted TRAF3 as enhanced TRAF3 abolished the effects of miR-202-5p in the OGD/Rinduced neuronal injury. In vivo, lentivirus with a short hairpin (sh)-circEfnb2 inhibited cerebral injury, when injected into cerebral cortex in MCAO mice (p > 0.05). Conclusion: Our results suggest that circEfnb2 deficiency may decrease OGD/R-induced HT22 cell damage by modulating the miR-202-5p/TRAF3 axis. This explanation may provide a new direction for cerebral infarction potential therapeutic targets. [ABSTRACT FROM AUTHOR]

Thrombolytic therapy with the tissue plasminogen activator (tPA) is a therapeutic option for acute ischemic stroke. However, this approach is subject to several limitations, particularly the increased risk of hemorrhagic transformation (HT). Lithium salts show neuroprotective effects in stroke, but their effects on HT mechanisms are still unknown. In our study, we use the models of photothrombosis (PT)-induced brain ischemia and oxygen-glucose deprivation (OGD) to investigate the effect of Li+ on tPA-induced changes in brain and endothelial cell cultures. We found that tPA did not affect lesion volume or exacerbate neurological deficits but disrupted the blood–brain barrier. We demonstrate that poststroke treatment with Li+ improves neurological status and increases blood–brain barrier integrity after thrombolytic therapy. Under conditions of OGD, tPA treatment increased MMP-2/9 levels in endothelial cells, and preincubation with LiCl abolished this MMP activation. Moreover, we observed the effect of Li+ on glycolysis in tPA-treated endothelial cells, which we hypothesized to have an effect on MMP expression. [ABSTRACT FROM AUTHOR]

Neonatal hypoxia-ischemia (HI) affects 2–3 per 1000 live births in developed countries and up to 26 per 1000 live births in developing countries. It is estimated that of the 750,000 infants experiencing a hypoxic-ischemic event during birth per year, more than 400,000 will be severely affected. As treatment options are limited, rapidly identifying new therapeutic avenues is critical, and repurposing drugs already in clinical use offers a fast-track route to clinic. One emerging avenue for therapeutic intervention in neonatal HI is to target mitochondrial dysfunction, which occurs early in the development of brain injury. Mitochondrial dynamics are particularly affected, with mitochondrial fragmentation occurring at the expense of the pro-fusion protein Optic Atrophy (OPA)1. OPA1, together with mitofusins (MFN)1/2, are required for membrane fusion, and therefore, protecting their function may also safeguard mitochondrial dynamics. Leflunomide, an FDA-approved immunosuppressant, was recently identified as an activator of MFN2 with partial effects on OPA1 expression. We, therefore, treated C17.2 cells with Leflunomide before or after oxygen-glucose deprivation, an in vitro mimic of HI, to determine its efficacy as a neuroprotection and inhibitor of mitochondrial dysfunction. Leflunomide increased baseline OPA1 but not MFN2 expression in C17.2 cells. However, Leflunomide was unable to promote cell survival following OGD. Equally, there was no obvious effect on mitochondrial morphology or bioenergetics. These data align with studies suggesting that the tissue and mitochondrial protein profile of the target cell/tissue are critical for taking advantage of the therapeutic actions of Leflunomide. [ABSTRACT FROM AUTHOR]

Luteolin-7-O-β-d-glucuronide (LGU) is a major active flavonoid glycoside compound that is extracted from Ixeris sonchifolia (Bge.) Hance, and it is a Chinese medicinal herb mainly used for the treatment of coronary heart disease, angina pectoris, cerebral infarction, etc. In the present study, the neuroprotective effect of LGU was investigated in an oxygen glucose deprivation (OGD) model and a middle cerebral artery occlusion (MCAO) rat model. In vitro, LGU was found to effectively improve the OGD-induced decrease in neuronal viability and increase in neuronal death by a 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and a lactate dehydrogenase (LDH) leakage rate assay, respectively. LGU was also found to inhibit OGD-induced intracellular Ca2+ overload, adenosine triphosphate (ATP) depletion, and mitochondrial membrane potential (MMP) decrease. By Western blotting analysis, LGU significantly inhibited the OGD-induced increase in expressions of receptor-interacting serine/threonine-protein kinase 3 (RIP3) and mixed lineage kinase domain-like protein (MLKL). Moreover, molecular docking analysis showed that LGU might bind to RIP3 more stably and firmly than the RIP3 inhibitor GSK872. Immunofluorescence combined with confocal laser analyses disclosed that LGU inhibited the aggregation of MLKL to the nucleus. Our results suggest that LGU ameliorates OGD-induced rat primary cortical neuronal injury via the regulation of the RIP3/MLKL signaling pathway in vitro. In vivo, LGU was proven, for the first time, to protect the cerebral ischemia in a rat middle cerebral artery occlusion (MCAO) model, as shown by improved neurological deficit scores, infarction volume rate, and brain water content rate. The present study provides new insights into the therapeutic potential of LGU in cerebral ischemia. [ABSTRACT FROM AUTHOR]

Stroke is the second leading cause of death worldwide. Understanding of gene expression dynamics could bring new approaches in diagnostics and therapy of stroke. Small noncoding molecules termed 'microRNA' represent the most flexible network of gene expression regulators. To screen out miRNAs that are mainly regulated during reperfusion in mechanically embolized patients, and study their mechanisms of action in reperfusion injury after thrombectomy, in order to find new therapeutic targets for mechanically embolized patients. Serums from 30 patients with moderate to severe stroke after mechanical thrombectomy (MT) were collected to measure miRNA expressions. Clinical information of patients was analyze, and patients were divided into poor prognosis and good prognosis. Factors affecting prognosis was classified, and independent risk factors for poor prognosis were determined. Prognostic value of National Institutes of Health Stroke Scale (NIHSS) score on admission to patients with MT was assessed. ROC (receiver operating characteristic) curves were drawn, and Kaplan-Merier method determined whether different NIHSS scores at admission had any difference in the in-hospital survival rate of consistency index/random consistency index (CI/RI) patients treated with MT. An oxygen-glucose deprivation/reperfusion (OGD/R) cell model and an middle cerebral artery occlusion (MCAO)/reperfusion mouse model were established, in which miR-298 expression was tested. In OGD/R cells, proliferation, apoptosis, and autophagy were assessed after intervention with miR-298 and/or autophagy related gene 5 (ATG5). In MCAO mice, the infarct area was calculated, and neurological function was assessed. The relationship between miR-298 and ATG5 was explored and validated. Age, diabetes, hypertension, hemorrhage transformation, NIHSS score at admission, leukocyte, neutrophil count and neutrophil to lymphocyte ratio (NLR) level were associated with patient's prognosis. Diabetes, NIHSS score at admission, and hemorrhagic transformation were independent risk factors for predicting poor prognosis in patients treated with MT. NIHSS score on admission had a predictive value on patient's prognosis. miR-298 was upregulated in acute cerebral ischemia patients with MT (p<0.05), especially in those with poor prognosis. miR-298 was elevated in both cell and mouse models (p<0.05). Apoptosis and autophagy of cells were weakened after miR-298 knockdown, and infarction in the mouse brain tissues was reduced. ATG5 was a target of miR-298. Overexpressing ATG5 rescued miR-298-induced apoptosis and autophagy. In conclusion: regulation of miR-298 and ATG5 attenuates neuronal apoptosis and autophagy, providing a new strategy for brain injury after reperfusion in patients with MT. [ABSTRACT FROM AUTHOR]

Background: Vascular dementia (VaD) is a prevalent neurodegenerative disease characterized by cognitive impairment due to cerebrovascular factors, affecting a significant portion of the aging population and highlighting the critical need to understand specific targets and mechanisms for effective prevention and treatment strategies. We aimed to identify pathways and crucial genes involved in the progression of VaD through bioinformatics analysis and subsequently validate these findings. Methods: We conducted differential expression analysis, Weighted Gene Co-expression Network Analysis (WGCNA), Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis, and Protein-Protein Interaction (PPI) analysis. We utilized pheochromocytoma 12 (PC12) cells to create an in vitro oxygen-glucose deprivation (OGD) model. We investigated the impact of overexpression and interference of adrenoceptor alpha 1D (ADRA1D) on OGD PC12 cells using TdT-mediated dUTP nick-end labeling (TUNEL), reverse transcription-quantitative polymerase chain reaction (RT-qPCR), western blot (WB), and Fluo-3-pentaacetoxymethyl ester (Fluo-3 AM) analysis. Results: We found 187 differentially expressed genes (DEGs) in the red module that were strongly associated with VaD and were primarily enriched in vasoconstric-tion, G protein-coupled amine receptor activity, and neuroactive ligand-receptor interaction, mitogen-activated protein kinase (MAPK) signaling pathway, and cell adhesion. Among these pathways, we identified ADRA1D as a gene shared by vasoconstriction, G protein-coupled amine receptor activity, and neuroactive ligand-receptor interaction. The TUNEL assay revealed a significant decrease in PC12 cell apoptosis with ADRA1D overexpression (p<0.01) and a significant increase in apoptosis upon silencing ADRA1D (p < 0.01). RT-qPCR and WB analysis revealed elevated ADRA1D expression (p < 0.001) and decreased phospholipase C beta (PLCß) and inositol 1,4,5-trisphosphate receptor (IP3R) expression (p < 0.05) with ADRA1D overexpression. Moreover, the Fluo-3 AM assessment indicated significantly lower intracellular Ca2+ levels with ADRA1D overexpression (p < 0.001). Conversely, interference with ADRA1D yielded opposite results. Conclusion: Our study provides a new perspective on the pathogenic mechanisms of VaD and potential avenues for therapeutic intervention. The results highlight the role of ADRA1D in modulating cellular responses to OGD and VaD, suggesting its potential as a target for VaD treatment. [ABSTRACT FROM AUTHOR]

Hypoxic–ischemic brain injury induces metabolic dysfunction that ultimately leads to neuronal cell death. Astrocytes, a type of glial cell, play a key role in brain metabolism; however, their response to hypoxic–ischemic brain injury is not fully understood. Microglia were removed from murine primary mixed glial cultures to enrich astrocytes. Next, we explored genes whose expression is altered following oxygen–glucose deprivation using a microarray. Microarray analysis revealed that the expression of Nr4a1 and Nr4a3 is markedly increased in astrocyte-enriched cultures after 15 h of oxygen–glucose deprivation. The expression of both Nr4a1 and Nr4a3 was regulated by HIF-1α. At the protein level, NR4A1 was translocated from the nucleus to the cytoplasm following oxygen–glucose deprivation and co-localized with mitochondria in apoptotic cells; however, its localization was restored to the nucleus after reoxygenation. Oxygen–glucose deprivation causes an increase in NR4A1 mRNA in astrocytes as well as its nuclear to cytoplasmic transfer. Furthermore, reoxygenation enhances NR4A1 transcription and promotes its nuclear translocation. [ABSTRACT FROM AUTHOR]

Ischemic stroke is typified by hypoxia and a cascade of pathophysiological events, including metabolic dysfunction, ionic dysregulation, excitotoxicity, inflammatory infiltration, and oxidative stress. These ultimately result in neuronal apoptosis or necrosis with constrained neuroregenerative capabilities. In this study, neural stem cells (NSCs) under conditions of oxygen–glucose deprivation (OGD) in vitro and following middle cerebral artery occlusion (MCAO) in vivo were explored. Transcriptome sequencing revealed a decline in NSC differentiation and neurogenesis after OGD exposure, which was related to cellular senescence. This observation was corroborated by increased senescence markers in the MCAO mouse model, reduction in NSC numbers, and decline in neurogenesis. Importantly, iMSC-sEVs (induced mesenchymal stem cells-small extracellular vesicles) have the therapeutic potential to alleviate NSC senescence and rejuvenate their regenerative capacities both in vitro and in vivo. Moreover, iMSC-sEVs contribute to the recovery of cognitive function and synapse loss caused by MCAO. [ABSTRACT FROM AUTHOR]

Myocardial infarction (MI) is an extremely severe cardiovascular disease, which ranks as the leading cause of sudden death worldwide. Studies have proved that cardiac injury following MI can cause cardiomyocyte apoptosis and myocardial fibrosis. Bilobalide (Bilo) from Ginkgo biloba leaves have been widely reported to possess excellent cardioprotective effects. However, concrete roles of Bilo in MI have not been investigated yet. We here designed both in vitro and in vivo experiments to explore the effects of Bilo on MI-induced cardiac injury and the underlying mechanisms of its action. We conducted in vitro experiments using oxygen–glucose deprivation (OGD)-treated H9c2 cells. Cell apoptosis in H9c2 cells was assessed by conducting flow cytometry assay and evaluating apoptosis-related proteins with western blotting. MI mouse model was established by performing left anterior descending artery (LAD) ligation. Cardiac function of MI mice was determined by assessing ejection fraction (EF), fractional shortening (FS), left ventricular end-systolic diameter (LVESD), and left ventricular end-diastolic diameter (LVEDD). Histological changes were analyzed, infarct size and myocardial fibrosis were measured by hematoxylin and eosin (H&E) and Masson staining in cardiac tissues from the mice. The apoptosis of cardiomyocytes in MI mice was assessed by TUNEL staining. Western blotting was applied to detect the effect of Bilo on c-Jun N-terminal kinase (JNK)/p38 mitogen-activated protein kinases (p38 MAPK) signaling both in vitro and in vivo. Bilo inhibited OGD-induced cell apoptosis and lactate dehydrogenase (LDH) release in H9c2 cells. The protein levels of p-JNK and p-p38 were significantly downregulated by Bilo treatment. SB20358 (inhibitor of p38) and SP600125 (inhibitor of JNK) suppressed OGD-induced cell apoptosis as Bilo did. In MI mouse model, Bilo improved the cardiac function and significantly reduced the infarct size and myocardial fibrosis. Bilo inhibited MI-induced cardiomyocytes apoptosis in mice. Bilo suppressed the protein levels of p-JNK and p-p38 in cardiac tissues from MI mice. Bilo alleviated OGD-induced cell apoptosis in H9c2 cells and suppressed MI-induced cardiomyocyte apoptosis and myocardial fibrosis in mice via the inactivation of JNK/p38 MAPK signaling pathways. Thus, Bilo may be an effective anti-MI agent. [ABSTRACT FROM AUTHOR]

Our previous study found that rat bone marrow–derived neural crest cells (acting as Schwann cell progenitors) have the potential to promote long-distance nerve repair. Cell-based therapy can enhance peripheral nerve repair and regeneration through paracrine bioactive factors and intercellular communication. Nevertheless, the complex contributions of various types of soluble cytokines and extracellular vesicle cargos to the secretome remain unclear. To investigate the role of the secretome and extracellular vesicles in repairing damaged peripheral nerves, we collected conditioned culture medium from hypoxia-pretreated neural crest cells, and found that it significantly promoted the repair of sensory neurons damaged by oxygen-glucose deprivation. The mRNA expression of trophic factors was highly expressed in hypoxia-pretreated neural crest cells. We performed RNA sequencing and bioinformatics analysis and found that miR-21-5p was enriched in hypoxia-pretreated extracellular vesicles of neural crest cells. Subsequently, to further clarify the role of hypoxia-pretreated neural crest cell extracellular vesicles rich in miR-21-5p in axonal growth and regeneration of sensory neurons, we used a microfluidic axonal dissociation model of sensory neurons in vitro, and found that hypoxia-pretreated neural crest cell extracellular vesicles promoted axonal growth and regeneration of sensory neurons, which was greatly dependent on loaded miR-21-5p. Finally, we constructed a miR-21-5p-loaded neural conduit to repair the sciatic nerve defect in rats and found that the motor and sensory functions of injured rat hind limb, as well as muscle tissue morphology of the hind limbs, were obviously restored. These findings suggest that hypoxia-pretreated neural crest extracellular vesicles are natural nanoparticles rich in miRNA-21-5p. miRNA-21-5p is one of the main contributors to promoting nerve regeneration by the neural crest cell secretome. This helps to explain the mechanism of action of the secretome and extracellular vesicles of neural crest cells in repairing damaged peripheral nerves, and also promotes the application of miR-21-5p in tissue engineering regeneration medicine.

Abstract Background Brain activity governing cognition and behaviour depends on the fine-tuned microenvironment provided by a tightly controlled blood–brain barrier (BBB). Brain endothelium dysfunction is a hallmark of BBB breakdown in most neurodegenerative/neuroinflammatory disorders. Therefore, the identification of new endogenous molecules involved in endothelial cell disruption is essential to better understand BBB dynamics. Cortistatin is a neuroimmune mediator with anti-inflammatory and neuroprotective properties that exerts beneficial effects on the peripheral endothelium. However, its role in the healthy and injured brain endothelium remains to be evaluated. Herein, this study aimed to investigate the potential function of endogenous and therapeutic cortistatin in regulating brain endothelium dysfunction in a neuroinflammatory/neurodegenerative environment. Methods Wild-type and cortistatin-deficient murine brain endothelium and human cells were used for an in vitro barrier model, where a simulated ischemia-like environment was mimicked. Endothelial permeability, junction integrity, and immune response in the presence and absence of cortistatin were evaluated using different size tracers, immunofluorescence labelling, qPCR, and ELISA. Cortistatin molecular mechanisms underlying brain endothelium dynamics were assessed by RNA-sequencing analysis. Cortistatin role in BBB leakage was evaluated in adult mice injected with LPS. Results The endogenous lack of cortistatin predisposes endothelium weakening with increased permeability, tight-junctions breakdown, and dysregulated immune activity. We demonstrated that both damaged and uninjured brain endothelial cells isolated from cortistatin-deficient mice, present a dysregulated and/or deactivated genetic programming. These pathways, related to basic physiology but also crucial for the repair after damage (e.g., extracellular matrix remodelling, angiogenesis, response to oxygen, signalling, and metabolites transport), are dysfunctional and make brain endothelial barrier lacking cortistatin non-responsive to any further injury. Treatment with cortistatin reversed in vitro hyperpermeability, tight-junctions disruption, inflammatory response, and reduced in vivo BBB leakage. Conclusions The neuropeptide cortistatin has a key role in the physiology of the cerebral microvasculature and its presence is crucial to develop a canonical balanced response to damage. The reparative effects of cortistatin in the brain endothelium were accompanied by the modulation of the immune function and the rescue of barrier integrity. Cortistatin-based therapies could emerge as a novel pleiotropic strategy to ameliorate neuroinflammatory/neurodegenerative disorders with disrupted BBB.

Mitochondrial dysfunction is known to play a critical role in the development of cardiomyocyte death during acute myocardial infarction (AMI). However, the exact mechanisms underlying this dysfunction are still under investigation. Adenine nucleotide translocase 2 (ANT2) is a key functional protein in mitochondria. We aimed at exploring the potential benefits of ANT2 inhibition against AMI. We utilized an oxygen–glucose deprivation (OGD) cell model and an AMI mice model to detect cardiomyocyte injury. We observed elevated levels of reactive oxygen species (ROS), disrupted mitochondrial membrane potential (MMP), and increased apoptosis due to the overexpression of ANT2. Additionally, we discovered that ANT2 is involved in myocardial apoptosis by activating the mTOR (mechanistic target of rapamycin kinase)-dependent PGC-1α (PPARG coactivator 1 alpha) pathway, establishing a novel feedback loop during AMI. In our experiments with AC16 cells under OGD conditions, we observed protective effects when transfected with ANT2 siRNA and miR-1203. Importantly, the overexpression of ANT2 counteracted the protective effect resulting from miR-1203 upregulation in OGD-induced AC16 cells. All these results supported that the inhibition of ANT2 could alleviate myocardial cell injury under OGD conditions. Based on these findings, we propose that RNA interference (RNAi) technology, specifically miRNA and siRNA, holds therapeutic potential by activating the ANT2/mTOR/PGC-1α feedback loop. This activation could help mitigate mitochondria-mediated injury in the context of AMI. These insights may contribute to the development of future clinical strategies for AMI.

Reactive oxygen species (ROS) are considered a primary source of damage during ischemic stroke. However, the precise timing of ROS production (during hypoxia or reperfusion) remains unclear. Cellular 3D spheroids are often proposed as an optimal alternative to both 2D cell cultures and animal models in modeling disease conditions. Here we report live imaging of hydrogen peroxide dynamics during the acute phase of hypoxia and reperfusion in human iPSC-derived neural spheroids, stably expressing fluorescent biosensor HyPer7. Contrary to previous reports, we did not observe a hydrogen peroxide production burst neither during hypoxia nor in course of reperfusion. Our data suggest either lack of oxidative stress during ischemia-reperfusion in spheroids or existence of different mechanisms of oxidative damage. [Display omitted] • HyPer7-iPSCs differentiate into 3D neural tissue and physiologically mature neurons. • Real-time dynamics of H 2 O 2 were recorded in neural spheroids during ischemia-reperfusion modeling. • ROS-induced oxidative stress is not triggered in HyPer7-neurospheroids during IR modeling. [ABSTRACT FROM AUTHOR]

Several volatile anesthetics have presented neuroprotective functions in ischemic injury. This study investigates the effect of desflurane (Des) on neurons following oxygen–glucose deprivation (OGD) challenge and explores the underpinning mechanism. Mouse neurons HT22 were subjected to OGD, which significantly reduced cell viability, increased lactate dehydrogenase release, and promoted cell apoptosis. In addition, the OGD condition increased oxidative stress in HT22 cells, as manifested by increased ROS and MDA contents, decreased SOD activity and GSH/GSSG ratio, and reduced nuclear protein level of Nrf2. Notably, the oxidative stress and neuronal apoptosis were substantially blocked by Des treatment. Bioinformatics suggested potassium voltage-gated channel subfamily A member 1 (Kcna1) as a target of Des. Indeed, the Kcna1 expression in HT22 cells was decreased by OGD but restored by Des treatment. Artificial knockdown of Kcna1 negated the neuroprotective effects of Des. By upregulating Kcna1, Des activated the Kv1.1 channel, therefore enhancing K+ currents and inducing neuronal repolarization. Pharmacological inhibition of the Kv1.1 channel reversed the protective effects of Des against OGD-induced injury. Collectively, this study demonstrates that Des improves electrical activity of neurons and alleviates OGD-induced neuronal injury by activating the Kcna1-dependent Kv1.1 channel. [ABSTRACT FROM AUTHOR]

AIM: To investigate the effect of gastrodin (GAS) on microglia-mediated inflammatory response after hypoxic-ischemic brain damage (HIBD) neonatal mice by regulating the expression of JAK1/STAT1 pathway through C-C chemokine recepeor 5(CCR5) . METHODS: Forty-eight C57BL/6J mice at about 10 days after birth were randomly divided into sham group, HIBD model group and HIBD+GAS group. BV-2 microglia were divided into control (Con) group, oxygen glucose deprivation (OGD) group, oxygen glucose deprivation with gastrodin intervention (OGD+GAS) group, GAS group, Maraviroc (MVC) group, OGD+MVC group, and OGD+MVC+GAS group. The mRNA expression of CCL4 and CCR5 were detected by RT-qPCR. The protein expression of CCR5，p-JAK1, p-STAT1, tumor necrosis factorα(TNF-α) and interleukin-1β(IL-1β) were detected by Western blot. The expression of CCR5, p-JAK1 and p-STAT1 in cells were observed by immunofluorescence staining. RESULTS：(1) Compared with sham group, the expression levels of CCL4 and CCR5 mRNA, and CCR5, p-JAK1 and pSTAT1 proteins were significantly higher in the ischemic side of the corpus callosum in HIBD group (P<0. 05) . (2) Compared with Con group, the protein levels of CCR5, p-JAK1 and pSTAT1 significantly increased in BV-2 cells of OGD group (P<0. 05) . The protein levels of CCR5, p-JAK1 and p-STAT1 in BV-2 cells of OGD+GAS group were significantly lower than those of OGD group (P<0. 05) . (3) Maraviroc did not cause significant BV-2 cell death in the 0~80 μmol/L range. The p-JAK1 and p-STAT1 protein levels in MVC+OGD group were significantly lowered compared with OGD group (P<0. 05), but no significant difference was found between MVC+ OGD and OGD+MVC+GAS groups. CONCLUSION: Gastrodin can exert neuroprotective effects via CCR5/JAK1/STAT1 signaling pathway. [ABSTRACT FROM AUTHOR]

This study compared the neuroprotective efficacy of three antioxidants—the plant-derived carnosic acid (CA), and two synthetic free radical scavengers: edaravone (ED) and ebselen (EB)—in in vitro models of neuronal cell damage. Results showed that CA protected mouse primary neuronal cell cultures against hydrogen peroxide-induced damage more efficiently than ED or EB. The neuroprotective effects of CA were associated with attenuation of reactive oxygen species level and increased mitochondrial membrane potential but not with a reduction in caspase-3 activity. None of the tested substances was protective against glutamate or oxygen-glucose deprivation-evoked neuronal cell damage, and EB even increased the detrimental effects of these insults. Further experiments using the human neuroblastoma SH-SY5Y cells showed that CA but not ED or EB attenuated the cell damage induced by hydrogen peroxide and that the composition of culture medium is the critical factor in evaluating neuroprotective effects in this model. Our data indicate that the neuroprotective potential of CA, ED, and EB may be revealed in vitro only under specific conditions, with their rather narrow micromolar concentrations, relevant cellular model, type of toxic agent, and exposure time. Nevertheless, of the three compounds tested, CA displayed the most consistent neuroprotective effects. [ABSTRACT FROM AUTHOR]

Objective：To discuss the protective effect of gingerone on the hippocampal neuron HT22 cells after oxygen-glucose deprivation/reoxygenation (OGD/R), and to clarify the related mechanism. Methods：The HT22 cells were cultured, and the OGD/R cell injury model was established by setting the gradient of OGD/R time. The HT22 cells were divided into control group, OGD/R group, OGD/R+ 1 μmol·L－1 gingerone group, OGD/R + 10 μmol·L－1 gingerone group, OGD/R+100 μmol·L－1 gingerone group, and OGD/R+0. 2% dimethyl sulfoxide (DMSO) group. The viability of the cells in various groups was detected by CCK-8 assay； the survival rates of the cells in various groups were calculated to determine the optimal drug concentration of gingerone. The cells were divided into control, OGD/R group, OGD/R+ gingerone, and OGD/R+gingerone+nuclear factor erythroid-2-related factor 2 (Nrf2) inhibitor (ML385) groups. The cells in OGD/R + gingerone group were treated with gingerone for 4 h before OGD treatment for 8 h followed by reoxygenation for 8 h, and the cells in OGD/R+gingerone+ ML385 group were treated with 10 μmol·L－1 ML385 for 6 h before gingerone treatment. The viability of the cells in various groups was detected by CCK-8 assay；the expression levels of Nrf2, heme oxygenase－1 (HO－1 ), B-cell lymphoma-2 (Bcl-2), and Bcl-2-associated X protein (Bax) proteins in the cells in various groups were detected by Western blotting method；the activity of superoxide dismutase (SOD) and the level of malondialdehyde (MDA) in the cell culture supernatant in various groups were detected by enzyme-linked immunosorbent assay (ELISA) method. Results：Compared with control group, the survival rate of the HT22 cells was below 50% after treated with OGD for 8 h and reoxygenation for 8 h, so the HT22 cell OGD/R model was established by treated with OGD for 8 h and reoxygenation for 8 h. Compared with OGD/R group, the survival rates of the cells in OGD/R+different doses of gingerone groups were increased to various extents, and the survival rate of the cells in OGD/R+ 100 μmol·L－1 gingerone group was significantly increased (P<0. 01) ； so 100 μmol·L－1 gingerone was used for the subsequent experiment. Compared with control group, the viability of the cells in OGD/R group was significantly decreased (P<0. 01), and the expression levels of Nrf2, HO－1 , and Bax proteins in the cells were significantly increased (P<0. 01), while the expression level of Bcl-2 protein in the cells was significantly decreased (P<0. 05), and the SOD activity in the cell culture supernatant was significantly decreased (P<0. 01), and the level of MDA was significantly increased (P<0. 01) ； compared with OGD/R group, the viability of the cells in OGD/R + gingerone group was significantly increased (P< 0. 01), and the expression levels of Nrf2, HO－1 , and Bcl-2 proteins in the cells were significantly increased (P<0. 05 or P<0. 01), while the expression level of Bax protein in the cells was decreased (<0. 05), the SOD activity in the cell culture supernatant was significantly increased (P<0. 01), and the level of MDA was significantly decreased (P<0. 01) ； compared with OGD/R + gingerone group, the viability of the cells in OGD/R + gingerone + ML385 group was significantly decreased (P<0. 01), and the expression levels of Nrf2, HO－1 , and Bcl-2 proteins were significantly decreased (P<0. 01), while the expression level of Bax protein in the cells was significantly increased (P<0. 01）, the SOD activity in the cell culture supernatant was significantly decreased (P<0. 01), and the level of MDA was significantly increased (P<0. 05）. Conclusion : Gingerone alleviates the oxidative stress damage, and thereby plays an inhibiory effect on the apoptosis of the HT22 neurons by activating the Nrf2/HO-1 signaling pathway after OGD/R. [ABSTRACT FROM AUTHOR]

To investigate the effect and its mechanism of oleuropein on the oxidative damage and apoptosis of astrocytes induced by oxygen-glucose deprivation, and its effect on circ_TTC3 and microRNA-138-5p. Astrocytes were divided into control group, oxygen-glucose deprivation group, oxygen-glucose deprivation+oleuropein 25 μmol/l, 50 μmol/l, 100 μmol/l groups, oxygen-glucose deprivation+si-negative control, si-circ_TTC3 group, oxygen-glucose deprivation+oleuropein+plasmid cloning deoxyribonucleic acid plasmid cloning deoxyribonucleic acid and plasmid cloning deoxyribonucleic acid-circ_TTC3 group. In comparison with the control group, the inhibiting rate of cell growth, apoptosis rate, malondialdehyde, lactate dehydrogenase levels and circ_TTC3 expression of astrocytes in the oxygen-glucose deprivation group were increased, while superoxide dismutase activity and miR-138-5p expression were declined (p<0.05). Compared to the oxygen-glucose deprivation group, the proliferative inhibiting rate, apoptosis rate, malondialdehyde, lactate dehydrogenase levels and circ_TTC3 expression of astrocytes in the oxygen-glucose deprivation+oleuropein 25 μmol/l group, oxygen-glucose deprivation+oleuropein 50 μmol/l group, and oxygen-glucose deprivation+oleuropein 100 μmol/l group were all weakened, superoxide dismutase activity and microRNA-138-5p expression were enhanced (p<0.05), and all were concentration-dependent. After silencing circ_TTC3, circ_TTC3 expression, malondialdehyde, lactate dehydrogenase levels, cell proliferation inhibition rate and apoptosis rate in astrocytes in the oxygen-glucose deprivation+si-circ_TTC3 group were all lower than those in oxygen-glucose deprivation+si-negative control group, while the superoxide dismutase activity was higher than that in oxygen-glucose deprivation+si-negative control group (p<0.05). Circ_TTC3 targeted and regulated microRNA- 138-5p. Circ_TTC3 level, malondialdehyde and lactate dehydrogenase levels, cell proliferation inhibition rate, and apoptosis rate in astrocytes in the oxygen-glucose deprivation+oleuropein+ plasmid cloning deoxyribonucleic acid-circ_TTC3 group were all higher than those in oxygen-glucose deprivation+oleuropein+plasmid cloning deoxyribonucleic acid group, and there was a decrease of superoxide dismutase activity in the oxygen-glucose deprivation +oleuropein+plasmid cloning deoxyribonucleic acid-circ_TTC3 group compared with oxygen-glucose deprivation+oleuropein+plasmid cloning deoxyribonucleic acid group (p<0.05). Oleuropein could reduce oxidative stress and apoptosis, thereby protecting astrocytes from damage induced by oxygen-glucose deprivation by upregulating circ_TTC3 to target microRNA-138-5p. [ABSTRACT FROM AUTHOR]

Cellular senescence refers to the permanent and irreversible cessation of the cell cycle. Recently, it has gained significant interest as a promising target for preventing cardiovascular diseases. Aldehyde dehydrogenase 2 (ALDH2) is a mitochondrial enzyme that has been closely linked with an increased risk of cardiovascular diseases. In this study, bioinformatics analysis revealed that the signaling pathway for fibroblast senescence is significantly activated in mice after myocardial infarction (MI), and that ALDH2 might be a crucial molecule responsible for inducing this change. Therefore, we created an NIH3T3 fibroblast cell line oxygen-glucose deprivation (OGD) model to replicate the conditions of MI in vitro. We further revealed that decreased ALDH2 enzyme activity is a critical factor that affects fibroblast senescence after OGD, and the activation of ALDH2 can improve the mitochondrial damage caused by OGD. We identified Heat Shock 70-kDa Protein 8 (HSPA8) as an interacting protein of ALDH2 through co-immunoprecipitation (Co-IP) and mass spectrometry (MS) detection. Subsequently, our studies showed that HSPA8 translocates to the mitochondria after OGD, potentially binding to ALDH2 and inhibiting its enzyme activity. By transfecting siRNA to inhibit HSPA8 expression in cells, it was found that ALDH2 enzyme activity can be significantly increased, and the senescence characteristics induced by OGD in NIH3T3 cells can be improved. In conclusion, the data from this study suggest that HSPA8, in conjunction with ALDH2, could regulate fibroblast senescence after oxygen-glucose deprivation, providing a new direction and foundation for effectively intervening in fibroblast senescence after myocardial infarction. [ABSTRACT FROM AUTHOR]

The obstructed coronary artery undergoes a series of pathological changes due to ischemic-hypoxic shocks during acute myocardial infarction (AMI). However, the altered DNA methylation levels in endothelial cells under these conditions and their implication for the etiopathology of AMI have not been investigated in detail. This study aimed to explore the relationship between DNA methylation and pathologically altered gene expression profile in human umbilical vein endothelial cells (HUVECs) subjected to oxygen-glucose deprivation (OGD), and its clinical implications in AMI patients. The Illumina Infinium MethylationEPIC BeadChip assay was used to explore the genome-wide DNA methylation profile using the Novaseq6000 platform for mRNA sequencing in 3 pairs of HUVEC-OGD and control samples. GO and KEGG pathway enrichment analyses, as well as correlation, causal inference test (CIT), and protein-protein interaction (PPI) analyses identified 22 hub genes that were validated by MethylTarget sequencing as well as qRT-PCR. ELISA was used to detect four target molecules associated with the progression of AMI. A total of 2,524 differentially expressed genes (DEGs) and 22,148 differentially methylated positions (DMPs) corresponding to 6,642 differentially methylated genes (DMGs) were screened (|Δβ|>0.1 and detection p < 0.05). After GO, KEGG, correlation, CIT, and PPI analyses, 441 genes were filtered. qRT-PCR confirmed the overexpression of VEGFA, CCL2, TSP-1, SQSTM1, BCL2L11, and TIMP3 genes, and downregulation of MYC, CD44, BDNF, GNAQ, RUNX1, ETS1, NGFR, MME, SEMA6A, GNAI1, IFIT1, and MEIS1. DNA fragments BDNF_1_ (r = 0.931, p < 0.0001) and SQSTM1_2_NEW (r = 0.758, p = 0.0043) were positively correlated with the expressions of corresponding genes, and MYC_1_ (r = -0.8245, p = 0.001) was negatively correlated. Furthermore, ELISA confirmed TNFSF10 and BDNF were elevated in the peripheral blood of AMI patients (p = 0.0284 and p = 0.0142, respectively). Combined sequencing from in vitro cellular assays with clinical samples, aiming to establish the potential causal chain of the causal factor (DNA methylation) - mediator (mRNA)-cell outcome (endothelial cell ischemic-hypoxic injury)- clinical outcome (AMI), our study identified promising OGD-specific genes, which provided a solid basis for screening fundamental diagnostic and prognostic biomarkers of coronary endothelial cell injury of AMI. Moreover, it furnished the first evidence that during ischemia and hypoxia, the expression of BNDF was regulated by DNA methylation in endothelial cells and elevated in peripheral blood. [ABSTRACT FROM AUTHOR]

Hypoxia triggers reactive microglial inflammation and lipid droplet (LD) accumulation under stroke conditions, although the mutual interactions between these two processes are insufficiently understood. Hence, the involvement of transforming growth factor (TGF)-β1 in inflammation and LD accumulation in cultured microglia exposed to hypoxia were analyzed herein. Primary microglia were exposed to oxygen-glucose deprivation (OGD) injury and lipopolysaccharide (LPS) stimulation. For analyzing the role of TGF-β1 patterns under such conditions, a TGF-β1 siRNA and an exogenous recombinant TGF-β1 protein were employed. Further studies applied Triacsin C, an inhibitor of LD formation, in order to directly assess the impact of LD formation on the modulation of inflammation. To assess mutual microglia-to-neuron interactions, a co-culture model of these cells was established. Upon OGD exposure, microglial TGF-β1 levels were significantly increased, whereas LPS stimulation yielded decreased levels. Elevating TGF-β1 expression proved highly effective in suppressing inflammation and reducing LD accumulation in microglia exposed to LPS. Conversely, inhibition of TGF-β1 led to the promotion of microglial cell inflammation and an increase in LD accumulation in microglia exposed to OGD. Employing the LD formation inhibitor Triacsin C, in turn, polarized microglia towards an anti-inflammatory phenotype. Such modulation of both microglial TGF-β1 and LD levels significantly affected the resistance of co-cultured neurons. This study provides novel insights by demonstrating that TGF-β1 plays a protective role against microglia-mediated neuroinflammation through the suppression of LD accumulation. These findings offer a fresh perspective on stroke treatment, suggesting the potential of targeting this pathway for therapeutic interventions. [ABSTRACT FROM AUTHOR]

Following a stroke, the emergence of amygdala-related disorders poses a significant challenge, with severe implications for post-stroke mental health, including conditions such as anxiety and depression. These disorders not only hinder post-stroke recovery but also elevate mortality rates. Despite their profound impact, the precise origins of aberrant amygdala function after a stroke remain elusive. As a target of reduced brain pH in ischemia, acid-sensing ion channels (ASICs) have been implicated in synaptic transmission after ischemia, hinting at their potential role in reshaping neural circuits following a stroke. This study delves into the intriguing relationship between post-stroke alterations and ASICs, specifically focusing on postsynaptic ASIC1a enhancement in the amygdala following prefrontal cortex (PFC) ischemia induced by endothelin-1 (ET-1) injection. Our findings intriguingly illustrate that mPFC ischemia not only accentuates the PFC to the amygdala circuit but also implicates ASIC1a in fostering augmented synaptic plasticity after ischemia. In contrast, the absence of ASIC1a impairs the heightened induction of long-term potentiation (LTP) in the amygdala induced by ischemia. This pivotal research introduces a novel concept with the potential to inaugurate an entirely new avenue of inquiry, thereby significantly enhancing our comprehension of the intricate mechanisms underlying post-stroke neural circuit reconfiguration. Importantly, these revelations hold the promise of paving the way for groundbreaking therapeutic interventions. [ABSTRACT FROM AUTHOR]

Abstract Background The pathological basis for many retinal diseases, retinal ischemia is also one of the most common causes of visual impairment. Numerous ocular diseases have been linked to Endoplasmic reticulum(ER)stress. However, there is still no clear understanding of the relationship between ER stress and Müller glial cells during retinal ischemia and hypoxia. This study examined the effects of ER stress on autophagy and apoptosis-related proteins, as well as the microtubule-related protein tau in rMC-1 cells. Methods rMC-1 cells were cultured in vitro. RT-PCR、immunofluorescence and Western blotting revealed the expression levels of associated mRNAs and proteins, and the CCK-8 and flow cytometry assays detected cell apoptosis. Results The results showed that under OGD(Oxygen-glucose deprivation) conditions, the number of rMC-1 cells was decreased, the PERK/eIF2a pathway was activated, and the expressions of p-tau, LC3、Beclin1 and Caspase-12 proteins were increased. After the PERK knockout, the expression of the above proteins was decreased, and the apoptosis was also decreased. Conclusion According to the findings of this study, specific downregulation of PERK expression had an anti-apoptotic effect on OGD-conditioned rMC-1 cells. There is a possibility that this is one of the mechanisms of MG cell apoptosis during retinal ischemic injury.

Objective Spinal cord injury (SCI) is a severe type of neurological trauma. N6-methyladenosine (m6A) modification is one of the most common internal modifications of RNA. The role of METTL3, the predominant methylation enzyme of m6A modification, in SCI remains unclear. This study aimed to investigate the role of methyltransferase METTL3 in SCI. Methods After establishing the oxygen-glucose deprivation (OGD) model of PC12 cells and rat spinal cord hemisection model, we found that the expression of METTL3 and the overall m6A modification level were significantly increased in neurons. The m6A modification was identified on B-cell lymphoma 2 (Bcl-2) messenger RNA (mRNA) by bioinformatics analysis, and m6A-RNA immunoprecipitation and RNA immunoprecipitation. In addition, METTL3 was blocked by the specific inhibitor STM2457 and gene knockdown, and then apoptosis levels were measured. Results In different models, we found that the expression of METTL3 and the overall m6A modification level were significantly increased in neurons. After inducing OGD, inhibition of METTL3 activity or expression increased the mRNA and protein levels of Bcl-2, inhibited neuronal apoptosis, and improved neuronal viability in the spinal cord. Conclusion Inhibition of METTL3 activity or expression can inhibit the apoptosis of spinal cord neurons after SCI through the m6A/Bcl-2 signaling pathway.

Thrombolytic therapy with the tissue plasminogen activator (tPA) is a therapeutic option for acute ischemic stroke. However, this approach is subject to several limitations, particularly the increased risk of hemorrhagic transformation (HT). Lithium salts show neuroprotective effects in stroke, but their effects on HT mechanisms are still unknown. In our study, we use the models of photothrombosis (PT)-induced brain ischemia and oxygen-glucose deprivation (OGD) to investigate the effect of Li+ on tPA-induced changes in brain and endothelial cell cultures. We found that tPA did not affect lesion volume or exacerbate neurological deficits but disrupted the blood–brain barrier. We demonstrate that poststroke treatment with Li+ improves neurological status and increases blood–brain barrier integrity after thrombolytic therapy. Under conditions of OGD, tPA treatment increased MMP-2/9 levels in endothelial cells, and preincubation with LiCl abolished this MMP activation. Moreover, we observed the effect of Li+ on glycolysis in tPA-treated endothelial cells, which we hypothesized to have an effect on MMP expression.

Epigenetic regulators present novel opportunities for both ischemic stroke research and therapeutic interventions. While previous work has implicated that they may provide neuroprotection by potentially influencing coordinated sets of genes and pathways, most of them remain largely uncharacterized in ischemic conditions. In this study, we used the oxygen-glucose deprivation (OGD) model in the immortalized mouse hippocampal neuronal cell line HT-22 and carried out an RNAi screen on epigenetic regulators. PRMT5 was identified as a novel negative regulator of neuronal cell survival after OGD, which presented a phenotype of translocation from the cytosol to the nucleus upon oxygen and energy depletion both in vitro and in vivo. PRMT5 bound to the chromatin and a large number of promoter regions to repress downstream gene expression. Silencing Prmt5 significantly dampened the OGD-induced changes for a large-scale of genes, and gene ontology analysis showed that PRMT5-target genes were highly enriched for Hedgehog signaling. Encouraged by the above observation, mice were treated with middle cerebral artery occlusion with the PRMT5 inhibitor EPZ015666 and found that PRMT5 inhibition sustains protection against neuronal death in vivo. Together, these findings revealed a novel epigenetic mechanism of PRMT5 in cerebral ischemia and uncovered a potential target for neuroprotection.

Objective: To prepare gelatin hydrogel patches loaded with basic fibroblast growth factor(bFGF)and to detect the effect of bFGF on dendritic cells(DCs)under oxygen glucose deprivation(OGD)condition. Methods: Gelatin hydrogel patches were prepared by glutaraldehyde cross-linking method, and the swelling rate was detected. The cytotoxicity of hydrogel patches was detected by CCK-8 method. Western blot was used to detect the expression of inflammatory factors in DC2.4 cells. The secretion of inflammatory factors was detected by ELISA. The activity and contents of LDH, MDA and SOD in cell supernatant were detected by micromethod. Results: The bFGF-loaded hydrogel patches we prepared had moderate swelling rate and low cytotoxicity. The bFGF-loaded hydrogel patch could significantly reduce the expression and secretion of inflammatory factors in DC2.4 cells under OGD conditions, increase the activity of SOD in DC2.4 cells, reduce the contents of MDA and LDH in the supernatant of DC2.4 cells, and improve the oxidative stress response of DC2.4 cells under OGD condition. Conclusion: The bFGF-loaded hydrogel can improve the expression of inflammatory factors and oxidative stress response of DCs under OGD condition. [ABSTRACT FROM AUTHOR]

Background: Brain activity governing cognition and behaviour depends on the fine-tuned microenvironment provided by a tightly controlled blood–brain barrier (BBB). Brain endothelium dysfunction is a hallmark of BBB breakdown in most neurodegenerative/neuroinflammatory disorders. Therefore, the identification of new endogenous molecules involved in endothelial cell disruption is essential to better understand BBB dynamics. Cortistatin is a neuroimmune mediator with anti-inflammatory and neuroprotective properties that exerts beneficial effects on the peripheral endothelium. However, its role in the healthy and injured brain endothelium remains to be evaluated. Herein, this study aimed to investigate the potential function of endogenous and therapeutic cortistatin in regulating brain endothelium dysfunction in a neuroinflammatory/neurodegenerative environment. Methods: Wild-type and cortistatin-deficient murine brain endothelium and human cells were used for an in vitro barrier model, where a simulated ischemia-like environment was mimicked. Endothelial permeability, junction integrity, and immune response in the presence and absence of cortistatin were evaluated using different size tracers, immunofluorescence labelling, qPCR, and ELISA. Cortistatin molecular mechanisms underlying brain endothelium dynamics were assessed by RNA-sequencing analysis. Cortistatin role in BBB leakage was evaluated in adult mice injected with LPS. Results: The endogenous lack of cortistatin predisposes endothelium weakening with increased permeability, tight-junctions breakdown, and dysregulated immune activity. We demonstrated that both damaged and uninjured brain endothelial cells isolated from cortistatin-deficient mice, present a dysregulated and/or deactivated genetic programming. These pathways, related to basic physiology but also crucial for the repair after damage (e.g., extracellular matrix remodelling, angiogenesis, response to oxygen, signalling, and metabolites transport), are dysfunctional and make brain endothelial barrier lacking cortistatin non-responsive to any further injury. Treatment with cortistatin reversed in vitro hyperpermeability, tight-junctions disruption, inflammatory response, and reduced in vivo BBB leakage. Conclusions: The neuropeptide cortistatin has a key role in the physiology of the cerebral microvasculature and its presence is crucial to develop a canonical balanced response to damage. The reparative effects of cortistatin in the brain endothelium were accompanied by the modulation of the immune function and the rescue of barrier integrity. Cortistatin-based therapies could emerge as a novel pleiotropic strategy to ameliorate neuroinflammatory/neurodegenerative disorders with disrupted BBB. [ABSTRACT FROM AUTHOR]