Oxygen And Glucose Deprivation Research Articles

Lipid nanoparticle-encapsulated lncRNA DLX6-AS1 knockdown ameliorates cerebral ischemic injury via the Nrf2/HO-1/NLRP3 axis

ABSTRACT Objective Cerebral ischemia is a neurological disorder that leads to permanent disability. This research focuses on exploring the ameliorative effects of lipid nanoparticle (LNP)-encapsulated lncRNA DLX6-AS1 knockdown in cerebral ischemic injury via the Nrf2/HO-1/NLRP3 axis. Methods LNP-encapsulated lncRNA DLX6-AS1 was prepared. Cerebral ischemic injury mouse models were established utilizing middle cerebral artery occlusion (MCAO). The mice were treated by intravenous injection of LNP-encapsulated lncRNA DLX6-AS1. The neurological deficits, Inflammatory factor levels, pathological characteristics were observed. In vitro N2a cell oxygen and glucose deprivation (OGD) models were established, and the cells were treated with LNP-encapsulated lncRNA DLX6-AS1 or Nrf2 inhibitor (ML385). Cell viability and apoptosis were tested. DLX6-AS1, Nrf2, HO-1, and NLRP3 expression levels were assessed. Results LncRNA DLX6-AS1 levels were elevated in the brain tissues of mice with cerebral ischemic injury and OGD-induced N2a cells. LNP-encapsulated DLX6-AS1 siRNA (si-DLX6-AS1) improved neurological deficit scores, reduced the levels of inflammatory factors, improved brain tissue pathological damage, and raised the number of survival neurons in CA1. LNP-encapsulated si-DLX6-AS1 ameliorated the OGD-induced N2a cell viability decrease and apoptosis rate increase, and ML385 (Nrf2 inhibitor) reversed the ameliorative effects of LNP-encapsulated si-DLX6-AS1. In cerebral ischemic injury mice and OGD-induced N2a cells, Nrf2 and HO-1 levels were reduced and NLRP3 levels were increased. LNP-encapsulated si-DLX6-AS1 raised Nrf2 and HO-1 levels and reduced NLRP3 levels. Nrf2 inhibitor ML385 treatment reversed the ameliorative effects of LNP-encapsulated si-DLX6-AS1 on OGD-induced N2a cell viability and apoptosis. Conclusion Lipid nanoparticle-encapsulated si-DLX6-AS1 ameliorates cerebral ischemic injury via the Nrf2/HO-1/NLRP3 axis.

Overwintering Induces Ischemia-Tolerance of Synaptic Function in the Forebrain of Bullfrogs

Healthy brain function requires the continuous delivery of oxygen and glucose to nerve cells. Interruptions result in rapid cessation of normal circuit function in most vertebrates. The American bullfrog ( Lithobates catesbeianus) is an interesting model, as we have shown that brainstem synapses increase their capacity to function during hypoxia and ischemia by roughly 20-30 fold following aquatic overwintering (cold-acclimated, CA) compared to control conditions (warm-acclimated, WA). Here, we tested the hypothesis that other synapses, specially those in the forebrain, also improve their function to determine if extreme metabolic plasticity is specific to the brainstem, or a generalized response throughout the central nervous system. We used the synapse that carries thalamic input to dorsal pallium, which is involved in sensory processing and associative memory. To measure synaptic function, we stimulated thalamic axons and recorded evoked excitatory postsynaptic currents (eEPSCs) in the medial dorsal pallium, the proposed homolog to the mammalian hippocampus, in normoxic conditions and in oxygen and glucose deprivation (OGD). eEPSC amplitude at a baseline frequency of 0.5 Hz was depressed at OGD onset in WA frogs (paired t-test, p=0.0002, n=12) but not in CA animals (paired t-test, p=0.1602, n=6). This suggests that unlike WA frogs, overwintering allows maintained synaptic function in OGD within the forebrain, as well as the brainstem. Mechanistically, the maintenance of synaptic function in OGD appeared to be associated with dynamic increase in the probability of neurotransmitter release. Paired-pulse experiments to assess dynamics in neurotransmitter release in response to high-frequency stimulus pairs showed that synapses depressed in normoxic conditions in both WA (9 of 11 cells) and CA (4 out of 6 cells) groups. In OGD, synaptic depression was maintained in WA frogs (paired t-test, p=0.9192); however, synaptic depression reversed, whereby paired-pulse stimulation instead led to synaptic facilitation in 5 out of 6 neurons (paired t-test, p=0.0113). This suggests that synapses may alter the probability of glutamate release to act as a countermeasure to maintain forebrain function during energetic stress associated with emergence. Overall, these results show that metabolic plasticity to restart critical behaviors in hypoxia following hibernation is preserved across the brain and that altered synaptic dynamics may play a role in this response. This research was funded by the National Institutes of Health (R01NS114514, R15NS112920-01A1). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

SDF-1/CXCR4 axis participants in the pathophysiology of adult patients with moyamoya disease

BackgroundMoyamoya disease (MMD) is characterized by an abundance of moyamoya vessels; however, the precise mechanism driving the spontaneous angiogenesis of these compensatory vessels remains unclear. Previous research has established a link between the stromal cell-derived factor-1 (SDF-1)/ CXC receptor 4 (CXCR4) axis and angiogenesis under hypoxic conditions. Nevertheless, the alterations in this axis within the cerebrospinal fluid, arachnoid membranes and vascular tissue of MMD patients have not been fully investigated. MethodsOur study enrolled 66 adult MMD patients and 61 patients with atherosclerotic vascular disease (ACVD). We investigated the SDF-1 concentration in cerebrospinal fluid (CSF) and CXCR4 expression level on the arachnoid membranes and vascular tissue. We utilized enzyme-linked immunosorbent assay and immunohistochemistr. Additionally, we cultured and stimulated human brain microvascular endothelial cells (HBMECs) and smooth muscle cells (SMCs) under oxygen and glucose deprivation (OGD) conditions followed by reoxygenation, to examine any changes in the SDF-1/CXCR4 axis. ResultsThe results demonstrated an elevation in the level of SDF-1 in CSF among MMD patients compared to those with ACVD. Moreover, the expression of CXCR4 in arachnoid membranes and vascular tissue showed a similar trend. Furthermore, the content of CXCR4 in HBMECs and SMCs increased with the duration of ischemia and hypoxia. However, it was observed that the expression of CXCR4 decreased at OGD/R 24h compared to OGD 24h. The temporal pattern of SDF-1 expression in HBMECs and SMCs mirrored that of CXCR4 expression. ConclusionThese findings indicate a critical role for the SDF-1/CXCR4 axis in the angiogenesis of moyamoya disease.

Enhancing 7-dehydrocholesterol suppresses brain ferroptosis and tissue injury after neonatal hypoxia–ischemia

Neonatal hypoxic-ischemic brain injury (HIBI) results in part from excess reactive oxygen species and iron-dependent lipid peroxidation (i.e. ferroptosis). The vitamin D precursor 7-dehydrocholesterol (7-DHC) may inhibit iron-dependent lipid peroxidation. Primary neurons underwent oxygen and glucose deprivation (OGD) injury and treatment with 7-DHC-elevating medications such as cariprazine (CAR) or vehicle. Postnatal day 9 mice underwent sham surgery or carotid artery ligation and hypoxia and received intraperitoneal CAR. In neurons, CAR administration resulted in significantly increased cell survival compared to vehicle controls, whether administered 48 h prior to or 30 min after OGD, and was associated with increased 7-DHC. In the mouse model, malondialdehyde and infarct area significantly increased after HIBI in the vehicle group, which were attenuated by post-treatment with CAR and were negatively correlated with tissue 7-DHC concentrations. Elevating 7-DHC concentrations with CAR was associated with improved cellular and tissue viability after hypoxic-ischemic injury, suggesting a novel therapeutic avenue.

Exploring the neuroprotection of the combination of astragaloside A, chlorogenic acid and scutellarin in treating chronic cerebral ischemia via network analysis and experimental validation

Chronic cerebral ischemia (CCI) primarily causes cognitive dysfunction and other neurological impairments, yet there remains a lack of ideal therapeutic medications. The preparation combination of Astragalus membranaceus (Fisch.) Bunge and Erigeron breviscapus (Vant.) Hand.-Mazz have been utilized to ameliorate neurological dysfunction following cerebral ischemia, but material basis of its synergy remains unclear. The principal active ingredients and their optimal proportions in this combination have been identified through the oxygen and glucose deprivation (OGD) cell model, including astragaloside A, chlorogenic acid and scutellarin (ACS), and its efficacy in enhancing the survival of OGD PC12 cells surpasses that of the combination preparation. Nevertheless, mechanism of ACS against CCI remains elusive. In this study, 63 potential targets of ACS against CCI injury were obtained by network pharmacology, among which AKT1, CASP3 and TNF are the core targets. Subsequent analysis utilizing KEGG and GO suggested that PI3K/AKT pathway may play a crucial role for ACS in ameliorating CCI injury. Then, a right unilateral common carotid artery occlusion (rUCCAO) mouse model and an OGD PC12 cell model were established to replicate the pathological processes of CCI in vivo and in vitro. These models were utilized to explore the anti-CCI effects of ACS and its regulatory mechanisms, particularly focusing on PI3K/AKT pathway. The results showed that ACS facilitated the restoration of cerebral blood flow in CCI mice, enhanced the function of the central cholinergic nervous system, protected against ischemic nerve cell and mitochondrial damage, and improved cognitive function and other neurological impairments. Additionally, ACS upregulated the expression of p-PI3K, p-AKT, p-GSK3β and Bcl-2, and diminished the expression of Cyto-c, cleaved Caspase-3 and Bax significantly. However, the PI3K inhibitor (LY294002) partially reversed the downregulation of Bax, Cyto-c and cleaved Caspase-3 expression as well as the upregulation of p-AKT/AKT, p-GSK3β/GSK3β, and Bcl-2/Bax ratios. These findings suggest that ACS against neuronal damage in cerebral ischemia may be closely related to the activation of PI3K/AKT pathway. These results declared first time ACS may become an ideal candidate drug against CCI due to its neuroprotective effects, which are mediated by the activated PI3K/AKT pathway mitigates mitochondrial damage and prevents cell apoptosis.

Transcriptome analysis revealed the new mechanism of the intra-myocardial injectable alginate-hydrogel in the treatment of ventricular function degradation.

Myocardial infarction (MI) is one of the most lethal cardiovascular diseases. The loss of cardiomyocytes and the degradation of the extracellular matrix leads to high ventricular wall stress, which further drives the pathological thinning of the ventricular wall during MI. Injecting biomaterials to thicken the infarct ventricular wall provides mechanical support, thereby inhibiting the continued expansion of the heart. As an injectable biomaterial, alginate hydrogel has achieved exciting results in clinical trials, but further research needs to be conducted to determine whether it can improve cardiac function in addition to providing mechanical support. This study sought to explore these mechanisms in an animal model of MI. A MI model was established in male C57BL/6J mice by ligation of the proximal left anterior descending (LAD) coronary artery. Intramyocardial injections (hydrogel or saline group) were performed in the proximal wall regions bordering the infarct area (with one 20-µL injection). Four weeks after MI, RNA sequencing revealed that 342 messenger RNAs (mRNAs) from the infarcted hearts were differentially expressed between the saline group and hydrogel group. We subsequently conducted a Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis to analyze the RNA sequencing data. In addition, we employed both western blotting and quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) techniques to verify a number of genes that were differentially expressed and could potentially affect cardiac function after MI. Subsequently, we confirmed these findings through in vitro experiments. We found that compared with hydrogel treatment group, 250 mRNAs were upregulated and 92 mRNAs were downregulated in saline group (P<0.05). And by exploring the GO and KEGG signaling pathways as well as the protein-protein interaction (PPI) network, we found that administration of alginate hydrogel modulated cardiomyocyte inflammation-associated proteins as well as chemokine-related proteins during the inflammatory response phase after MI. In addition, our analysis at both the protein and RNA level revealed that B2M was effective in improving cardiac function after MI in the hydrogel treatment group, which was consistent in the myocardium oxygen and glucose deprivation (OGD) injury model. We explored the transcriptome changes of infarcted hearts after alginate-hydrogel injection during the inflammatory response period. Our findings suggest that the injectable hydrogel directly alters the inflammatory response and the chemokine-mediated signaling pathway of cardiomyocytes, ultimately improving cardiac function.

Lactate Protects Microglia and Neurons from Oxygen-Glucose Deprivation/Reoxygenation.

Lactate has received attention as a potential therapeutic intervention for brain diseases, particularly those including energy deficit, exacerbated inflammation, and disrupted redox status, such as cerebral ischemia. However, lactate roles in metabolic or signaling pathways in neural cells remain elusive in the hypoxic and ischemic contexts. Here, we tested the effects of lactate on the survival of a microglial (BV-2) and a neuronal (SH-SY5Y) cell lines during oxygen and glucose deprivation (OGD) or OGD followed by reoxygenation (OGD/R). Lactate signaling was studied by using 3,5-DHBA, an exogenous agonist of lactate receptor GPR81. Inhibition of lactate dehydrogenase (LDH) or monocarboxylate transporters (MCT), using oxamate or 4-CIN, respectively, was performed to evaluate the impact of lactate metabolization and transport on cell viability. The OGD lasted 6h and the reoxygenation lasted 24h following OGD (OGD/R). Cell viability, extracellular lactate concentrations, microglial intracellular pH and TNF-ɑ release, and neurite elongation were evaluated. Lactate or 3,5-DHBA treatment during OGD increased microglial survival during reoxygenation. Inhibition of lactate metabolism and transport impaired microglial and neuronal viability. OGD led to intracellular acidification in BV-2 cells, and reoxygenation increased the release of TNF-ɑ, which was reverted by lactate and 3,5-DHBA treatment. Our results suggest that lactate plays a dual role in OGD, acting as a metabolic and a signaling molecule in BV-2 and SH-SY5Y cells. Lactate metabolism and transport are vital for cell survival during OGD. Moreover, lactate treatment and GPR81 activation during OGD promote long-term adaptations that potentially protect cells against secondary cell death during reoxygenation.

Kaempferol regulates apoptosis and migration of neural stem cells to attenuate cerebral infarction by O-GlcNAcylation of β-catenin.

Ischemic stroke remains a major cause of disability and death. Kaempferol (Kae) is a neuroprotective flavonoid compound. Thus, this study aimed to explore the impact of Kae on cerebral infarction. We generated the middle cerebral artery occlusion (MCAO) mouse model to study the effects of Kae on infarction volume and neurological function. The oxygen and glucose deprivation (OGD)/reoxygenation (R) model of neural stem cells (NSCs) was established to study the effects of Kae on cell viability, migration, and apoptosis. Cell processes were assessed by cell counting kit-8, Transwell assay, flow cytometry, and TUNEL analysis. The molecular mechanism was assessed using the Western blot. The results indicated that Kae attenuated MCAO-induced cerebral infarction and neurological injury. Besides, Kae promoted cell viability and migration and inhibited apoptosis of OGD/R-treated NSCs. Moreover, OGD/R suppressed total O-GlcNAcylation level and O-GlcNAcylation of β-catenin, thereby suppressing the Wnt/β-catenin pathway, whereas Kae reversed the suppression. Inactivation of the Wnt/β-catenin pathway abrogated the biological functions of NSCs mediated by Kae. In conclusion, Kae suppressed cerebral infarction by facilitating NSC viability, migration, and inhibiting apoptosis. Mechanically, Kae promoted O-GlcNAcylation of β-catenin to activate the Wnt/β-catenin pathway. Kae may have a lessening effect on ischemic stroke.

Disrupting PIAS3-mediated SUMOylation of MLK3 ameliorates poststroke neuronal damage and deficits in cognitive and sensorimotor behaviors

Activated small ubiquitin-like modifiers (SUMOs) have been implicated in neuropathological processes following ischemic stroke. However, the target proteins of SUMOylation and their contribution to neuronal injury remain to be elucidated. MLK3 (mixed-lineage kinase 3), a member of the mitogen-activated protein kinase kinase kinase (MAPKKK) family, is a critical regulator of neuronal lesions following cerebral ischemia. Here, we found that SUMOylation of MLK3 increases in both global and focal ischemic rodent models and primary neuronal models of oxygen and glucose deprivation (OGD). SUMO1 conjugation at the Lys401 site of MLK3 promoted its activation, stimulated its downstream p38/c-Jun N-terminal kinase (JNK) cascades, and led to cell apoptosis. The interaction of MLK3 with PIAS3, a SUMO ligase, was elevated following ischemia and reperfusion. The PINIT domain of PIAS3 was involved in direct interactions with MLK3. Overexpression of the PINIT domain of PIAS3 disrupted the MLK3-PIAS3 interaction, inhibited SUMOylation of MLK3, suppressed downstream signaling, and reduced cell apoptosis and neurite damage. In rodent ischemic models, the overexpression of the PINIT domain reduced brain lesions and alleviated deficits in learning, memory, and sensorimotor functions. Our findings demonstrate that brain ischemia-induced MLK3 SUMOylation by PIAS3 is a potential target against poststroke neuronal lesions and behavioral impairments.

Floralozone regulates MiR-7a-5p expression through AMPKα2 activation to improve cognitive dysfunction in vascular dementia

BackgroundThe pathogenesis of vascular dementia (VD) is complex, and currently, no effective treatments have been recommended. Floralozone is a colorless liquid first discovered in Lagotis Gaertn. Recently, its medicinal value has been increasingly recognized. Our previous study has demonstrated that Floralozone can improve cognitive dysfunction in rats with VD by regulating the transient receptor potential melastatin 2 (TRPM2) and N-methyl-D-aspartate receptor (NMDAR) signaling pathways. However, the mechanism by which Floralozone regulates TRPM2 and NMDAR to improve VD remains unclear. AMP-activated protein kinase (AMPK) is an energy regulator in vivo; however, its role of AMPK activation in stroke remains controversial. MiR-7a-5p has been identified to be closely related to neuronal function. PurposeTo explore whether Floralozone can regulate the miR-7a-5p level in vivo through AMPKα2 activation, affect the TRPM2 and NR2B expression levels, and improve VD symptoms. MethodsThe VD model was established by a modified bilateral occlusion of the common carotid arteries (2-VO) of Sprague-Dawley (SD) rats and AMPKα2 KO transgenic (AMPKα2−/−) mice. Primary hippocampal neurons were modeled using oxygen and glucose deprivation (OGD). Morris water maze (MWM) test, hematoxylin-eosin staining (HE staining), and TUNEL staining were used to investigate the effects of Floralozone on behavior and hippocampal morphology in rats. Minichromosome maintenance complex component 2(MCM2) positive cells were used to investigate the effect of Floralozone on neurogenesis. Immunofluorescence staining, qRT-PCR, and western blot analysis were used to investigate the effect of Floralozone on the expression levels of AMPKα2, miR-7a-5p, TRPM2, and NR2B. ResultsThe SD rat experiment revealed that Floralozone improved spatial learning and memory, improved the morphology and structure of hippocampal neurons, reduced apoptosis of hippocampal neurons and promoted neurogenesis in VD rats. Floralozone could increase the miR-7a-5p expression level, activate AMPKα2 and NR2B expressions, and inhibit TRPM2 expression in hippocampal neurons of VD rats. The AMPKα2 KO transgenic (AMPKα2−/−) mice experiment demonstrated that Floralozone could regulate miR-7a-5p, TRPM2, and NR2B expression levels through AMPKα2 activation. The cell experiment revealed that the TRPM2 and NR2B expression levels were regulated by miR-7a-5p, whereas the AMPKα2 expression level was not. ConclusionFloralozone could regulate miR-7a-5p expression level by activating the protein expression of AMPKα2, control the protein expression of TRPM2 and NR2B, improve the morphology and structure of hippocampus neurons, reduce the apoptosis of hippocampus neurons, promote neurogenesis and improve the cognitive dysfunction.

Therapeutic Polymer-Based Cannabidiol Formulation: Tackling Neuroinflammation Associated with Ischemic Events in the Brain.

Cannabidiol (CBD) is the most relevant nonpsychostimulant phytocompound found in Cannabis sativa. CBD has been extensively studied and has been proposed as a therapeutic candidate for neuroinflammation-related conditions. However, being a highly lipophilic drug, it has several drawbacks for pharmaceutical use, including low solubility and high permeability. Synthetic polymers can be used as drug delivery systems to improve CBD's stability, half-life, and biodistribution. Here, we propose using a synthetic polymer as a nanoparticulate vehicle for CBD (NPCBD) to overcome the pharmacological drawbacks of free drugs. We tested the NPCBD-engineered system in the context of ischemic events in a relevant oxygen and glucose deprivation (OGD) model in primary cortical cells (PCC). Moreover, we have characterized the inflammatory response of relevant cell types, such as THP-1 (human monocytes), HMC3 (human microglia), and PCC, to NPCBD and observed a shift in the inflammatory state of the treated cells after the ischemic event. In addition, NPCBD exhibited a promising ability to restore mitochondrial function after OGD insult in both HMC3 and PCC cells at low doses of 1 and 0.2 μM CBD. Taken together, these results suggest the potential for preclinical use.

Glutathione supplementation improves fat graft survival by inhibiting ferroptosis via the SLC7A11/GPX4 axis

BackgroundAutologous fat grafting is hampered by unpredictable graft survival, which is potentially regulated by ferroptosis. Glutathione (GSH), a powerful antioxidant used in tissue preservation, has ferroptosis-regulating activity; however, its effects on fat grafts are unclear. This study investigated the effects and mechanisms of GSH in fat graft survival.MethodsHuman lipoaspirates were transplanted subcutaneously into the backs of normal saline-treated (control) or GSH-treated nude mice. Graft survival was evaluated by magnetic resonance imaging and histology. RNA sequencing was performed to identify differentially expressed genes and enriched pathways. GSH activity was evaluated in vitro using an oxygen and glucose deprivation (OGD) model of adipose-derived stem cells.ResultsCompared with control group, GSH induced better outcomes, including superior graft retention, appearance, and histological structures. RNA sequencing suggested enhanced negative regulation of ferroptosis in the GSH-treated grafts, which showed reduced lipid peroxides, better mitochondrial ultrastructure, and SLC7A11/GPX4 axis activation. In vitro, OGD-induced ferroptosis was ameliorated by GSH, which restored cell proliferation, reduced oxidative stress, and upregulated ferroptosis defense factors.ConclusionsOur study confirms that ferroptosis participates in regulating fat graft survival and that GSH exerts a protective effect by inhibiting ferroptosis. GSH-assisted lipotransfer is a promising therapeutic strategy for future clinical application.

YAP promotes the healing of ischemic wounds by reducing ferroptosis in skin fibroblasts through inhibition of ferritinophagy

The impaired healing of chronic wounds is often attributed to the ischemic and hypoxic microenvironment, leading to increased cell death. Ferroptosis, a novel form of cell death unveiled in recent years, could potentially be linked with the process of wound healing. In this study, we explored the significance and mechanism of ferroptosis in ischemic wounds. Using transmission electron microscopy, Western blot, flow cytometry, immunofluorescence, and glutathione (GSH) assay, we observed that the death of primary mouse skin fibroblasts induced by oxygen and glucose deprivation (OGD) was associated with ferroptosis. Specifically, we observed elevated intracellular Fe2+ and lipid peroxidation levels and decreased GSH levels in vitro, indicative of ferroptosis. Importantly, we found that ferroptosis in OGD-treated skin fibroblasts was dependent on autophagy, as the autophagy inhibitor chloroquine phosphate (CHQ) significantly reduced ferroptosis induced by OGD. Moreover, our study revealed that NCOA4-mediated ferritinophagy significantly contributed to the occurrence of ferroptosis induced by OGD in skin fibroblasts. Additionally, we identified the involvement of YAP in the regulation of ferritinophagy, with YAP suppressing NCOA4 expression in OGD-treated skin fibroblasts, thereby reducing ferroptosis. Furthermore, in ischemic wound models in mice, both inhibitors of ferroptosis and autophagy promoted wound healing, while a YAP inhibitor, verteporfin, delayed wound healing. In conclusion, these findings indicate that ferroptosis, regulated by YAP through ferritinophagy inhibition, presents a novel mechanism responsible for the delayed healing of ischemic wounds. Understanding this process could offer promising therapeutic targets to improve wound healing in ischemic conditions.

Kaempferol-3-O-(2″-O-galloyl-β-D-glucopyranoside): a novel neuroprotective agent from Diospryros kaki against cerebral ischemia-induced brain injury.

Our previous study demonstrated neuroprotective and therapeutic effects of a standardized flavonoid extract from leaves of Diospyros kaki L.f. (DK) on middle cerebral artery occlusion-and-reperfusion (MCAO/R)-induced brain injury and its underlying mechanisms. This study aimed to clarify flavonoid components responsible for the effects of DK using in vitro and in vivo transient brain ischemic models. Organotypic hippocampal slice cultures (OHSCs) subjected to oxygen- and glucose-deprivation (OGD) were performed to evaluate in vitro neuroprotective activity of DK extract and nine isolated flavonoid components. MCAO/R mice were employed to elucidate in vivo neuroprotective effects of the flavonoid component that exhibited the most potent neuroprotective effect in OHSCs. DK extract and seven flavonoids [quercetin, isoquercetin, hyperoside, quercetin-3-O-(2″-O-galloyl-β-D-galactopyranoside), kaempferol, astragalin, and kaempferol-3-O-(2″-O-galloyl-β-D-glucopyranoside) compound (9)] attenuated OGD-induced neuronal cell damage and compound (9) possessed the most potent neuroprotective activity in OHSCs. The MCAO/R mice showed cerebral infarction, massive weight loss, characteristic neurological symptoms, and deterioration of neuronal cells in the brain. Compound (9) and a reference drugs, edaravone, significantly attenuated these physical and neurological impairments. Compound (9) mitigated the blood-brain barrier dysfunction and the change of glutathione and malondialdehyde content in the MCAO mouse brain. Edaravone suppressed the oxidative stress but did not significantly affect the blood-brain barrier permeability. The present results indicated that compound (9) is a flavonoid constituent of DK with a potent neuroprotective activity against transient ischemia-induced brain damage and this action, at least in part, via preservation of blood-brain barrier integrity and suppression of oxidative stress caused by ischemic insult.

MicroRNA-126-3P targets PIK3R2 to ameliorate autophagy and apoptosis of cortex in hypoxia-reoxygenation treated neonatal rats.

Here, we explored a possible mechanism of microRNA-126-3p (miR-126-3p) on neonatal rats with hypoxia-reoxygenation injury (HI). After administering HI to newborn Sprague-Dawley rats, the expression of miR-126-3p in the brain injury was assessed by RT-PCR. A miR-126-3p mimic and inhibitor were treated in the HI neonatal rats. The water maze test, TTC, HE, Nissl and TUNEL staining were separately implemented to test the effects of miR-126-3p on the HI-treated neonatal rats. At the same time, the phosphoinositide-3-kinase regulatory subunit 2 (PIK3R2) expression in the damaged cortex region was analyzed. In vitro, cortical neurons were cultured and treated with oxygen and glucose deprivation (OGD), then transfected miR-126-3p mimic, PIK3R2 overexpression lentivirus vector or silence of PIK3R2. The cell viability was observed by CCK-8. The autophagy of neurons was detected by acridine orange staining. In contrast to the sham-operated rats, the miR-126-3p expression significantly decreased, but PIK3R2 expression markedly rose in the cortex of HI rats. Injection of miR-126-3p mimic raised the learning and memory abilities through down-regulating the cerebral ischemic area, improving pathological damage of the cortex, reducing the neurons apoptosis of the cortex and down-regulating the autophagy-related and apoptosis-related proteins. Overexpression of PIK3R2, a miR-126-3p target, may reduce cell viability and boost autophagy and apoptosis. Silence of PIK3R2 promoted cell viability and inhibited cell apoptosis and autophagy. The consequences of miR-126-3p were comparable to those of PIK3R2 silencing. A new therapeutic target for HI injury in newborn rats is provided by the overexpression of miR-126-3p, which inhibits autophagy and death of cortical neurons by targeting PIK3R2 in HI-treated neonatal rats.

Homer1 Protects against Retinal Ganglion Cell Pyroptosis by Inhibiting Endoplasmic Reticulum Stress-Associated TXNIP/NLRP3 Inflammasome Activation after Middle Cerebral Artery Occlusion-Induced Retinal Ischemia.

Retinal ischemia, after cerebral ischemia, is an easily overlooked pathophysiological problem in which inflammation is considered to play an important role. Pyroptosis is a kind of cell death pattern accompanied by inflammation. Homer scaffold protein 1 (Homer1) has anti-inflammation properties and protects against ischemic injury. However, little is known about pyroptosis following middle cerebral artery occlusion (MCAO)-induced retinal ischemia and the regulatory mechanisms involved by Homer1 for the development of pyroptosis. In the present study, retinal ischemic injury was induced in mice by permanent MCAO in vivo, and retinal ganglion cells (RGCs) were subjected to Oxygen and Glucose Deprivation (OGD) to establish an in vitro model. It was shown that TXNIP/NLRP3-mediated pyroptosis was located predominantly in RGCs, which gradually increased after retinal ischemia and peaked at 24 h after retinal ischemia. Interestingly, the RGCs pyroptosis occurred not only in the cell body but also in the axon. Notably, the occurrence of pyroptosis coincided with the change of Homer1 expression in the retina after retinal ischemia and Homer1 also co-localized with RGCs. It was demonstrated that overexpression of Homer1 not only alleviated RGCs pyroptosis and inhibited the release of pro-inflammatory factors but also led to the increase in phosphorylation of AMPK, inhibition of ER stress, and preservation of visual function after retinal ischemia. In conclusion, it was suggested that Homer1 may protect against MCAO-induced retinal ischemia and RGCs pyroptosis by inhibiting endoplasmic reticulum stress-associated TXNIP/NLRP3 inflammasome activation after MCAO-induced retinal ischemia.

Neuron-secreted NLGN3 ameliorates ischemic brain injury via activating Gαi1/3-Akt signaling

We here tested the potential activity and the underlying mechanisms of neuroligin-3 (NLGN3) against ischemia-reperfusion-induced neuronal cell injury. In SH-SY5Y neuronal cells and primary murine cortical neurons, NLGN3 activated Akt-mTOR and Erk signalings, and inhibited oxygen and glucose deprivation (OGD)/re-oxygenation (OGD/R)-induced cytotoxicity. Akt activation was required for NLGN3-induced neuroprotection. Gαi1/3 mediated NLGN3-induced downstream signaling activation. NLGN3-induced Akt-S6K1 activation was largely inhibited by Gαi1/3 silencing or knockout. Significantly, NLGN3-induced neuroprotection against OGD/R was almost abolished by Gαi1/3 silencing or knockout. In vivo, the middle cerebral artery occlusion (MCAO) procedure induced NLGN3 cleavage and secretion, and increased its expression and Akt activation in mouse brain tissues. ADAM10 (A Disintegrin and Metalloproteinase 10) inhibition blocked MCAO-induced NLGN3 cleavage and secretion, exacerbating ischemic brain injury in mice. Neuronal silencing of NLGN3 or Gαi1/3 in mice also inhibited Akt activation and intensified MCAO-induced ischemic brain injury. Conversely, neuronal overexpression of NLGN3 increased Akt activation and alleviated MCAO-induced ischemic brain injury. Together, NLGN3 activates Gαi1/3-Akt signaling to protect neuronal cells from ischemia-reperfusion injury.

Resveratrol Prevents Cell Swelling Through Inhibition of SUR1 Expression in Brain Micro Endothelial Cells Subjected to OGD/Recovery.

The SUR1-TRPM4-AQP4 complex is overexpressed in the initial phase of edema induced after cerebral ischemia, allowing the massive internalization of Na+ and water within the brain micro endothelial cells (BMEC) of the blood-brain barrier. The expression of the Abcc8 gene encoding SUR1 depends on transcriptional factors that are responsive to oxidative stress. Because reactive oxygen species (ROS) are generated during cerebral ischemia, we hypothesized that antioxidant compounds might be able to regulate the expression of SUR1. Therefore, the effect of resveratrol (RSV) on SUR1 expression was evaluated in the BMEC cell line HBEC-5i subjected to oxygen and glucose deprivation (OGD) for 2 h followed by different recovery times. Different concentrations of RSV were administered. ROS production was detected with etidine, and protein levels were evaluated by Western blotting and immunofluorescence. Intracellular Na+ levels and cellular swelling were detected by imaging; cellular metabolic activity and rupture of the cell membrane were detected by MTT and LDH release, respectively; and EMSA assays measured the activity of transcriptional factors. OGD/recovery increased ROS production induced the AKT kinase activity and the activation of SP1 and NFκB. SUR1 protein expression and intracellular Na+ concentration in the HBEC-5i cells increased after a few hours of OGD. These effects correlated with cellular swelling and necrotic cell death, responses that the administration of RSV prevented. Our results indicate that the ROS/AKT/SP1-NFκB pathway is involved in SUR1 expression during OGD/recovery in BMEC of the blood-brain barrier. Thus, RSV prevented cellular edema formation through modulation of SUR1 expression.

Endothelial cell-derived RSPO3 activates Gαi1/3-Erk signaling and protects neurons from ischemia/reperfusion injury

The current study explores the potential function and the underlying mechanisms of endothelial cell-derived R-spondin 3 (RSPO3) neuroprotection against ischemia/reperfusion-induced neuronal cell injury. In both neuronal cells (Neuro-2a) and primary murine cortical neurons, pretreatment with RSPO3 ameliorated oxygen and glucose deprivation (OGD)/re-oxygenation (OGD/R)-induced neuronal cell death and oxidative injury. In neurons RSPO3 activated the Akt, Erk and β-Catenin signaling cascade, but only Erk inhibitors reversed RSPO3-induced neuroprotection against OGD/R. In mouse embryonic fibroblasts (MEFs) and neuronal cells, RSPO3-induced LGR4-Gab1-Gαi1/3 association was required for Erk activation, and either silencing or knockout of Gαi1 and Gαi3 abolished RSPO3-induced neuroprotection. In mice, middle cerebral artery occlusion (MCAO) increased RSPO3 expression and Erk activation in ischemic penumbra brain tissues. Endothelial knockdown or knockout of RSPO3 inhibited Erk activation in the ischemic penumbra brain tissues and increased MCAO-induced cerebral ischemic injury in mice. Conversely, endothelial overexpression of RSPO3 ameliorated MCAO-induced cerebral ischemic injury. We conclude that RSPO3 activates Gαi1/3-Erk signaling to protect neuronal cells from ischemia/reperfusion injury.

VEGF-A ameliorates ischemia hippocampal neural injury via regulating autophagy and Akt/CREB signaling in a rat model of chronic cerebral hypoperfusion

ObjectiveChronic cerebral hypoperfusion (CCH) can cause a series of pathophysiological processes, including neuronal autophagy and apoptosis. VEGF-A has been reported to affect angiogenesis and neurogenesis in many CNS diseases. However, its effects on neuronal autophagy and apoptosis, as well as the underlying mechanisms in CCH remain unclear. MethodsTo address these issues, the CCH model was established by permanent bilateral common carotid artery occlusion (2VO). Rats were sacrificed at different stages of CCH. Hippocampal morphological and ultrastructural changes were detected using HE staining and electron microscopy. The immunoreactivities of microtubule-associated protein 1 light chain 3 (LC3) and phospho-cAMP response element binding protein (p-CREB) were examined by immunofluorescence staining. The neuronal apoptosis was detected via TUNEL staining. The levels of LC3-II, Beclin-1, Akt, p-Akt, CREB, p-CREB, Caspase-3, and Bad were accessed by Western blotting. Furthermore, mouse hippocampal HT22 neurons received the oxygen and glucose deprivation (OGD) treatment, VEGF-A treatment, and GSK690693 (an Akt inhibitor) treatment, respectively. ResultsLC3-II protein started to increase at 3 days of CCH, peaked at 4 weeks of CCH, then decreased. CCH increased the levels of LC3-II, Caspase-3, and Bad, and decreased the levels of p-Akt, CREB, and p-CREB, which were reversed by VEGF-A treatment. VEGF-A also improved CCH-induced neuronal ultrastructural injuries and apoptosis in the hippocampus in vitro. In HT22, the anti-apoptosis and pro-phosphorylation of VEGF-A were reversed by GSK690693. ConclusionPresent results provide a novel neuroprotective effect of VEGF-A in CCH that is related to the inhibition of neuronal autophagy and activation of the Akt/CREB signaling, suggesting a potential therapeutic strategy for ischemic brain damage.