N6022 attenuates cerebral ischemia/reperfusion injury-induced microglia ferroptosis by promoting Nrf2 nuclear translocation and inhibiting the GSNOR/GSTP1 axis.

Stroke poses a significant risk of mortality, particularly among the elderly population. The pathophysiological process of ischemic stroke is complex, and it is crucial to elucidate its molecular mechanisms and explore potential protective drugs. Ferroptosis, a newly recognized form of programmed cell death distinct from necrosis, apoptosis, and autophagy, is closely associated with the pathophysiology of ischemic stroke. N6022, a selective inhibitor of S-nitrosoglutathione reductase (GSNOR), is a "first-in-class" drug for asthma with potential therapeutic applications. However, it remains unclear whether N6022 exerts protective effects in ischemic stroke, and the precise mechanisms of its action are unknown. This study aimed to investigate whether N6022 mitigates cerebral ischemia/reperfusion (I/R) injury by reducing ferroptosis and to elucidate the underlying mechanisms. Accordingly, we established an oxygen-glucose deprivation/reperfusion (OGD/R) cell model and a middle cerebral artery occlusion/reperfusion (MCAO/R) mouse model to mimic cerebral I/R injury. Our data, both in vitro and in vivo, demonstrated that N6022 effectively protected against I/R-induced brain damage and neurological deficits in mice, as well as OGD/R-induced BV2 cell damage. Mechanistically, N6022 promoted Nrf2 nuclear translocation, enhancing intracellular antioxidant capacity of SLC7A11-GPX4 system. Furthermore, N6022 interfered with the interaction of GSNOR with GSTP1, thereby boosting the antioxidant capacity of GSTP1 and attenuating ferroptosis. These findings provide novel insights, showing that N6022 attenuates microglial ferroptosis induced by cerebral I/R injury through the promotion of Nrf2 nuclear translocation and inhibition of the GSNOR/GSTP1 axis.

Therapeutic strategies targeting the NLRP3­mediated inflammatory response and pyroptosis in cerebral ischemia/reperfusion injury (Review).

Ischemic stroke poses a major threat to human health. Therefore, the molecular mechanisms of cerebral ischemia/reperfusion injury (CIRI) need to be further clarified, and the associated treatment approaches require exploration. The NOD­like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome serves an important role in causing CIRI, and its activation exacerbates the underlying injury. Activation of the NLRP3 inflammasome triggers the maturation and production of the inflammatory molecules IL­1ß and IL­18, as well as gasdermin­D­mediated pyroptosis and CIRI damage. Thus, the NLRP3 inflammasome may be a viable target for the treatment of CIRI. In the present review, the mechanisms of the NLRP3 inflammasome in the intense inflammatory response and pyroptosis induced by CIRI are discussed, and the therapeutic strategies that target the NLRP3­mediated inflammatory response and pyroptosis in CIRI are summarized. At present, certain drugs have already been studied, highlighting future therapeutic perspectives.

Lycium Barbarum polysaccharides ameliorates hyperglycemia-exacerbated cerebral ischemia/reperfusion injury via protecting blood-brain barrier.

BACKGROUND: Hyperglycemia exacerbates brain damage in cerebral ischemia/reperfusion injury. Previous study found that Lycium barbarum polysaccharides (LBP) has a neuroprotective effect on hyperglycemia-aggravated ischemic brain injury, which raising the possibility for treatment of neurodegenerative diseases. However, the underlying mechanism of LBP-induced protection by ameliorating hyperglycemia-aggravated ischemia/reperfusion injury needs to be tested. This study aimed to investigate the effects of LBP on blood-brain barrier (BBB) integrity with a hyperglycemia-aggravated cerebral ischemia/reperfusion injury model. METHODS: Sprague-Dawley male rats were randomly divided into three groups: normoglycemic (NG), hyperglycemic (HG), and LBP-pretreated hyperglycemic (HG + LBP). Animals underwent middle cerebral artery occlusion (MCAO) for 30 min, followed by 1-, 3-, and 7-day of reperfusion. RESULTS: Our results showed that the neurological deficit, infarct volume, cell apoptosis, and IgG leakage in the HG group significantly increased separately, compared with that of the NG group, (p < 0.05). Pre-treatment with LBP reversed these injury indicators (p < 0.05). And much more severe degree of swelling endothelium, swollen astrocyte, and decreased tight junctions in the micro-vessel were detected in the HG group comparing to that of the NG group. In addition, increased degree of basement membrane degradation, dissociation between the astrocyte endfeet and basement membrane, and tight junction's protein degradation was found in the HG group compared with the NG group (p < 0.05). However, when exposure to LBP therapy could reverse the above alterations (p < 0.05). CONCLUSIONS: These results demonstrated that LBP could ameliorate hyperglycemia-exacerbated cerebral ischemia/reperfusion injury via protecting the blood-brain barrier.

A plant-based mutant huntingtin model-driven discovery of impaired expression of GTPCH and DHFR.

Pathophysiology associated with Huntington's disease (HD) has been studied extensively in various cell and animal models since the 1993 discovery of the mutant huntingtin (mHtt) with abnormally expanded polyglutamine (polyQ) tracts as the causative factor. However, the sequence of early pathophysiological events leading to HD still remains elusive. To gain new insights into the early polyQ-induced pathogenic events, we expressed Htt exon1 (Httex1) with a normal (21), or an extended (42 or 63) number of polyQ in tobacco plants. Here, we show that transgenic plants accumulated Httex1 proteins with corresponding polyQ tracts, and mHttex1 induced protein aggregation and affected plant growth, especially root and root hair development, in a polyQ length-dependent manner. Quantitative proteomic analysis of young roots from severely affected Httex1Q63 and unaffected Httex1Q21 plants showed that the most reduced protein by polyQ63 is a GTP cyclohydrolase I (GTPCH) along with many of its related one-carbon (C1) metabolic pathway enzymes. GTPCH is a key enzyme involved in folate biosynthesis in plants and tetrahydrobiopterin (BH4) biosynthesis in mammals. Validating studies in 4-week-old R6/2 HD mice expressing a mHttex1 showed reduced levels of GTPCH and dihydrofolate reductase (DHFR, a key folate utilization/alternate BH4 biosynthesis enzyme), and impaired C1 and BH4 metabolism. Our findings from mHttex1 plants and mice reveal impaired expressions of GTPCH and DHFR and may contribute to a better understanding of mHtt-altered C1 and BH4 metabolism, and their roles in the pathogenesis of HD.

Prenatal GABAB Receptor Agonist Administration Corrects the Inheritance of Autism-Like Core Behaviors in Offspring of Mice Prenatally Exposed to Valproic Acid.

This study was performed to evaluate the effects of prenatal baclofen (a GABAB receptor agonist) treatment on the inheritance of autism-like behaviors in valproic acid (VPA)-exposed mice. VPA model mice (first generation, F1) that were prenatally exposed to VPA exhibited robust core autism-like behaviors, and we found that oral administration of baclofen to F1 mice corrected their autism-like behavioral phenotypes at an early age. Based on a previous epigenetics study, we mated the F1 male offspring with litter females to produce the second generation (F2). The F2 male mice showed obvious inheritance of autism-like phenotypes from F1 mice, implying the heritability of autism symptoms in patients with prenatal VPA exposure. Furthermore, we found prenatal baclofen administration was associated with beneficial effects on the autism-like phenotype in F2 male mice. This may have involved corrections in the density of total/mature dendritic spines in the hippocampus (HC) and medial prefrontal cortex (mPFC), normalizing synaptic plasticity. In this research, GABAB receptor agonist administration corrected the core autism-like behaviors of F1 mice and protected against the inheritance of neurodevelopmental disorders in the offspring of F1 mice, suggesting the potential of early intervention with GABAB receptor agonists in the treatment of neurodevelopmental disorders.

The GABAB receptor agonist STX209 reverses the autism­like behaviour in an animal model of autism induced by prenatal exposure to valproic acid.

Autism spectrum disorder (ASD) is a lifelong neurodevelopmental condition characterized by impaired social interaction, compromised communication, and restrictive or stereotyped behaviours and interests. Due to the complex pathophysiology of ASD, there are currently no available medical therapies for improving the associated social deficits. Consequently, the present study investigated the effects of STX209, a selective γ­aminobutyric acid type B receptor (GABABR2) agonist, on an environmental rodent model of autism. The mouse model of autism induced by prenatal exposure to valproic acid (VPA) was used to assess the therapeutic potential of STX209 on autism­like behaviour in the present study. This study investigated the effects of STX209 on VPA model mice via behavioral testing and revealed a significant reversal of core/associated autism­like behavior, including sociability and preference for social novelty, novelty recognition, locomotion and exploration activity and marble­burying deficit. This may be associated with STX209 correcting dendritic arborization, spine density and GABABR2 expression in hippocampus of VPA model mice. However, expression of glutamic acid decarboxylase 65/67 in the hippocampus were not altered by STX209. The present results demonstrated that STX209 administration ameliorated autism­like symptoms in mice exposed to VPA prenatally, suggesting that autism­like symptoms in children with a history of prenatal VPA exposure may also benefit from treatment with the GABABR2 agonist STX209.

Oxymatrine Alleviates Hyperglycemic Cerebral Ischemia/Reperfusion Injury via Protecting Microvessel.

Hyperglycemia aggravates cerebral ischemia/reperfusion (I/R) injury via vascular injury. There is still a lack of effective pharmaceutical preparations for cerebral I/R injury under hyperglycemia. This study aimed to investigate the effects of oxymatrine (OMT) on hyperglycemia-exacerbated cerebral I/R injury in vitro and in vivo. The middle cerebral artery occlusion (MCAO) and reperfusion was established in the rats under hyperglycemia. Meanwhile, oxygen-glucose deprivation and reoxygenation (OGD/R) with high glucose was used as an in vitro model of hyperglycemic cerebral I/R injury. The results showed that the neurological deficit score, mortality, infarct volume and penumbra apoptosis in hyperglycemia group were significantly higher than those in normal glucose group. OMT pre-treated obviously reduced the degree of neurological deficit, mortality, infarct volume, improve cerebral blood flow after I/R in rats with hyperglycemia, and increase the survival rate of human brain microvascular endothelial cells (HBMECs) in high glucose and OGD/R group. OMT significantly improved the ultrastructure changes of endothelial cells, and maintain the migration and angiogenesis potency of HBMECs in high glucose and OGD/R group. OMT obviously alleviated the down-regulating CD31 and CD105 expression in cerebral microvessels caused by hyperglycemia. It is concluded that OMT treatment might alleviate cerebral I/R injury under hyperglycemia via protecting microvessels.

A Novel Plant-Produced Asialo-rhuEPO Protects Brain from Ischemic Damage Without Erythropoietic Action.

Mammalian cell-produced recombinant human erythropoietin (rhuEPOM) has been shown to be a multimodal neuroprotectant targeting an array of key pathological mechanisms in experimental stroke models. However, the rhuEPOM clinical trials were terminated due to increased risk of thrombosis, largely ascribed to its erythropoietic function. We recently took advantage of a plant-based expression system lacking sialylation capacity to produce asialo-rhuEPOP, a rhuEPO derivative without sialic acid residues. In the present study, we proved that asialo-rhuEPOP is non-erythropoietic by repeated intravenous injection (44 µg/kg bw) in mice showing no increase in hemoglobin levels and red blood cell counts, and confirmed that it is non-immunogenic by measuring humoral response after immunizing the mice. We demonstrate that it is neuroprotective in a cerebral ischemia and reperfusion (I/R) mouse model, exhibiting ~ 50% reduction in cerebral infarct volume and edema, and significant improvement in neurological deficits and histopathological outcome. Our studies further revealed that asialo-rhuEPOP, like rhuEPOM, displays pleiotropic neuroprotective effects, including restoring I/R-interrupted mitochondrial fission and fusion proteins, preventing I/R injury-induced increase in mitophagy and autophagy markers, and inhibiting apoptosis to benefit nerve cell survival. Most importantly, asialo-rhuEPOP lacking erythropoietic activity and immunogenicity holds great translational potential as a multimodal neuroprotectant for stroke treatment.

Uncoupling Protein 2 Deficiency Enhances NLRP3 Inflammasome Activation Following Hyperglycemia-Induced Exacerbation of Cerebral Ischemia and Reperfusion Damage In Vitro and In Vivo.

Mitochondrial uncoupling protein 2 (UCP2) deficiency exacerbates brain damage following cerebral ischemia/reperfusion (I/R). The Nod-like receptor protein-3 (NLRP3) inflammasome also plays a vital role in cerebral I/R damage. However, the effect of UCP2 on NLRP3 inflammasome-mediated hyperglycemia and I/R damage is not clear. In the present study, UCP2-knockout (UCP2-/-) and wild-type (WT) mice were used to establish a model of middle cerebral artery occlusion (MCAO) and reperfusion under normo- and hyperglycemic conditions. HT22 cells were established as a model of oxygen-glucose deprivation and reoxygenation (OGD/R) with high glucose to mimic hyperglycemia and I/R in vitro. HT22 cells were treated with/without different concentrations of the UCP2-specific inhibitor genipin for different periods of time. The results showed that UCP2 deficiency significantly increased histopathological changes and apoptosis after cerebral I/R damage in hyperglycemic mice. Moreover, UCP2 deficiency enhanced NLRP3 inflammasome activation in neurons when cerebral I/R damage was exacerbated by hyperglycemia. Furthermore, UCP2 deficiency enhanced NLRP3 inflammasome activation and reactive oxygen species (ROS) production in HT22 cells under OGD/R and high-glucose conditions. UCP2 deficiency aggravated hyperglycemia-induced exacerbation of cerebral I/R damage. UCP2 deficiency also enhanced NLRP3 inflammasome activation and ROS production in neurons in vitro and in vivo. These findings suggest that UCP2 deficiency enhances NLRP3 inflammasome activation following hyperglycemia-induced exacerbation of cerebral I/R damage in vitro and in vivo. UCP2 may be a potential therapeutic target for hyperglycemia-induced exacerbation of cerebral I/R damage.

Deletion of mitochondrial uncoupling protein 2 exacerbates mitophagy and cell apoptosis after cerebral ischemia and reperfusion injury in mice.

Objective: Uncoupling protein 2 (UCP2) is a member of inner mitochondrial membrane proteins and deletion of UCP2 exacerbates brain damage after cerebral ischemia/reperfusion (I/R). Nevertheless, its functional role during cerebral I/R is not entirely understood. The objective of present study was to explore the influence of UCP2 deletion on mitochondrial autophagy (mitophagy) and mitochondria-mediated cell death pathway after cerebral I/R. Methods: UCP2-/- and wildtype (WT) mice were subjected to 60 min middle cerebral artery occlusion (MCAO) and allowed reperfusion for 24 hours. Infarct volume and histological outcomes were assessed, reactive oxygen species (ROS) and autophagy markers were measured, and mitochondrial ultrastructure was examined. Results: Deletion of UCP2 enlarged infarct volume, increased numbers of necrotic and TUNEL positive cells, and significantly increased pro-apoptotic protein levels in UCP2-/- mice compared with WT mice subjected to the same duration of I/R. Further, deletion of UCP2 increased ROS production, elevated LC3, Beclin1 and PINK1, while it suppressed p62 compared with respective WT ischemic controls. Electron microscopic study demonstrated the number of autophagosomes was higher in the UCP2-/- group, compared with the WT group. Conclusions: It is concluded that deletion of UCP2 exacerbates cerebral I/R injury via reinforcing mitophagy and cellular apoptosis in mice.

Deletion of Mitochondrial Uncoupling Protein 2 Exacerbates Mitochondrial Damage in Mice Subjected to Cerebral Ischemia and Reperfusion Injury under both Normo- and Hyperglycemic Conditions.

Deletion of mitochondrial uncoupling protein 2 (UCP2) has been shown to aggravate ischemic damage in the brain. However, the underlying mechanisms are not fully understood. The objective of this study is to explore the impact of homozygous UCP2 deletion (UCP2-/-) on mitochondrial fission and fusion dynamic balance in ischemic mice under normo- and hyperglycemic conditions. UCP2-/- and wildtype mice were subjected to a 60 min middle cerebral artery occlusion (MCAO) and allowed reperfusion for 6h, 24h and 72h. Our results demonstrated that deletion of UCP2 enlarged infarct volumes and increased numbers of cell death in both normo- and hyperglycemic ischemic mice compared with their wildtype counterparts subjected to the same duration of ischemia and reperfusion. The detrimental effects of UCP deletion were associated with increased ROS production, elevated mitochondrial fission markers Drp1 and Fis1 and suppressed fusion markers Opa1 and Mfn2 in UCP2-/- mice. Electron microscopic study demonstrated a marked mitochondrial swolling after 6h of reperfusion in UCP2-/- mice, contrasting to a mild mitochondrial swolling in wildtype ischemic animals. It is concluded that the exacerbating effects of UCP2-/- on ischemic outcome in both normo- and hyperglycemic animals are associated with increased ROS production, disturbed mitochondrial dynamic balance towards fission and early damage to mitochondrial ultrastructure.

Coenzyme Q10 protects astrocytes from ROS-induced damage through inhibition of mitochondria-mediated cell death pathway.

Coenzyme Q10 (CoQ10) acts by scavenging reactive oxygen species to protect neuronal cells against oxidative stress in neurodegenerative diseases. The present study was designed to examine whether CoQ10 was capable of protecting astrocytes from reactive oxygen species (ROS) mediated damage. For this purpose, ultraviolet B (UVB) irradiation was used as a tool to induce ROS stress to cultured astrocytes. The cells were treated with 10 and 25 µg/ml of CoQ10 for 3 or 24 h prior to the cells being exposed to UVB irradiation and maintained for 24 h post UVB exposure. Cell viability was assessed by MTT conversion assay. Mitochondrial respiration was assessed by respirometer. While superoxide production and mitochondrial membrane potential were measured using fluorescent probes, levels of cytochrome C (cyto-c), cleaved caspase-9, and caspase-8 were detected using Western blotting and/or immunocytochemistry. The results showed that UVB irradiation decreased cell viability and this damaging effect was associated with superoxide accumulation, mitochondrial membrane potential hyperpolarization, mitochondrial respiration suppression, cyto-c release, and the activation of both caspase-9 and -8. Treatment with CoQ10 at two different concentrations started 24 h before UVB exposure significantly increased the cell viability. The protective effect of CoQ10 was associated with reduction in superoxide, normalization of mitochondrial membrane potential, improvement of mitochondrial respiration, inhibition of cyto-c release, suppression of caspase-9. Furthermore, CoQ10 enhanced mitochondrial biogenesis. It is concluded that CoQ10 may protect astrocytes through suppression of oxidative stress, prevention of mitochondrial dysfunction, blockade of mitochondria-mediated cell death pathway, and enhancement of mitochondrial biogenesis.