Oxygen-glucose Deprivation/reoxygenation Research Articles

Mitochondrial membrane potential and oxidative stress interact to regulate Oma1-dependent processing of Opa1 and mitochondrial dynamics.

Mitochondrial form and function are regulated by the opposing forces of mitochondrial dynamics: fission and fusion. Mitochondrial dynamics are highly active and consequential during neuronal ischemia/reperfusion (I/R) injury. Mitochondrial fusion is executed at the mitochondrial inner membrane by Opa1. The balance of long (L-Opa1) and proteolytically cleaved short (S-Opa1) isoforms is critical for efficient fusion. Oma1 is the predominant stress-responsive protease for Opa1 processing. In neuronal cell models, we assessed Oma1 and Opa1 regulation during mitochondrial stress. In an immortalized mouse hippocampal neuron line (HT22), Oma1 was sensitive to mitochondrial membrane potential depolarization (rotenone, FCCP) and hyperpolarization (oligomycin). Further, oxidative stress was sufficient to increase Oma1 activity and necessary for depolarization-induced proteolysis. We generated Oma1 knockout (KO) HT22 cells that displayed normal mitochondrial morphology and fusion capabilities. FCCP-induced mitochondrial fragmentation was exacerbated in Oma1 KO cells. However, Oma1 KO cells were better equipped to perform restorative fusion after fragmentation, presumably due to preserved L-Opa1. We extended our investigations to a combinatorial stress of neuronal oxygen-glucose deprivation and reoxygenation (OGD/R), where we found that Opa1 processing and Oma1 activation were initiated during OGD in an ROS-dependent manner. These findings highlight a novel dependence of Oma1 on oxidative stress in response to depolarization. Further, we demonstrate contrasting fission/fusion roles for Oma1 in the acute response and recovery stages of mitochondrial stress. Collectively, our results add intersectionality and nuance to the previously proposed models of Oma1 activity.

Knockdown of IGF2BP3 Down-Regulates PDCD4 Levels to Attenuate Hypoxic-Ischemic Brain Damage

Background: Hypoxic-ischemic brain damage (HIBD) is a prevalent brain injury with high mortality and morbidity. It results from hypoxia and ischemia of the brain due to various perinatal factors. A previous study showed that knockdown of programmed cell death factor 4 (PDCD4) could reduce infarction injury resulting from ischemia/reperfusion injury. However, exact mechanism by which PDCD4 acts in HIBD is not yet understood. Our aim in present investigation was to investigate the function and mechanism of PDCD4 in alleviating HIBD. Methods: An HIBD model was developed using neonatal rats. After 48 h of modeling, short-term neurological function was evaluated and the brain tissue removed for assessment of cerebral infarct volume and brain water content (BWC). A cell model of oxygen glucose deprivation/reoxygenation (OGD/R) was also constructed. Overexpression or knockdown of insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3) or PDCD4 was performed in pretreated cells. Results: The geotaxis reflex time, cerebral infarct volume, and BWC all increased after HIBD in this neonatal rat model. Additionally, the levels of PDCD4 and of the N6-Methyladenosine (m6A) reader protein IGF2BP3 were increased in HIBD rats and OGD/R-stimulated pheochromocytoma (PC12) cells relative to controls. Moreover, OGD/R-stimulated pheochromocytoma PC12 cells showed decreased cell viability, increased apoptosis, and elevated Interleukin 6 (IL-6), Interleukin 1 β (IL-1β), and tumor necrosis factor-α (TNF-α) contents. These features were reversed after knocking down IGF2BP3. The interaction between IGF2BP3 protein and PDCD4 mRNA was confirmed by RNA immunoprecipitation and RNA pull-down assays. Furthermore, knockdown of IGF2BP3 in OGD/R-stimulated PC12 cells reduced cell damage via down-regulation of PDCD4. Finally, the IGF2BP3/PDCD4 axis alleviated OGD/R-induced cell injury in primary cortical neurons (PCNs). Conclusions: PDCD4 and m6A reader protein IGF2BP3 were up-regulated in an HIBD neonatal rat model. Knockdown of IGF2BP3 in OGD/R-stimulated PC12 cells or PCNs alleviated cell damage through reducing PDCD4.

Ubiquitination of ATAD3A by TRIM25 exacerbates cerebral ischemia-reperfusion injury via regulating PINK1/Parkin signaling pathway-mediated mitophagy

BackgroundCerebral ischemia-reperfusion injury (CI/RI) is a complex process leading to neuronal damage and death, with mitophagy implicated in its pathogenesis. However, the significance of mitophagy in CI/RI remains debated. HypothesisWe hypothesized that TRIM25 reduces ATAD3A expression by ubiquitinating ATAD3A, promoting mitophagy via the PINK1/Parkin pathway, and aggravating CI/RI. Study designRat middle cerebral artery occlusion (MCAO) followed by reperfusion and oxygen-glucose deprivation and reoxygenation (OGD/R) in PC12 cells were used as animal and cell models, respectively. MethodsTo evaluate the success of the CI/R modeling, TTC and HE staining were employed. The determination of serum biochemical indexes was carried out using relative assay kits. The Western Blot analysis was employed to assess the expression of ATAD3A, TRIM25, as well as mitophagy-related proteins (PINK1, Parkin, P62, and LC3II/LC3I). The mRNA levels were detected using QRT-PCR. Mitochondrial membrane potential was assessed through JC-1 staining. Mitosox Red Assay Kit was utilized to measure mitochondrial reactive oxygen species levels in PC12 cells. Additionally, characterization of the mitophagy structure was performed using transmission electron microscopy (TEM). ResultsOur findings showed down-regulation of ATAD3A and up-regulation of TRIM25 in both in vivo and in vitro CI/RI models. Various experimental techniques such as Western Blot, JC-1 staining, Mitosox assay, Immunofluorescence assay, and TEM observation supported the occurrence of PINK1/Parkin signaling pathway-mediated mitophagy in both models. ATAD3A suppressed mitophagy, while TRIM25 promoted it during CI/RI injury. Additionally, the results indicated that TRIM25 interacted with and ubiquitinated ATAD3A via the proteasome pathway, affecting ATAD3A protein stability and expression. ConclusionTRIM25 promoted Pink1/Parkin-dependent excessive mitophagy by destabilizing ATAD3A, exacerbating CI/RI. Targeting TRIM25 and ATAD3A may offer therapeutic strategies for mitigating CI/RI and associated neurological damage.

Inhibition of Phosphodiesterase 4 Suppresses Neuronal Ferroptosis After Cerebral Ischemia/Reperfusion.

We have previously shown that inhibition of phosphodiesterase 4 (PDE4) protects against cerebral ischemia/reperfusion injury. However, it remains unclear whether and how PDE4 affects ferroptosis under cerebral ischemia/reperfusion conditions. In this study, we found that overexpression of PDE4B in HT-22 cells exacerbated the detrimental effects of oxygen-glucose deprivation/reoxygenation (OGD/R), including a decrease in cell viability and glutathione (GSH) levels and an increase in Fe2+ content. PDE4B knockdown mitigated the effects of OGD/R, as evidenced by decreased oxidative stress, lactate dehydrogenase (LDH) release, Fe2+ content, and nuclear receptor coactivator 4 (NCOA4) expression. PDE4B knockdown also enhanced the levels of GSH, ferroportin (FPN), and ferritin heavy chain 1 (FTH1). Consistently, inhibition of PDE4 by roflumilast (Roflu) produced similar effects as PDE4B knockdown. Roflu also ameliorated the morphology and membrane potential of the mitochondria. Glutathione peroxidase 4 (GPX4) knockdown blocked the effects of Roflu on cell viability and lipid peroxidation. Moreover, we found that nuclear factor erythroid 2-related factor 2 (Nrf-2) knockdown decreased GPX4 expression. In addition, Nrf-2 knockdown led to enhanced lipid peroxidation, LDH release, and iron levels, while the GSH and FPN levels decreased. More crucially, PDE4 inhibition decreased infarct volume, alleviated oxidative stress, and restored the expression levels of ferroptosis-associated proteins in middle cerebral artery occlusion/reperfusion (MCAO/R) rats. Interestingly, the GPX4 inhibitor RSL3 blocked the neuroprotective effects of Roflu in rats subjected to MCAO/R. Thus, PDE4 inhibition significantly inhibits neuronal ferroptosis by activating the Nrf-2/GPX4 pathway. These data indicate the existence of a novel mechanism underlying the neuroprotective effects of PDE4 inhibition.

The alpha2-adrenoceptor agonist clonidine protects against hypoxic-ischemic brain damage in neonatal mice through the Nrf2/NF-κB signaling pathway

Neonatal hypoxic-ischemic brain damage (HIBD) is a severe condition closely associated with neuroinflammation and oxidative stress. Clonidine, a selective α2-adrenergic receptor agonist, is known for its anti-inflammatory and antioxidant properties. Despite these recognized therapeutic benefits, the exact mechanisms by which clonidine exerts its effects in the context of HIBD are not fully understood. This study was designed to thoroughly investigate the impact of clonidine on HIBD-induced neuronal injury and to clarify its underlying mechanism of action. We employed a neonatal mouse model of HIBD to meticulously assess the effects of clonidine on neuronal injury, apoptosis, inflammation, and oxidative stress markers. In addition, we conducted extensive in vitro studies to evaluate the neuroprotective effects of clonidine on primary hippocampal neuronal cells, utilizing advanced techniques such as the Cell Counting Kit-8 (CCK-8), flow cytometry, enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, and western blotting. Furthermore, we explored the regulatory effects of clonidine on the nuclear factor erythroid 2-related factor (Nrf2)/nuclear factor-κB (NF-κB) signaling pathway through a combination of in vivo and in vitro experiments. The results showed that clonidine significantly reduced cerebral infarction, neuronal damage, and apoptosis in HIBD mice. It also alleviated neuroinflammation and oxidative stress, improved cell viability, and reduced neuronal injury following oxygen-glucose deprivation/reoxygenation (OGD/R). The neuroprotective effects of clonidine were linked to the activation of the Nrf2/heme oxygenase-1 (HO-1) pathway and the inhibition of the NF-κB pathway. Overall, clonidine exhibited neuroprotective properties in HIBD by reducing neuroinflammation and oxidative stress, likely through the modulation of the Nrf2/NF-κB signaling pathway.

Bioactivity-Guided Isolation of Antistroke Compounds from Gymnadenia conopsea (L.) R. Br.

A bioactivity-guided separation strategy was used to identify novel antistroke compounds from Gymnadenia conopsea (L.) R. Br., a medicinal plant. As a result, 4 undescribed compounds (1-2, 13, and 17) and 13 known compounds, including 1 new natural product (3), were isolated from G. conopsea. The structures of these compounds were elucidated through comprehensive spectroscopic techniques, such as 1D/2D nuclear magnetic resonance (NMR) spectroscopy, high-resolution electrospray ionization mass spectrometry (HRESIMS), and quantum chemical calculations. An oxygen-glucose deprivation/reoxygenation (OGD/R)-injured rat pheochromocytoma (PC12) cell model was used to evaluate the antistroke effects of the isolates. Compounds 1-2, 10-11, 13-15, and 17 provided varying degrees of protection against OGD/R injury in the PC12 cells at concentrations of 12.5, 25, and 50 µM. Among the tested compounds, compound 17 demonstrated the most potent neuroprotective effect, which was equivalent to that of the positive control drug (edaravone). Then, transcriptomic and bioinformatics analyses were conducted to reveal the regulatory effect of compound 17 on gene expression. In addition, quantitative real-time PCR (qPCR) was performed to verify the results of the transcriptomic and bioinformatics analyses. These results suggest that the in vitro antistroke effect of compound 17 may be associated with the regulation of the Col27a1 gene. Thus, compound 17 is a promising candidate for the development of novel antistroke drugs derived from natural products, and this topic should be further studied.

Protective effects of tetramethylpyrazine on myocardial ischemia/reperfusion injury involve NLRP3 inflammasome suppression by autophagy activation

Tetramethylpyrazine (TMP) belongs to the active ingredients of the traditional Chinese medicine Chuanxiong, which has a certain protective effect in myocardial ischemia–reperfusion (I/R) injury. It can improve postoperative cardiac function and alleviate ventricular remodeling in acute myocardial infarction patients. However, its specific protective mechanism is still unclear. In this study, a certain concentration of TMP was introduced into I/R mice or H9C2 cells after oxygen-glucose deprivation/reoxygenation (OGD/R) treatment to observe the effects of TMP on cardiomyocyte activity, cytotoxicity, apoptosis, autophagy, pyroptosis, and NLRP3 inflammasome activation. The results displayed that TMP intervention could reduce OGD/R and I/R-induced cardiomyocyte apoptosis, accelerate cellular activity and autophagy levels, and ameliorate myocardial tissue necrosis in I/R mice in a dose-dependent manner. Further, TMP prevented the formation of NLRP3 inflammasomes to suppress pyroptosis by increasing the level of cardiomyocyte autophagy after I/R and OGD/R modelling, the introduction of chloroquine to suppress autophagic activity in vivo and in vitro was further analyzed to confirm whether TMP inhibits NLRP3 inflammasome activation and pyroptosis by increasing autophagy, and we found the inhibitory effect of TMP on NLRP3 inflammasomes and its protective effect against myocardial injury were blocked when autophagy was inhibited with chloroquine. In conclusion, this experiment demonstrated that TMP unusually attenuated I/R injury in mice, and this protective effect was achieved by inhibiting the activation of NLRP3 inflammasomes through enhancing autophagic activity.

Mitofusin 2 Mediates the Protective Effect of NR6A1 Silencing Against Neuronal Injury in Experimental Stroke Models.

An abnormal increase in the expression of nuclear receptor subfamily 6 group A member 1 (NR6A1) in the hippocampus has been reported to result in depressive-like behavior in mice. However, the role of NR6A1 in the progression of neuronal death induced by ischemic stroke remains unknown. In this study, we observed an increase in NR6A1 in neurons in both in vivo and in vitro cerebral ischemic models. We found that knocking down NR6A1 in HT-22 neuronal cells subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) attenuated mitochondrial dysfunction and endoplasmic reticulum (ER) stress. Conversely, NR6A1 overexpression exacerbated neuronal damage following OGD/R. NR6A1 hindered the transcription of mitonfusin 2 (MFN2), leading to a decrease in its expression. In contrast, MFN2 conferred the protective effect of NR6A1 silencing against both mitochondrial dysfunction and ER stress. In addition, NR6A1 silencing also attenuated brain infarction, ER stress, neuronal apoptosis, and loss of MFN2 in mice subjected to middle cerebral artery occlusion/reperfusion. These findings indicate that NR6A1 is a promising target for the treatment of neuronal death following cerebral ischemia. Furthermore, these results confirm the involvement of MFN2 in the effects of NR6A1 silencing. Therefore, targeting NR6A1 has potential as a viable strategy for the treatment of ischemic stroke.

SMP30 alleviates cerebral ischemia/reperfusion-induced neuronal injury by inhibiting HDAC4/PSD-95 to preserve mitochondrial function

Abstract Ischemic stroke is a major cause of global death and permanent disability. Major consequences of ischemic stroke include neuronal mitochondrial dysfunction. We investigated the effects of senescence marker protein 30 (SMP30) on mitochondria-mediated apoptosis and histone deacetylase 4 (HDAC4)/postsynaptic density-95 (PSD-95) signaling in stroke models in vivo and in vitro. Rats with middle cerebral artery occlusion/reperfusion (MCAO/R) were used to simulate cerebral ischemia/reperfusion (I/R) injury. SMP30 was downregulated in the brain tissues of rats after I/R induction. SMP30 overexpression decreased MCAO/R-induced infarct volumes and improved neurologic function and histopathological changes. Increasing SMP30 expression suppressed neuronal apoptosis and reduced mitochondrial dysfunction. SMP30 overexpression in SH-SY5Y and PC12 cells treated with oxygen-glucose deprivation/reoxygenation (OGD/R) decreased HDAC4 and PSD-95 expression; PSD-95 could bind to HDAC4. Furthermore, HDAC4 upregulation abolished the effects of SMP30 overexpression on OGD/R-induced apoptosis and mitochondrial dysfunction in SH-SY5Y cells. Together, these findings indicate that SMP30 alleviates cerebral I/R-induced neuronal injury by inhibiting HDAC4/PSD-95 to preserve mitochondrial function. These interactions might provide new treatment methods for patients with ischemic stroke.

Tyrobp deficiency blocks NLRP3-mediated inflammation and pyroptosis to alleviate myocardial ischemia-reperfusion injury through regulating Syk

PurposeThe present study aims to investigate the biological function of Tyrobp in myocardial ischemia-reperfusion injury (MIRI) and to clarify its potential reaction mechanisms. MethodsAC16 cells were induced by oxygen-glucose deprivation/reoxygenation (OGD/R) to simulate the MIRI in vitro. The cell transfection technology was used to downregulate Tyrobp, followed by assessment of cell damage, apoptosis and cytokines production via Cell Counting Kit (CCK)-8 assay, lactate dehydrogenase (LDH) release assay, TUNEL and ELISA assays, respectively. Immunofluorescence assay was performed to assess GSDMD. Corresponding proteins were detected via western blotting, and Co-immunoprecipitation (Co-IP) assay was used to validate proteins interaction. ResultsTyrobp was upregulated in OGD/R-exposed AC16 cells, and Tyrobp deficiency significantly alleviated OGD/R-caused cell viability loss, LDH release and cell apoptosis in AC16 cells. Meanwhile, Tyrobp deficiency inhibited the activation of NLRP3 inflammasome, reduced the production of cytokines and inhibited GSDMD intensity and GSDMD-N expression. Additionally, Tyrobp could interact with Syk and regulate Syk/NF-κB signaling. The rescue experiments showed that the above effects of Tyrobp deficiency on OGD/R-exposed AC16 cells were partly weakened by Syk overexpression. ConclusionTyrobp deficiency alleviated MIRI by inhibiting NLRP3-mediated inflammation and pyroptosis through regulating Syk, providing a novel target for the treatment of MIRI.

The Extract of Camellia japonica L. Protects Against Mice Cerebral Ischemia/Reperfusion Injury via Promoting the H2S-BKCa Pathway

Background and Purpose The effect and mechanism of the extract of Camellia japonica L. (ECJ) on brain injury following cerebral ischemia/reperfusion (I/R) were demonstrated in the present study. Methods We detected mice’s brain damage after cerebral I/R and tested neuronal injury following oxygen-glucose deprivation/re-oxygenation (OGD/R) to evaluate the neuroprotection of ECJ. Besides, we tested the expressions of hydrogen sulfide (H2S) synthase cystathionine-β-synthase (CBS) and α subunit of large-conductance Ca2+-activated K+ channels (BKα) both in brain tissues and culture neurons. Importantly, the roles of iberiotoxin (IbTX), BKCa channel inhibitor, and CBS inhibitor aminooxyacetic acid (AOAA) on ECJ-mediated neuroprotection were assessed to explore the neuroprotective mechanism of ECJ. Results ECJ treatment could alleviate the mice’s brain injury following cerebral I/R and reduce the neuronal damage caused by OGD/R in vitro, which was inhibited by IbTX and AOAA. In addition, ECJ could increase the expression of CBS and the α subunit of BKCa channel (BKα) in mice brain tissues and the culture neurons, as well as improve the H2S production. Furthermore, exogenous H2S donor NaHS also improved the BKα expression in OGD/R neurons. Importantly, NaHS alleviated the neuronal injury in vitro, which was inhibited by IbTX as well. Conclusion ECJ can protect against mouse cerebral I/R injury, the mechanism of which is correlated with promoting the CBS/H2S-BKCa pathway.

Astilbin exerts a neuroprotective effect by upregulating the signaling of nuclear NF-E2-related factor 2 in vitro

ObjectiveThe present study aims to evaluate the impact of Astilbin (AST) on cortical neuron survival in vitro under conditions of oxygen-glucose deprivation and reoxygenation (OGD/R) and determine the role of NF-E2-related factor 2 (Nrf2) in this process. MethodsPrimary neurons were pre-treated with various concentrations of AST for 8 h before OGD induction. Cell viability and lactate dehydrogenase (LDH) leakage were assessed to determine the optimal concentration. Biomarkers related to oxidative stress, antioxidant enzyme activities, and apoptosis were evaluated at 24 h post-OGD/R. To investigate the involvement of Nrf2 in AST-mediated neuroprotection, we conducted molecular docking and microscale thermophoresis analyses, as well as examined the expression levels of Nrf2 and its regulatory genes including heme oxygenase-1(HO-1), (NAD(P)H: quinone oxidoreductase 1 (NQO-1), and peroxiredoxin 1 (Prdx1). Additionally, lentivirus-mediated knockdown of Nrf2 and overexpression of Nrf2 with L-sulforaphane (SFN) were performed, followed by an assessment of cell viability, oxidative stress, antioxidant enzyme activities and apoptosis. ResultsPre-treatment with AST reduced oxidative stress levels while increasing antioxidant enzyme activities and mitigating neuronal apoptosis. After OGD/R exposure, AST upregulated nuclear Nrf2 expression and increased the expression of HO-1, NQO-1 and Prdx1 in the cytoplasm. However, the knockdown of Nrf2 abolished the antioxidative and protective effects exerted by AST treatment. Conversely, combining AST with the Nrf2 agonist SFN demonstrated an enhancement in the protective effects provided by AST. These results demonstrate that Nrf2-dependent antioxidant responses contribute to AST-induced tolerance against neuronal injury caused by OGD/R injury. ConclusionsOverall findings support the ability of AST to protect primary neurons from OGD/R-induced damage through activation of Nrf2-dependent antioxidant responses.

HIF-1α knockdown attenuates inflammation and oxidative stress in ischemic stroke male rats via CXCR4/NF-κB pathway.

Hypoxia inducible factor-1α (HIF-1α) is a sensitive indicator of oxygen homeostasis, of which the expression elevates following hypoxia/ischemia. This study reveals the specific mechanisms underlying the effects of HIF-1α on ischemic stroke (IS). IS model was established using middle cerebral artery occlusion (MCAO)-modeled male rats and oxygen glucose deprivation/reoxygenation (OGD/R)-treated mice hippocampal cells HT22, followed by the silencing of HIF-1α and the overexpression of C-X-C motif chemokine receptor 4 (CXCR4) and nuclear factor-kappa B (NF-κB). Following the surgery, Garcia's grading scale was applied for neurological evaluation. Cerebral infarcts and injuries were visualized using 2,3,5-triphenyltetrazolium chloride and hematoxylin-eosin staining. The levels of tumor necrosis factor-α, Interleukin (IL)-6, IL-1β, malondialdehyde, and 8-hydroxy-2'-deoxyguanosine, were calculated via ELISA. MTT assay and lactate dehydrogenase (LDH) assay kit were adopted to determine the viability and cytotoxicity of OGD/R-modeled cells. Reactive oxygen species (ROS) generation was evaluated using a 2'-7'dichlorofluorescin diacetate (DCFH-DA) probe. The levels of HIF-1α, CXCR4, and NF-κB p65 were quantified via Western blot and immunofluorescence, respectively. HIF-1α knockdown improved Garcia's score, attenuated the cerebral infarct, inflammation, and ROS generation, and alleviated the levels of inflammatory cytokines and CXCR4/NF-κB p65 in MCAO-modeled rats. Such effects were reversed following the overexpression of CXCR4 and NF-κB. Also, in OGD/R-treated HT22 cells, HIF-1α silencing diminished the cytotoxicity and ROS production and reduced the expressions of CXCR4/NF-κB p65, while promoting viability. However, CXCR4/NF-κB p65 overexpression did the opposite. HIF-1α knockdown alleviates inflammation and oxidative stress in IS through the CXCR4/NF-κB pathway.

E3 ubiquitin ligase MARCH1 reduces inflammation and pyroptosis in cerebral ischemia-reperfusion injury via PCSK9 downregulation.

Pyroptosis has been regarded as caspase-1-mediated monocyte death that induces inflammation, showing a critical and detrimental role in the development of cerebral ischemia-reperfusion injury (IRI). MARCH1 is an E3 ubiquitin ligase that exerts potential anti-inflammatory functions. Therefore, the study probed into the significance of MARCH1 in inflammation and pyroptosis elicited by cerebral IRI. Middle cerebral artery occlusion/reperfusion (MCAO/R)-treated mice and oxygen glucose deprivation/reoxygenation (OGD/R)-treated hippocampal neurons were established to simulate cerebral IRI in vivo and in vitro. MARCH1 and PCSK9 expression was tested in MCAO/R-operated mice, and their interaction was identified by means of the cycloheximide assay and co-immunoprecipitation. The functional roles of MARCH1 and PCSK9 in cerebral IRI were subsequently determined by examining the neurological function, brain tissue changes, neuronal viability, inflammation, and pyroptosis through ectopic expression and knockdown experiments. PCSK9 expression was increased in the brain tissues of MCAO/R mice, while PCSK9 knockdown reduced brain damage and neurological deficits. Additionally, inflammation and pyroptosis were inhibited in OGD/R-exposed hippocampal neurons upon PCSK9 knockdown, accompanied by LDLR upregulation and NLRP3 inflammasome inactivation. Mechanistic experiments revealed that MARCH1 mediated ubiquitination and degradation of PCSK9, lowering PCSK9 protein expression. Furthermore, it was demonstrated that MARCH1 suppressed inflammation and pyroptosis after cerebral IRI by downregulating PCSK9 both in vivo and in vitro. Taken together, the present study demonstrate the protective effect of MARCH1 against cerebral IRI through PCSK9 downregulation, which might contribute to the discovery of new therapies for improving cerebral IRI.

Neuroprotection on ischemic brain injury by Mg2+/H2 released from endovascular Mg implant

Most acute ischemic stroke patients with large vessel occlusion require stent implantation for complete recanalization. Yet, due to ischemia-reperfusion injury, over half of these patients still experience poor prognoses. Thus, neuroprotective treatment is imperative to alleviate the ischemic brain injury, and a proof-of-concept study was conducted on “biodegradable neuroprotective stent”. This concept is premised on the hypothesis that locally released Mg2+/H2 from Mg metal within the bloodstream could offer synergistic neuroprotection against reperfusion injury in distant cerebral ischemic tissues. Initially, the study evaluated pure Mg's neuroactive potential using oxygen-glucose deprivation/reoxygenation (OGD/R) injured neuron cells. Subsequently, a pure Mg wire was implanted into the common carotid artery of the transient middle cerebral artery occlusion (MCAO) rat model to simulate human brain ischemia/reperfusion injury. In vitro analyses revealed that pure Mg extract aided mouse hippocampal neuronal cell (HT-22) in defending against OGD/R injury. Additionally, the protective effects of the Mg wire on behavioral abnormalities, neural injury, blood-brain barrier disruption, and cerebral blood flow reduction in MCAO rats were verified. Conclusively, Mg-based biodegradable neuroprotective implants could serve as an effective local Mg2+/H2 delivery system for treating distant cerebral ischemic diseases.

NDRG2 regulates glucose metabolism and ferroptosis of OGD/R-treated astrocytes by the Wnt/β-catenin signaling.

Ischemic stroke is one main type of cerebrovascular disorders with leading cause of death and disability worldwide. Astrocytes are the only nerve cell type storing glycogen in the brain, which regulate the glucose metabolism and handle the energy supply and survive of neurons. Astrocyte ferroptosis contributes to neuron injury in brain disorders. N-myc downstream-regulated gene 2 (NDRG2) has been implicated in the progression of brain diseases, including ischemic stroke. However, whether NDRG2 could affect the glucose metabolism and ferroptosis of astrocytes during ischemic stroke remains largely unknown. Mouse astrocytes were treated with oxygen-glucose deprivation/reoxygenation (OGD/R) to establish the in vitro model. Glial fibrillary acidic protein, NDRG2, Wnt3a and β-catenin expression levels were detected by immunofluorescence staining and western blot analyses. Glucose metabolism was investigated by glucose uptake, lactate production, nicotinamide adenine dinucleotide phosphate hydrogen/nicotinamide adenine dinucleotide phosphate (NADPH/NADP+), ATP and glycolysis enzymes (HK2, PKM2 and lactate dehydrogenase A [LDHA]) levels. Ferroptosis was assessed via reactive oxygen species (ROS), glutathione (GSH), iron and ferroptosis-related markers (GPX4 and PTGS2) contents. Glycolysis enzymes and ferroptosis-related markers levels were measured via western blot. NDRG2 expression was elevated in OGD/R-induced astrocytes. NDRG2 overexpression aggravated OGD/R-induced loss of glucose metabolism through reducing glucose uptake, lactate production, NADPH/NADP+ and ATP levels. NDRG2 upregulation exacerbated OGD/R-caused reduction of glycolysis enzymes (HK2, PKM2 and LDHA) levels. NDRG2 promoted OGD/R-induced ferroptosis of astrocytes by increasing ROS, iron and PTGS2 levels and decreasing GSH and GPX4 levels. NDRG2 overexpression enhanced OGD/R-induced decrease of Wnt/β-catenin signaling activation by reducing Wnt3a and β-catenin expression. NDRG2 silencing played an opposite effect. Inhibition of Wnt/β-catenin signaling activation by IWR-1 attenuated the influences of NDRG2 knockdown on glucose metabolism, glycolysis enzymes levels and ferroptosis. These findings demonstrated that NDRG2 contributes to OGD/R-induced inhibition of glucose metabolism and promotion of ferroptosis in astrocytes through inhibiting Wnt/β-catenin signaling activation, which might be associated with ischemic stroke progression.

Sufentanil-induced Nrf2 protein ameliorates cerebral ischemia-reperfusion injury through suppressing neural ferroptosis

As an oxidative stress and inflammation-related disease, cerebral ischemia-reperfusion injury (CIRI) is a prevalent pathogenic factor of ischemic stroke (IS) and seriously degrades the life quality of human beings. As an opioid analgesic for anesthesia, Sufentanil (SUF) can activate the Nrf2 protein-induced anti-oxidant effects, which indicate that SUF may be used as alternative drug for CIRI therapy, but little is known regarding to its molecular mechanisms. Thus, this research aimed to examine whether SUF pre-treatment alleviated CIRI through the modulation of Nrf2 protein-mediated antioxidant activity. Our research revealed that middle cerebral artery occlusion/reperfusion (MCAO/R)-treated rats exhibited apparent CIRI-related symptoms and induced damages in rats' brain, which were all notably mitigated in the MCAO/R rats. The subsequent in vitro cellular experiments verified that oxygen-glucose deprivation/reoxygenation (OGD/R)-induced cytotoxicity were apparently reversed by SUF co-treatment in HT22 and BV2 cells, and it was also validated that SUF was capable of suppressing inflammation and ferroptosis in CIRI models by inhibiting oxidative stress-related damages. Mechanistically, the Akt/GSK-3β pathway was excessively activated by SUF to promote Nrf2 protein expressions and enhance Nrf2-meidated anti-oxidant effects, and it was found that SUF-induced protective effects during CIRI progression were all abrogated by co-treating cells with MK2206 (Akt inhibitor), NP-12 (GSK-3β inhibitor), or ML385 (Nrf2 inhibitor). In conclusion, SUF activated the Akt/GSK-3β pathway to initiate Nrf2 protein-mediated antioxidant effects, which further suppressed oxidative stress-related inflammation and ferroptosis to ameliorate CIRI progression, and SUF could potentially be used as novel therapeutic agent for CIRI treatment in clinic.

The mechanism of hypoxia-inducible factor-1α enhancing the transcriptional activity of transferrin ferroportin 1 and regulating the Nrf2/HO-1 pathway in ferroptosis after cerebral ischemic injury

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.

β-Caryophyllene mitigates ischemic stroke-induced white matter lesions by inhibiting pyroptosis

β-Caryophyllene (BCP), a selective agonist for cannabinoid receptor 2 (CB2R), has demonstrated promising protective effects in various pathological conditions. However, the neuroprotective effects of BCP on white matter damage induced by ischemic stroke have not been elucidated previously. In this study, we find that BCP not only improves sensorimotor and cognitive function via CB2R but also mitigates white matter lesions in mice following ischemic stroke. Furthermore, BCP enhances the viability of MO3.13 oligodendrocytes after oxygen-glucose deprivation and reoxygenation (OGD/R), attenuating OGD/R-induced cellular damage and pyroptosis. Notably, these protective effects of BCP are partially enhanced by the NLRP3 inhibitor MCC950 and counteracted by the NLRP3 activator nigericin. In addition, nigericin significantly exacerbates neurological outcomes and increases white matter lesions following BCP treatment in middle cerebral artery occlusion (MCAO) mice. These results suggest that BCP may ameliorate neurological deficits and white matter damage induced by cerebral ischemia through inhibiting NLRP3-mediated pyroptosis.

Activation of GPR30 Ameliorates Cerebral Ischemia-Reperfusion Injury by Suppressing Ferroptosis Through Nrf2/GPX4 Signaling Pathway.

The newly identified estrogen receptor, G protein-coupled receptor 30 (GPR30), is prevalent in the brain and has been shown to provide significant neuroprotection. Recent studies have linked ferroptosis, a newly characterized form of programmed cell death, closely with cerebral ischemia-reperfusion injury (CIRI), highlighting it as a major contributing factor. Consequently, our research aimed to explore the potential of GPR30 targeting in controlling neuronal ferroptosis and lessening CIRI impacts. Results indicated that GPR30 activation not only improved neurological outcomes and decreased infarct size in a mouse model but also lessened iron accumulation and malondialdehyde formation post-middle cerebral artery occlusion (MCAO). This protective effect extended to increased levels of Nrf2 and GPX4 proteins. Similar protective results were replicated in PC12 cells subjected to Oxygen Glucose Deprivation and Reoxygenation (OGD/R) using the GPR30-specific agonist G1. Importantly, inhibition of Nrf2 with ML385 curtailed the neuroprotective effects of GPR30 activation, suggesting that GPR30 mitigates CIRI primarily through inhibition of neuronal ferroptosis via upregulation of Nrf2 and GPX4.