Oxygen-glucose Deprivation/reoxygenation Research Articles

Transmembrane p24 trafficking protein 10 (TMED10) inhibits mitochondrial damage and protects neurons in ischemic stroke via the c-Jun N-terminal kinase (JNK) signaling pathway.

Stroke, a type of acute cerebrovascular disease, is a global disease with high mortality. Neuronal ischemia and hypoxia are closely related to occurrence and development of cognitive impairment. Transmembrane p24 trafficking protein 10 (TMED10) as a transmembrane protein involves in vesicle protein transport in the secretory pathways. However, the function and mechanism of TMED10 on ischemic stroke and cognitive impairments remain unclear. In current study, TMED10 was highly expressed in cerebral ischemic penumbra of middle cerebral artery occlusion (MCAO) mouse model. Downregulation of TMED10 suppressed cell survival and facilitated apoptosis in primary cortical neurons, which were grown under oxygen glucose deprivation/reoxygenation (OGD/R) condition. Upregulation of TMED10 protected neurons form apoptosis induced by OGD/R. Further research indicated that the decrease of TMED10 resulted in neuronal mitochondrial injury through increasing reactive oxygen species (ROS) production. Meanwhile, TMED10 reduction induced neuronal apoptosis and mitochondrial damage through activating the c-Jun N-terminal kinase (JNK) pathway. Moreover, the knockdown of TMED10 increased cerebral infarction area, aggravated neuronal injury and promoted neuronal apoptosis through activating the JNK pathway in the cerebral ischemic penumbra of MCAO mouse model. Additionally, Morris water maze test verified that the severity of cognitive impairment increased with the decline of TMED10. Collectively, this study reveals that TMED10 inhibits mitochondrial damage, and protects neurons from apoptosis in MCAO-induced ischemic stroke and cognitive impairment via blocking the JNK pathway.

Exosomal miR-133a-3p Derived from BMSCs Alleviates Cerebral Ischemia-Reperfusion Injury via Targeting DAPK2

Cerebral ischemia-reperfusion (CI/R) injury is a subtype of complication after treatment of ischemic stroke. It has been reported that exosomes derived from BMSCs could play an important role in CI/R injury. However, whether BMSCs-derived exosomes could regulate CI/R injury via carrying miRNAs remains to be further explored. RNA sequencing was performed to identify the differentially expressed miRNAs. To mimic CI/R in vitro, SH-SY5Y cells were exposed to oxygen glucose deprivation/reoxygenation (OGD/R). The viability of SH-SY5Y cells was tested by CCK8 assay, and TUNEL staining was performed to detect the cell apoptosis. MiR-133a-3p was identified to be reduced in exosomes derived from the plasma of patients with IS. Upregulation of miR-133a-3p significantly reversed OGD/R-induced SH-SY5Y cell growth inhibition. Consistently, BMSCs-derived exosomal miR-133a-3p could restore OGD/R-decreased SH-SY5Y cell proliferation via inhibiting apoptosis. Meanwhile, DAPK2 was a direct target of miR-133a-3p. In addition, OGD/R notably upregulated the level of DAPK2 and weakened the expressions of p-Akt and p-mTOR in SH-SY5Y cells, whereas exosomal miR-133a-3p derived from BMSCs notably reversed these phenomena. Exosomal miR-133a-3p derived from BMSCs could reverse OGD/R-induced cell apoptosis via inhibiting autophagy. Furthermore, exosomal miR-133a-3p derived from BMSCs markedly alleviated the symptom of CI/R injury in vivo. Exosomal miR-133a-3p derived from BMSCs alleviates CI/R injury via targeting DAPK2/Akt signaling. Thus, our study might shed new light on discovering new strategies against CI/R injury.

Protective effects of alpha-mangostin encapsulated in cyclodextrin-nanoparticle on cerebral ischemia.

The production of reactive oxygen species (ROS) during and after the onset of an ischemic stroke induces neuronal cell death and severely damages brain function. Therefore, reducing ROS by administrating antioxidant compounds is a promising approach to improving ischemic symptoms. Alpha-mangostin (α-M) is an antioxidant compound extracted from the pericarp of the mangosteen fruit. Reportedly, α-M decreases neuronal toxicity in primary rat cerebral cortical neurons. In this study, we investigated the neuroprotective activity of α-M in both in vitro and in vivo assays. Pretreatment with α-M inhibited excessive cellular ROS production after oxygen-glucose deprivation/reoxygenation (OGD/R) in vitro using an SH-SY5Y (human neuroblastoma) cell line. In addition, α-M maintained mitochondrial membrane potential and suppressed mitochondrial-specific ROS production induced by OGD/R. Meanwhile, the low bioavailability of α-M due to its poor water solubility has been an insuperable obstruction impeding extensive investigations of the biological functions of α-M and its medical applications. To overcome this problem, we synthesized a cyclodextrin-based nanoparticle (CDNP) that is known to increase the loading efficiency and binding constant of α-M, compared with cyclodextrins themselves. This nano-formulated α-M (α-M/CDNP) was optimized for an in vivo ischemic stroke model. Our results indicated that α-M/CDNP (25mg/kg/injection) reduced infarct volume and improved neurological behavior (p=0.036 and p=0.046, respectively). These in vivo results suggest that α-M appears to cross the blood-brain barrier (BBB) with the help of a nano-formulation with CDNP. Combining an in vitro BBB model and a physicochemical binding assay between α-M and albumin, it is speculated that α-M released from CDNP would interact with albumin during its prolonged circulation in the blood, and the resultant α-M/albumin complex may cross the BBB through the absorptive-mediated transcytosis pathway. These findings suggest the potential clinical application of α-M in ischemic stroke treatment.

Physalin A exerts neuroprotective effects: inhibition of OGD/R-induced cellular pyroptosis and inflammatory responses in nerve cells

A prevalent cerebrovascular disease like the acute cerebral infarction is caused by cerebral thrombosis or arteriosclerosis that can result in ischemic necrosis of brain tissues. Treatment drugs of higher efficiency are required to be developed for acute cerebral infarction. Physalin A (PA) is a major compound from Physalis alkekengi L. and has pharmacological properties. However, the neuroprotective effects of PA are less reported and the mechanism remains unclear. The acute cerebral infarction cell model was constructed by treating PC12 cells with oxygen-glucose deprivation/reoxygenation (OGD/R). The impacts of PA on cell viability were determined by performing 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) and lactate dehydrogenase (LDH) releasing assays. Immunoblot and Enzyme-linked Immunosorbent assay (ELISA) were conducted to examine PA effects on inflammation. PA effects on pyroptosis were detected by performing immunoblot and immunostaining. Moreover, immunoblot was further conducted to confirm the mechanism. In this study, the survival and inhibition of the pyroptosis of OGD/R-induced PC12 cells were regulated by PA. Furthermore, PA suppressed the inflammation. PA inhibited mitogen-activated protein kinase (MAPK) pathway activation, and the activation of MAPK pathway reversed the neuroprotective effect of PA. Therefore, PA exerts neuroprotective effects by suppressing OGD/R-induced cellular pyroptosis and inflammatory responses in nerve cells.

Geraniol-Mediated Suppression of Endoplasmic Reticulum Stress Protects against Cerebral Ischemia-Reperfusion Injury via the PERK-ATF4-CHOP Pathway.

Endoplasmic reticulum (ER) stress plays an important role in cerebral ischemia-reperfusion injury (CIRI). Geraniol has antioxidant, antibacterial, and anti-inflammatory activities. Studies have shown that geraniol has a protective effect against CIRI in rats, but the exact mechanism is unclear. Purpose: The aim of this study was to investigate the protective mechanism of geraniol against CIRI. We established a middle cerebral artery occlusion reperfusion model in rats and a PC12 cell oxygen-glucose deprivation/reoxygenation (OGD/R) model to observe the neuroprotective effects of geraniol. Neurological scoring, 2,3,5-triphenyltetrazolium chloride staining, and hematoxylin and eosin staining were used to evaluate the neuroprotective effects of geraniol against CIRI. ER-stress-related and apoptosis-related protein expression was detected via Western blotting and immunofluorescence. Apoptosis was also detected via TUNEL assays and flow cytometry. The fluorescent detection of intracellular calcium was achieved using fluorescent calcium-binding dyes, and transmission electron microscopy was used to assess the neuronal ultrastructure. Geraniol effectively attenuated cerebral infarction and pathological injury after CIRI, had a protective effect against CIRI, significantly reduced the expression of the ER-stress-related proteins P-PERK, ATF4, CHOP, and GRP78 and the pro-apoptotic protein BAX, increased the expression of the anti-apoptotic protein BCL-2, and reduced the occurrence of apoptosis. In the OGD/R model in PC12 cells, the protective effect of geraniol was the same as that in vivo. Our results suggest that geraniol has a protective effect against ischemic stroke by a mechanism possibly related to ER stress via the PERK-ATF4-CHOP pathway.

Inhibition of the Glycolysis Prevents the Cerebral Infarction Progression Through Decreasing the Lactylation Levels of LCP1

Cerebral infarction (CI), also known as ischemic stroke, has a high incidence rate and mortality rate. The purpose of this study was to investigate the potential effect and mechanism of Lymphocyte cytosolic protein 1 (LCP1) in the CI progression. The middle cerebral artery occlusion (MCAO) treated rats and oxygen–glucose deprivation/reoxygenation (OGD/R) stimulated PC12 cells were used to establish CI model in vivo and in vitro. The cell proliferation and apoptosis was determined by CCK-8 assay and flow cytometry, respectively. Immunoprecipitation and western blot was performed to test the lactylation levels of LCP1. The cells were treated with cycloheximide to determined the protein stability of LCP1. The glucose uptake and lactate production was determined with commercial kits. The extracellular acidification rate were evaluated by Seahorse. The results showed that LCP1 was upregulated in the MCAO rats and OGD/R stimulated PC12 cells. LCP1 knockdown dramatically decreased the neurological score, infarct volume and the brain water content of MCAO rats. Besides, LCP1 knockdown promoted the cell viability while decreased the apoptosis rate of the OGD/R stimulated PC12 cells. Additionally, the global lactylation and lactylation levels of LCP1 was prominently enhanced in vivo and in vitro in cerebral infarction. 2-DG treatment prominently decreased it. In conclusion, inhibiting the glycolysis decreased the lactylation levels of LCP1 and resulted in the degradation of LCP1, which eventually relieved the CI progression.

Protective effects of Gypenoside XVII against cerebral ischemia/reperfusion injury via SIRT1-FOXO3A- and Hif1a-BNIP3-mediated mitochondrial autophagy

BackgroundMitochondrial autophagy maintains mitochondrial function and cellular homeostasis and plays a critical role in the pathological process of cerebral ischemia/reperfusion injury (CIRI). Whether Gypenoside XVII (GP17) has regulatory effects on mitochondrial autophagy against CIRI remains unclear. The purpose of this study was to investigate the pharmacodynamic effects and mechanisms of GP17 on mitochondrial autophagy after CIRI.MethodsA rat middle cerebral artery occlusion/reperfusion (MCAO/R) model was used to assess the effects of GP17 against CIRI and to explore the underlying mechanisms. An oxygen-glucose deprivation/reoxygenation (OGD/R) cell model was used to verify the ameliorative effects on mitochondrial damage and to probe the autophagy pathways involved in combating neural injuries.ResultsThe in vivo results showed that GP17 significantly improved mitochondrial metabolic functions and suppressed cerebral ischemic injury, possibly via the autophagy pathway. Further research revealed that GP17 maintains moderate activation of autophagy under ischemic and OGD conditions, producing neuroprotective effects against CIRI, and that the regulation of mitochondrial autophagy is associated with crosstalk between the SIRT1-FOXO3A and Hif1a-BNIP3 signalling pathway that is partially eliminated by the specific inhibitors AGK-7 and 2-ME.ConclusionOverall, this work offers new insights into the mechanisms by which GP17 protects against CIRI and highlights the potential of therapy with Notoginseng leaf triterpene compounds as a novel clinical strategy in humans.

Neuroprotective effects of Chrysanthemum morifolium on cerebral ischemia- reperfusion injury contributes to the oxidative stress suppression and related Keap1/Nrf2 pathway

ABSTRACT Background Ischemic stroke, the cause of death and disability worldwide, is closely related to oxidative stress damage. Chrysanthemum has profound antiantioxidant activity. We aimed to verify whether Chrysanthemum morifolium extract (CME) influences brain injury in cerebral ischemia-reperfusion injury (CR/RI) model. Methods In vitro, rat hippocampal H19-7 neurons were pretreated with CME, CR/RI was simulated with oxygen glucose deprivation/reoxygenation (OGD/R). The cell viability, apoptosis, lactate dehydrogenase release, reactive oxygen species (ROS) generation, malonaldehyde (MDA) content and superoxide dismutase（SOD） activity were detected. In vivo, middle cerebral artery occlusion (MCAO) model rats were pre-administered with CME, and then behavioral test, triphenyltetrazolium chloride (TTC), hematoxylin-eosin staining (HE), terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end labeling (TUNEL), ROS immunofluorescence, MDA and SOD activity were tested. Furthermore, Keap1/Nrf2 signaling of CME in CI/RI was investigated. Results In OGD/R induced in H19-7 cells, CME increased OGD/R-induced cell viability and reduced cell apoptosis, which was reversed by siNrf2 transfection . In MCAO rats, CME improved the neurological deficits and alleviated brain injury. However, co-treatment with MLK385 counteracted these neuroprotective effects of CME on MCAO rats. Conclusion CME could significantly reduce oxidative stress and nerve injury in vitro and in vivo models of CI/RI by regulating the Keap1/Nrf2 pathway.

Asiaticoside attenuates oxygen-glucose deprivation/reoxygenation-caused injury of cardiomyocytes by inhibiting autophagy.

Asiaticoside is a natural triterpene compound derived from Centella asiatica, possessing confirmed cardioprotective property. However, the roles of asiaticoside in regulating oxygen-glucose deprivation/reoxygenation (OGD/R)-caused cardiomyocyte dysfunction remain largely obscure. Human cardiomyocyte AC16 cells were stimulated with OGD/R to mimic myocardial ischemia/reperfusion injury and treated with asiaticoside. Cytotoxicity was investigated by CCK-8 assay and lactate dehydrogenase (LDH) release analysis. Autophagy- and Wnt/β-catenin signaling-related protein levels were measured via western blotting. Asiaticoside (0-20 μM) did not induce cardiomyocyte cytotoxicity. Asiaticoside (20 μM) mitigated OGD/R-induced autophagy, cytotoxicity, oxidative stress, and myocardial injury. Rapamycin, an autophagy inductor, reversed the influences of asiaticoside on autophagy, cytotoxicity, oxidative stress, and myocardial injury, whereas 3-methyadanine, an autophagy inhibitor, played an opposite effect. Asiaticoside (20 μM) attenuated OGD/R-induced Wnt/β-catenin signaling inactivation, which was reversed after transfection with si-β-catenin. Transfection with si-β-catenin attenuated the influences of asiaticoside on autophagy, cytotoxicity, oxidative stress, and myocardial injury. In conclusion, asiaticoside protected against OGD/R-induced cardiomyocyte cytotoxicity, oxidative stress, and myocardial injury via blunting autophagy through activating the Wnt/β-catenin signaling, indicating the therapeutic potential of asiaticoside in myocardial ischemia/reperfusion injury.

Up-regulation of lncRNA NEAT1 in cerebral ischemic stroke promotes activation of astrocytes by modulation of miR-488-3p/RAC1

We aim to research the molecular mechanism of lncRNA NEAT1 in the activation of astrocytes in a cerebral ischemia-reperfusion injury model. Mouse model of cerebral ischemia-reperfusion injury was constructed, and shNEAT1 was transfected. The infarct area, brain water content, and neurological deficiency were detected. Immunofluorescence detection and fluorescence in situ hybridization (FISH) assay were processed to detect glial fibrillary acidic protein (GFAP) expression. Astrocyte cells were cultured for oxygen-glucose deprivation/re-oxygenation (OGD)/re-oxygenation model construction. After treatment by shNEAT1, miR-488-3p mimic, miR-488-3p inhibitor, Q-PCR assay, western blot and ELISA were undertaken to detect the expressions of NEAT1, miR-488-3p, RAC1, inflammatory cytokines, RAC1 and GFAP. Dual luciferase reporter assay and RNA-binding protein immunoprecipitation (RIP) assay were used to verify the binding of NEAT1, miR-488-3p and RAC1. The expression of NEAT1 in brain tissue was significantly higher than that in Sham operation group. Knockdown of NEAT1 inhibited the brain damage caused by middle cerebral artery occlusion (MCAO) treatment, reduced the inflammatory response, and suppressed the activation of astrocytes. By constructing an in vitro OGD/R cell model, it was found that NEAT1 knockdown also inhibited the activation of astrocytes caused by OGD/R. Knockdown of NEAT1 caused the up-regulation of miR-488-3p and the down-regulation of RAC1. Knockdown of miR-488-3p or over-expression of RAC1 reversed the inhibitory effect of shNEAT1 on OGD/R-induced astrocyte activation. Over-expression of NEAT1 in cerebral ischemic stroke promotes activation of astrocytes by modulation miR-488-3p/RAC1, which is proved in vitro. Our study may provide a new idea for the diagnosis and treatment of MCAO.

Gallic acid inhibits neuroinflammation and reduces neonatal hypoxic-ischemic brain damages.

Neuroinflammation is a leading cause of secondary neuronal injury in neonatal hypoxic-ischemic encephalopathy (HIE). Regulation of neuroinflammation may be beneficial for treatment of HIE and its secondary complications. Gallic acid (GA) has been shown to have anti-inflammatory and antioxidant effects. In this report we found that oxygen-glucose deprivation and/reoxygenation (OGD/R)-induced cell death, and the generation of excessive reactive oxygen species (ROS) and inflammatory cytokines by microglia were inhibited by GA treatment. Furthermore, GA treatment reduced neuroinflammation and neuronal loss, and alleviated motor and cognitive impairments in rats with hypoxic-ischemic brain damage (HIBD). Together, our results reveal that GA is an effective regulator of neuroinflammation and has potential as a pharmaceutical intervention for HIE therapy.

Resveratrol Inhibits Oxidative Stress and Regulates M1/M2-Type Polarization of Microglia via Mediation of the Nrf2/Shh Signaling Cascade after OGD/R Injury In Vitro.

Microglia are closely related to the occurrence and development of oxidative stress. Cerebral ischemia leads to abnormal activation of microglia. Resveratrol can regulate M1/M2-type microglia polarization, but the underlying mechanism is not well understood, although the Nrf2 and Shh signaling pathways may be involved. Given that resveratrol activates Shh, the present study examined whether this is mediated by Nrf2 signaling. N9 microglia were pretreated with drugs before oxygen-glucose deprivation/reoxygenation (OGD/R). HT22 neurons were also used for conditional co-culture with microglia. Cell viability was measured by CCK-8 assay. MDA levels and SOD activity in the supernatant were detected by TBA and WST-1, respectively. Immunofluorescence detected Nrf2 and Gli1 nuclear translocation. The levels of CD206, Arg1, iNOS, TNF-α, Nrf2, HO-1, NQO1, Shh, Ptc, Smo, Gli1 protein and mRNA were measured by Western blotting or RT-qPCR. Annexin V-FITC Flow Cytometric Analysis detected apoptosis. Resveratrol and Nrf2 activator RTA-408 enhanced the viability of microglia, reduced oxidative stress, promoted M2-type microglia polarization and activated Nrf2 and Shh signaling. ML385, a selective inhibitor of Nrf2, decreased the viability of microglia, aggravated oxidative stress, promoted M1-type microglia polarization and inhibited Nrf2 and Shh signaling. Moreover, resveratrol and RTA-408-treated microglia can reduce the apoptosis and increase the viability of HT22 neurons, while ML385-treated microglia aggravated the apoptosis and weakened the viability of HT22 neurons. These results demonstrated that resveratrol may inhibit oxidative stress, regulate M1/M2-type polarization of microglia and decrease neuronal injury in conditional co-culture of neurons and microglia via the mediation of the Nrf2/Shh signaling cascade after OGD/R injury in vitro.

Chebulic Acid Prevents Hypoxia Insult via Nrf2/ARE Pathway in Ischemic Stroke.

Excessive reactive oxygen species (ROS) production contributes to brain ischemia/reperfusion (I/R) injury through many mechanisms including inflammation, apoptosis, and cellular necrosis. Chebulic acid (CA) isolated from Terminalia chebula has been found to have various biological effects, such as antioxidants. In this study, we investigated the mechanism of the anti-hypoxic neuroprotective effect of CA in vitro and in vivo. The results showed that CA could protect against oxygen-glucose deprivation/reoxygenation (OGD/R) induced neurotoxicity in SH-SY5Y cells, as evidenced by the enhancement of cell viability and improvement of total superoxide dismutase (T-SOD) in SH-SY5Y cells. CA also attenuated OGD/R-induced elevations of malondialdehyde (MDA) and ROS in SH-SY5Y cells. Nuclear factor-E2-related factor 2 (Nrf2) is one of the key regulators of endogenous antioxidant defense. CA acted as antioxidants indirectly by upregulating antioxidant-responsive-element (ARE) and Nrf2 nuclear translocation to relieve OGD/R-induced oxidative damage. Furthermore, the results showed that CA treatment resulted in a significant decrease in ischemic infarct volume and improved performance in the motor ability of mice 24 h after stroke. This study provides a new niche targeting drug to oppose ischemic stroke and reveals the promising potential of CA for the control of ischemic stroke in humans.

Activin A alleviates neuronal injury through inhibiting cGAS-STING-mediated autophagy in mice with ischemic stroke

Activin A plays an essential role in ischemic stroke as a well-known neuroprotective factor. We previously reported that Activin A could promote white matter remyelination. However, the exact molecular mechanism of Activin A in neuronal protection post-stroke is still unclear. In this study, the middle cerebral artery occlusion/reperfusion (MCAO/R)-induced ischemic stroke mouse model and oxygen-glucose deprivation/reoxygenation (OGD/R)-treated primary neurons were used to explore the molecular mechanism of Activin A-mediated neuroprotection against ischemic injuries. We found that Activin A significantly inhibits cGAS-STING-mediated excessive autophagy through the PI3K-PKB pathway, but not mTOR-dependent autophagy. Consequently, Activin A protected neurons against OGD/R-induced ischemic injury and improved cell survival in a dose-dependent manner. In addition, Activin A improved neurological functions and reduced infarct size of mice with MCAO/R-induced ischemic stroke by inhibiting autophagy. Furthermore, Activin A depended on ACVR1C receptor to exert neuroprotective effects in 1 h MCAO/R treated mice. Our findings showed that Activin A alleviated neuronal ischemic injury through inhibiting cGAS-STING-mediated excessive autophagy in mice with ischemic stroke, which may suggest a potential therapeutic target for ischemic stroke.

Artemisinin Alleviates Cerebral Ischemia/Reperfusion-Induced Oxidative Damage via Regulating PHB2-Mediated Autophagy in the Human Neuroblastoma SH-SY5Y Cell Line.

Oxidative stress plays a key role in cerebral ischemia/reperfusion injury. Artemisinin (ART) has antioxidative stress activity in addition to its powerful antimalarial effects. In this article, we investigated the effect of ART on OGD/R-induced oxidative stress injury and its underlying mechanisms. We used oxygen-glucose deprivation/reoxygenation (OGD/R) to establish an in vitro model of cerebral ischemia/reperfusion (I/R) injury. CCK-8 and lactate dehydrogenase (LDH) release were used to assess cellular damage. Measurement of reactive oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH), and mitochondrial membrane potential (MMP) estimates oxidative stress-induced damage and protection from ART effect. OGD/R treatment aggravated oxidative stress damage, whereas ART reversed the effects of OGD/R. Autophagy is closely related to oxidative stress; in order to confirm whether the antioxidative stress effect of ART is related to PHB2-mediated autophagy, we examined the protein expression of prohibitin 2 (PHB2), TOMM20, p62, and the conversion of microtubule-associated protein light chain 3I (LC3I) to LC3II and found that the protein expression of PHB2, TOMM20, p62, and LC3II/LC3I was significantly correlated with OGD/R treatment. The colocalization of PHB2 and LC3, TOMM20, and LC3 was reduced after OGD/R treatment, and ART reversed this change. After silencing PHB2, the protective effect of ART against OGD/R-induced oxidative stress injury was reduced, the protein expressions of PHB2, TOMM20 and LC3II/LC3I and the colocalization of PHB2 and LC3, TOMM20, and LC3 were decreased. We used chloroquine to block the lysosomal pathway and found that ART increased the conversion of LC3I to LC3II, silencing PHB2 which inhibited the conversion of LC3I to LC3II, and impaired mitophagy. Our findings showed that ART attenuated OGD/R-induced oxidative stress damage through PHB2-mediated mitophagy. To the current knowledge, our study is the first to demonstrate that ART attenuates OGD/R-induced oxidative stress injury through PHB2-mediated autophagy in the human neuroblastoma SH-SY5Y cell line, which provided new insights into the treatment of OGD/R injury.

TJ-M2010-5, a novel CNS drug candidate, attenuates acute cerebral ischemia-reperfusion injury through the MyD88/NF-κB and ERK pathway.

Background: Cerebral ischemia-reperfusion injury (CIRI) inevitably occurs after vascular recanalization treatment for ischemic stroke. The accompanying inflammatory cascades have a major impact on outcome and regeneration after ischemic stroke. Evidences have demonstrated that TLR/MyD88/NF-κB signaling contributes to CIRI. This study aimed to investigate the druggability of MyD88 in the central nervous system (CNS) and the neuroprotective and anti-neuroinflammatory effects of the MyD88 inhibitor TJ-M2010-5 on CIRI. Methods: A middle cerebral artery occlusion (MCAO) model was used to simulate CIRI in mice. BV-2 cells were stimulated with oxygen glucose deprivation/reoxygenation (OGD/R) or lipopolysaccharide, and SH-SY5Y cells were induced by OGD/R in vitro. Neurological deficit scores and cerebral infarction volumes were evaluated. Immunofluorescence staining was performed to measure neuronal damage and apoptosis in the brain. The anti-neuroinflammatory effect of TJ-M2010-5 was evaluated by analyzing the expression of inflammatory cytokines, activation of microglia, and infiltration of peripheral myeloid cells. The expression of proteins of the MyD88/NF-κB and ERK pathway was detected by Simple Western. The concentrations of TJ-M2010-5 in the blood and brain were analyzed by liquid chromatography-mass spectrometry. Results: The cerebral infarction volume decreased in mice treated with TJ-M2010-5, with the most prominent decrease being approximately 80% of the original infarction volume. Neuronal loss and apoptosis were reduced following TJ-M2010-5 treatment. TJ-M2010-5 inhibited the infiltration of peripheral myeloid cells and the activation of microglia. TJ-M2010-5 also downregulated the expression of inflammatory cytokines and inhibited the MyD88/NF-κB and ERK pathway. Furthermore, TJ-M2010-5 showed good blood-brain barrier permeability and no neurotoxicity. Conclusion: TJ-M2010-5 has an excellent therapeutic effect on CIRI as a novel CNS drug candidate by inhibiting excessive neuroinflammatory responses.

The lncRNA NEAT1 Mediates Neuronal Cell Autophagy and Related Protein Expression After Cerebral Ischemia‒Reperfusion Injury.

The present study focuses on the role of the long noncoding RNA (lncRNA) NEAT1 in regulating autophagy during the ischemia‒reperfusion (I/R) injury process and its possible regulatory mechanism based on the results of laboratory experiments. Neuro-2a (N2a) cells and BV-2 microglial cells were cultured separately, and oxygen-glucose deprivation/reoxygenation (OGD/R) was induced in vitro to mimic cerebral I/R injury. The expression of lncRNA NEAT1 was measured after reoxygenation for different durations, and the results showed that NEAT1 expression was significantly different after OGD/R for 12h; thus, cell models of NEAT1 overexpression and knockdown were constructed. Knockdown of NEAT1 effectively relieved reperfusion injury. In an N2a and BV-2 cell coculture system, knockdown of NEAT1 reduced autophagic flow in neuronal cells after reperfusion. To clarify the mechanism of NEAT1 after neuronal I/R injury, label-free quantitative proteomics (LFQ) was used to identify the differentially expressed proteins (DEPs) in NEAT1 knockdown neurons after OGD/R for 12h. Additionally, Gene Ontology (GO) enrichment, protein‒protein interaction (PPI) network and parallel-reaction monitoring (PRM) quantitative analyses were carried out; the results showed that the expression levels of the autophagy-related proteins Gaa, Glb1, Prkaa1, Kif23, Sec24a and Vps25 were significantly reduced and that these proteins interact. In summary, this study shows that NEAT1 can regulate the interactions between autophagy-related proteins after neuronal I/R injury, reducing the level of autophagy and relieving neuronal reperfusion injury.

Leptin alleviates endoplasmic reticulum stress induced by cerebral ischemia/reperfusion injury via the PI3K/Akt signaling pathway.

Cerebral ischemic/reperfusion injury (CIRI) is a key factor for the prognosis of ischemic stroke (IS), the leading disease in terms of global disability and fatality rates. Recent studies have shown that endoplasmic reticulum stress (ERS) may be a target against CIRI and that leptin, a peptide hormone, has neuroprotective activity to mitigate CIRI. An in vitro CIRI model was induced in primary cortical neurons by oxygen-glucose deprivation and reoxygenation (OGD/R) after pretreatment with LY294002 (10 µmol/L) and/or leptin (0.4 mg/L), and cell viability, neuronal morphology and endoplasmic reticulum (ER) dysfunction were evaluated. An in vivo CIRI model was established in rats by middle cerebral artery occlusion and reperfusion (MCAO/R) after the injection of LY294002 (10 μmol/L) and/or leptin (1 mg/kg), and neurological function, infarct volume, cerebral pathological changes, the expression of ERS-related proteins and cell apoptosis were examined. In vitro, leptin treatment improved the cell survival rate, ameliorated neuronal pathological morphology and alleviated OGD/R-induced ERS. In vivo, administration of leptin significantly reduced the infarct volume, neurological deficit scores and neuronal apoptosis as well as pathological alterations. In addition, leptin suppressed MCAO/R-induced ERS and may decrease apoptosis by inhibiting ERS-related death and caspase 3 activation. It also regulated expression of the antiapoptotic protein Bcl-2 and the proapoptotic protein Bax in the cortex. Furthermore, the inhibitory effect of leptin on ERS was significantly decreased by the effective phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. These results confirm that ERS plays an important role in CIRI and that leptin can inhibit the activation of ERS through the PI3K/Akt pathway, thereby alleviating CIRI. These findings provide novel therapeutic targets for IS.

LncRNA PEG11as aggravates cerebral ischemia/reperfusion injury after ischemic stroke through miR-342-5p/PFN1 axis

AimLncRNAs are highly expressed in the CNS and regulate pathophysiological processes. However, the potential role of lncRNAs inischemic stroke (IS) remains unknown. In this study, we investigated the functions and possible molecular mechanism of lncRNA paternal expressed gene 11 antisense (PEG11as) in this process. MethodsMiddle cerebral artery occlusion/reperfusion (MCAO/R) mice model and N2a cells model from oxygen-glucose deprivation/reoxygenation (OGD/R) were used to simulate cerebral I/R in vivo and in vitro. High-throughput sequencing (RNA-Seq) was used todetect differential expression of lncRNAs in cerebral I/R. QRT-PCR was used to detect the expression of PEG11as and miR-342-5p. Bioinformatics analysis, FISH, luciferase reporter assay, RIP, Western blot, and immunofluorescence were used to detect the interaction between PEG11as, miR-342-5p and PFN1. The effect on neuronal apoptosis was analyzed using loss-of-function combined with TUNEL, Hoechst, and caspase3 activity assays. Key findings254 lncRNAs were differentially expressed in MCAO1h/R6h mice. Among them, PEG11as was significantly up-regulated. PEG11as down-regulated could markedly attenuate the brain infarct volume, alleviate neurological deficit in vivo, and effectively promote neuron survival, attenuate neuronal apoptosis both in vivo and in vitro. FISH assay discovered that PEG11as was mainly located in the cytoplasm. Furthermore, we demonstrated that PEG11as was able to bind miR-342-5p to inhibit miR-342-5p activity, whereas the down-regulated of miR-342-5p resulted in profilin 1 (PFN1) overexpression and thus promoting apoptosis. SignificanceThis study suggests that PEG11as regulates neuronal apoptosis by miR-342-5p/PFN1 axis, which may contribute to our understanding of pathogenesis and provide a potential therapeutic option for cerebral I/R.

OIP5-AS1 Inhibits Oxidative Stress and Inflammation in Ischemic Stroke Through miR-155-5p/IRF2BP2 Axis.

Ischemic stroke is a very dangerous disease with high incidence, fatality and disability rate in human beings. Massive evidence has indicated that oxidative stress and inflammation are intimately correlated with progression of ischemic stroke. Additionally, LncRNAs were reported to be involved in ischemic stroke. Here, we aim to explore the effects and molecular mechanism of lncRNA OIP5-AS1 on oxidative stress and inflammation in ischemic stroke. HMC3 and SH-SY5Y cells were under the condition of oxygen-glucose deprivation/reoxygenation (OGD/R) treatment to establish cell models of ischemic stroke. Commercial kits were employed to detect the indicators of oxidative stress including ROS, MDA and SOD. The expression of OIP5-AS1, miR-155-5p and IRF2BP2 mRNA was determined using RT-qPCR. The protein levels of inflammatory factors including TNF-α, IL-1β and IL-6 and IRF2BP2 were assessed by western blot and/or ELISA. Luciferase activity assay was employed to validate their correlations among OIP5-AS1, miR-155-5p and IRF2BP2. In OGD/R-induced HMC3 and SH-SY5Y cells, the expression of OIP5-AS1 and IRF2BP2 was reduced while miR-155-5p was elevated. OGD/R induction promoted oxidative stress and inflammatory response in HMC3 and SH-SY5Y cells, while OIP5-AS1 or IRF2BP2 sufficiency as well as miR-155-5p inhibitor attenuated OGD/R-induced these influences. In addition, IRF2BP2 knockdown abolished the suppressive impacts of OIP5-AS1 overexpression on oxidative stress and inflammatory response in OGD/R-induced HMC3 and SH-SY5Y cells. Mechanistically, OIP5-AS1 enhanced IRF2BP2 expression via sponging miR-155-5p. OIP5-AS1 suppressed oxidative stress and inflammatory response to alleviate cell injury caused by OGD/R induction in HMC3 and SH-SY5Y cells through regulating miR-155-5p/IRF2BP2 axis, which might offer novel targeted molecules for ischemic stroke therapy.