Deciphering the neuroprotective mechanisms of RACK1 in cerebral ischemia-reperfusion injury: Pioneering insights into mitochondrial autophagy and the PINK1/Parkin axis.

INTRODUCTION: Cerebral ischemia-reperfusion injury (CIRI) is a common and debilitating complication of cerebrovascular diseases such as stroke, characterized by mitochondrial dysfunction and cell apoptosis. Unraveling the molecular mechanisms behind these processes is essential for developing effective CIRI treatments. This study investigates the role of RACK1 (receptor for activated C kinase 1) in CIRI and its impact on mitochondrial autophagy. METHODS: We utilized high-throughput transcriptome sequencing and weighted gene co-expression network analysis (WGCNA) to identify core genes associated with CIRI. In vitro experiments used human neuroblastoma SK-N-SH cells subjected to oxygen and glucose deprivation (OGD) to simulate ischemia, followed by reperfusion (OGD/R). RACK1 knockout cells were created using CRISPR/Cas9 technology, and cell viability, apoptosis, and mitochondrial function were assessed. In vivo experiments involved middle cerebral artery occlusion/reperfusion (MCAO/R) surgery in rats, evaluating neurological function and cell apoptosis. RESULTS: Our findings revealed that RACK1 expression increases during CIRI and is protective by regulating mitochondrial autophagy through the PINK1/Parkin pathway. In vitro, RACK1 knockout exacerbated cell apoptosis, while overexpression of RACK1 reversed this process, enhancing mitochondrial function. In vivo, RACK1 overexpression reduced cerebral infarct volume and improved neurological deficits. The regulatory role of RACK1 depended on the PINK1/Parkin pathway, with RACK1 knockout inhibiting PINK1 and Parkin expression, while RACK1 overexpression restored them. CONCLUSION: This study demonstrates that RACK1 safeguards against neural damage in CIRI by promoting mitochondrial autophagy through the PINK1/Parkin pathway. These findings offer crucial insights into the regulation of mitochondrial autophagy and cell apoptosis by RACK1, providing a promising foundation for future CIRI treatments.

Huanglian Jiedu decoction alleviates ischemia-induced cerebral injury in rats by mitigating NET formation and activiting GABAergic synapses.

Huanglian Jiedu decoction (HLJD) has been used to treat ischemic stroke in clinic. However, the detailed protective mechanisms of HLJD on ischemic stroke have yet to be elucidated. The aim of this study is to elucidate the underlying pharmacological mechanisms of HLJD based on the inhibition of neuroinflammation and the amelioration of nerve cell damage. A middle cerebral artery occlusion reperfusion (MCAO/R) model was established in rats and received HLJD treatment. Effects of HLJD on neurological function was assessed based on Bederson's score, postural reflex test and asymmetry score. 2, 3, 5-Triphenyltetrazolium chloride (TTC) staining, Hematein and eosin (HE) and Nissl staining were used to observe the pathological changes in brain. Then, transcriptomics was used to screen the differential genes in brain tissue in MCAO/R model rats following HLJD intervention. Subsequently, the effects of HLJD on neutrophil extracellular trap (NET) formation-related neuroinflammation, gamma-aminobutyric acid (GABA)ergic synapse activation, nerve cell damage and proliferation were validated using immunofluorescence, western blot and enzyme-linked immunosorbent assay (ELISA). Our results showed that HLJD intervention reduced the Bederson's score, postural reflex test score and asymmetry score in MCAO/R model rats. Pathological staining indicated that HLJD treatment decreased the cerebral infarction area, mitigated neuronal damage and increased the numbers of Nissl bodies. Transcriptomics suggested that HLJD affected 435 genes in MCAO/R rats. Among them, several genes involving in NET formation and GABAergic synapses pathways were dysregulated. Subsequent experimental validation showed that HLJD reduced the MPO+CitH3+ positive expression area, reduced the protein expression of PAD4, p-P38/P38, p-ERK/ERK and decreased the levels of IL-1ß, IL-6 and TNF-α, reversed the increase of Iba1+TLR4+, Iba1+p65+ and Iba1+NLRP3+ positive expression area in brain. Moreover, HLJD increased GABA levels, elevated the protein expression of GABRG1 and GAT3, decreased the TUNEL positive expression area and increased the Ki67 positive expression area in brain. HLJD intervention exerts a multifaceted positive impact on ischemia-induced cerebral injury in MCAO/R rats. This intervention effectively inhibits neuroinflammation by mitigating NET formation, and concurrently improves nerve cell damage and fosters nerve cell proliferation through activating GABAergic synapses.

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.

Astrocyte-derived lactate aggravates brain injury of ischemic stroke in mice by promoting the formation of protein lactylation.

Aim: Although lactate supplementation at the reperfusion stage of ischemic stroke has been shown to offer neuroprotection, whether the role of accumulated lactate at the ischemia phase is neuroprotection or not remains largely unknown. Thus, in this study, we aimed to investigate the roles and mechanisms of accumulated brain lactate at the ischemia stage in regulating brain injury of ischemic stroke. Methods and Results: Pharmacological inhibition of lactate production by either inhibiting LDHA or glycolysis markedly attenuated the mouse brain injury of ischemic stroke. In contrast, additional lactate supplement further aggravates brain injury, which may be closely related to the induction of neuronal death and A1 astrocytes. The contributing roles of increased lactate at the ischemic stage may be related to the promotive formation of protein lysine lactylation (Kla), while the post-treatment of lactate at the reperfusion stage did not influence the brain protein Kla levels with neuroprotection. Increased protein Kla levels were found mainly in neurons by the HPLC-MS/MS analysis and immunofluorescent staining. Then, pharmacological inhibition of lactate production or blocking the lactate shuttle to neurons showed markedly decreased protein Kla levels in the ischemic brains. Additionally, Ldha specific knockout in astrocytes (Aldh1l1 CreERT2; Ldha fl/fl mice, cKO) mice with MCAO were constructed and the results showed that the protein Kla level was decreased accompanied by a decrease in the volume of cerebral infarction in cKO mice compared to the control groups. Furthermore, blocking the protein Kla formation by inhibiting the writer p300 with its antagonist A-485 significantly alleviates neuronal death and glial activation of cerebral ischemia with a reduction in the protein Kla level, resulting in extending reperfusion window and improving functional recovery for ischemic stroke. Conclusion: Collectively, increased brain lactate derived from astrocytes aggravates ischemic brain injury by promoting the protein Kla formation, suggesting that inhibiting lactate production or the formation of protein Kla at the ischemia stage presents new therapeutic targets for the treatment of ischemic stroke.

Exosomal miR-486 derived from bone marrow mesenchymal stem cells promotes angiogenesis following cerebral ischemic injury by regulating the PTEN/Akt pathway.

Bone marrow mesenchymal stem cell-derived exosomes (BMSC-Exos) have been shown to promote angiogenesis after ischemic stroke, in which microRNAs (miRs) are believed to play an important role in exosome-mediated therapeutic effects, though the mechanism is still not clear. In this study, a series of molecular biological and cellular assays, both in vitro and in vivo, were performed to elucidate the role of exosomal miR-486 in angiogenesis following cerebral ischemic and its molecular mechanisms. Our results revealed that BMSC-Exos significantly improved neurological function and increased microvessel density in ischemic stroke rats. In vitro assays showed that BMSC-Exos promoted the proliferation, migration, and tube formation ability of oxygen-glucose deprivation/reoxygenation (OGD/R) injured rat brain microvascular endothelial cells (RBMECs). Importantly, BMSC-Exos increased the expression of miR-486 and phosphorylated protein kinase B (p-Akt) and down-regulated the protein level of phosphatase and tensin homolog (PTEN) in vivo and in vitro. Mechanistic studies demonstrated that transfection with miR-486 mimic enhanced RBMECs angiogenesis and increased p-Akt expression, while inhibited PTEN expression. On the other hand, the miR-486 inhibitor induced an opposite effect, which could be blocked by PTEN siRNA. It was thus concluded that exosomal miR-486 from BMSCs may enhance the functional recovery by promoting angiogenesis following cerebral ischemic injury, which might be related to its regulation of the PTEN/Akt pathway.

A2 reactive astrocyte-derived exosomes alleviate cerebral ischemia-reperfusion injury by delivering miR-628.

Ischemia and hypoxia activate astrocytes into reactive types A1 and A2, which play roles in damage and protection, respectively. However, the function and mechanism of A1 and A2 astrocyte exosomes are unknown. After astrocyte exosomes were injected into the lateral ventricle, infarct volume, damage to the blood-brain barrier (BBB), apoptosis and the expression of microglia-related proteins were measured. The dual luciferase reporter assay was used to detect the target genes of miR-628, and overexpressing A2-Exos overexpressed and knocked down miR-628 were constructed. qRT-PCR, western blotting and immunofluorescence staining were subsequently performed. A2-Exos obviously reduced the infarct volume, damage to the BBB and apoptosis and promoted M2 microglial polarization. RT-PCR showed that miR-628 was highly expressed in A2-Exos. Dual luciferase reporter assays revealed that NLRP3, S1PR3 and IRF5 are target genes of miR-628. After miR-628 was overexpressed or knocked down, the protective effects of A2-Exos increased or decreased, respectively. A2-Exos reduced pyroptosis and BBB damage and promoted M2 microglial polarization through the inhibition of NLRP3, S1PR3 and IRF5 via the delivery of miR-628. This study explored the mechanism of action of A2-Exos and provided new therapeutic targets and concepts for treating cerebral ischemia.

Nrf1 Reduces COX-2 Expression and Maintains Cellular Homeostasis After Cerebral Ischemia/Reperfusion By Targeting IL-6/TNF-α Protein Production.

Neuroinflammation has been considered involved in the process of cerebral ischemia-reperfusion injury (CIRI). Transcription factors play a crucial role in regulating gene transcription and the expressions of specific proteins during the progression of various neurological diseases. Evidence showed that transcription factor nuclear factor erythroid 2-related factor 1 (NFE2L1, also known as Nrf1) possessed strong biological activities including antioxidant, anti-inflammatory and neuroprotective properties. However, its role and potential molecular mechanisms in CIRI remain unclear. In our study, we observed a significant elevation of Nrf1 in the cerebral cortex following cerebral ischemia-reperfusion in rats. The Nrf1 downregulation markedly raised COX-2, TNF-α, IL-1ß, and IL-6 protein levels during middle cerebral artery occlusion/reperfusion in rats, which led to worsened neurological deficits, higher cerebral infarct volume, and intensified cortical histopathological damage. In subsequent in vitro studies, the expression of Nrf1 protein increased following oxygen-glucose deprivation/reperfusion treatment on neurons. Subsequently, Nrf1 knockdown resulted in a significant upregulation of inflammatory factors, leading to a substantial increase in the cell death rate. Through analyzing the alterations in the expression of inflammatory factors under diverse interventions, it is indicated that Nrf1 possesses the capacity to discern variations in inflammatory factors via specific structural domains. Our findings demonstrate the translocation of the Nrf1 protein from the cytoplasm to the nucleus, thereby modulating the protein expression of IL-6/TNF-α and subsequently reducing the expression of multiple inflammatory factors. This study signifies, for the first time, that during cerebral ischemia-reperfusion, Nrf1 translocases to the nucleus to regulate the protein expression of IL-6/TNF-α, consequently suppressing COX-2 expression and governing cellular inflammation, ultimately upholding cellular homeostasis.

Application and Development of Cell Membrane Functionalized Biomimetic Nanoparticles in the Treatment of Acute Ischemic Stroke.

Ischemic stroke is a serious neurological disease involving multiple complex physiological processes, including vascular obstruction, brain tissue ischemia, impaired energy metabolism, cell death, impaired ion pump function, and inflammatory response. In recent years, there has been significant interest in cell membrane-functionalized biomimetic nanoparticles as a novel therapeutic approach. This review comprehensively explores the mechanisms and importance of using these nanoparticles to treat acute ischemic stroke with a special emphasis on their potential for actively targeting therapies through cell membranes. We provide an overview of the pathophysiology of ischemic stroke and present advances in the study of biomimetic nanoparticles, emphasizing their potential for drug delivery and precision-targeted therapy. This paper focuses on bio-nanoparticles encapsulated in bionic cell membranes to target ischemic stroke treatment. It highlights the mechanism of action and research progress regarding different types of cell membrane-functionalized bi-onic nanoparticles such as erythrocytes, neutrophils, platelets, exosomes, macrophages, and neural stem cells in treating ischemic stroke while emphasizing their potential to improve brain tissue's ischemic state and attenuate neurological damage and dysfunction. Through an in-depth exploration of the potential benefits provided by cell membrane-functionalized biomimetic nanoparticles to improve brain tissue's ischemic state while reducing neurological injury and dysfunction, this study also provides comprehensive research on neural stem cells' potential along with that of cell membrane-functionalized biomimetic nanoparticles to ameliorate neurological injury and dysfunction. However, it is undeniable that there are still some challenges and limitations in terms of biocompatibility, safety, and practical applications for clinical translation.

Bruton's tyrosine kinase inhibition ameliorated neuroinflammation during chronic white matter ischemia.

Chronic cerebral hypoperfusion (CCH), a disease afflicting numerous individuals worldwide, is a primary cause of cognitive deficits, the pathogenesis of which remains poorly understood. Bruton's tyrosine kinase inhibition (BTKi) is considered a promising strategy to regulate inflammatory responses within the brain, a crucial process that is assumed to drive ischemic demyelination progression. However, the potential role of BTKi in CCH has not been investigated so far. In the present study, we elucidated potential therapeutic roles of BTK in both in vitro hypoxia and in vivo ischemic demyelination model. We found that cerebral hypoperfusion induced white matter injury, cognitive impairments, microglial BTK activation, along with a series of microglia responses associated with inflammation, oxidative stress, mitochondrial dysfunction, and ferroptosis. Tolebrutinib treatment suppressed both the activation of microglia and microglial BTK expression. Meanwhile, microglia-related inflammation and ferroptosis processes were attenuated evidently, contributing to lower levels of disease severity. Taken together, BTKi ameliorated white matter injury and cognitive impairments induced by CCH, possibly via skewing microglia polarization towards anti-inflammatory and homeostatic phenotypes, as well as decreasing microglial oxidative stress damage and ferroptosis, which exhibits promising therapeutic potential in chronic cerebral hypoperfusion-induced demyelination.

Hypomethylated leptin receptor reduces cerebral ischaemia-reperfusion injury by activating the JAK2/STAT3 signalling pathway.

OBJECTIVE: To investigate the cerebroprotective effects of leptin in vitro and in vivo via the Janus kinase-2 (JAK2)/transcription factor signal transducer and activators of transcription-3 (STAT3) pathway and leptin receptors (LEPR). METHODS: The study used the cellular oxygen-glucose deprivation (OGD) model in PC12 cells and the middle cerebral artery occlusion (MCAO) rat model of cerebral ischaemia-reperfusion injury (CIRI) to assess changes in gene expression and protein levels following leptin pretreatment. The methylated DNA immunoprecipitation (MeDIP) assay measured DNA methylation levels. RESULTS: The optimal leptin concentration for exerting neuroprotective effects against ischaemia-reperfusion injury in PC12 cells was 200 ng/ml in vitro, but excessive leptin diminished this effect. Leptin pretreatment in the MCAO rat model demonstrated a similar effect to previously reported leptin administration post-CIRI. In addition to regulating the expression of inflammation-related cytokines, Western blot analysis showed that leptin pretreatment upregulated BCL-2 and downregulated caspase 3 levels. The MeDIP analysis demonstrated that DNA methylation regulated LEPR gene expression in the MCAO rat model when leptin pretreatment was used. CONCLUSION: Exogenous leptin might bind to extra-activated LEPR by reducing the methylation level of the LEPR gene promoter region, which leads to an increase in phosphorylated JAK2/STAT3 and apoptotic signalling pathways.

[Exogenous leptin improves cerebral ischemia-reperfusion-induced glutamate excitotoxic injury in mice by up-regulating GLT-1 and GLAST expression in astrocytes].

OBJECTIVE: To investigate the protective effect of exogenous leptin against focal cerebral ischemia-reperfusion (I/R) injury in mice and explore the underlying mechanism. METHODS: A total of 100 C57BL/6 mice were randomly divided into 5 groups, including a sham-operated group, cerebral I/R model group, and 3 leptin treatment groups with intraperitoneal injections of 0.5, 1.0 or 2.0 leptin immediately after occlusion of the internal carotid artery. At 24 h after reperfusion, neurological function scores of the mice were assessed, and TTC staining was used to determine the area of cerebral infarction. The pathological changes in the cortical brain tissue of the mice were observed using HE staining, and degenerative damage of the cortical neurons were assessed with Fluoro-Jade C staining. The expression of glial fibrillary acidic protein in cortical brain tissues was detected using immunohistochemistry and Western blotting. In another 45 C57BL/6 mice with sham operation, I/R modeling, or leptin (1 mg/kg) treatment, glutamic acid in the cortical brain tissue was detected using glutamate assay, and cortical glutamate-aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1) protein expressions were detected using immunohistochemistry. RESULTS: Compared with the I/R model mice, the leptin-treated mice had significantly lower neurological deficit scores, smaller cerebral infarct area, milder pathologies in the cortical brain tissue, and lessened cortical neuronal damage with normal morphology and less excessive proliferation of the astrocytes. Leptin treatment significantly up-regulated the expressions of GLT-1 and GLAST and lowered the content of glutamic acid in the brain tissue of the I/R mice. CONCLUSION: Exogenous leptin has obvious neuroprotective effect against cerebral I/R injury in mice, mediated probably by controlling excessive astrocyte proliferation and up-regulating cortical GLT-1 and GLAST expressions to reduce glutamate-mediated excitotoxic injury of the astrocytes.

The effects and mechanisms of AM1241 in alleviating cerebral ischemia-reperfusion injury.

OBJECTIVE: Research has shown that cerebral ischemia-reperfusion injury (CIRI) involves a series of physiological and pathological mechanisms, including inflammation, oxidative stress, and cell apoptosis. The cannabinoid receptor 2 agonist AM1241 has been found to have anti-inflammatory and anti-oxidative stress effects. However, it is unclear whether AM1241 has a protective effect against brain ischemia-reperfusion injury, and its underlying mechanisms are not yet known. METHODS: In this study, we investigated the anti-inflammatory, anti-oxidative stress, and anti-apoptotic effects of AM1241 and its mechanisms in BV2 cells stimulated with H2O2 and in a C57BL/6 mouse model of CIRI in vitro and in vivo, respectively. RESULTS: In vitro, AM1241 significantly inhibited the release of pro-inflammatory cytokines TNF-α and IL-6, reactive oxygen species (ROS), and the increase in Toll-like receptor 4/myeloid differentiation protein 2 (MD2/TLR4) complex induced by H2O2. Under H2O2 stimulation, MD2 overexpression resulted in increased levels of MD2/TLR4 complex, TNF-α, IL-6, NOX2, BAX, and Cleaved-Caspase3 (C-Caspase3), as well as the activation of the MAPK pathway and NF-κB, which were reversed by AM1241. In addition, molecular docking experiments showed that AM1241 directly interacted with MD2. Surface Plasmon Resonance (SPR) experiments further confirmed the binding of AM1241 to MD2. In vivo, AM1241 significantly attenuated neurofunctional impairment, brain edema, increased infarct volume, oxidative stress levels, and neuronal apoptosis in CIRI mice overexpressing MD2. CONCLUSION: Our study demonstrates for the first time that AM1241 alleviates mouse CIRI by inhibiting the MD2/TLR4 complex, exerting anti-inflammatory, anti-oxidative stress and anti-apoptotic effects.

[Mechanism study of 6-shogaol alleviating cerebral ischemia/reperfusion injury by regulating microRNA-26a-5p/death-associated protein kinase 1].

OBJECTIVE: To investigate whether 6-shogaol (6-SH) alleviates oxygen-glucose deprivation/reoxygenation (OGD/R)-induced neuronal autophagy and calcium overload by promoting the expression of microRNA-26a-5p (miR-26a-5p) and inhibiting death-associated protein kinase 1 (DAPK1), and to explore its potential mechanisms. METHODS: Primary cultured logarithmic growth phase mouse hippocampal neurons HT22 cells were taken and cell counting kit-8 (CCK-8) was used to detect cell viability, searching for the optimal concentration of Na2S2O4. HT22 cells were divided into blank control group (NC group), OGD/R group (sugar-free culture medium + 10 mmol/L Na2S2O4 treatment for 1.5 hours followed by normal culture medium for 4 hours), 6-SH intervention group (cultured with 10 µmol/L 6-SH for 4 hours after OGD), negative control inhibitor pretreatment group (transfected with negative control inhibitor for 48 hours followed by OGD, then cultured with 6-SH for 4 hours), and miR-26a-5p inhibitor pretreatment group (transfected with miR-26a-5p inhibitor for 48 hours followed by OGD, then cultured with 6-SH for 4 hours). Cell viability of each group was detected by CCK-8 method; cell ultrastructure was observed under transmission electron microscopy; real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the gene expressions of DAPK1 and miR-26a-5p; molecular docking were used to verify the interaction between 6-SH and miR-26a-5p; dual-luciferase assay was used to verify the targeting relationship between DAPK1 and miR-26a-5p; flow cytometry was used to determine the levels of intracellular Ca2+; Western blotting was used to detect the protein expressions of phosphorylated-glutamate receptor 2B (p-NMDAR2B) Ser1303, DAPK1, autophagy related protein Beclin1, light chain 3 (LC3), and p-DAPK1 Ser308; immunofluorescence was used to detect the expression of LC3 and Beclin1. RESULTS: The results of the CCK-8 assay showed that the cell viability of the 6-SH intervention group was significantly increased compared to the OGD/R group, while the cell viability of the miR-26a-5p inhibitor pretreatment group was significantly decreased compared to the 6-SH intervention group. Transmission electron microscopy revealed that the number of autophagosomes in the 6-SH intervention group was significantly reduced compared to the OGD/R group, while the number of autophagosomes in the miR-26a-5p inhibitor pretreatment group was significantly increased compared to the 6-SH intervention group. RT-qPCR results showed that compared with the OGD/R group, the expression of miR-26a-5p was significantly upregulated and the expression of DAPK1 mRNA was significantly downregulated in the 6-SH intervention group; compared with the 6-SH intervention group, the expression of miR-26a-5p was significantly downregulated and the expression of DAPK1 mRNA was significantly upregulated in the miR-26a-5p inhibitor pretreatment group. Molecular docking verified the interaction between 6-SH and miR-26a-5p. Dual-luciferase reporter gene assay showed that compared with the negative control group, mmu-miR-26a-5p significantly downregulated the luciferase expression of m-DAPK1-3UTR-WT, indicating a binding interaction between them. Flow cytometry results showed that compared with the OGD/R group, the level of intracellular Ca2+; was significantly decreased in the 6-SH intervention group; compared with the 6-SH intervention group, the level of Ca2+ was significantly increased in the miR-26a-5p inhibitor pretreatment group. Western blotting results showed that compared with the OGD/R group, the protein expressions of p-NMDAR2B Ser1303, DAPK1, Beclin1, and LC3 were significantly decreased in the 6-SH intervention group (p-NMDAR2B Ser1303/ß-actin: 2.34±0.27 vs. 4.78±0.39, DAPK1/ß-actin: 1.40±0.13 vs. 2.37±0.21, Beclin1/ß-actin: 2.61±0.32 vs. 4.32±0.29, LC3/ß-actin: 2.52±0.45 vs. 5.09±0.18, all P < 0.05), while the protein expression of p-DAPK1 Ser308 was significantly increased (p-DAPK1 Ser308/ß-actin: 0.66±0.09 vs. 0.40±0.02, P < 0.05); compared with the 6-SH intervention group, the protein expressions of p-NMDAR2B Ser1303, DAPK1, Beclin1, and LC3 were significantly increased in the miR-26a-5p inhibitor pretreatment group (p-NMDAR2B Ser1303/ß-actin: 4.08±0.14 vs. 2.34±0.27, DAPK1/ß-actin: 1.96±0.15 vs. 1.40±0.13, Beclin1/ß-actin: 3.92±0.31 vs. 2.61±0.32, LC3/ß-actin: 4.33±0.33 vs. 2.52±0.45, all P < 0.05), while the expression of p-DAPK1 Ser308 protein was significantly decreased (p-DAPK1 Ser308/ß-actin: 0.33±0.12 vs. 0.66±0.09, P < 0.05); immunofluorescence staining showed that compared with the OGD/R group, the fluorescence intensity of LC3 and Beclin1 was significantly decreased in the 6-SH intervention group; compared with the 6-SH intervention group, the fluorescence intensity of LC3 and Beclin1 was significantly increased in the miR-26a-5p inhibitor pretreatment group. CONCLUSIONS: 6-SH can alleviate neuronal damage by regulating miR-26a-5p/DAPK1 to reduce autophagy and calcium overload in cells.

Impact of acupuncture on ischemia/reperfusion injury: Unraveling the role of miR-34c-5p and autophagy activation.

We have previously reported that the expression of miR-34c-5p was up-regulated during acupuncture treatment in the setting of a cerebral ischemia/reperfusion injury (CIRI), indicating that miR-34c-5p plays an important role in healing from a CIRI-induced brain injury. This study sought to evaluate the effects of acupuncture on miR-34c-5p expression and autophagy in the forward and reverse directions using a rat focal cerebral ischemia/reperfusion model. After 120 minutes of middle cerebral artery occlusion and reperfusion, rats were treated with acupuncture at the "Dazhui" (DU20), "Baihui" (DU26) and "Renzhong" (DU14) points. Neurologic function deficit score, cerebral infarct area ratio, neuronal apoptosis and miR-34c-5p expression were evaluated 72 hr after treatment. The autophagy agonist RAPA and the antagonist 3MA were used to evaluate the neuro protective effects of autophagy-mediated acupuncture. We found that acupuncture treatment improved autophagy in the brain tissue of CIRI rats. Acupuncture reversed the negative effects of 3MA on CIRI, and acupuncture combined with RAPA further enhanced autophagy. We also found that acupuncture could increase miR-34c-5p expression in hippocampal neurons after ischemia/reperfusion. Acupuncture and a miR-34c agomir were able to enhance autophagy, improve neurologic deficits, and reduce the cerebral infarct area ratio and apoptosis rate by promoting the expression of miR-34c-5p. Silencing miR-34c resulted in a significantly reduced activating effect of acupuncture on autophagy and increased apoptosis, neurologic deficit symptoms, and cerebral infarct area ratio. This confirms that acupuncture can upregulate miR-34c-5p expression, which is beneficial in the treatment of CIRI.

Thymol improves ischemic brain injury by inhibiting microglia-mediated neuroinflammation.

BACKGROUND: Microglia-mediated inflammation is a critical factor in the progression of ischemic stroke. Consequently, mitigating excessive microglial activation represents a potential therapeutic strategy for ischemic injury. Thymol, a monophenol derived from plant essential oils, exhibits diverse beneficial biological activities, including anti-inflammatory and antioxidant properties, with demonstrated protective effects in various disease models. However, its specific effects on ischemic stroke and microglial inflammation remain unexplored. METHODS: Rodent transient middle cerebral artery occlusion (tMCAO) model was established to simulate ischemic stroke. TTC staining, modified neurological function score (mNSS), and behavioral tests were used to assess the severity of neurological damage. Then immunofluorescence staining and cytoskeleton analysis were used to determine activation of microglia. Lipopolysaccharide (LPS) was utilized to induce the inflammatory response of primary microglia in vitro. Quantitative real-time polymerase chain reaction (qRT-PCR), western blot, and enzyme-linked immunosorbent assay (ELISA) were performed to exam the expression of inflammatory cytokines. And western blot was used to investigate the mechanism of the anti-inflammatory effect of thymol. RESULTS: In this study, we found that thymol treatment could ameliorate post-stroke neurological impairment and reduce infarct volume by mitigating microglial activation and pro-inflammatory response (IL-1ß, IL-6, and TNF-α). Mechanically, thymol could inhibit the phosphorylation of phosphatidylinositol-3-kinase (PI3K), sink serine/threonine kinase (Akt), and mammalian target of rapamycin (mTOR), thereby suppressing the activation of nuclear factor-κB (NF-κB). CONCLUSIONS: Our study demonstrated that thymol could reduce the microglial inflammation by targeting PI3K/Akt/mTOR/NF-κB signaling pathway, ultimately alleviating ischemic brain injury. These findings suggest that thymol is a promising candidate as a neuroprotective agent against ischemic stroke.

Modulating the RPS27A/PSMD12/NF-κB pathway to control immune response in mouse brain ischemia-reperfusion injury.

BACKGROUND: Investigating immune cell infiltration in the brain post-ischemia-reperfusion (I/R) injury is crucial for understanding and managing the resultant inflammatory responses. This study aims to unravel the role of the RPS27A-mediated PSMD12/NF-κB axis in controlling immune cell infiltration in the context of cerebral I/R injury. METHODS: To identify genes associated with cerebral I/R injury, high-throughput sequencing was employed. The potential downstream genes were further analyzed using Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Protein-Protein Interaction (PPI) analyses. For experimental models, primary microglia and neurons were extracted from the cortical tissues of mouse brains. An in vitro cerebral I/R injury model was established in microglia using the oxygen-glucose deprivation/reoxygenation (OGD/R) technique. In vivo models involved inducing cerebral I/R injury in mice through the middle cerebral artery occlusion (MCAO) method. These models were used to assess neurological function, immune cell infiltration, and inflammatory factor release. RESULTS: The study identified RPS27A as a key player in cerebral I/R injury, with PSMD12 likely acting as its downstream regulator. Silencing RPS27A in OGD/R-induced microglia decreased the release of inflammatory factors and reduced neuron apoptosis. Additionally, RPS27A silencing in cerebral cortex tissues mediated the PSMD12/NF-κB axis, resulting in decreased inflammatory factor release, reduced neutrophil infiltration, and improved cerebral injury outcomes in I/R-injured mice. CONCLUSION: RPS27A regulates the expression of the PSMD12/NF-κB signaling axis, leading to the induction of inflammatory factors in microglial cells, promoting immune cell infiltration in brain tissue, and exacerbating brain damage in I/R mice. This study introduces novel insights and theoretical foundations for the treatment of nerve damage caused by I/R, suggesting that targeting the RPS27A and downstream PSMD12/NF-κB signaling axis for drug development could represent a new direction in I/R therapy.

Single-nucleus RNA sequencing reveals glial cell type-specific responses to ischemic stroke in male rodents.

Neuroglia critically shape the brain´s response to ischemic stroke. However, their phenotypic heterogeneity impedes a holistic understanding of the cellular composition of the early ischemic lesion. Here we present a single cell resolution transcriptomics dataset of the brain´s acute response to infarction. Oligodendrocyte lineage cells and astrocytes range among the most transcriptionally perturbed populations and exhibit infarction- and subtype-specific molecular signatures. Specifically, we find infarction restricted proliferating oligodendrocyte precursor cells (OPCs), mature oligodendrocytes and reactive astrocytes, exhibiting transcriptional commonalities in response to ischemic injury. OPCs and reactive astrocytes are involved in a shared immuno-glial cross talk with stroke-specific myeloid cells. Within the perilesional zone, osteopontin positive myeloid cells accumulate in close proximity to CD44+ proliferating OPCs and reactive astrocytes. In vitro, osteopontin increases the migratory capacity of OPCs. Collectively, our study highlights molecular cross talk events which might govern the cellular composition of acutely infarcted brain tissue.

ß-hydroxybutyrate and ischemic stroke: roles and mechanisms.

Stroke is a significant global burden, causing extensive morbidity and mortality. In metabolic states where glucose is limited, ketone bodies, predominantly ß-hydroxybutyrate (BHB), act as alternative fuel sources. Elevated levels of BHB have been found in the ischemic hemispheres of animal models of stroke, supporting its role in the pathophysiology of cerebral ischemia. Clinically, higher serum and urinary BHB concentrations have been associated with adverse outcomes in ischemic stroke, highlighting its potential utility as a prognostic biomarker. In both animal and cellular models, exogenous BHB administration has exhibited neuroprotective effects, reduction of infarct size, and improvement of neurological outcomes. In this review, we focus on the role of BHB before and after ischemic stroke, with an emphasis on the therapeutic potential and mechanisms of ketone administration after ischemic stroke.

Therapeutic Strategies for Ischemic Stroke: Modulating the Adult Neural Stem Cell Niche through the Wnt/ß-catenin Pathway.

Stroke is a prominent contributor to mortality and impairment on a global scale. Ischemic stroke accounts for approximately 80% of stroke cases and is caused by occlusion of cerebral blood vessels. Enhancing neurogenesis through the modulation of the neural stem cell niche in the adult brain is a promising therapeutic strategy for individuals afflicted with ischemic stroke. Neurogenesis results in the generation of newborn neurons that serve as replacements for deceased neural cells within the ischemic core, thereby playing a significant role in the process of neural restoration subsequent to cerebral ischemia. Research has shown that activation of the Wnt/ß-catenin pathway can augment neurogenesis following cerebral ischemia, suggesting that this pathway is a potentially beneficial therapeutic target for managing ischemic stroke. This review provides an extensive analysis of the current knowledge regarding the involvement of the Wnt/ß-catenin pathway in promoting neurogenesis, thereby offering a promising avenue for therapeutic intervention in the context of ischemic stroke or other neurological impairments.

Glutamate acts on acid-sensing ion channels to worsen ischaemic brain injury.

Glutamate is traditionally viewed as the first messenger to activate NMDAR (N-methyl-D-aspartate receptor)-dependent cell death pathways in stroke1,2, but unsuccessful clinical trials with NMDAR antagonists implicate the engagement of other mechanisms3-7. Here we show that glutamate and its structural analogues, including NMDAR antagonist L-AP5 (also known as APV), robustly potentiate currents mediated by acid-sensing ion channels (ASICs) associated with acidosis-induced neurotoxicity in stroke4. Glutamate increases the affinity of ASICs for protons and their open probability, aggravating ischaemic neurotoxicity in both in vitro and in vivo models. Site-directed mutagenesis, structure-based modelling and functional assays reveal a bona fide glutamate-binding cavity in the extracellular domain of ASIC1a. Computational drug screening identified a small molecule, LK-2, that binds to this cavity and abolishes glutamate-dependent potentiation of ASIC currents but spares NMDARs. LK-2 reduces the infarct volume and improves sensorimotor recovery in a mouse model of ischaemic stroke, reminiscent of that seen in mice with Asic1a knockout or knockout of other cation channels4-7. We conclude that glutamate functions as a positive allosteric modulator for ASICs to exacerbate neurotoxicity, and preferential targeting of the glutamate-binding site on ASICs over that on NMDARs may be strategized for developing stroke therapeutics lacking the psychotic side effects of NMDAR antagonists.