Middle Cerebral Artery Occlusion Mice Research Articles

JLX001 ameliorates cerebral ischemia injury by modulating microglial polarization and compromising NLRP3 inflammasome activation via the NF-κB signaling pathway

Ischemic stroke is a devastating disease with high morbidity and mortality rates, and the proinflammatory microglia-mediated inflammatory response directly affects stroke outcome. Previous studies have reported that JLX001, a novel compound with a structure similar to that of cyclovirobuxine D (CVB-D), exerts antiapoptotic, anti-inflammatory and antioxidative effects on ischemia-induced brain injury. However, the role of JLX001 in microglial polarization and nucleotide-binding oligomerization domain (NOD)-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome regulation after ischemic stroke has not been fully investigated. In this study, we used the middle cerebral artery occlusion (MCAO) method to establish a focal cerebral ischemia model and found that JLX001 attenuated the brain infarct size and improved cerebral damage. Moreover, the expression levels of proinflammatory cytokines (interleukin [IL]-1β and tumor necrosis factor [TNF]-α) were significantly reduced while those of the anti-inflammatory cytokine IL-10 were increased in the JLX001-treated group. Immunofluorescence staining and flow cytometry revealed an increased number of anti-inflammatory phenotypic microglia and a reduced number of proinflammatory phenotypic microglia in JLX001-treated MCAO mice. Western blotting analysis showed that JLX001 inhibited the expression of NLRP3 and proteins related to the NLRP3 inflammasome axis in vivo. Furthermore, JLX001 reduced the number of NLRP3/Iba1 cells in ischemic penumbra tissues. Finally, mechanistic analysis revealed that JLX001 significantly inhibited the expression of proteins related to the NF-κB signaling pathway. Additionally, pyrrolidine dithiocarbamate (PDTC), an NF-κB inhibitor, ameliorated cerebral ischemia–reperfusion injury by suppressing microglial polarization towards the proinflammatory phenotype and NLRP3 activation in vivo, further suggesting that these protective effects of JLX001 were mediated by inhibition of the NF-κB signaling pathway. These results suggest that JLX001 is a promising therapeutic approach for ischemic stroke.

Serum small extracellular vesicles promote M1 activation of microglia after cerebral ischemia/reperfusion injury

Microglial M1 activation is detrimental to stroke outcomes. Recent studies have shown that circulating small extracellular vesicles (sEVs) can deliver miRNAs to target cells and regulate recipient cell functions. Herein, we tested the hypothesis that miRNA delivery by serum sEVs after cerebral ischemia/reperfusion (I/R) injury promote microglial M1 activation, demonstrating that serum sEVs from middle cerebral artery occlusion (MCAO) mice promoted proliferation and M1 activation of BV2 microglia. To explore the underlying mechanism of serum sEVs-mediated microglial activation in the early phase of cerebral I/R injury, we examined the effects of ischemic brain injury on the serum sEVs miRNAs profile in a mouse MCAO model using small RNAseq. Of the 1257 detected miRNA replications, the levels of 72 were significantly modulated. Bioinformatics analysis revealed that a panel of miRNAs was closely associated with inflammation, and in vitro experiments demonstrated that serum sEVs from MCAO mice could effectively transfer inflammatory miRNAs to BV2 microglia. Collectively, our data suggested that miRNAs delivered by serum sEVs after cerebral I/R injury promoted microglial M1 activation. The identification of microglial activation regulators in future studies will give rise to more effective treatments for stroke.

WITHDRAWN: Foxp3 attenuates cerebral ischemia/reperfusion injury through microRNA-150-5p-modified NCS1

Cerebral ischemia/reperfusion injury (CI/RI) is a pathological process involving complicated molecular mechanisms. We investigated forkhead box P3 (Foxp3)-related mechanism in CI/RI with particular focus on microRNA (miR)-150-5p/nucleobase cation symporter-1 (NCS1) axis. A mouse model was constructed by middle cerebral artery occlusion (MCAO) method. Levels of Foxp3, miR-150-5p and NCS1 were assessed in brain tissues of MCAO mice. By determining the neurological behavior function, neurological deficits, brain tissue pathological characteristics, neuronal apoptosis, inflammatory factors, and oxidative stress-related factors, the functional role of Foxp3, miR-150-5p and NCS1 were evaluated in MCAO mice. The feedback loop was analyzed among Foxp3, miR-150-5p and NCS1. The level of Foxp3 and NCS1 were reduced and that of miR-150-5p was augmented in MCAO mice. Foxp3 bound to miR-150-5p to target NCS1. Up-regulating Foxp3 or NCS1 or suppressing miR-150-5p improved neurological behavior function and neurological deficits, and reduced brain tissue pathological damage, neuronal apoptosis, inflammatory and oxidative stress reactions in MCAO mice. Silencing miR-150-5p or elevating NCS1 decreased Foxp3 silencing-mediated ischemic injury in MCAO mice. Foxp3 is neuroprotective in CI/RI through binding to miR-150-5p to promote NCS1 expression.

MiR-191-5p Disturbed the Angiogenesis in a Mice Model of Cerebral Infarction by Targeting Inhibition of BDNF.

miRNAs are crucial regulators of angiogenesis, but there have been no detailed studies on the role of miR-191-5p in cerebral infarct angiogenesis. Here, we investigated the role of miR-191-5p in regulating cerebral infarction angiogenesis. Mice were injected intracerebroventricularly with antagomir negative control (NC-antagomir), miR-191-5p antagomir, or pcDNA-BDNF 2 h before middle cerebral artery occlusion (MCAO), followed by neurobehavioral score and foot-fault test. The cerebral infarct volume was performed by TTC staining. The microvessel density was detected by FITC-dextran. RT-qPCR was used to detect the levels of miR-191-5p and its target gene BDNF. Western blotting was applied to detect the protein levels of BDNF. The luciferase reporter assay verified that miR-191-5p targeted BDNF. We found an increased level of miR-191-5p in the brain tissue of mice to MCAO. Down-regulation of miR-191-5p reduced the infarct volume and ameliorated neurological deficits in MCAO mice. Further investigation showed that miR-191-5p directly targeted BDNF and that the protective effect of miR-191-5p inhibition in angiogenesis was achieved by regulating BDNF. Our results indicated that miR-191-5p disturbed the angiogenesis in the mouse models of cerebral infarction by inhibiting BDNF.

Protective multi‑target effects of DL‑3‑n‑butylphthalide combined with 3‑methyl‑1‑phenyl‑2‑pyrazolin‑5‑one in mice with ischemic stroke.

DL-3-n-butylphthalide (NBP) and 3-methyl-1- phenyl-2-pyrazolin-5-one (edaravone) are acknowledged neuroprotective agents that protect against ischemic stroke. However, the underlying mechanisms of a combination therapy with NBP and edaravone have not yet been fully clarified. The aim of the present study was to explore whether the co-administration of NBP and edaravone had multi-target protective effects on the neurovascular unit (NVU) of mice affected by ischemic stroke. Male C57BL/6 mice were randomly divided into the following three groups: i) Sham operation control, ii) middle cerebral artery occlusion (MCAO) and reperfusion, iii) and MCAO/reperfusion with the co-administration of NBP (40 mg/kg) and edaravone (6 mg/kg) delivered via intraperitoneal injection at 0 and 4 h after reperfusion (NBP + edaravone). After ischemia and reperfusion, infarct volumes and neurological deficits were evaluated. The immunoreactivity of the NVU, comprising neurons, endothelial cells and astrocytes, was determined using immunofluorescence staining of neuronal nuclei (NeuN), platelet and endothelial cell adhesion molecule 1 (CD31) and glial fibrillary acidic protein (GFAP). Western blotting was used to detect the expression levels of apoptosis-related proteins. The infarct volume, neurological function scores and cell damage were increased in the MCAO group compared with the sham operation group. Furthermore, the MCAO mice had reduced NeuN and CD31 expression and increased GFAP expression compared with the sham group. By contrast, the NBP + edaravone group exhibited reduced cell damage and consequently lower infarct volume and neurological deficit scores compared with the MCAO group. The NBP + edaravone group exhibited increased NeuN and CD31 expression and decreased GFAP expression compared with the MCAO group. Furthermore, the expression levels of Bax and cleaved caspase-3 in the NBP + edaravone group were decreased significantly compared with the MCAO group, while the expression levels of Bcl-2 and mitochondrial cytochrome c were increased. In conclusion, the results of the present study demonstrated that NBP and edaravone effectively prevented ischemic stroke damage with multi-target protective effects. In addition, NBP + edaravone may be a promising combination therapy for ischemic stroke.

In vivo Imaging of Cannabinoid Type 2 Receptors: Functional and Structural Alterations in Mouse Model of Cerebral Ischemia by PET and MRI

PurposeStroke is one of the most prevalent vascular diseases. Non-invasive molecular imaging methods have the potential to provide critical insights into the temporal dynamics and follow alterations of receptor expression and metabolism in ischemic stroke. The aim of this study was to assess the cannabinoid type 2 receptor (CB2R) levels in transient middle cerebral artery occlusion (tMCAO) mouse models at subacute stage using positron emission tomography (PET) with our novel tracer [18F]RoSMA-18-d6 and structural imaging by magnetic resonance imaging (MRI).ProceduresOur recently developed CB2R PET tracer [18F]RoSMA-18-d6 was used for imaging neuroinflammation at 24 h after reperfusion in tMCAO mice. The RNA expression levels of CB2R and other inflammatory markers were analyzed by quantitative real-time polymerase chain reaction using brain tissues from tMCAO (1 h occlusion) and sham-operated mice. [18F]fluorodeoxyglucose (FDG) was included for evaluation of the cerebral metabolic rate of glucose (CMRglc). In addition, diffusion-weighted imaging and T2-weighted imaging were performed for anatomical reference and delineating the lesion in tMCAO mice.ResultsmRNA expressions of inflammatory markers TNF-α, Iba1, MMP9 and GFAP, CNR2 were increased to 1.3–2.5 fold at 24 h after reperfusion in the ipsilateral compared to contralateral hemisphere of tMCAO mice, while mRNA expression of the neuronal marker MAP-2 was markedly reduced to ca. 50 %. Reduced [18F]FDG uptake was observed in the ischemic striatum of tMCAO mouse brain at 24 h after reperfusion. Although higher activity of [18F]RoSMA-18-d6 in ex vivo biodistribution studies and higher standard uptake value ratio (SUVR) were detected in the ischemic ipsilateral compared to contralateral striatum in tMCAO mice, the in vivo specificity of [18F]RoSMA-18-d6 was confirmed only in the CB2R-rich spleen.ConclusionsThis study revealed an increased [18F]RoSMA-18-d6 measure of CB2R and a reduced [18F]FDG measure of CMRglc in the ischemic striatum of tMCAO mice at subacute stage. [18F]RoSMA-18-d6 might be a promising PET tracer for detecting CB2R alterations in animal models of neuroinflammation without neuronal loss.

Baoyuan Capsule promotes neurogenesis and neurological functional recovery through improving mitochondrial function and modulating PI3K/Akt signaling pathway

BackgroundBao Yuan Capsule (BYC) is a patented Chinese medicinal formula for health promotion but its application for ischemic stroke remains unknown. In this study, we proposed the hypothesis that BYC could promote neurogenesis and neurological functional recovery through promoting mitochondrial function and activating PI3K/Akt signaling pathway. MethodsWe firstly performed chemical identification studies by using QIT-TOF-MS technology. Then, we investigated the effects of BYC (1 g/kg, 2 g/kg, 4 g/kg per day) on improving the recovery of the neurological functions in transient middle cerebral artery occlusion (MCAO) ischemic mice. ResultsWe tentatively characterized 36 compounds from the BYC extractions. At dosage of 4 g/kg, BYC effectively improved locomotor ability, attenuated anxiety-like behaviors, and enhanced the exploring behaviors, learning and memory capability in the transient MCAO ischemic mice. BYC treatment promoted neural stem cell differentiations in the subventricular zone (SVZ) and subgranular zone (SGZ) of the MCAO mice. BYC also up-regulated the expression of Aconitase 2 (ACO2), Succinate dehydrogenase complex, subunit A (SDHA), phosphorylation of AMP-activated protein kinase (p-AMPK), protein kinase B (p-Akt) and glycogen synthase kinase 3β (p-GSK3β) in the hippocampus of the MCAO mice. BYC (200 µg/ml) significantly improved the mitochondrial functions in cultured mouse multipotent neural stem like C17.2 cells. BYC treatment also promoted neuronal differentiations in the C17.2 cells under oxygen-glucose deprivation (OGD) condition. The neurogenetic effects were abolished by co-treatments of ATP synthesis inhibitor oligomycin and PI3K/Akt inhibitor wortmannin. Moreover, Akt phosphorylation was dramatically reduced by oligomycin. ConclusionBYC could promote neurogenesis and neurological functional recovery in post ischemic brains by regulating the mitochondrial functions and Akt signaling pathway.

Targeted Treatment of Ischemic Stroke by Bioactive Nanoparticle-Derived Reactive Oxygen Species Responsive and Inflammation-Resolving Nanotherapies.

Stroke is a primary cause of death and disability worldwide, while effective and safe drugs remain to be developed for its clinical treatment. Herein, we report bioactive nanoparticle-derived multifunctional nanotherapies for ischemic stroke, which are engineered from a pharmacologically active oligosaccharide material (termed as TPCD) prepared by covalently conjugating a radical-scavenging compound (Tempol) and a hydrogen-peroxide-eliminating moiety of phenylboronic acid pinacol ester (PBAP) on β-cyclodextrin. Of note, combined functional moieties of Tempol and PBAP on β-cyclodextrin contribute to antioxidative and anti-inflammatory activities of TPCD. Cellularly, TPCD nanoparticles (i.e., TPCD NPs) reduced oxygen-glucose deprivation-induced overproduction of oxidative mediators, increased antioxidant enzyme expression, and suppressed microglial-mediated inflammation, thereby inhibiting neuronal apoptosis. After intravenous (i.v.) delivery, TPCD NPs could efficiently accumulate at the cerebral ischemic injury site of mice with middle cerebral artery occlusion (MCAO), showing considerable distribution in cells relevant to the pathogenesis of stroke. Therapeutically, TPCD NPs significantly decreased infarct volume and accelerated recovery of neurological function in MCAO mice. Mechanistically, efficacy of TPCD NPs is achieved by its antioxidative, anti-inflammatory, and antiapoptotic effects. Furthermore, TPCD NPs can function as a reactive oxygen species labile nanovehicle to efficiently load and triggerably release an inflammation-resolving peptide Ac2-26, giving rise to an inflammation-resolving nanotherapy (i.e., ATPCD NP). Compared to TPCD NP, ATPCD NP demonstrated notably enhanced in vivo efficacies, largely resulting from its additional inflammation-resolving activity. Consequently, TPCD NP-derived nanomedicines can be further developed as promising targeted therapies for stroke and other inflammation-associated cerebrovascular diseases.

Integrated Bioinformatics Analysis of Potential mRNA and miRNA Regulatory Networks in Mice With Ischemic Stroke Treated by Electroacupuncture.

Background: The complicated molecular mechanisms underlying the therapeutic effect of electroacupuncture (EA) on ischemic stroke are still unclear. Recently, more evidence has revealed the essential role of the microRNA (miRNA)–mRNA networks in ischemic stroke. However, a systematic analysis of novel key genes, miRNAs, and miRNA–mRNA networks regulated by EA in ischemic stroke is still absent.Methods: We established a middle cerebral artery occlusion (MCAO) mouse model and performed EA therapy on ischemic stroke mice. Behavior tests and measurement of infarction area were applied to measure the effect of EA treatment. Then, we performed RNA sequencing to analyze differentially expressed genes (DEGs) and functional enrichment between the EA and control groups. In addition, a protein–protein interaction (PPI) network was built, and hub genes were screened by Cytoscape. Upstream miRNAs were predicted by miRTarBase. Then hub genes and predicted miRNAs were verified as key biomarkers by RT-qPCR. Finally, miRNA–mRNA networks were constructed to explore the potential mechanisms of EA in ischemic stroke.Results: Our analysis revealed that EA treatment could significantly alleviate neurological deficits in the affected limbs and reduce infarct area of the MCAO model mice. A total of 174 significant DEGs, including 53 upregulated genes and 121 downregulated genes, were identified between the EA and control groups. Functional enrichment analysis showed that these DEGs were associated with the FOXO signaling pathway, NF-kappa B signaling pathway, T-cell receptor signaling pathway, and other vital pathways. The top 10 genes with the highest degree scores were identified as hub genes based on the degree method, but only seven genes were verified as key genes according to RT-qPCR. Twelve upstream miRNAs were predicted to target the seven key genes. However, only four miRNAs were significantly upregulated and indicated favorable effects of EA treatment. Finally, comprehensive analysis of the results identified the miR-425-5p-Cdk1, mmu-miR-1186b-Prc1, mmu-miR-434-3p-Prc1, and mmu-miR-453-Prc1 miRNA–mRNA networks as key networks that are regulated by EA and linked to ischemic stroke. These networks might mainly take place in neuronal cells regulated by EA in ischemic stroke.Conclusion: In summary, our study identified key DEGs, miRNAs, and miRNA–mRNA regulatory networks that may help to facilitate the understanding of the molecular mechanism underlying the effect of EA treatment on ischemic stroke.

Sustained Baclofen-Induced Activation of GABA B Receptors After Cerebral Ischemia Restores Receptor Expression and Function and Limits Progressing Loss of Neurons.

One important function of GABAB receptors is the control of neuronal activity to prevent overexcitation and thereby excitotoxic death, which is a hallmark of cerebral ischemia. Consequently, sustained activation of GABAB receptors with the selective agonist baclofen provides neuroprotection in in vitro and in vivo models of cerebral ischemia. However, excitotoxic conditions severely downregulate the receptors, which would compromise the neuroprotective effectiveness of baclofen. On the other hand, recent work suggests that sustained activation of GABAB receptors stabilizes receptor expression. Therefore, we addressed the question whether sustained activation of GABAB receptors reduces downregulation of the receptor under excitotoxic conditions and thereby preserves GABAB receptor-mediated inhibition. In cultured neurons subjected to oxygen and glucose deprivation (OGD), to mimic cerebral ischemia, GABAB receptors were severely downregulated. Treatment of the cultures with baclofen after OGD restored GABAB receptor expression and reduced loss of neurons. Restoration of GABAB receptors was due to enhanced fast recycling of the receptors, which reduced OGD-induced sorting of the receptors to lysosomal degradation. Utilizing the middle cerebral artery occlusion (MCAO) mouse model of cerebral ischemia, we verified the severe downregulation of GABAB receptors in the affected cortex and a partial restoration of the receptors after systemic injection of baclofen. Restored receptor expression recovered GABAB receptor-mediated currents, normalized the enhanced neuronal excitability observed after MCAO and limited progressive loss of neurons. These results suggest that baclofen-induced restoration of GABAB receptors provides the basis for the neuroprotective activity of baclofen after an ischemic insult. Since GABAB receptors regulate multiple beneficial pathways, they are promising targets for a neuroprotective strategy in acute cerebral ischemia.

Knockdown of lncRNA SNHG15 Ameliorates Oxygen and Glucose Deprivation (OGD)-Induced Neuronal Injury via Regulating the miR-9-5p/TIPARP Axis.

Stroke is a cerebrovascular disease with impaired nerve function. Long non-coding RNA (lncRNA) is considered to be an important regulator of various diseases. Nevertheless, the role of lncRNA small nucleolar RNA host gene 15 (SNHG15) in cerebral ischemia injury induced by stroke is still unclear. Cell-counting kit 8 assay and flow cytometry were used to detect cell viability and apoptosis, respectively. The caspase3 activity of cells was measured using Caspase3 Activity Assay Kit. Besides, the protein levels of apoptosis markers and TCCD-induced poly (ADP)-ribose polymerase (TIPARP) were determined using western blot analysis. Moreover, quantitative real-time polymerase chain reaction was employed to examine the relative expression of SNHG15 and miR-9-5p. Furthermore, dual-luciferase reporter assay was used to assess the interaction between miR-9-5p and SNHG15 or TIPARP. In addition, biotin-labeled RNA pull-down assay was performed to evaluate the interaction between miR-9-5p and SNHG15 further. Middle cerebral artery occlusion (MCAO) model was constructed to further explore the role of SNHG15 in neuronal injury in vivo. Our data showed that oxygen and glucose deprivation (OGD) could induce N-2a cell injury and enhance SNHG15 expression. Silenced SNHG15 could promote the viability and suppress the apoptosis of OGD-induced N-2a cells. Also, SNHG15 knockdown also could alleviate the neuronal injury of MCAO mice. Mechanistically, SNHG15 could sponge miR-9-5p, and miR-9-5p could target TIPARP. Further experiments revealed that miR-9-5p inhibition or TIPARP overexpression could reverse the suppressive effect of SNHG15 knockdown on OGD-induced N-2a cell injury. Our findings indicated that SNHG15 knockdown inhibited neuronal injury through the miR-9-5p/TIPARP axis, suggesting that SNHG15 might be a potential target for cerebral ischemia injury induced by stroke.

Baicalein ameliorates ischemic brain damage through suppressing proinflammatory microglia polarization via inhibiting the TLR4/NF-κB and STAT1 pathway

Microglial polarization mediated neuroinflammation plays an important role in the pathological process of stroke. The aim of this study is to determine whether baicalein indirectly ameliorates neuronal injury through modulating microglial polarization after stroke and if so, then by what mechanism. The effects of baicalein on microglial polarization were revealed through the middle cerebral artery occlusion mouse model (MCAO, n = 6), the lipopolysaccharide (LPS) + interferon-γ (IFN-γ) and oxygen-glucose deprivation (OGD) induced neuroinflammatory microglia model (BV2, n = 3), respectively. Mice were treated with baicalein (100 mg/kg, i.g.) after reperfusion, and followed by daily administrations for 3 days. Results showed that the infarct volumes at 3 d in vehicle and baicalein-treated MCAO mice were 91.18 ± 4.02% and 55.36 ± 4.10%. Baicalein improved sensorimotor functions (p < 0.01) after MCAO. Real-time PCR revealed that baicalein decreased proinflammatory markers expression (p < 0.05), while elevated the anti-inflammatory markers (p < 0.05) in vivo and in vitro. Both western blot and immunofluorescent staining further confirmed that baicalein reduced proinflammatory marker CD16 levels (p < 0.01) and enhanced anti-inflammatory marker CD206 or Arg-1 levels (p < 0.05). Notably, baicalein suppressed the release of proinflammatory cytokines (p < 0.05) and nitric oxide (NO, p < 0.001). Mechanistically, baicalein prevented increases in TLR4 protein levels (p < 0.001), the phosphorylation of IKBα and p65 (p < 0.01), and the nuclear translocation of NF-κB p65 (p < 0.05). The NF-κB inhibitor, BAY 11-7085, enhanced the inhibitory effect of baicalein on the proinflammatory microglial polarization. Baicalein also inhibited the phosphorylation of signal transducer and activator of transcription 1 (STAT1, p < 0.001). A microglia-neuron co-culture system revealed that baicalein driven neuroprotection against OGD induced neuronal damage through modulating microglial polarization (p < 0.05). Baicalein indirectly ameliorates neuronal injury after stroke by polarizing microglia toward the anti-inflammatory phenotype via inhibition of the TLR4/NF-κB pathway and down-regulation of phosphorylated STAT1, suggesting that baicalein might serve a potential therapy for stroke.

C-myc protects mice from ischemia stroke through elevating microRNA-200b-5p-regulated SIRT1 expression

Objectivec-myc has been reported to attenuate ischemia stroke (IS). We initiated the research to uncover the molecular mechanism of c-myc with regard to microRNA (miR)-200b-5p/Sirtuin1 (SIRT1) axis. MethodsAn IS mouse model was prepared by middle cerebral artery occlusion (MCAO). Measurements of c-myc, miR-200b-5p and SIRT1 levels in MCAO mice were conducted. c-myc, miR-200b-5p and SIRT1 expression levels in MCAO mice were detected. The neurological function, production of inflammatory cytokines, neuronal apoptosis, brain tissue pathology and neuronal survival of MCAO mice were observed. Resultsc-myc and SIRT1 levels went downward while miR-200b-5p expression went upward in MCAO mice. Elevation of c-myc or suppression of miR-200b-5p improved neurological function, reduced inflammation and neuronal apoptosis, and attenuated brain tissue pathology and neuronal survival of MCAO mice. Enhancement of miR-200b-5p or knockdown of SIRT1 weakened c-myc-induced protection against MCAO-induced brain injury in mice. ConclusionOverall, c-myc protects mice from IS through elevating miR-200b-5p-targeted SIRT1 expression.

Long non-coding RNA Gm11974 aggravates oxygen–glucose deprivation-induced injury via miR-122-5p/SEMA3A axis in ischaemic stroke

Long non-coding RNAs (lncRNAs) play important roles in ischaemic stroke. This study aimed to investigate the role and potential mechanism of lncRNA Gm11974 in ischaemic stroke. Mouseneuroblastoma N2a cells were treated with oxygen-glucose deprivation (OGD). The levels of Gm11974, microRNA-122-5p (miR-122-5p) and semaphorin 3A (SEMA3A) were detected by quantitative real-time PCR (qRT-PCR) or western blot. Cell viability and apoptosis were determined by 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, Caspase-3 Assay Kit and flow Cytometry. The levels of oxidative stress indicators were measured by using commercial kits. The relationship between miR-122-5p and Gm11974 or SEMA3A was verified by dual-luciferase reporter, RNA immunoprecipitation and RNA pull-down assays. Middle cerebral artery occlusion (MCAO) in mice was used to mimic ischaemic stroke. Gm11974 and SEMA3A were up-regulated, while miR-122-5p was down-regulated in OGD-treated N2a cells and MCAO mice. Down-regulation of Gm11974 ameliorated OGD-mediated N2a cell damage by increasing cell viability and reducing cell apoptosis and oxidative stress. Gm11974 promoted OGD-induced injury in N2a cells via negatively regulating miR-122-5p. Also, miR-122-5p alleviated OGD-resulted N2a cell injury by targeting SEMA3A. Moreover, silencing of Gm11974 decreased infarct volume and neurological score in MCAO mice. Knockdown of Gm11974 attenuated neuronal injury in ischaemic stroke by regulating miR-122-5p/SEMA3A signaling pathway.

Protective effects of Dimethyl malonate on neuroinflammation and blood-brain barrier after ischemic stroke.

After ischemic stroke, microglia will be activated and play a key role in neuroinflammation and the destruction of the blood-brain barrier (BBB), and activated microglia could polarize into pro-inflammation M1 phenotype and anti-inflammation M2 phenotype. Dimethyl malonate (DMM) could reduce reactive oxygen species and we speculate DMM could regulate microglia to protect ischemic brain. We used transient middle cerebral artery occlusion (tMCAO) mouse model to simulate ischemic stroke and adult male C57BL/6 mice were used in our study. 2,3,5-triphenyltetrazolium chloride staining was used to measure infarct volume. Evans Blue and Brain water content were used to evaluate the destruction of BBB. We used a five-point scale to assess the neurologic function of mice. Western blot and Immunofluorescence were used to measure microglia, pericytes and the expression of related proteins. DMM reduced cerebral infarct volume, Evans blue leakage, brain water content and improved neurologic deficits after tMCAO. The number of activated microglia and M1 microglia were decreased and the number of M2 microglia and pericytes were increased after DMM treatment. The expression of tumor necrosis factor-α was reduced while protein levels of IL-10 and ZO-1 were increased through DMM treatment. DMM could regulate activation and polarization of microglia to inhibit neuroinflammation and protect BBB.

Janus Kinase Inhibition Ameliorates Ischemic Stroke Injury and Neuroinflammation Through Reducing NLRP3 Inflammasome Activation via JAK2/STAT3 Pathway Inhibition

BackgroundInflammatory responses play a multiphase role in the pathogenesis of cerebral ischemic stroke (IS). Ruxolitinib (Rux), a selective oral JAK 1/2 inhibitor, reduces inflammatory responses via the JAK2/STAT3 pathway. Based on its anti-inflammatory and immunosuppressive effects, we hypothesized that it may have a protective effect against stroke. The aim of this study was to investigate whether inhibition of JAK2 has a neuroprotective effect on ischemic stroke and to explore the potential molecular mechanisms.MethodsRux, MCC950 or vehicle was applied to middle cerebral artery occlusion (MCAO) mice in vivo and an oxygen-glucose deprivation/reoxygenation (OGD/R) model in vitro. After 3 days of reperfusion, neurological deficit scores, infarct volume and brain water content were assessed. Immunofluorescence staining and western blots were used to measure the expression of NLRP3 inflammasome components. The infiltrating cells were investigated by flow cytometry. Proinflammatory cytokines were assessed by RT-qPCR. The expression of the JAK2/STAT3 pathway was measured by western blots. Local STAT3 deficiency in brain tissue was established with a lentiviral vector carrying STAT3 shRNA, and chromatin immunoprecipitation (ChIP) assays were used to investigate the interplay between NLRP3 and STAT3 signaling.ResultsRux treatment improved neurological scores, decreased the infarct size and ameliorated cerebral edema 3 days after stroke. In addition, immunofluorescence staining and western blots showed that Rux application inhibited the expression of proteins related to the NLRP3 inflammasome and phosphorylated STAT3 (P-STAT3) in neurons and microglia/macrophages. Furthermore, Rux administration inhibited the expression of proinflammatory cytokines, including TNF-α, IFN-γ, HMGB1, IL-1β, IL-2, and IL-6, suggesting that Rux may alleviate IS injury by inhibiting proinflammatory reactions via JAK2/STAT3 signaling pathway regulation. Infiltrating macrophages, B, T, cells were also reduced by Rux. Local STAT3 deficiency in brain tissue decreased histone H3 and H4 acetylation on the NLRP3 promoter and NLRP3 inflammasome component expression, indicating that the NLRP3 inflammasome may be directly regulated by STAT3 signaling. Rux application suppressed lipopolysaccharide (LPS)-induced NLRP3 inflammasome secretion and JAK2/STAT3 pathway activation in the OGD/R model in vitro.ConclusionJAK2 inhibition by Rux in MCAO mice decreased STAT3 phosphorylation, thus inhibiting the expression of downstream proinflammatory cytokines and the acetylation of histones H3 and H4 on the NLRP3 promoter, resulting in the downregulation of NLRP3 inflammasome expression.

Glycocalyx is critical for blood-brain barrier integrity by suppressing caveolin1-dependent endothelial transcytosis following ischemic stroke.

The breakdown of the blood‐brain barrier (BBB) is related to the occurrence and deterioration of neurological dysfunction in ischemic stroke, which leads to the extravasation of blood‐borne substances, resulting in vasogenic edema and increased mortality. However, a limited understanding of the molecular mechanisms that control the restrictive properties of the BBB hinders the manipulation of the BBB in disease and treatment. Here, we found that the glycocalyx (GCX) is a critical factor in the regulation of brain endothelial barrier integrity. First, endothelial GCX displayed a biphasic change pattern, of which the timescale matched well with the biphasic evolution of BBB permeability to tracers within the first week after t‐MCAO. Moreover, GCX destruction with hyaluronidase increased BBB permeability in healthy mice and aggravated BBB leakage in transient middle cerebral artery occlusion (t‐MCAO) mice. Surprisingly, ultrastructural observation showed that GCX destruction was accompanied by increased endothelial transcytosis at the ischemic BBB, while the tight junctions remained morphologically and functionally intact. Knockdown of caveolin1 (Cav1) suppressed endothelial transcytosis, leading to reduced BBB permeability, and brain edema. Lastly, a coimmunoprecipitation assay showed that GCX degradation enhanced the interaction between syndecan1 and Src by promoting the binding of phosphorylated syndecan1 to the Src SH2 domain, which led to rapid modulation of cytoskeletal proteins to promote caveolae‐mediated endocytosis. Overall, these findings demonstrate that the dynamic degradation and reconstruction of GCX may account for the biphasic changes in BBB permeability in ischemic stroke, and reveal an essential role of GCX in suppressing transcellular transport in brain endothelial cells to maintain BBB integrity. Targeting GCX may provide a novel strategy for managing BBB dysfunction and central nervous system drug delivery.

Silencing of lncRNA XIST impairs angiogenesis and exacerbates cerebral vascular injury after ischemic stroke

The aim of this study was to investigate the function and regulatory mechanism of long non-coding RNA (lncRNA) X-inactive-specific transcript (XIST) in cerebral ischemic stroke (CIS). The impact of lncRNA XIST on CIS was evaluated in acute CIS patients, middle cerebral artery occlusion (MCAO) mice, and oxygen-glucose deprivation and restoration brain endothelial cells. Our results demonstrated that the expression of lncRNA XIST decreased during the early stages of CIS but then increased in the later stages in CIS patients and ischemic models in vivo and in vitro. In addition, the serum levels of lncRNA XIST negatively correlated with severity of neurological impairment of CIS patients. Further studies exhibited that lncRNA XIST regulated the expression of proangiogenic factor-integrin α5 (Itgα5) and anti-inflammation factor-Kruppel-like transcription factor 4 (KLF4) by targeting microRNA-92a (miR-92a). Silencing of lncRNA XIST impaired angiogenesis and exacerbated cerebral vascular injury following CIS, leading to larger infarcts and worse neurological deficits in transient MCAO mice. Mechanistic analysis revealed that lncRNA XIST modulated angiogenesis and alleviated cerebral vascular injury following CIS through mediating the miR-92a/Itgα5 or KLF4 axis, respectively. These data indicate that lncRNA XIST confers protection against CIS, providing a valuable target for future prevention and treatment of CIS.

Long non-coding RNA MIAT impairs neurological function in ischemic stroke via up-regulating microRNA-874-3p-targeted IL1B

ObjectiveLong non-coding RNAs (lncRNAs) have diagnostic and therapeutic values in the setting of ischemic stroke (IS). Here, we evaluated the value of myocardial infarction-associated transcript (MIAT) in IS with the involvement of microRNA (miR)-874-3p/interleukin (IL) 1B. MethodsMIAT, miR-874-3p and IL1B levels in serum of patients with IS were measured. A middle cerebral artery occlusion (MCAO) model was established in mice. MCAO mice were injected with Agomir of miR-874-3p, shRNA or overexpression vector of MIAT or siRNA of IL1B. Subsequently, behavioral activities and neurological function of mice were assessed. The number of Nissl bodies, brain damage, neuronal apoptosis and inflammatory factors in brain tissues of mice were measured. The targeting relationship between MIAT and miR-874-3p, as well as that between miR-874-3p and IL1B was explored. ResultsIn patients with IS, MIAT and IL1B were up-regulated and miR-874-3p was down-regulated. MIAT absorbed miR-874-3p while miR-874-3p targeted IL1B. Silencing of MIAT or IL1B, or promotion of miR-874-3p improved behavioral activities and neurological function of mice, reduced the number of Nissl bodies, as well as improved brain damage, neuronal apoptosis and inflammation. Overexpression of miR-874-3p abrogated up-regulated MIAT-mediated influence on MCAO mice. ConclusionShortly, this study figures out that MIAT impairs neurological function in IS via up-regulating miR-874-3p-targeted IL1B.

Lipidomic Profiling of Ipsilateral Brain and Plasma after Celastrol Post-Treatment in Transient Middle Cerebral Artery Occlusion Mice Model.

Celastrol, a pentacyclic triterpene isolated from the traditional Chinese medicine Tripterygium wilfordii Hook. F., exhibits effectiveness in protection against multiple central nervous system (CNS) diseases such as cerebral ischemia, but its influence on lipidomics still remains unclear. Therefore, in the present study, the efficacy and potential mechanism of celastrol against cerebral ischemia/reperfusion (I/R) injury were investigated based on lipidomics. Middle cerebral artery occlusion (MCAO) followed by reperfusion was operated in mice to set up a cerebral I/R model. TTC staining and TUNEL staining were used to evaluate the therapeutic effect of celastrol. Ultra-performance liquid chromatography-tandem mass spectrometry (UPLC/MS) was employed for lipidomics analysis in ipsilateral hemisphere and plasma. Celastrol remarkably reduced cerebral infarct volume and apoptosis positive cells in tMCAO mice. Furthermore, lipidomics analysis showed that 14 common differentially expressed lipids (DELs) were identified in brain and five common DELs were identified in plasma between the Sham, tMCAO and Celastrol-treated tMCAO groups. Through enrichment analysis, sphingolipid metabolism and glycerophospholipid metabolism were demonstrated to be significantly enriched in all the comparison groups. Among the DELs, celastrol could reverse cerebral I/R injury-induced alteration of phosphatidylcholine, phosphatidylethanolamine and sulfatide, which may be responsible for the neuroprotective effect of celastrol. Our findings suggested the neuroprotection of celastrol on cerebral I/R injury may be partially associated with its regulation of lipid metabolism.