Electro-scalp acupuncture regulates the expression of CYP27a1/b1, CYP24a and related inflammatory cytokines in ischemic cortex of rats with middle cerebral artery occlusion. / ç”µå¤´é’ˆè°ƒæŽ§å¤§è„‘ä¸­åŠ¨è„‰æ “å¡žå¤§é¼ ç¼ºè¡€å¤§è„‘çš®å±‚åŒºCYP27a1/b1、CYP24aåŠç›¸å ³ç‚Žæ€§ç»†èƒžå› å­è¡¨è¾¾çš„ç ”ç©¶.

OBJECTIVES: To observe the effect of electro-scalp acupuncture (ESA) on the expression of cytochrome P450a1/b1 (CYP27a1/b1), cytochrome P45024a (CYP24a), signal transducer and activator of transcription (STAT)4, STAT6, tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß and IL-4 in ischemic cerebral cortex of rats with acute ischemic stroke, so as to explore its mechanism in alleviating inflammatory reaction of ischemic stroke. METHODS: Sixty SD rats were randomly divided into sham-operation, model, vitamin D3 and ESA groups, with 15 rats in each group. The middle cerebral artery occlusion rat model was established with thread ligation according to Zea-Longa's method. Rats in the vitamin D3 group were given 1, 25-VitD3 solution (3 ng·100 g-1·d-1) by gavage, once daily for 7 days. Rats in the ESA group were treated at bilateral anterior parietotemporal slash (MS6) with ESA (2 Hz/100 Hz, 1 mA), 30 min a day for 7 days. Before and after interventions, the neurological deficit score and neurobehavioral score were evaluated. TTC staining was used to detect the volume of cerebral infarction in rats. The positive expressions of CYP24a, CYP27a1 and CYP27b1 in the cerebral cortex of ischemic area were detected by immunofluorescence. The mRNA expressions of STAT4 and STAT6 in the cerebral cortex of ischemic area were detected by quantitative real-time PCR. The protein expression levels of TNF-α, IL-1ß and IL-4 in the cerebral cortex of ischemic area were detected by Western blot. RESULTS: Compared with the sham-operation group, the neurological deficit score, neurobehavioral score, the percentage of cerebral infarction volume, the positive expression level of CYP24a and mRNA expression level of STAT4, protein expression levels of TNF-α and IL-1ß in cerebral cortex were increased (P<0.01), while the positive expression levels of CYP27a1/b1 and STAT6 mRNA, protein expression level of IL-4 were decreased (P<0.01) in the model group. After the treatment and compared with the model group, the neurological deficit score, neurobehavioral score, the percentage of cerebral infarction volume, the positive expression level of CYP24a and mRNA expression level of STAT4, protein expression levels of TNF-α and IL-1ß in cerebral cortex were decreased (P<0.01), while the positive expression levels of CYP27a1/b1 and STAT6 mRNA expression level, protein expression level of IL-4 were increased (P<0.01) in the ESA and vitamin D3 groups. CONCLUSIONS: ESA can alleviate the inflammatory response in ischemic stroke, which maybe related to its function in regulating the balance between CYP27a1/b1 and CYP24a, converting vitamin D into active vitamin D3, inhibiting vitamin D3 degradation, and regulating Th1/Th2 balance.

Effect of Naoluoxintong formula and its split prescriptions on cerebral vascular regeneration in rats with the cerebral ischemia-reperfusion.

OBJECTIVE: To observe regulatory effect of Naoluoxintong formula (, NLXT) and its split prescriptions on vascular regeneration of rats suffering from cerebral ischemia-reperfusion (IR) syndrome of Qi deficiency with blood stasis (QDBS). METHODS: NLXT is the representative prescription of Yiqi Huoxue Tongluo decoction, and NLXT is divided into Yiqi herbs and Huoxue Tongluo herbs according to their efficacies. One hundred and eight specific-pathogen-free, clean-grade, Sprague-Dawley male rats were selected to prepare the classical rat model with QDBS due to middle artery ischemia-reperfusion using the multi-factor compound simulation approach. The animals were classified into sham operation (S), model (M), Nimodping (NMDP), NLXT, YQ and HXTL groups, each having 18 rats. Cerebral ischemia was reperfused after 2 h, and 24 h later, they were administered traditional Chinese medicine treatment for 14 d twice a day. Angiogenesis changes after NLXT administration to middle cerebral artery occlusion-reperfusion (MCAO/R) rats with QDBS were analyzed using the neurological deficit score and hematoxylin-eosin staining. Cerebral infarct area by 2,3,5-Triphenyltetrazolium chloride was detected, and the ultrastructure of the blood vessel in the ischemic frontoparietal cortex was observed by transmission electron microscopy. Angiopoietin 1 (Ang1), angiopoietin 2 (Ang2), vascular endothelial growth factor A (VEGFA), vascular endothelial growth factor receptor 2 (VEGFR2), platelet endothelial cell adhesion molecule-1 (CD31), angiopoietin receptor 2 (Tie2), and P38 mitogen-activated protein kinase (MAPK) protein levels in the frontal and parietal cortex were quantified by immunofluorescence, reverse transcription-polymerase chain reaction, and Western blotting assays. RESULTS: Relative to the S group, VEGFA and VEGFR2 levels in the frontal and parietal cortex of group M were increased, and Ang1, Ang2, Tie2, CD31, and p38 MAPK levels remarkably increased (P < 0.05); cerebral infarct area was significant and pathological morphology and ultrastructure damage was obvious. Relative to the group M, VEGFA, VEGFR2, CD31, Ang1, Ang2, and Tie2 expression of group NLXT and NMDP remarkably elevated (P < 0.05) and infarct focus, pathological morphology and ultrastructure were significantly improved; VEGFA and VEGFR2 levels in the groups YQ and HXTL increased, and Ang1, Ang2, CD31, and Tie2 levels remarkably increased (P < 0.05); p38 MAPK levels in the three treatment groups decreased (P < 0.05). Relative to the group NLXT, the expression levels of p38 MAPK in group YQ and group HXTL were significantly increased, and the expression levels of other indicators were significantly decreased (P < 0.05). CONCLUSION: NLXT can promote the angiogenesis of the rat model of MCAO/R with QDBS by activating VEGFA and inhibiting P38 MAPK, and the effect is better than that of split prescription groups.

Competitive binding of circCCDC6 to microRNA-128-3p activates TXNIP/NLRP3 pathway and promotes cerebral ischemia-reperfusion defects.

OBJECTIVE: Circular RNAs (circRNAs) are enriched in the brain and involved in various central nervous system diseases. The potential role of circCCDC6 in cerebral ischemia-reperfusion defects was partly elucidated in the work. METHODS: A middle cerebral artery occlusion/reperfusion (MCAO/R) rat model and an oxygen-glucose deprivation and re-oxygenation (OGD/R)-treated SH-SY5Y cell model were constructed. CircCCDC6 expression in the two models was examined, and circCCDC6-involved mechanisms in neuronal pyroptosis and inflammation were analyzed through loss- and gain-of-function assays. RESULTS: MCAO/R rat brain tissues and OGD/R-treated SH-SY5Y cells exhibited upregulated circCCDC6. Silencing circCCDC6 attenuated neuronal pyroptosis and inflammation in the brain tissue of MCAO/R rats. Overexpressing circCCDC6 or inhibiting miR-128-3p stimulated OGD/R-induced pyroptosis and inflammation in SH-SY5Y cells, while upregulating miR-128-3p attenuated OGD/R injury. CircCCDC6 silencing-induced effects on SH-SY5Y cells were antagonized by TXNIP overexpression. CONCLUSION: Mechanistically, circCCDC6 mediates miR-128-3p and activates TXNIP/NLRP3, thereby promoting OGD/R-induced neuronal pyroptosis and inflammation. CircCCDC6 may provide a new strategy for the treatment of MCAO/R.

ADAR1 exacerbates ischemic brain injury via astrocyte-mediated neuron apoptosis.

Astrocytes affect stroke outcomes by acquiring functionally dominant phenotypes. Understanding molecular mechanisms dictating astrocyte functional status after brain ischemia/reperfusion may reveal new therapeutic strategies. Adenosine deaminase acting on RNA (ADAR1), an RNA editing enzyme, is not normally expressed in astrocytes, but highly induced in astrocytes in ischemic stroke lesions. The expression of ADAR1 steeply increased from day 1 to day 7 after middle cerebral artery occlusion (MCAO) for 1 h followed by reperfusion. ADAR1 deficiency markedly ameliorated the volume of the cerebral infarction and neurological deficits as shown by the rotarod and cylinder tests, which was due to the reduction of the numbers of activated astrocytes and microglia. Surprisingly, ADAR1 was mainly expressed in astrocytes while only marginally in microglia. In primary cultured astrocytes, ADAR1 promoted astrocyte proliferation via phosphatidylinositol 3-kinase (PI3K)/Akt pathway. Furthermore, ADAR1 deficiency inhibited brain cell apoptosis in mice with MCAO as well as in activated astrocyte-conditioned medium-induced neurons in vitro. It appeared that ADAR1 induces neuron apoptosis by secretion of IL-1ß, IL-6 and TNF-α from astrocytes through the production of reactive oxygen species. These results indicated that ADAR1 is a novel regulator promoting the proliferation of the activated astrocytes following ischemic stroke, which produce various inflammatory cytokines, leading to neuron apoptosis and worsened ischemic stroke outcome.

Essential genes Ptgs2, Tlr4, and Ccr2 regulate neuro-inflammation during the acute phase of cerebral ischemic in mice.

Ischemic stroke (IS) is associated with changes in gene expression patterns in the ischemic penumbra and extensive neurovascular inflammation. However, the key molecules related to the inflammatory response in the acute phase of IS remain unclear. To address this knowledge gap, conducted a study using Gene Set Enrichment Analysis (GSEA) on two gene expression profiles, GSE58720 and GSE202659, downloaded from the GEO database. We screened differentially expressed genes (DEGs) using GEO2R and analyzed 170 differentially expressed intersection genes for Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment and Gene Ontology (GO) analysis. We also used Metascape, DAVID, STRING, Cytoscape, and TargetScan to identify candidate miRNAs and genes. The targeted genes and miRNA molecule were clarified using the mice middle cerebral artery occlusion-reperfusion (MCAO/R) model. Our findings revealed that 170 genes were correlated with cytokine production and inflammatory cell activation, as determined by GO and KEGG analyses. Cluster analysis identified 11 hub genes highly associated with neuroinflammation: Ccl7, Tnf, Ccl4, Timp1, Ccl3, Ccr1, Sele, Ccr2, Tlr4, Ptgs2, and Il6. TargetScan results suggested that Ptgs2, Tlr4, and Ccr2 might be regulated by miR-202-3p. In the MCAO/R model, the level of miR-202-3p decreased, while the levels of Ptgs2, Tlr4, and Ccr2 increased compared to the sham group. Knockdown of miR-202-3p exacerbated ischemic reperfusion injury (IRI) through neuroinflammation both in vivo and in vitro. Our study also demonstrated that mRNA and protein levels of Ptgs2, Tlr4, and Ccr2 increased in the MCAO/R model with miR-202-3p knockdown. These findings suggest that differentially expressed genes, including Ptgs2, Tlr4, and Ccr2 may play crucial roles in the neuroinflammation of IS, and their expression may be negatively regulated by miR-202-3p. Our study provides new insights into the regulation of neuroinflammation in IS.

CircCRIM1/microRNA-141-3p/thioredoxin-binding protein axis mediates neuronal apoptosis after cerebral ischemia-reperfusion.

Numerous studies have indicated enrichment of circular RNA (circRNA) in the brain takes on a momentous role in cerebral ischemia-reperfusion (CIR) injury. A recent study discovered a novel circCRIM1, was highly expressed in the middle cerebral artery occlusion-reperfusion (MCAO/R) model. Nevertheless, its specific biological function remained unknown. The study was to explore circCRIM1 in CIR-induced neuronal apoptosis. As measured, circCRIM1 and TXNIP were up-regulated, while miR-141-3p was down-regulated in MCAO/R mouse model and OGD/R SH-SY5Y cells. Depleting circCRIM1 reduced the number of apoptotic neurons in MCAO/R rats, increased the number of Nissl bodies, prevented reactive oxygen species production and oxidative stress imbalance in brain tissues, repressed cleaved caspase-3, Bax, and Cyto C protein levels and increased Bcl-2 levels. Overexpression of circCRIM1 further repressed neuronal activity and accelerated apoptosis in OGD/R model, disrupted redox balance. Depleting circCRIM1 had the opposite effect in OGD/R model. Knocking down miR-141-3p or TXNIP weakened the effects of knocking down circCRIM1 or overexpressing circCRIM1, separately. Mechanistically, circCRIM1 exerted an active role in CIR injury via miR-141-3p to mediate TXNIP. All in all, the circCRIM1/miR-141-3p/TXNIP axis might be a latent therapeutic target for CIR injury.

Long non-coding RNA HOXA11-AS regulates ischemic neuronal death by targeting miR-337-3p/YBX1 signaling pathway: protective effect of dexmedetomidine.

Cerebral ischemia/reperfusion (I/R) is a common neurological disease. Homeobox A11 antisense RNA (HOXA11-AS), a long non-coding RNA (lncRNA), has been demonstrated as an important regulator in diverse human cancers. However, its function and regulatory mechanism in ischemic stroke remains largely unknown. Dexmedetomidine (Dex) have received wide attraction because of its neuroprotective effects. This study aimed to explore the possible link between Dex and HOXA11-AS in protecting neuronal cells from by ischemia/reperfusion-induced apoptosis. We used oxygen-glucose deprivation and reoxygenation (OGD/R) in mouse neuroblastoma Neuro-2a cells and middle cerebral artery occlusion (MACO) mouse model to test the link. We found that Dex significantly alleviated OGD/R-induced DNA fragmentation, cell viability and apoptosis, and rescued the decreased HOXA11-AS expression after ischemic damage in Neuro-2a cells. Gain-/loss-of-function studies revealed that HOXA11-AS promoted proliferation, inhibited apoptosis in Neuro-2a cells exposed to OGD/R. Knockdown of HOXA11-AS decreased the protective effect of Dex on OGD/R cells. HOXA11-AS was found to transcriptionally regulate microRNA-337-3p (miR-337-3p) expression as evidenced by luciferase reporter assay, while miR-337-3p expression was upregulated following ischemia in vitro and in vivo. Besides, knockdown of miR-337-3p protected OGD/R-induced apoptotic death of Neuro-2a cells. Furthermore, HOXA11-AS functioned as a competing endogenous RNA (ceRNA) and competed with Y box protein 1 (Ybx1) mRNA for directly binding to miR-337-3p, which protected ischemic neuronal death. Dex treatment protected against ischemic damage and improved overall neurological functions in vivo. Our data suggest a novel mechanism of Dex neuroprotection for ischemic stroke through regulating lncRNA HOXA11-AS by targeting the miR-337-3p/Ybx1 signaling pathway, which might help develop new strategies for the therapeutic interventions in cerebral ischemic stroke.

Loss of lncRNA UCA1 ameliorates the injury managed by cerebral ischemia-reperfusion by sponging miR-18a-5p.

INTRODUCTION: Acute ischemic stroke (AIS) is a disease with high morbidity and mortality in the clinic. The current experiments aimed to study the effects of UCA1 interfering miR-18a-5p on cerebral ischemia-reperfusion (CI/R). MATERIAL AND METHODS: For rat models undergoing middle cerebral artery infarction (MCAO) surgery, the expression of UCA1 and miR-18a-5p was evaluated by qRT-PCR, and underlying function was identified by detecting infarct size, neurological scores, and inflammation. Luciferase report was applied to verify the relationship between UCA1 and miR-18a-5p. In the cell models, the impacts of UCA1 and miR-18a-5p were validated by CCK-8 assay, flow cytometry analysis, and ELISA. In patients with AIS, Pearson correlation was carried out to unveil the association between UCA1 and miR-18a-5p. RESULTS: The expression of UCA1 was at high levels and miR-18a-5p was at low levels in AIS patients. UCA1 knockdown showed a protective role in infarct size, neurofunction, and inflammation via binding miR-18a-5p. MiR-18a-5p participated in the regulation of UCA1 on cell viability, cell apoptosis, lactate dehydrogenase (LDH) levels, and inflammation. In patients with AIS, overexpression of UCA1 and underexpression of miR-18a-5p had a reverse correlation. CONCLUSIONS: Elimination of UCA1 was favourable to the recovery of the rat model and cells from CI/R damage by efficaciously sponging miR-18a-5p.

lncfos/miR-212-5p/CASP7 Axis-Regulated miR-212-5p Protects the Brain Against Ischemic Damage.

miR-212-5p has been reported to be involved in many biological processes. However, the role of miR-212-5p in ischemic stroke remains unclear. This study explored the biological role and potential mechanism of miR-212-5p in ischemic stroke by investigating the lncfos/miR-212-5p/CASP7 axis. A total of 32 patients with ischemic stroke and 32 age- and sex-matched healthy controls (HCs) were enrolled in this study. In addition, 336 rats were used in this study. The rats were subjected to middle cerebral artery occlusion (MCAO) and intracerebroventricular injection of a microRNA (miRNA) agomir, a miRNA antagomir, a short hairpin RNA (shRNA) lentiviral vector, or a negative control. The neurological deficit score was calculated; the infarct volume was measured; histopathological assays were performed; the neuronal apoptosis rate was determined; and the lncfos, miR-212-5p, and CASP7 expression levels in the peri-infarct area were assessed. In this study, we found that the expression level of miR-212-5p was significantly downregulated in the peri-infarct area and blood of the MCAO model rats and the blood of patients with ischemic stroke. A double-luciferase experiment showed that CASP7 was a direct target gene of miR-212-5p and that miR-212-5p was a target miRNA of lncfos. Lateral ventricular injection of the miR-212-5p agomir effectively inhibited the apoptosis induced by ischemic brain damage, reduced the infarct volume, attenuated the neurological deficit symptoms, and downregulated the expression of CASP7 in the peri-infarct area of the MCAO model rats. Suppressing lncfos with sh-fos led to the upregulated expression of miR-212-5p and played a neuroprotective role in the rat MCAO models. We concluded that miR-212-5p plays a neuroprotective role in ischemic stroke and that its function is regulated by the lncfos/miR-212-5p/CASP7 axis. Moreover, miR-212-5p may be a potential biomarker and therapeutic target for ischemic stroke.

MiR-342-5p protects neurons from cerebral ischemia induced-apoptosis through regulation of Akt/NF-κB pathways by targeting CCAR2.

OBJECTIVES: Ischemic stroke causes high morbidity, mortality and health burden in the world. MiR-342-5p was associated with Alzheimer's disease and cardio-protection. Herein, we aimed to reveal effects of miR-342-5p on cerebral ischemia injury as well as novel targets for stroke. MATERIALS AND METHODS: AgomiR-342-5p was intracerebroventricularly injected into the middle cerebral artery occlusion (MCAO) mouse models to evaluate functions of miR-342-5p on cerebral ischemia. RT-qPCR and western blot assays were used to evaluate genes expression. Oxygen-glucose deprivation (OGD) was used as an in vitro model for ischemia. Viability and apoptosis ratio of neurons was evaluated by CCK-8, LDH release detection, and flow cytometry. The potential targets of miR-342-5p were predicted by Targetscan, and their interaction was confirmed by luciferase assay. RESULTS: The intervention of miR-342-5p effectively attenuated ischemic injury in MCAO mice. MiR-342-5p overexpression could protect neurons against OGD-induced injury, as revealed by increased cell viability and BCL2 expression, and decreased LDH release, apoptosis ratio, and BAX expression in OGD-induced neurons. Mechanically, miR-342-5p could directly bound with CCAR2 to inhibit its expression. Overexpressing CARR2 aggravated the OGD-induced injury of neurons, which was partly restrained by overexpressing miR-342-5p reversed. Furthermore, miR-342-5p/CARR2 axis regulates Akt/NF-κB signaling pathway in vitro as well as in vivo cerebral ischemia models. CONCLUSIONS: MiR-342-5p inhibited neuron apoptosis by regulating Akt/NF-kB signaling pathway via CCAR2 suppression. Our findings revealed the neuroprotection of miR-342-5p in cerebral ischemia.

Taurine-Upregulated Gene 1 Attenuates Cerebral Angiogenesis following Ischemic Stroke in Rats.

Objective: Angiogenesis is one of the therapeutic targets of cerebral infarction. Long noncoding RNAs (lncRNAs) can regulate the pathological process of angiogenesis following ischemic stroke. Taurine-upregulated gene 1 (TUG1), an lncRNA, is correlated to ischemic stroke. We intended to determine the effect of TUG1 on angiogenesis following an ischemic stroke. Materials and Methods: Middle cerebral artery occlusion (MCAO) was adopted to build a focal ischemic model of the rat brain, and pcDNA-TUG1 and miR-26a mimics were injected into rats. Neurological function was estimated through modified neurological severity scores. The volume of focal brain infarction was calculated through 2,3,5-triphenyltetrazolium chloride staining. The level of TUG1 and miR-26a was measured by PCR. The expression of vascular endothelial growth factor (VEGF) and CD31 was checked using immunohistochemistry and western blot. The correlation between miR-26a and TUG1 was verified through a luciferase reporter assay. Results: TUG1 increased noticeably while miR-26a was markedly reduced in MCAO rats. Overexpression of miR-26a improved neurological function recovery and enhanced cerebral angiogenesis in MCAO rats. TUG1 overexpression aggravated neurological deficits and suppressed cerebral angiogenesis in MCAO rats. Bioinformatics analysis revealed that miR-26a was one of the predicted targets of TUG1. Furthermore, TUG1 combined with miR-26a to regulate angiogenesis. TUG1 overexpression antagonized the role of miR-26a in neurological recovery and angiogenesis in MCAO rats. Conclusions: TUG1/miR-26a, which may act as a regulatory axis in angiogenesis following ischemic stroke, can be considered a potential target for cerebral infarction therapy.

MiR-145 enriched exosomes derived from bone marrow-derived mesenchymal stem cells protects against cerebral ischemia-reperfusion injury through downregulation of FOXO1.

BACKGROUND: Mesenchymal stem cells-derived exosomes (MSCs-Exo) were able to exert neuroprotective effects in brain injury after ischemic stroke (IS). In addition, exosomes containing microRNAs (miRNAs) can be transported to recipient cells to mediate intercellular communication. It has been shown that the level of miR-145 was significantly downregulated in brain tissues of rats subjected to middle cerebral artery occlusion (MCAO). However, the role of MSCs-derived exosomal miR-145 in IS progression remains largely unknown. METHODS: Microglial BV2 cell exposed to oxygen-glucose deprivation/reperfusion (OGD/R) was applied to mimic cerebral ischemia/reperfusion (I/R) injury conditions in vitro. In addition, a rat model of MCAO was established to induce I/R injury. Meanwhile, exosomes were isolated from miR-145-transfected bone marrow MSCs, and then these isolated exosomes were used to treat OGD/R-stimulated BV-2 cell and rats subject to MCAO/R. RESULTS: In this study, we found that miR-145 could be transferred from MSCs to BV2 cells via exosomes. In addition, exosomal miR-145-derived from MSCs was able to shift microglia polarization toward anti-inflammatory M2 phenotype in OGD/R-stimulated BV2 cells. Moreover, exosomal miR-145 markedly suppressed the apoptosis, cell cycle arrest and oxidative stress in OGD/R-treated BV2 cells. Additionally, exosomal miR-145 notably decreased the expression of FOXO1 in BV2 cell exposed to OGD/R and in brain tissues of MCAO rats. Furthermore, exosomal miR-145 remarkably decreased infarct area in MCAO rats. CONCLUSION: Collectively, exosomal miR-145-derived from MSCs was able to attenuate cerebral I/R injury through downregulation of FOXO1. These studies may serve as a potential approach for treating of cerebral I/R injury.

Mesenchymal stem cell aggregation mediated by integrin α4/VCAM-1 after intrathecal transplantation in MCAO rats.

BACKGROUND: Mesenchymal stem cells (MSCs) have shown immense therapeutic potential for various brain diseases. Intrathecal administration of MSCs may enhance their recruitment to lesions in the central nervous system, but any impact on cerebrospinal fluid (CSF) flow remains unclear. METHODS: Rats with or without middle cerebral artery occlusion (MCAO) received intrathecal injections of 2D cultured MSCs, 3D cultured MSCs or an equal volume of artificial cerebrospinal fluid (ACSF). Ventricle volume was assessed by MRI on Days 2 and 14 post-MCAO surgery. A beam walking test was used to assess fine motor coordination and balance. Aggregation of MSCs was evaluated in CSF and frozen brain tissue. Differential expression of cell adhesion molecules was evaluated by RNA-Seq, flow cytometry and immunofluorescence analyses. The influence of VCAM-1 blockade in mediating the aggregation of 2D MSCs was investigated in vitro by counting cells that passed through a strainer and in vivo by evaluating ventricular dilation. RESULTS: MSC expanded in 2D culture formed aggregates in the CSF and caused ventricular enlargement in both MCAO and normal rats. Aggregates were associated with impaired motor function. 2D MSCs expressed higher levels of integrin α4 and VCAM-1 than 3D MSCs. Blockade of VCAM-1 in 2D MSCs reduced their aggregation in vitro and reduced lateral ventricular enlargement after intrathecal infusion. 3D MSCs exhibited lower cell aggregation and reduced cerebral ventricular dilation after intrathecal transplantation CONCLUSIONS: The aggregation of 2D MSCs, mediated by the interaction of integrin α4 and VCAM-1, is a potential risk for obstruction of CSF flow after intrathecal transplantation.

Endothelial caveolin-1 regulates cerebral thrombo-inflammation in acute ischemia/reperfusion injury.

BACKGROUND: Thrombo-inflammation is an important checkpoint that orchestrates infarct development in ischemic stroke. However, the underlying mechanism remains largely unknown. Here, we explored the role of endothelial Caveolin-1 (Cav-1) in cerebral thrombo-inflammation. METHODS: The correlation between serum Cav-1 level and clinical outcome was analyzed in acute ischemic stroke patients with successful recanalization. Genetic manipulations by endothelial-specific adeno-associated virus (AAV) and siRNA were applied to investigate the effects of Cav-1 in thrombo-inflammation in a transient middle cerebral artery occlusion (tMCAO) model. Thrombo-inflammation was analyzed by microthrombosis formation, myeloid cell infiltration, and endothelial expression of adhesion molecules as well as inflammatory factors. FINDINGS: Reduced circulating Cav-1, with the potential to predict microembolic signals, was more frequently detected in recanalized stroke patients without early neurological improvement. At 24 h after tMCAO, serum Cav-1 was consistently reduced in mice. Endothelial Cav-1 was decreased in the peri-infarct region. Cav-1-/- endothelium, with prominent barrier disruption, displayed extensive microthrombosis, accompanied by increased myeloid cell inflammatory infiltration after tMCAO. Specific enhanced expression of endothelial Cav-1 by AAV-Tie1-Cav-1 remarkably reduced infarct volume, attenuated vascular hyper-permeability and alleviated thrombo-inflammation in both wild-type and Cav-1-/- tMCAO mice. Transcriptome analysis after tMCAO further designated Rxrg as the most significantly changed molecule resulting from the knockdown of Cav-1. Supplementation of RXR-γ siRNA reversed AAV-Tie1-Cav-1-induced amelioration of thrombo-inflammation without affecting endothelial tight junction. INTERPRETATION: Endothelial Cav-1/RXR-γ may regulate infarct volume and neurological impairment, possibly through selectively controlling thrombo-inflammation coupling, in cerebral ischemia/reperfusion. FUNDING: This work was supported by National Natural Science Foundation of China.

Bone marrow-derived mesenchymal stem cells overexpressed with miR-182-5p protects against brain injury in a mouse model of cerebral ischemia.

BACKGROUND: Toll-like receptor 4 (TLR4) plays a critical role in ischemic brain injury by mediating the inflammatory response. The microRNA miR-185-5p suppresses inflammatory signaling by targeting TLR4. This study investigates whether overexpressing miR-182-5p in bone marrow-derived mesenchymal stem cells (BM-MSCs) could potentiate the neuroprotective effects of BM-MSCs in a mouse model of ischemic brain injury. METHODS: We isolated BM-MSCs from mice, transfected the cells with miR-182-5p mimic, determined their MSC lineage through flow cytometry analysis of surface markers, examined miR-182-5p and TLR4 expression levels, and injected them into mice undergone middle cerebral artery occlusion (MCAO). MSC transplanted mice were subjected to behavior assays to determine cognitive and motor functions and biochemical analysis to determine neuroinflammation and TLR4/NF-κB in the ischemic hemisphere. RESULTS: We found that BM-MSCs overexpressing miR-182-5p showed reduced TLR4 expression without affecting their MSC lineage. Mice transplanted with miR-182-5p overexpressing BM-MSCs after MCAO showed significantly improved cognitive and motor functions and reduced neuroinflammation, including suppressed microglial M1 polarization, reduced inflammatory cytokines, and inhibited TLR4/ NF-κB signaling. CONCLUSION: Our findings suggest that overexpressing miR-182-5p in BM-MSCs can enhance the neuroprotective effects of BM-MSCs against ischemic brain injury by suppressing TLR4-mediated inflammatory response.

Bone marrow mesenchymal stem cell-derived exosomes carrying long noncoding RNA ZFAS1 alleviate oxidative stress and inflammation in ischemic stroke by inhibiting microRNA-15a-5p.

BACKGROUND/AIM: Bone marrow mesenchymal stem cell (BMSC)-derived exosomes can prevent oxidative stress and inflammation in cerebral ischemia-reperfusion injury. This study intended to assess influences of BMSC-released exosomes on oxidative stress and inflammation following ischemic stroke. METHODS: In vitro and in vivo models were developed using oxygen-glucose deprivation/reperfusion (OGD/R) and middle cerebral artery occlusion (MCAO), respectively. After exosome isolation, co-culture experiments of BMSCs or BMSC-derived exosomes and OGD/R-treated BV-2 cells were implemented to evaluate the impacts of BMSCs or BMSC-secreted exosomes on proliferation, inflammation, oxidative stress, and apoptosis. The gain-of-function experiments of ZFAS1 or microRNA (miR)-15a-5p were conducted to investigate the associated mechanisms. Besides, MCAO mice were injected with exosomes from BMSCs overexpressing ZFAS1 for in vivo verification. The binding of ZFAS1 to miR-15a-5p was assessed through dual-luciferase reporter gene assay. RESULTS: Co-culture with BMSCs accelerated proliferation and downregulated IL-1ß, IL-6, and TNF-α in OGD/R-exposed BV-2 cells, accompanied by increased SOD level and decreased MDA level and apoptosis, all of which were nullified by inhibiting exosome secretion. Mechanistically, ZFAS1 bound to miR-15a-5p to negatively orchestrate its expression. In addition, BMSC-released exosomes or BMSC-secreted exosomal ZFAS1 augmented proliferation but reduced oxidative stress, apoptosis, and inflammation in OGD/R-exposed BV-2 cells, whereas these impacts of BMSC-released exosomal ZFAS1 were nullified by overexpressing miR-15a-5p. Moreover, BMSC-derived exosomal ZFAS1 diminished MCAO-induced oxidative stress, cerebral infarction, and inflammation in mice. CONCLUSIONS: Conclusively, BMSC-released exosomes might carry long noncoding RNA ZFAS1 to curb oxidative stress and inflammation related to ischemic stroke, which was possibly realized through miR-15a-5p inhibition.

LncRNA SNHG14 is beneficial to oxygen glucose deprivation/reoxygenation-induced neuro-2a cell injury via mir-98-5p sequestration-caused BCL2L13 upregulation.

BACKGROUND: The deregulation of long non-coding RNA (lncRNA) is associated with diverse human disorders, including cerebral ischemia/reperfusion injury (CI/RI). LncRNA SNHG14 was reported to function in CI/RI. Whereas, molecular mechanisms regulated by SNHG14 are not fully unveiled. METHODS: Mice subjected to middle cerebral artery occlusion/reperfusion (MCAO/R) were used as CI/RI animal models. Neuro-2a (N2A) cells subjected to oxygen glucose deprivation/reoxygenation (OGD/R) were used as CI/RI cell models. The expression of SNHG14, miR-98-5p and BCL2 like 13 (BCL2L13) was examined using quantitative real-time PCR (qPCR) or western blot. Apoptosis was monitored by flow cytometry assay. Apoptosis-related markers and endoplasmic reticulum (ER) stress-related markers were quantified by western blot. Inflammatory factors and oxidative stress were detected using matched commercial kits. The predicted relationship between miR-98-5p and SNHG14 or BCL2L13 was validated by dual-luciferase reporter assay, RIP assay and pull-down assay. RESULTS: The high expression of SNHG14 was monitored in MCAO/R-treated mice and OGD/R-treated N2A cells. OGD/R-induced N2A cell apoptosis, ER stress, inflammation and oxidative stress were attenuated by SNHG14 knockdown. SNHG14 targeted miR-98-5p to positively regulate BCL2L13 expression. Inhibition of miR-98-5p recovered cell apoptosis, ER stress, inflammation and oxidative stress that were repressed by SNHG14 knockdown. Overexpression of BCL2L13 enhanced cell apoptosis, ER stress, inflammation and oxidative stress that were repressed by miR-98-5p enrichment. CONCLUSIONS: SNHG14 knockdown alleviated OGD/induced N2A cell apoptosis, ER stress, inflammation and oxidative stress by depleting BCL2L13 via increasing miR-98-5p.

Exosomes Released from Bone-Marrow Stem Cells Ameliorate Hippocampal Neuronal Injury Through transferring miR-455-3p.

BACKGROUND: The neuroprotective roles of mesenchymal stem cells (MSCs) in brain injury are elicited at least partially through the secretion exosomes containing microRNAs (miRNAs). We herein investigate the protective function of bone marrow MSCs (BMSCs)-derived exosomes harboring miR-455-3p against hippocampal neuronal injury in mouse and N2a cell damage model. METHODS: First, BMSC surface markers were detected by flow cytometry, followed by extraction of BMSCs-derived exosomes (BMSCs-Exos). A mouse model of neuronal injury was induced by middle cerebral artery occlusion/reperfusion (MCAO/R), and N2a cells were exposed to oxygen-glucose deprivation/reoxygenation (OGD/R) for in vitro experiments. BMSCs-Exos were administrated in mice and N2a cells. We subsequently determined viability- and apoptosis-features using EdU staining, CCK-8, flow cytometry and Caspase-3 kits. Subsequently, we used RT-qPCR to assess miR-455-3p expression in brain tissues as well as N2a cells, and bioinformatic tools to predict the targeting mRNA of miR-455-3p, which was validated by dual-luciferase assays. RESULTS: BMSCs-Exos improved hippocampal neuronal injury in MCAO/R-treated mice and OGD/R-induced injury to N2a cells. BMSCs-Exos upregulated miR-455-3p expression in brain tissues of mice and OGD/R-treated N2a cells. miR-455-3p targeted and conversely regulated PDCD7 expression. The protective effect of BMSCs-Exos on OGD/R-treated N2a cells was markedly mitigated following miR-455-3p downregulation. Moreover, overexpression of miR-455-3p contributed to increased N2a cell activity and decreased apoptosis, while the rescue experiment results were opposite. CONCLUSION: MSCs-derived exosomal miR-455-3p targeted PDCD7 to alleviate hippocampal neuronal injury in MCAO/R-treated mice and injury of OGD/R-treated N2a cells.

CircRNA CTNNB1 (circCTNNB1) ameliorates cerebral ischemia/reperfusion injury by sponging miR-96-5p to up-regulate scavenger receptor class B type 1 (SRB1) expression.

Emerging studies show that circRNA catenin beta 1 (circCTNNB1) plays a critical role in cancer. However, the expression and function of circCTNNB1 in cerebral ischemia/reperfusion injury (IRI) have not been reported. The present study discovered that circCTNNB1 and scavenger receptor class B type 1 (SRB1) expression levels were significantly down-regulated in mouse astrocytes (mAS) treated with oxygen glucose deprivation and reperfusion (OGD/R), and similar results were observed in a mouse middle cerebral artery occlusion model. Overexpression of circCTNNB1 alleviated cell apoptosis, oxidative stress and the inflammatory response induced by OGD/R in vitro. Up-regulation of circCTNNB1 increased SRB1 expression levels to protect mAS cells from OGD/R-induced damage. CircCTNNB1 and SRB1 interacted with miR-96-5p, and the overexpression of miR-96-5p efficiently reversed the function of circCTNNB1 in OGD/R-treated mAS cells. CircCTNNB1 protected against cerebral ischemia-reperfusion injury by up-regulating SRB1 in vivo. In conclusion, our findings suggest that circCTNNB1 acts as a competitive endogenous RNA for miR-96-5p to alleviate cerebral IRI, which provides novel evidence that circCTNNB1 and SRB1 may be biomarkers and therapeutic targets for cerebral IRI.

Early Aerobic Exercise Promotes Neurological Function Recovery of Rats after Cerebral Ischemia/Reperfusion by Upregulating the Expression of Heat Shock Protein A5.

OBJECTIVE: The neuroprotective function of heat shock protein A5 (HSPA5) in ischemic stroke has been confirmed. This study aimed to investigate the effects of early aerobic exercise on neurological function recovery from cerebral ischemia/reperfusion and to determine whether these effects are associated with the expression level of HSPA5 in the ischemic penumbra. METHODS: A total of 72 male Sprague-Dawley rats were randomly assigned to the ischemia and exercise group [middle cerebral artery occlusion (MCAO)-Ex, n=18], ischemia and sedentary group (MCAO-St, n=18), sham-surgery and exercise group (Sham-Ex, n=18), or sham-surgery and sedentary group (Sham-St, n=18). The MCAO-Ex and MCAO-St groups were subjected to MCAO for 60 min, whereas the Sham-Ex and Sham-St groups were subjected to an identical operation without MCAO. Rats in the MCAO-Ex and Sham-Ex groups then ran on a treadmill for 30 min once a day for 5 consecutive days. After reperfusion, the motor function of the rats was scored by the Bederson neurological function test, balance beam test, and screen test. Nissl staining was conducted to assess morphological and structural change of nerve cells in the ischemic penumbra. The reverse transcription-quantitative polymerase chain reaction was applied to detect the mRNA expression of HSPA5. Western blot analysis was conducted to determine the protein expression of HSPA5. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining was carried out in the ischemic penumbra after MCAO. RESULTS: Rats receiving early treadmill exercise had lower Bederson neurological function, balance beam, and screen test scores on the 3rd, 7th, and 14th days after MCAO; in addition, more neurons survived in the ischemic penumbra after MCAO, and higher mRNA and protein expression of HSPA5 and fewer TUNEL-positive stained cells were observed. CONCLUSION: Our study demonstrated that early aerobic exercise can improve neurological function recovery after ischemia/reperfusion. Furthermore, the increased level of HSPA5 in the ischemic penumbra might be one of the mechanisms of enhanced neurological function recovery.