Exploring the molecular mechanism of dexmedetomidine in alleviating blood-brain barrier disruption in rats with cerebral ischemia reperfusion injury based on network pharmacology.

The mechanism contributing to blood-brain barrier (BBB) disruption, involved in poststroke edema and hemorrhagic transformation, is important but elusive. We investigated microRNA-21 (miR-21)-mediated mechanism in the disruption of BBB following cerebral ischemia-reperfusion (I/R) injury. Rats with cerebral I/R injury were prepared after middle cerebral artery occlusion and subsequent reperfusion. The underlying regulatory mechanisms of miR-382 were investigated with treatment of miR-382 mimics, miR-382 inhibitors, or SB203580 (an inhibitor of the MAPK signaling pathway) prior to I/R modeling. Compared with sham-operated rats, rats following I/R showed increased Longa's scores, ischemic hemisphere volume, cerebral infarct volume, EB content in brain tissues, enhanced levels of p38, iNOS, and MMP-9. The ectopic expression of miR-21 by mimics and MAPK signaling inhibition by SB203580 reduced Longa's scores, ischemic hemisphere volume, cerebral infarct volume, EB content in brain tissues, decreased levels of p38, MAP2K3, iNOS, and MMP-9. The luciferase activity determination showed miR-21 bound to MAP2K3 in its 3'UTR. miR-21 downregulation mediated by inhibitors appeared to yield an opposed trend. We also found that MAPK signaling inhibition by SB203580 could rescue rats with treatment of miR-382 inhibitors. The study highlights the neuroprotective role of MiR-21 during cerebral I/R injury and its preventive effect against BBB disruption by blocking the MAPK signaling pathway via targeted inhibition of MAP2K3, potentially opening a novel therapeutic avenue for the treatment of cerebral ischemia.

XingNaoJing injections protect against cerebral ischemia/reperfusion injury and alleviate blood-brain barrier disruption in rats, through an underlying mechanism of NLRP3 inflammasomes suppression

This study aimed to systematically investigate the therapeutic effects and underlying mechanisms of ASIV against cerebral ischemia-reperfusion injury (CIRI) using an integrated approach combining network pharmacology and experimental validation. Potential targets of Astragaloside IV (ASIV) were predicted using SwissTargetPrediction, PharmMapper and Comparative Toxicogenomics Database (CTD). Ischemic stroke-related targets were collected from GeneCards, DisGeNET and DrugBank. Overlapping targets were used to construct a protein-protein interaction (PPI) network via STRING and visualized in Cytoscape. Functional enrichment analysis of Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways was performed using DAVID. The neuroprotective effects of ASIV were evaluated in a mouse model of middle cerebral artery occlusion/reperfusion (MCAO/R), assessing neurological function, infarct volume, blood brain barrier (BBB) integrity, inflammatory markers, and PI3K/Akt/NF-κB pathway activity. In vitro, LPS-stimulated BV-2 microglia were used to examine the effects of ASIV on cell viability, inflammatory cytokine expression, and PI3K/Akt/NF-κB signaling. Network pharmacology analysis identified 166 overlapping targets, and enrichment analysis emphasized the PI3K/Akt pathway as a key mechanism. In MCAO/R mice, ASIV significantly improved neurological function, reduced infarct volume and neuronal apoptosis, decreased Evans blue leakage, attenuated MMP-9 expression, and restored ZO-1 and Occludin levels. ASIV also suppressed the mRNA levels of IL-6, IL-1β, and TNF-α both in vivo and in LPS-stimulated BV-2 cells. Additionally, ASIV effectively upregulated the expression of Anti-inflammatory cytokines (IL-10 and Arg-1) in LPS-induced BV2 cells. Mechanistically, ASIV suppresses NF-κB activation through the stimulation of the PI3K/Akt signaling pathway. ASIV exerts neuroprotective effects against CIRI by inhibiting neuroinflammation and preserving BBB integrity likely through modulation of the PI3K/Akt/NF-κB signaling pathway.

Background: Danhong injection (DHI), a standardized traditional Chinese medicine formulation, has shown clinical benefits in treating cerebrovascular diseases. Blood-brain barrier (BBB) disruption is a key pathological feature of ischemic stroke, but its modulation by DHI under hyperlipidemic conditions remains unclear. This study aimed to investigate the protective effects and mechanisms of DHI in cerebral ischemia/reperfusion injury (CI/RI) under hyperlipidemia, focusing on BBB integrity and the Wnt/β-catenin signaling pathway. Methods: Rats were divided into control, ischemic, hyperlipidemic, and treatment subgroups to evaluate DHI's dose-dependent effects and pathway specificity using DKK1 inhibition. Assessments included neurological scores, TTC and Nissl staining, TEM, and molecular analyses (qRT-PCR/Western blot/immunofluorescence/immunohistochemistry). Results: DHI significantly improved neurological function, reduced cerebral infarct size, and alleviated cortical damage. DHI treatment upregulated the expression of tight junction proteins (Claudin-5, Occludin, ZO-1) and downregulated MMP-9 expression. Mechanistically, DHI promoted the nuclear translocation of β-catenin and increased the expression of Wnt3α, p-GSK-3β, and Cyclin D1, thereby activating the Wnt/β-catenin pathway. Additionally, DHI treatment increased the count of NeuN-positive neurons, suppressed astrocyte activation, and markedly reduced IgG infiltration in the ischemic cerebral cortex. These effects were reversed by DKK1. Conclusions: The results indicate that DHI protects BBB integrity and alleviates CI/RI in hyperlipidemic rats independently of direct lipid-lowering activity. Specifically, DHI activates the Wnt/β-catenin pathway by enhancing β-catenin nuclear translocation, which in turn mediates the upregulation of tight junction proteins and suppression of MMP-9, ultimately preserving BBB integrity. These findings support its therapeutic potential in ischemic stroke with comorbid hyperlipidemia.

Cerebral ischemia-reperfusion injury (CIRI) is a prevalent clinical complication associated with reperfusion following ischemic stroke resulting in neuronal damage and cognitive impairment. Dexmedetomidine (DEX), a highly selective α2-adrenoceptor agonist with sedative, and analgesic properties, is frequently utilized as a sedative anesthetic in clinical surgeries, and believed to play a crucial role in the prognosis of patients suffering from CIRI. However, the mechanism underlying DEX in CIRI remains to be determined. This study aimed to investigate the neuroprotective effects of Dex in rats suffering from CIRI. In the treatment group, DEX (50 µg/kg) was administered intraperitoneally 30 min prior to surgery. Middle cerebral artery occlusion (MCAO) used as a model of CIRI occurred with cerebral artery occlusion for 2 h was followed by reperfusion with blood for 24, 72, 120 or 168 h. Neurological function as assessed by the Longa neurological function score test demonstrated significantly reduced neurological scores and increased % infarct size in MCAO group which was blocked by DEX suggesting that DEX might be effective in treating ischemic stroke. In the MCAO animals, 2,3,5-triphenyltetrazolium chloride (TTC) showed large marked areas of cerebral infarction which were diminished in size by DEX. Using Western blot analysis, results showed that in MCAO rats protein expression levels of TNF-α and IL-6 were increased accompanied by reduced protein expression levels of PI3K/AKT signaling pathway. DEX pretreatment reversed the effects of MCAO as evidenced by decrease in protein expression levels of TNF-α and IL-6 associated with elevated protein expression levels of PI3K/AKT/NF-κB signaling pathway. Data demonstrated that DEX pretreatment improved the neuromotor performance and cognitive functions in animals suffering from consequences of MCAO by diminishing inflammation and activation of the PI3K/AKT/NF-κB signaling pathway.

XQ-1H regulates Wnt/GSK3β/β-catenin pathway and ameliorates the integrity of blood brain barrier in mice with acute ischemic stroke

To observe the effect of electroacupuncture (EA) on the expression of hypoxia-inducible factor 1α (HIF-1α), vascular endothelial growth factor (VEGF), matrix metalloproteinases 9 (MMP-9) and tight junction proteins in rats with cerebral ischemia-reperfusion injury (CI/RI) , so as to explore its potential mechanism in alleviating injury of neurological function and blood-brain barrier (BBB). A total of 84 male SD rats were used in the present study. The CIRI model was established by occlusion of the middle cerebral artery and reperfusion (MCAO/R), followed by dividing the CIRI rats into model group, EA group, and edaravone group (n=21 in each group). And another 21 normal rats were used as the sham operation group. For rats of the EA group, EA (2 Hz/15 Hz, 1 mA) was applied to "Baihui" (GV20) and "Zusanli" (ST36) on the affected limb for 20 min, once a day for 3 days. Rats of the edaravone group were intraperitoneally injected with edaravone injection (3 mg/kg), once a day for 3 days. The modified neurological severity score (mNSS) was used to evaluate the neurological behavior of rats. TTC staining was used to detect the percentage of cerebral infarction volume. HE staining was used to observe the pathological changes of cerebral cortex in ischemic area. Transmission electron microscopy was used to observe the changes of BBB ultrastructure in ischemic cortex. Evans blue (EB) staining was used to evaluate BBB permeability. Immunofluorescence was used to detect the expression of HIF-1α and VEGF protein in ischemic cortex. The protein and mRNA expressions of HIF-1α, VEGF, MMP-9, Zonula Occludens 1 (ZO-1), Occludin and Claudin-5 in the ischemic cortex were detected by Western blot or real-time PCR, respectively. Compared with the sham operation group, the neurological deficit score, the percentage of cerebral infarction volume, EB leakage, the positive expressions of HIF-1α and VEGF, the protein and mRNA expressions of HIF-1α, VEGF and MMP-9 of ischemic cortex were increased (P<0.01) in the model group , while the protein and mRNA expressions of ZO-1, Occludin and Claudin-5 were decreased (P<0.01). After the intervention and compared with the model group, the neurological deficit score, the percentage of cerebral infarction volume, EB leakage, the positive expressions of HIF-1α and VEGF, the protein and mRNA expressions of HIF-1α, VEGF and MMP-9 of ischemic cortex were decreased (P<0.01, P<0.05) in the EA and edaravone groups;the protein and mRNA expression of ZO-1, Occludin and Claudin-5 were increased (P<0.05, P<0.01) in the EA group;the protein and mRNA expression of Occludin were increased (P<0.01, P<0.05) and the mRNA expression of ZO-1 and Claudin-5 were increased (P<0.05, P<0.01) in the edaravone group. The ultrastructure of BBB was damaged in the model group, which were relatively milder in the EA and edaravone groups. EA intervention can reduce BBB injury and improve neurological dysfunction in rats with cerebral ischemia-reperfusion, and the underlying mechanism may be related to the regulation of HIF-1α/VEGF/MMP-9 signaling pathway.

Previous studies have demonstrated that inflammation and disruption of the blood-brain barrier (BBB) are important pathological processes during focal cerebral ischemia. Therefore, the present study evaluated the neuroprotective effects of resveratrol against brain damage, inflammation and BBB disruption in rats with focal cerebral ischemia and assessed the potential underlying molecular mechanisms. Sprague-Dawley rats underwent cerebral ischemia/reperfusion (IR) and then received intraperitoneal resveratrol (10 and 100 mg/kg) 2 h following the onset of ischemia. Following 24 h of ischemia, neurological deficit scores, cerebral infarctions, morphological characteristics, cerebral water content, myeloperoxidase (MPO) activity and Evans blue extravasation were assessed. Additionally, the protein expression levels of Toll-like receptor 4 (TLR4) and nuclear factor (NF)-κB p65 were detected using western blot analyses, the mRNA expression levels of cyclooxygenase-2 (COX-2) and matrix metalloproteinase-9 (MMP-9) were examined by reverse-transcription polymerase chain reaction, and tumor necrosis factor (TNF)-α and interleukin (IL)-1β blood levels were determined by ELISA. Resveratrol significantly reduced neurological deficit scores, cerebral infarct sizes, neuronal injury, MPO activity and EB content. Cerebral ischemia increased the expression levels of TLR4, NF-κB p65, COX-2, MMP-9, TNF-α and IL-1β, but all of these factors were reduced by resveratrol. In conclusion, the present data suggest that resveratrol reduces inflammation, BBB disruption and brain damage in rats following focal cerebral ischemia. Additionally, the neuroprotective effects of resveratrol against cerebral ischemia may be associated with downregulation of the TLR4 pathway.

Protection against stroke-induced blood-brain barrier disruption by Guanxinning injection and its active-component combination via TLR4/NF-κB/MMP9-mediated neuroinflammation.

Cerebral ischemia-reperfusion (IR) injury is associated with mitochondrial damage. Macrophage-stimulating 1 (MST1) reportedly stimulates mitochondrial apoptosis by suppressing BCL-2. We investigated whether MST1 promotes the progression of cerebral IR injury by inducing mitochondrial dysfunction in vivo and in vitro. Western blot analysis, quantitative polymerase chain reaction, immunofluorescence, and mitochondrial function assays were conducted in cells from wild-type and Mst1-knockout mice subjected to cerebral IR injury. MST1 expression in wild-type glial cells increased following cerebral IR injury. Cerebral IR injury reduced the mitochondrial membrane potential and mitochondrial metabolism in glial cells, while it enhanced mitochondrial reactive oxygen species generation and mitochondrial calcium levels in these cells. The deletion of Mst1 attenuated cerebral IR injury by improving mitochondrial function and reducing mitochondrial damage. The mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway was suppressed in wild-type glial cell upon cerebral IR injury but was reactivated in Mst1-knockout glial cell. Accordingly, blocking the MAPK/ERK pathway abolished the beneficial effects of Mst1 deletion during cerebral IR injury by inducing mitochondrial damage in glial cells. Our results suggest that cerebral IR injury is associated with MST1 upregulation in the brain, while the genetic ablation of Mst1 can attenuate mitochondrial damage and sustain brain function following cerebral IR injury.

Objective To investigate whether cerebral ischemia reperfusion (I/R) injury combined with diabetes mellitus (DM) could elevate concentrations of serum high mobility group box 1 (HMGB1) and its related inflammatory factors, and to explore the underlying mechanism of cerebral I/R injury combined with DM by blocking HMGB1. Methods One hundred and forty healthy male C57BL/6 mice were randomly divided into four groups: normoglycemia (NG) group (n=25), NG+I/R group (n=25), hyperglycemia (HG) group (n=25), and HG+I/R group (n=65); 40 mice in the HG+I/R group were chosen and divided into anti-HMGB1 group and IgG control group (n=20). High-fat feeding and i.p. injection of streptozotocin were used to establish HG mouse models, and then, middle cerebral artery occlusion was performed to establish the HG+I/R mouse models. Mice were treated with tail intravenous injection of 30 μg/g anti-HMGB1 polyclonal antibody or control IgG 1 h before ischemia as previously described. After accomplishment of animal models, serum HMGB1 concentrations were evaluated by ELISA, the permeability of blood brain barrier (BBB) was observed by Evans-blue fluorescence quantitative method, quantitative real-time PCR was introduced to detect the mRNA expressions of interleukin (IL)-1β, IL-6, and inducible nitric oxide synthase (iNOS). Morphology changes of damaged brains were observed by HE staining. Results The serum HMGB1 level in the HG+I/R group was significantly higher than that in the NG+I/R group (P<0.05). HG+I/R group had significantly higher BBB permeability than NG+I/R group (P<0.05), while anti-HMGB1 group had significantly lower BBB permeability than HG+I/R group (P<0.05). As compared with the NG group, the HG+I/R group had unclear cellular structures in the brain tissues with necrosis neurocytes and interstitial edema, while the cellular structures were obviously improved in the anti-HMGB1 group. Expressions of IL-1β, IL-6 and iNOS in the HG+I/R group were significantly elevated as compared with those in the NG+I/R group (P<0.05), while those in the anti-HMGB1 group had significantly decreased levels of IL-1β and iNOS as compared with those in the HG+I/R group (P<0.05), and the IL-6 level showed no significant difference between the two groups (P<0.05). Conclusion The pathogenesis of DM could increase concentration of serum HMGB1, and anti-HMGB1 mAb could alleviate brain injury by blocking HMGB1, and render a new promising therapeutic way for DM patients suffered with ischemic stroke. Key words: High mobility group box 1; Cerebral ischemia-reperfusion injury; Diabetes mellitus; Inflammation reaction

Background and purposeCerebral ischemia‒reperfusion injury causes significant harm to human health and is a major contributor to stroke-related deaths worldwide. Current treatments are limited, and new, more effective prevention and treatment strategies that target multiple cell components are urgently needed. Leucine-rich alpha-2 glycoprotein 1 (Lrg1) appears to be associated with the progression of cerebral ischemia‒reperfusion injury, but the exact mechanism of it is unknown.MethodsWild-type (WT) and Lrg1 knockout (Lrg1−/−) mice were used to investigate the role of Lrg1 after cerebral ischemia‒reperfusion injury. The effects of Lrg1 knockout on brain infarct volume, blood‒brain barrier permeability, and neurological score (based on 2,3,5-triphenyl tetrazolium chloride, evans blue dye, hematoxylin, and eosin staining) were assessed. Single-cell RNA sequencing (scRNA-seq), immunofluorescence, and microvascular albumin leakage tests were utilized to investigate alterations in various cell components in brain tissue after Lrg1 knockout.ResultsLrg1 expression was increased in various cell types of brain tissue after cerebral ischemia‒reperfusion injury. Lrg1 knockout reduced cerebral edema and infarct size and improved neurological function after cerebral ischemia‒reperfusion injury. Single-cell RNA sequencing analysis of WT and Lrg1−/− mouse brain tissues after cerebral ischemia‒reperfusion injury revealed that Lrg1 knockout enhances blood‒brain barrier (BBB) by upregulating claudin 11, integrin β5, protocadherin 9, and annexin A2. Lrg1 knockout also promoted an anti-inflammatory and tissue-repairing phenotype in microglia and macrophages while reducing neuron and oligodendrocyte cell death.ConclusionsOur results has shown that Lrg1 mediates numerous pathological processes involved in cerebral ischemia‒reperfusion injury by altering the functional states of various cell types, thereby rendering it a promising therapeutic target for cerebral ischemia‒reperfusion injury.

The mechanism of Bovis Culus Sativus protecting BBB damage in stroke: Insights from network pharmacology, bioinformatics, and experiments.

Objectives: Intra-artery infusion of recombinant human erythropoietin (rhEPO) has recently been reported to confer neuroprotection against cerebral ischemia-reperfusion injury in animal models; however, the molecular mechanisms are still under investigation. The present study focused on the specific mechanism involved in blood–brain barrier (BBB) disruption. Methods: Thirty-six male and nine female Sprague Dawley rats were subjected to middle cerebral artery (MCA) occlusion to induce focal cerebral ischemia, and administrated rhEPO at a dose of 800 U/kg through MCA infusion at the beginning of reperfusion. Neurobehavioral deficits, brain edema, and infarct volume were evaluated after 2 h of ischemia and 24 h of reperfusion. BBB permeability was assessed by quantifying the extravasation of Evans blue (EB) dye. The expression of tight junction proteins and matrix metalloproteinases (MMPs) (Claudin-5, Occludin, MMP-2, and MMP-9) in microvessels were detected by immunofluorescence and western blot. The activities of MMPs in the cerebral microvessels were determined by gelatin zymography. Results: Treatment with rhEPO through the MCA strongly alleviated infarct volume, brain edema, and improved neurobehavioral outcomes in male and female rats. In addition, rhEPO remarkably suppressed the EB extravasation induced by brain ischemia. Furthermore, rhEPO prevented degradation of Claudin-5 and Occludin, and reduced the expression and activity of MMP-2 and MMP-9 in isolated brain microvessels. Conclusions: Treatment with rhEPO through MCA infusion prevented brain edema formation and infarction through inhibition of MMP-mediated BBB disruption in acute ischemic stroke.

Fuzheng Jiedu Tongluo Granule mitigates blood-brain barrier disruption after ischemic stroke via inhibiting transcytosis in cerebral vascular endothelial cells.