Brain Water Content Research Articles

PD-1 mediates microglia polarization via the MAPK signaling pathway to protect blood-brain barrier function during cerebral ischemia/reperfusion

BackgroundCerebral ischemia is characterized by its rapid onset and high rates of recurrence, morbidity, and mortality, with blood-brain barrier (BBB) permeability playing a vital role in brain injury. Therefore, it is important to understand the molecular mechanism which regulates the BBB during cerebral ischemia. Materials and methodsAn in vitro model of oxygen-glucose deprivation (OGD) and an in vivo model of cerebral ischemia/reperfusion (I/R) were constructed. PD-1 overexpression vectors and vectors containing si-RNA were transfected and injected into in vitro and in vivo models. Western blotting, real-time quantitative PCR (qPCR), immunofluorescence (IF) analysis, and immunohistochemical staining were employed to evaluate the expression levels of programmed cell death-1 (PD-1), microglia M1 and M2 biomarkers, and tight junction proteins. Flow cytometry and ELISA were used to measure the levels of pro-inflammatory cytokines. The BBB permeability of brain tissues was evaluated by Evans blue dye (EBD) extravasation and transendothelial electrical resistance (TEER). Brain water content was measured to assess the extent of inflammatory exudation. The infarct volume and neurological severity score (NSS) were used to assess the severity of brain injury. Brain cell apoptosis was assessed by the TUNEL assay and hematoxylin-eosin (H&E) staining. ResultsPD-1 helped to convert the microglia M1 phenotype to the M2 phenotype and to reduce BBB permeability both in vitro and in vivo. Overexpression of PD-1 promoted a shift of the M1 phenotype to the M2 phenotype and reduced BBB permeability via the ERK and p38 MAPK signaling pathways. PD-1 reduced inflammatory exudation, BBB permeability, cell apoptosis, and brain injury in vivo. ConclusionOur present study verified that PD-1 exerts an anti-inflammatory effect by converting the microglia M1 phenotype to the M2 phenotype, reducing BBB permeability, and thereby relieves brain injury caused by cerebral ischemia. PD-1 is potential therapeutic target for brain injury caused by cerebral ischemia.

Fingolimod Alleviates Inflammation after Cerebral Ischemia via HMGB1/TLR4/NF-κB Signaling Pathway.

Clinically, ischemic reperfusion injury is the main cause of stroke injury. This study aimed to assess the effectiveness of fingolimod in suppressing inflammation caused by ischemic brain injury and explore its pharmacological mechanisms. In total, 75 male Sprague-Dawley rats were randomly and equally assigned to five distinct groups: sham, middle cerebral artery occlusion/reperfusion (MCAO/R) surgery, fingolimod low-dose (F-L), fingolimod medium-dose (F-M), and fingolimod high-dose (F-H). Neurobehavioral tests, 2,3,5-triphenyltetrazolium chloride staining, and the brain tissue drying-wet method were conducted to evaluate neurological impairment, cerebral infarction size, and brain water content. Enzyme-linked immunosorbent assay was employed to quantify pro-inflammatory cytokines interleukin (IL)-1β, IL-6, and tumor necrosis factor-alpha (TNF-α) protein levels. Western blotting and immunohistochemical staining were performed to assess high mobility group box 1 (HMGB1), toll-like receptor 4 (TLR4), and nuclear factor kappa-B p65 (NF-κBp65) levels. Rats in the F-L, F-M, and F-H groups exhibited lower Longa scores, reduced infarction volumes, and decreased brain edema than those in the MCAO/R group. Additionally, the F-L, F-M, and F-H groups exhibited lower serum levels of IL-1β, IL-6, and TNF-α than those of the MCAO/R group. Additionally, F-L, F-M, and F-H treatments resulted in decreased HMGB1, TLR4, and NF-κBp65 protein expression levels in the hippocampus of MCAO/R rats. Fingolimod was found to reduce ischemic brain injury in a dose-dependent manner. Moreover, it was also found to alleviate inflammation following ischemic brain injury via the HMGB1/TLR4/NF‑κB signaling pathway.

Genistein inhibits Nlrp3/caspase-1 signalling to alleviate traumatic brain injury-induced anxiety-like behaviours in rats.

Traumatic brain injury (TBI)-induced anxiety is a common but under-investigated disorder, for which neuroinflammation is a significant contributor. Here we aim to investigate the protective effects of genistein, a plant-derived anti-inflammatory drug, against TBI-induced anxiety, and the underlying mechanisms. A rat model of TBI was constructed using the lateral fluid percussion injury method. Genistein at the doses of 5, 10, and 20 mg/kg were used to treat rats at 30 min, 12 h, 24 h, 48 h, and 72 h up to 14 days after TBI. The evaluation of neurological deficit was performed preoperatively, on days 1, 3, 7, and 14 after TBI. The elevated plus maze test was carried out to assess anxiety and explorative behaviours, and the open field test was performed to assess locomotive activities. Brain injury was assessed by measuring brain water content and TdT-mediated dUTP Nick-End Labeling staining. Inflammatory responses were examined using enzyme-linked immunosorbent assay. The mRNA and protein expression were analysed using real-time polymerase chain reaction and Western blot, respectively. In the behavioural level, genistein treatment alleviated TBI-induced anxiety behaviours and neurological deficit in rats. In the meanwhile, brain oedema was also reduced by genistein treatment, showing alleviating effects of genistein at the pathological level. TUNEL staining also showed reduced apoptosis in rats treated with genistein. Genistein also inhibited Nlrp3/caspase-1 signalling, unveiling the effects of genistein in altering molecular pathways in brains with TBI. Genistein alleviates anxiety-like behaviours in TBI rats, which may be mediated via inhibiting Nlrp/caspase-1 signalling pathway.

The water content, apoptosis, and proliferation of the brain in marine medaka affected by seawater acidification.

A possible explanation for ocean acidification-induced changes in fish behavior is a systemic effect on the nervous system. Three biological barriers at the blood-brain interface effectively separate the brain from the body fluids. It is not known whether fish brain regions in contact with these barriers are affected by acidification. Here, we studied structural changes in medaka (Oryzias melastigma) brain regions contacting cerebrospinal fluid (CSF) after short-term (7 days) CO2 exposure. The brain water content decreased significantly and the superficial structure of the pia mater was changed, but there was no obvious damage to the internal structures of the brain after seawater acidification. Seawater acidification also led to an increase in apoptosis and a decrease in the number of proliferative cells in brain areas contacting CSF. These results indicate that the structure of CSF-contacting brain regions in medaka was affected by seawater acidification, and the brain responded to seawater acidification stress by increasing apoptosis and reducing proliferation.

Activation of NFE2L2 Alleviates Endoplasmic Reticulum Stress-Mediated Pyroptosis in Murine Hippocampus: A Bioinformatics Analysis and Experimental Validation.

Pyroptosis has been implicated in many pathologic processes, including endoplasmic reticulum stress (ERS). However, the underlying mechanisms and molecular targets of ERS affecting pyroptosis still need further exploration. We obtained gene sets associated with ERS and pyroptosis, and the common genes were regarded as crosstalk genes linking ERS and pyroptosis. Protein-protein interaction (PPI) network was constructed, and the hub genes were obtained via Cytoscape. Moreover, to validate the efficacy of the therapeutic target, neurological tests, brain water content measurements, Nissl staining, Western blot, ELISA, TUNEL analyses, and transmission electron microscopy were performed in a mouse model. A total of 13 crosstalk genes were acquired, and enrichment analysis revealed that these genes were mainly enriched in stress-associated cellular processes and pathways, including KEAP1-NFE2L2 pathway. The hub gene, NFE2L2, was identified by Cytoscape, and tert-butylhydroquinone (tBHQ) was screened as candidate drug to activate NFE2L2. Western blot and ELISA results showed that activation of NFE2L2 could attenuate the expression of ERS and pyroptosis-related proteins by promoting nuclear translocation of Nrf2 (encoded by NFE2L2). Pathological evaluation by Nissl staining and TUNEL assay reflected a similar trend. Furthermore, activation of NFE2L2 ameliorated neurological deficits and reduced brain edema. In conclusion, our bioinformatic analysis results established the theoretical foundation of NFE2L2 as a promising therapeutic target. Moreover, in the mouse model, tBHQ pretreatment further confirmed the effectiveness of this target. We hypothesized NFE2L2 may play a key role in the progression of ERS-mediated pyroptosis. These findings may inspire new ideas to treat neurological disorders.

Mailuo Shutong pills inhibit neuroinflammation by regulating glucose metabolism disorders to protect mice from cerebral ischemia-reperfusion injury

Ethnopharmacological relevanceMailuo Shutong Pill (MLST), a traditional Chinese medicine (TCM), has been widely used for clearing heat and detoxifying, eliminating stasis and dredging meridians, dispelling dampness and diminishing swelling. Earlier study found that MLST could improve cerebral ischemic-reperfusion injury, however, the potential mechanism has not been well evaluated. Aim of studyIn this study, a well established and widely used mice model of middle cerebral artery occlusion/reperfusion (MCAO/R) was preformed to evaluate the protective function of MLST on cerebral ischemic-reperfusion injury and further discuss the potential pharmacological mechanisms. Materials and methodsChemical profiling of MLST was analyzed based on Ultra-high-performance liquid chromatography electrospray ionization orbitrap tandem mass spectrometry. ICR mice were challenged by MCAO/R surgery. The protective effect of MLST on MCAO/R injury was evaluated by neurological deficit score, cerebral infarct rate, brain water content, H&E and nissl staining. The blood-brain barrier (BBB) integrity was detected by Evans blue staining. The potential pharmacological mechanism of MLST in treating MCAO/R injury was further elucidated by the methods of proteomics, central carbon targeted metabolomics, as well as Western blot. Immunohistochemistry was used to detect the microglia infiltration, enzyme linked immunosorbent assay (ELISA) kit was explored to evaluate the content of IL-1β, TNF-α and IL-6 in brain tissue, and Western blot was used to detect proteins expression in brain tissue. ResultsA total of 76 chemical compounds have been determined in MLST. MLST effectively protected mice from MCAO/R injury, which was confirmed by lower neurological deficit score, cerebral infarct rate, brain water content and nissl body loss, and improved brain pathology. Meanwhile, MLST upregulated the expression of ZO-1, Occludin and Claudin 5 by downregulating the ratio of TIMP1/MMP9 to suppress the entrance of Evans blue to brain tissue, indicating that MLST maintained the integrity of BBB. Further studies indicated that MLST inhibited the inflammatory level of brain tissue by inhibiting microglia infiltration and downregulating NLRP3 inflammasome signaling pathway. The results of proteomics, Western blot, and central carbon targeted metabolomics confirmed that MLST regulated Glycolysis/Gluconogenesis, Pyruvate metabolism and TCA cycle in brain tissue of mice with MCAO/R. ConclusionMLST inhibits neuroinflammation by regulating glucose metabolism disorders to interfere with immune metabolism reprogramming and inhibit the NLRP3 inflammasome signaling pathway, and finally improve cerebral ischemia-reperfusion injury. This study confirms that MLST is a potential drug for treating Cerebral ischemic stroke.

PEDF-34 attenuates neurological deficit and suppresses astrocyte-dependent neuroinflammation by modulating astrocyte polarization via 67LR/JNK/STAT1 signaling pathway after subarachnoid hemorrhage in rats

BackgroundReactive astrocytes participate in various pathophysiology after subarachnoid hemorrhage (SAH), including neuroinflammation, glymphatic–lymphatic system dysfunction, brain edema, BBB disruption, and cell death. Astrocytes transform into two new reactive phenotypes with changed morphology, altered gene expression, and secretion profiles, termed detrimental A1 and beneficial A2. This study investigates the effect of 67LR activation by PEDF-34, a PEDF peptide, on neuroinflammation and astrocyte polarization after the experimental SAH.MethodsA total of 318 male adult Sprague-Dawley rats were used in experiments in vivo, of which 272 rats were subjected to the endovascular perforation model of SAH and 46 rats underwent sham surgery. 67LR agonist (PEDF-34) was administrated intranasally 1 h after SAH. 67LR-specific inhibitor (NSC-47924) and STAT1 transcriptional activator (2-NP) were injected intracerebroventricularly 48 h before SAH. Short- and long-term neurological tests, brain water content, immunostaining, Nissl staining, western blot, and ELISA assay were performed. In experiments in vitro, primary astrocyte culture with hemoglobin (Hb) stimulation was used to mimic SAH. The expression of the PEDF-34/67LR signaling pathway and neuro-inflammatory cytokines were assessed using Western blot, ELISA, and immunohistochemistry assays both in vivo and in vitro.ResultsEndogenous PEDF and 67LR expressions were significantly reduced at 6 h after SAH. 67LR was expressed in astrocytes and neurons. Intranasal administration of PEDF-34 significantly reduced brain water content, pro-inflammatory cytokines, and short-term and long-term neurological deficits after SAH. The ratio of p-JNK/JNK and p-STAT1/STAT1 and the expression of CFB and C3 (A1 astrocytes marker), significantly decreased after PEDF-34 treatment, along with fewer expression of TNF-α and IL-1β at 24 h after SAH. However, 2-NP (STAT1 transcriptional activator) and NSC-47924 (67LR inhibitor) reversed the protective effects of PEDF-34 in vivo and in vitro by promoting A1 astrocyte polarization with increased inflammatory cytokines.ConclusionPEDF-34 activated 67LR, attenuating neuroinflammation and inhibiting astrocyte A1 polarization partly via the JNK/STAT1 pathway, suggesting that PEDF-34 might be a potential treatment for SAH patients.Graphical

Dental pulp mesenchymal stem cell-derived exosomes inhibit neuroinflammation and microglial pyroptosis in subarachnoid hemorrhage via the miRNA-197-3p/FOXO3 axis

BackgroundSubarachnoid hemorrhage (SAH) is a severe stroke subtype that lacks effective treatment. Exosomes derived from human dental pulp stem cells (DPSCs) are a promising acellular therapeutic strategy for neurological diseases. However, the therapeutic effects of DPSC-derived exosomes (DPSC-Exos) on SAH remain unknown. In this study, we investigated the therapeutic effects and mechanisms of action of DPSC-Exos in SAH.Materials and methodsSAH was established using 120 male Sprague-Dawley rats. One hour after SAH induction, DPSC-Exos were administered via tail vein injection. To investigate the effect of DPSC-Exos, SAH grading, short-term and long-term neurobehavioral assessments, brain water content, western blot (WB), immunofluorescence staining, Nissl staining, and HE staining were performed. The role of miR-197-3p/FOXO3 in regulating pyroptosis was demonstrated through miRNA sequencing, bioinformatics analysis, and rescue experiments. The SAH model in vitro was established by stimulating BV2 cells with hemoglobin (Hb) and the underlying mechanism of DPSC-Exos was investigated through WB and Hoechst/PI staining.ResultsThe expressions of pro-inflammatory cytokines (IL-1β, IL-6, and TNF-α) were increased after SAH. DPSC-Exos alleviated brain edema and neuroinflammation by inhibiting the expression of FOXO3 and reducing NLRP3 inflammasome activation, leading to improved neurobehavioral functions at 24 h after SAH. In vitro, the expression of the NLRP3 inflammasome components (NLRP3 and caspase1-p20), GSDMD-N, and IL-18 was inhibited in BV2 cells pretreated with DPSC-Exos. Importantly, DPSC-Exos overexpressing miR-197-3p had a more obvious protective effect than those from NC-transfected DPSCs, while those from DPSCs transfected with the miR-197-3p inhibitor had a weaker protective effect. Functional studies indicated that miR-197-3p bound to the 3ʹ-untranslated region of FOXO3, inhibiting its transcription. Furthermore, the overexpression of FOXO3 reversed the protective effects of miR-197-3p.ConclusionsDPSC-Exos inhibited activation of the NLRP3 inflammasome and related cytokine release via the miR-197-3p/FOXO3 pathway, alleviated neuroinflammation, and inhibited microglial pyroptosis. These findings suggest that using DPSC-Exos is a promising therapeutic strategy for SAH.Graphical

Neuroprotective Effect of Ganoderic Acid against Focal Ischemic Stroke Induced by Middle Cerebral Artery Occlusion in the Rats via Suppression of Oxidative Stress and Inflammation.

Swiss Wistar rats were used for the current study. The rats were subjected to middle cerebral artery occlusion (MCAO) to simulate transient focal ischemia, followed by reperfusion. Various neurological parameters, including infarct size, neurological deficit score, brain water content, Evans blue leakage, nitric oxide (NO), inducible nitric oxide synthase (iNOS), lactate dehydrogenase (LDH), antioxidant levels, inflammatory cytokines, apoptosis markers, inflammatory parameters, and matrix metalloproteinases (MMP) levels, were estimated. Additionally, mRNA expressions were evaluated in the brain tissue. Dose dependently treatment of GA significantly (P < 0.001) suppressed the infarct size, neurological deflects score, brain water, evans blue leakage, NO, iNOS, LDH, C-X-C chemokine receptor type 4 (CXCR-4), monocyte chemoattractant protein-1 (MCP-1), S100 calcium-binding protein B (S-100β) and K+-Cl- cotransporter 1 (KCC1) positive cells. GA altered the level of oxidative stress parameters like Total antioxidant capacity (T-AOC), 8-hydroxy-2'-deoxyguanosine (8-OhdG), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione (GSH), malonaldehyde (MDA); cytokines viz., tumor necrosis factor-α (TNF-α), interleukin-1 (IL-1), IL-1β, IL-6, IL-9, IL-10; inflammatory parameters such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), prostaglandin (PGE2), Nuclear factor kappa B (NF-κB); apoptosis parameters like B-cell leukemia/lymphoma 2 protein (Bcl-2), Bcl-2-associated protein x (Bax), Caspase-3; matrix metallopeptidase (MMP) parameters like MMP-2, MMP-3, and MMP-9, respectively. GA remarkably suppressed the mRNA expression of TRL-4, Syndecan-1, CSF, Aquaporin-1, OCT3, and RFX1. Ganoderic acid exhibited the protection against the cerebral ischemia reperfusion via multiple mechanism.

A meta-analysis of animal studies evaluating the effect of hydrogen sulfide on ischemic stroke: is the preclinical evidence sufficient to move forward?

Hydrogen sulfide (H2S) is a gasotransmitter that has been studied for its potential therapeutic effects, including its role in the pathophysiology and treatment of stroke. This systematic review and meta-analysis aimed to determine the sufficiency of overall preclinical evidence to guide the initiation of clinical stroke trials with H2S and provide tailored recommendations for their design. PubMed, Web of Science, Scopus, EMBASE, and MEDLINE were searched for studies evaluating the effect of any H2S donor on in vivo animal models of regional ischemic stroke, and 34 publications were identified. Pooling of the effect sizes using the random-effect model revealed that H2S decreased the infarct area by 34.5% (95% confidence interval (CI) 28.2-40.8%, p < 0.0001), with substantial variability among the studies (I2 = 89.8%). H2S also caused a 37.9% reduction in the neurological deficit score (95% CI 29.0-46.8%, p < 0.0001, I2 = 63.8%) and in the brain water content (3.2%, 95% CI 1.4-4.9%, p = 0.0014, I2 = 94.6%). Overall, the studies had a high risk of bias and low quality of evidence (median quality score 5/15, interquartile range 4-9). The majority of the included studies had a "high" or "unclear" risk of bias, and none of the studies overall had a "low" risk. In conclusion, H2S significantly improves structural and functional outcomes in in vivo animal models of ischemic stroke. However, the level of evidence from preclinical studies is not sufficient to proceed to clinical trials due to the low external validity, high risk of bias, and variable design of existing animal studies.

Unleashing the Neurotherapeutic Potential: The Crucial Role of miR-206-3p in Facilitating Hsp90aa1-Mediated Central Nervous System Injuries During Heat Stroke.

This study aims to explore the molecular mechanisms of miR-206-3p in regulating Hsp90aa1 and its involvement in the central nervous system (CNS) injury in heat stroke. Weighted gene co-expression network analysis (WGCNA) was performed on the GSE64778 dataset of heat stroke to identify module genes most closely associated with disease characteristics. Through the selection of key genes and predicting upstream miRNAs using RNAInter and miRWalk databases, the regulatory relationship between miR-206-3p and Hsp90aa1 was determined. Through in vitro experiments, various methods, including bioinformatics analysis, dual-luciferase reporter gene assay, RIP experiment, and RNA pull-down experiment, were utilized to validate this regulatory relationship. Furthermore, functional experiments, including CCK-8 assay to test neuron cell viability and flow cytometry to assess neuron apoptosis levels, confirmed the role of miR-206-3p. Transmission electron microscopy, real-time quantitative PCR, DCFH-DA staining, and ATP assay were employed to verify neuronal mitochondrial damage. Heat stroke rat models were constructed, and mNSS scoring and cresyl violet staining were utilized to assess neural functional impairment. Biochemical experiments were conducted to evaluate inflammation, brain water content, and histopathological changes in brain tissue using H&E staining. TUNEL staining was applied to detect neuronal apoptosis in brain tissue. RT-qPCR and Western blot were performed to measure gene and protein expression levels, further validating the regulatory relationship in vivo. Bioinformatics analysis indicated that miR-206-3p regulation of Hsp90aa1 may be involved in CNS injury in heat stroke. In vivo, animal experiments demonstrated that miR-206-3p and Hsp90aa1 co-localized in neurons of the rat hippocampal CA3 region, and with prolonged heat stress, the expression of miR-206-3p gradually increased while the expression of Hsp90aa1 gradually decreased. Further in vitro cellular mechanism validation and functional experiments confirmed that miR-206-3p could inhibit neuronal cell viability and promote apoptosis and mitochondrial damage by targeting Hsp90aa1. In vivo, experiments confirmed that miR-206-3p promotes CNS injury in heat stroke. This study revealed the regulatory relationship between miR-206-3p and Hsp90aa1, suggesting that miR-206-3p could regulate the expression of Hsp90aa1, inhibit neuronal cell viability, and promote apoptosis, thereby contributing to CNS injury in heat stroke.

Molecular mechanism of HDAC6-mediated pyroptosis in neurological function recovery after cardiopulmonary resuscitation in rats

Brain injury after cardiac arrest (CA) and cardiopulmonary resuscitation (CPR) is the leading cause of neurological dysfunction and death. This study aimed to explore the mechanism of histone deacetylase 6 (HDAC6) in neurofunctional recovery following CA/CPR in rats. A rat model was established by CA/CPR treatment. Adenovirus-packaged sh-HDAC6 was injected into the tail vein. To evaluate the neurofunction of rats, survival time, neurofunctional scores, serum NSE/S100B, and brain water content were measured and Morris water maze test was performed. HDAC6, microRNA (miR)-138-5p, Nod-like receptor protein 3 (NLRP3), and pyroptotic factor levels were determined by real-time quantitative polymerase chain reaction or Western blot assay. HDAC6 and H3K9ac enrichment on miR-138-5p promoter were examined by chromatin immunoprecipitation. miR-138-5p-NLRP3 binding was analyzed by dual-luciferase reporter assay. NLRP3 inflammasome was activated with nigericin sodium salt. After CPR treatment, HDAC6 was highly expressed, while miR-138-5p was downregulated. HDAC6 downregulation improved neurofunction and reduced pyroptosis. HDAC6 enrichment on the miR-138-5p promoter deacetylated H3K9ac, inhibiting miR-138-5p, and promoting NLRP3-mediated pyroptosis. Downregulating miR-138-5p partially reversed the protective effect of HDAC6 inhibition after CPR. In Conclusion, HDAC6 enrichment on miR-138-5p promoter deacetylated H3K9ac, inhibiting miR-138-5p expression and promoting NLRP3-mediated pyroptosis, worsening neurological dysfunction in rats after CPR.

Tamarixetin ameliorates cerebral ischemia-reperfusion injury via suppressing nicotinamide adenine dinucleotide phosphate oxidase 2/nucleotide-binding oligomerization domain like receptor family pyrin domain-containing 3 inflammasome activation.

Tamarixetin, a natural dietary flavone, exhibits remarkable potential for the treatment of ischemic stroke. The present article aimed to explore the impact of tamarixetin on ischemic stroke and elucidate the underlying mechanisms. Effects of tamarixetin on ischemic stroke were evaluated in rats using the middle cerebral artery occlusion and reperfusion (MCAO/R) model, by assessing the neurological deficit scores, brain water content, brain infraction, and neuronal damage. The levels of proinflammatory cytokines, NLRP3 inflammasome activation, reactive oxygen species (ROS) production, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase expression were measured in MCAO/R rats and lipopolysaccharide-stimulated cells. Tamarixetin administration improved the neurological dysfunction and neuronal loss in MCAO/R rats. In addition, tamarixetin reduced microglial hyperactivation and proinflammatory cytokines expression in vivo and in vitro. Tamarixetin attenuated NF-κB p65 phosphorylation and promoter activity, reduced NLRP3 expression and caspase-1 cleavage, and downregulated IL-1β and IL-18 secretions to suppress NLRP3 inflammasome activation. The levels of superoxide anion, hydrogen peroxide, and ROS were also suppressed by tamarixetin. The downregulation of NADP+ and NADPH levels, and gp91phox expression indicated the ameliorative effects of tamarixetin on NADPH oxidase activation. In the gp91phox knockdown cells treated with lipopolysaccharide, the effects of tamarixetin on NADPH oxidase activation, ROS generation, and NLRP3 inflammasome activation were diminished. Moreover, tamarixetin protects neurons against microglial hyperactivation in vitro. Our findings support the potential of tamarixetin as a therapeutic agent for ischemic stroke, and its mechanism of action involves the inhibition of NADPH oxidase-NLRP3 inflammasome signaling.

Step-down infusion of barbiturate improves neurofunction in a new rat model of rebleeding subarachnoid hemorrhage

Rebleeding from a ruptured aneurysm is a risk factor for fatal prognosis in subarachnoid hemorrhage (SAH). A novel barbiturate treatment, step-down infusion of barbiturate (sd-B), was previously developed; it showed beneficial effects on patients with severe traumatic brain injuries. The present study established a rebleeding SAH model in rats and evaluated the effect of sd-B. Fifty male Sprague-Dawley rats were divided into sham-operation with distilled water, sham-operation with barbiturate, SAH-rebleeding with distilled water, and SAH-rebleeding with barbiturate groups. For posttreatment with sd-B, thiamylal was intraperitoneally administered at 3 mg/kg/h on days 0–1, 2 mg/kg/h on days 1–2, and 1 mg/kg/h on days 2–3 after SAH using osmotic minipumps. We monitored neurofunction and case fatality as the primary endpoints and evaluated brain injuries, including brain edema and cortical neuronal cell death, as the secondary endpoints. Posttreatment with sd-B improved the modified Garcia test, reduced the brain water content, and inhibited the loss of neuronal cells and microglial expressions in the rat model. Our results revealed that sd-B ameliorated neurofunction and brain injuries on the rebleeding SAH model, suggesting that the novel treatment is a good candidate drug for patients with SAH with rebleeding.

Liangxue Tongyu prescription attenuates neuroinflammation by increasing cholecystokinin octapeptide in acute intracerebral hemorrhage rats

Inflammatory reactions after acute intracerebral hemorrhage (AICH) contribute significantly to a poor prognosis. Liangxue Tongyu Prescription (LTP) has been proven to be clinically effective in treating AICH. Numerous studies have shown that LTP suppresses brain inflammatory damage in AICH, while the internal mechanisms underlying its action remain unclear. The aim of this study was to verify the anti-inflammatory effects of LTP on an AICH rat model and investigate the potential mechanisms. The AICH rat models were created by injecting autologous blood into the right caudate nucleus. LTP markedly decreased cerebral hematoma and brain water content and recovered from neurological deficits. Meanwhile, LTP prevented microglial activation and reduced the inflammatory reaction caused by pro-inflammatory cytokines, such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6). Notably, the expression of cholecystokinin octapeptide (CCK-8) in the brain and intestine was increased by LTP or CCK-8 treatment. LTP further suppressed nuclear factor kappa B (NF-κB) in the brains of rats with AICH. Moreover, LTP increased the protein and mRNA expression of Occludin and Claudin-1 in the intestine and decreased the levels of lipopolysaccharide (LPS) and diamine oxidase (DAO) in serum. Furthermore, the results showed that LTP increased the protein and mRNA expression of Claudin-5 and zonula occludens-1 (ZO-1) in the brain. CCK-8 receptor antagonists increased the expression of NF-κB and the concentration of pro-inflammatory cytokines. These findings suggested that LTP attenuated neuroinflammation by increasing CCK-8 in the brain and intestine, and its mechanism might be related to alterations in the gut-brain axis (GBA).

Bezafibrate protects blood-brain barrier (BBB) integrity against traumatic brain injury mediated by AMPK

Bezafibrate (BEZ) has displayed a wide range of neuroprotective effects in different types of neurological diseases. However, its pharmacological function in traumatic brain injury (TBI) is still unknown. In the current study, a TBI model was constructed in mice to examine the potential beneficial roles of BEZ. After TBI, mice were daily dieted with BEZ or vehicle solution. The motor function, learning and memory, brain edema, vascular inflammatory factors, the integrity of the blood-brain barrier (BBB), and the expression of the tight junction zona occludens 1 (ZO-1) were assessed. The findings demonstrate that after TBI, BEZ treatment significantly promoted the recovery of motor function and cognitive function deficits. Moreover, BEZ attenuated brain edema by reducing the levels of brain water content. We also found that administration of BEZ alleviated cerebral vascular pro-inflammation by suppressing the expression of ICAM-1, VCAM-1, and E-selectin. Notably, BEZ improved the impaired BBB integrity in TBI mice by restoring the expression of the tight junction (TJ) protein ZO-1. Further in vitro experiments show that treatment with BEZ prevented the aggravation of endothelial permeability and restored the reduction of trans-epithelial electrical resistance (TEER) as well as the expression of ZO-1 in TBI-exposed brain bEnd.3 cells. Mechanistically, we prove that the protective effects of BEZ are mediated by AMPK. Based on these findings, we conclude that BEZ improves TBI-induced BBB injury and it might be considered for the treatment or management of TBI.

Luteolin-7-O-β-d-Glucuronide Attenuated Cerebral Ischemia/Reperfusion Injury: Involvement of the Blood-Brain Barrier.

Ischemic stroke is a common cerebrovascular disease with high mortality, high morbidity, and high disability. Cerebral ischemia/reperfusion injury seriously affects the quality of life of patients. Luteolin-7-O-β-d-glucuronide (LGU) is a major active flavonoid compound extracted from Ixeris sonchifolia (Bge.) Hance, a Chinese medicinal herb mainly used for the treatment of coronary heart disease, angina pectoris, cerebral infarction, etc. In the present study, the protective effect of LGU on cerebral ischemia/reperfusion injury was investigated in an oxygen-glucose deprivation/reoxygenation (OGD/R) neuronal model and a transient middle cerebral artery occlusion (tMCAO) rat model. In in vitro experiments, LGU was found to improve the OGD/R-induced decrease in neuronal viability effectively by the MTT assay. In in vivo experiments, neurological deficit scores, infarction volume rates, and brain water content rates were improved after a single intravenous administration of LGU. These findings suggest that LGU has significant protective effects on cerebral ischemia/reperfusion injury in vitro and in vivo. To further explore the potential mechanism of LGU on cerebral ischemia/reperfusion injury, we performed a series of tests. The results showed that a single administration of LGU decreased the content of EB and S100B and ameliorated the abnormal expression of tight junction proteins ZO-1 and occludin and metalloproteinase MMP-9 in the ischemic cerebral cortex of the tMCAO 24-h injury model. In addition, LGU also improved the tight junction structure between endothelial cells and the degree of basement membrane degradation and reduced the content of TNF-α and IL-1β in the brain tissue. Thereby, LGU attenuated cerebral ischemia/reperfusion injury by improving the permeability of the blood-brain barrier. The present study provides new insights into the therapeutic potential of LGU in cerebral ischemia.

A Novel Mechanism Linking Melatonin, Ferroptosis and Microglia Polarization via the Circodz3/HuR Axis in Subarachnoid Hemorrhage.

A subarachnoid hemorrhage (SAH) is life-threatening bleeding into the subarachnoid space that causes brain damage. Growing evidence has suggested that melatonin provides neuroprotection following SAH. Exploring the mechanisms underlying melatonin-mediated neuroprotection contributes to its clinical application in SAH. The plasma and cerebrospinal fluid (CSF) were collected from SAH patients, and SAH mice were established via pre-chiasmatic injection. Circodz3 expression, levels of IL-1β and TNF-α, brain water content, neurological and beam-waling scores were determined. Ferroptosis was evaluated by analyzing levels of iron, lipid ROS, MDA, and GSH. The colocalization of circodz3 and Iba-1 was analyzed by immunofluorescence staining. Interaction of circodz3 and HuR was determined with RNA pull-down and RNA immunoprecipitation assays. Herein, we found that circodz3 was highly abundant in SAH patients and mice. Colocalization of circodz3 and Iba-1 in the left hemisphere of SAH mice suggested the implication of circodz3 in regulating microglia activation following SAH. Melatonin alleviated brain edema, neurological impairment, and microglia activation and inhibited circodz3 expression in SAH mice. Moreover, melatonin inhibited M1 polarization, oxidative stress and ferroptosis and restrained circodz3 expression in primary microglia following SAH. These effects were abrogated by circodz3 overexpression. Circodz3 knockdown inhibited ferroptosis and M1 polarization of BV2 microglia after SAH. Circodz3 interacted with HuR to facilitate β-Trcp1-mediated ubiquitination and degradation, thus restraining the expression of SLC7A11 and GPX4. Collectively, melatonin exerted neuroprotection following SAH via inhibiting ferroptosis and M1 polarization through the circodz3/HuR axis. Our study suggests potential application of melatonin in the treatment of SAH.

CEBPD aggravates apoptosis and oxidative stress of neuron after ischemic stroke by Nrf2/HO-1 pathway

CCAAT enhancer binding protein delta (CEBPD) is a transcription factor and plays an important role in apoptosis and oxidative stress, which are the main pathogenesis of ischemic stroke. However, whether CEBPD regulates ischemic stroke through targeting apoptosis and oxidative stress is unclear. Therefore, to answer this question, rat middle cerebral artery occlusion (MCAO) reperfusion model and oxygen-glucose deprivation/reoxygenation (OGD/R) primary cortical neuron were established to mimic ischemic reperfusion injury. We found that CEBPD was upregulated and accompanied with increased neurological deficit scores and infarct size, and decreased neuron in MCAO rats. The siRNA targeted CEBPD inhibited CEBPD expression in rats, and meanwhile lentivirus system was used to blocked CEBPD expression in primary neuron. CEBPD degeneration decreased neurological deficit scores, infarct size and brain water content of MCAO rats. Knockdown of CEBPD enhanced cell viability and reduced apoptosis as well as oxidative stress in vivo and in vitro. CEBPD silencing promoted the translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) to the nucleus and the expression of heme oxygenase 1 (HO-1). Newly, CEBPD facilitated the transcription of cullin 3 (CUL3), which intensified ischemic stroke through Nrf2/HO-1 pathway that was proposed by our team in the past. In conclusion, targeting CEBPD-CUL3-Nrf2/HO-1 axis may be contributed to cerebral ischemia therapy.

Therapeutic Effects of Intranasal Administration of Resveratrol on the Rat Model of Brain Ischemia

BackgroundResveratrol is a natural phenolic compound widely found in plants. Previous studies have suggested its neuroprotective role in cerebral ischemia due to its anti-oxidative, anti-inflammatory, and anti-apoptotic effects. Intranasal administration of resveratrol enhances its capacity to penetrate the blood-brain barrier, increasing therapeutic efficacy and safety. ObjectiveWe aimed to examine the therapeutic potential of intranasal administration of resveratrol treatment in rats exposed to cerebral ischemia. MethodsSixty-four male rats were divided into three groups: the sham group, which was exposed to only surgical stress; the vehicle and resveratrol groups, which received intranasal vehicle or 50 mg/kg resveratrol for 7 days following middle cerebral artery occlusion, respectively. We assessed the modified neurologic severity scores, wire hanging tests, blood-brain barrier disruption, brain water content, and infarct volume. Levels of matrix metalloproteinase-9, nuclear factor-kappa B, B-cell lymphoma protein 2, and B-cell lymphoma protein 2-associated X messenger RNA expression were examined. ResultsAt 3- and 7-days post-ischemia, rats receiving intranasal resveratrol had lower modified neurological severity scores and a smaller brain infarct volume than the rats receiving vehicle. Additionally, the intranasal resveratrol-treated rats showed significantly prolonged wire-hanging performance at the 7-day mark post-ischemia compared to the vehicle group. The blood-brain barrier disruption and brain water content were significantly lower in the resveratrol group than in the vehicle group. Furthermore, the resveratrol-treated group displayed lower expression of Matrix Metalloproteinase-9 and Nuclear Factor-Kappa B in contrast to the vehicle group, while the difference in expression levels of B-cell lymphoma protein 2-associated X and B-cell lymphoma protein 2 were not significant. ConclusionIntranasal administration of resveratrol showed neuroprotective effects on ischemic stroke by improving neurobehavioral function, reducing blood-brain barrier disruption, cerebral edema, and infarct volume. This treatment also downregulated Matrix Metalloproteinase-9 and Nuclear Factor-Kappa B expression, indicating its potential as a therapeutic option for ischemic stroke.