Neuroprotection of rhubarb extract against cerebral ischaemia-reperfusion injury via the gut-brain axis pathway.

BACKGROUND: The gut-brain axis (GBA) plays a central role in cerebral ischaemia-reperfusion injury (CIRI). Rhubarb, known for its purgative properties, has demonstrated protective effects against CIRI. However, it remains unclear whether this protective effect is achieved through the regulation of the GBA. AIM: This study aims to investigate the mechanism by which rhubarb extract improves CIRI by modulating the GBA pathway. METHODS: We identified the active components of rhubarb extract using LC-MS/MS. The model of middle cerebral artery occlusion (MCAO) was established to evaluate the effect of rhubarb extract. We conducted 16S rDNA sequencing and untargeted metabolomics to analyze intestinal contents. Additionally, we employed HE staining, TUNEL staining, western blot, and ELISA to assess intestinal barrier integrity. We measured the levels of inflammatory cytokines in serum via ELISA. We also examined blood-brain barrier (BBB) integrity using Evans blue (EB) penetration, transmission electron microscopy (TEM), western blot, and ELISA. Neurological function scores and TTC staining were utilized to evaluate neurological outcomes. RESULTS: We identified twenty-six active components in rhubarb. Rhubarb extract enhanced α-diversity, reduced the abundance of Enterobacteriaceae, and partially rectified metabolic disorders in CIRI rats. It also ameliorated pathological changes, increased the expressions of ZO-1, Occludin, and Claudin 1 in the colon, and reduced levels of LPS and d-lac in serum. Furthermore, it lowered the levels of IL-1ß, IL-6, IL-10, IL-17, and TNF-α in serum. Rhubarb extract mitigated BBB dysfunction, as evidenced by reduced EB penetration and improved hippocampal microstructure. It upregulated the expressions of ZO-1, Occludin, Claudin 1, while downregulating the expressions of TLR4, MyD88, and NF-κB. Similarly, rhubarb extract decreased the levels of IL-1ß, IL-6, and TNF-α in the hippocampus. Ultimately, it reduced neurological function scores and cerebral infarct volume. CONCLUSION: Rhubarb effectively treats CIRI, potentially by inhibiting harmful bacteria, correcting metabolic disorders, repairing intestinal barrier function, alleviating BBB dysfunction, and ultimately improving neurological outcomes.

Experimental Study: The Effect Of 20% Intralipid Emulsion Therapy In Cerebral Ischemic Reperfusion.

BACKGROUND: This experimental study was conducted to investigate the effect of 20% Intralipid Emulsion (ILE) treatment on Cerebral Ischemia Reperfusion Injury (CIRI) after reperfusion in acute ischemic stroke. METHODS: In this experimental study, seven rats without any intervention (control group), seven rats (sham group) for which CIRI was created after the common carotid artery was ligated for 2 hours, and seven rats who were treated with 20% ILE after CIRI (CIRI + ILE group) were sacrificed after 24 hours, and histopathological findings were investigated. RESULTS: In rats that were not treated after CIRI, 52.7% had level-1, 32.7% had level-2. and 14.5% had level-3. histopathological findings. While 72.2% of the rats treated with ILE had level-1 and 27.8% had level-2 findings, no level-3 histopathological findings were detected in any of the rats. While no signs of coagulative necrosis, spongiosis of surrounding tissue and polymorphonuclear leukocytes were observed histopathological in any of the rats given ILE, there was no macrophages finding in 85.6% of the rats. ILE treatment also reduced the histopathological findings of eosinophilic neurons, astrogliosis, neovascularization, vascular thrombosis and mononuclear inflammatory cells. CONCLUSION: This study showed that 20% ILE treatment reduces the histopathological damage seen in cerebral ischemia and CIRI.

Synergistic effects and molecular mechanisms of DL-3-n-butylphthalide combined with dual antiplatelet therapy in acute ischemic stroke.

DL-3-n-butylphthalide (NBP) is isolated from the seeds of Apium graveolens L., and has been recently used as a neuroprotective agent for acute ischemic stroke. The present study aimed to determine the efficacy and safety of the combined use of dual antiplatelet therapy (DAPT) and NBP for treating of acute ischemic stroke in rats and to explore the synergistic mechanism of this treatment strategy in rat middle cerebral artery occlusion models. The efficacy of DAPT combined with NBP was evaluated by determining neurological deficits, infarction status, and histological changes. Changes in body weight, blood glucose level, blood count, and serum biochemical parameters were detected to evaluate the safety. To explore the synergistic pharmacological mechanism, the mRNA expression and protein levels of key proteins in the pyroptosis-inflammatory pathway, and the pyroptosis ratio of microglias were examined. Compared with the administration of NBP or DAPT alone, combination of them significantly improved neurological deficits, reduced infarct area, and repaired tissue injury and inflammation after cerebral ischemia. No hepatorenal toxicity was observed. The mRNA expression and protein levels of key proteins in the pyroptosis-inflammation pathway, and the pyroptosis ratio of microglias were significantly downregulated in the combined administration group than in the monotherapy group. We demonstrated that the combined use of NBP and DAPT exhibits better efficacy and high safety and plays a synergistic role by inhibiting the pyroptosis-inflammation pathway in the brain tissues, particularly in microglial cells.

Salvianolic acid A provides neuroprotective effects on cerebral ischemia-reperfusion injury in rats via PKA/CREB/c-Fos signaling pathway.

BACKGROUND: Cerebral ischemia-reperfusion injury (CIRI) is a phenomenon that pathological injury of ischemic brain tissue is further aggravated after the restoration of blood supply. The complex pathological mechanism of CIRI has led to the failure of multiple neuroprotective agents in clinical studies. Salvianolic acid A (SAA) is a neuroprotective extract from Salvia miltiorrhiza Bge., with significant pharmacological activities in the treatment of brain injury. However, the neuroprotective mechanisms of SAA remain unclear. PURPOSE: To explore the potential protective effect of SAA on CIRI and its mechanism, and to provide experimental basis for the research of new drugs for CIRI. STUDY DESIGN: A model of transient middle cerebral artery occlusion (tMCAO) in rats was used to simulate clinical CIRI, and the neuroprotective effect of SAA on tMCAO rats was investigated within 14 days after reperfusion. The improvement effects of SAA on cognitive impairment of tMCAO rats were investigated by behavioral tests from days 7-14. Finally, the neuroprotective mechanism of SAA was investigated on day 14. METHODS: The neuroprotective effects and mechanism of SAA were investigated by behavioral tests, HE and TUNEL staining, RNA sequence (RNA-seq) analysis and Western blot in tMCAO rats. RESULTS: The brain protective effects of SAA were achieved by alleviating cerebral infarction, cerebral edema, cerebral atrophy and nerve injury in tMCAO rats. Meanwhile, SAA could effectively improve the cognitive impairment and pathological damage of hippocampal tissue, and inhibit cell apoptosis in tMCAO rats. Besides, SAA could provide neuroprotective effects by up-regulating the expression of Bcl-2, inhibiting the activation of Caspase 3, and regulating PKA/CREB/c-Fos signaling pathway. CONCLUSION: SAA can significantly improve brain injury and cognitive impairment in CIRI rats, and this neuroprotective effect may be achieved through the anti-apoptotic effect and the regulation of PKA/CREB/c-Fos signaling pathway.

Nuciferine reduces inflammation induced by cerebral ischemia-reperfusion injury through the PI3K/Akt/NF-κB pathway.

BACKGROUND: Cerebral ischemia has the characteristics of high incidence, mortality, and disability, which seriously damages people's health. Cerebral ischemia-reperfusion injury is the key pathological injury of this disease. However, there is a lack of drugs that can reduce cerebral ischemia-reperfusion injury in clinical practice. At present, a few studies have provided some evidence that nuciferine can reduce cerebral ischemia-reperfusion injury, but its specific mechanism of action is still unclear, and further research is still needed. OBJECTIVE: In this study, PC12 cells and SD rats were used to construct OGD/R and MCAO/R models, respectively. Combined with bioinformatics methods and experimental verification methods, the purpose of this study was to conduct a systematic and comprehensive study on the effect and mechanism of nuciferine on reducing inflammation induced by cerebral ischemia-reperfusion injury. RESULTS: Nuciferine can improve the cell viability of PC12 cells induced by OGD/R, reduce apoptosis, and reduce the expression of inflammation-related proteins; it can also improve the cognitive and motor dysfunction of MCAO/R-induced rats by behavioral tests, reduce the area of cerebral infarction, reduce the release of inflammatory factors TNF-α and IL-6 in serum and the expression of inflammation-related proteins in brain tissue. CONCLUSION: Nuciferine can reduce the inflammatory level of cerebral ischemia-reperfusion injury in vivo and in vitro models by acting on the PI3K/Akt/NF-κB signaling pathway, and has the potential to be developed as a drug for the treatment of cerebral ischemia-reperfusion injury.

Transcutaneous electrical acupoint stimulation attenuated neuroinflammation and oxidative stress by activating SIRT1-induced signaling pathway in MCAO/R rat models.

BACKGROUND: Silent information regulator 1 (SIRT1) plays a beneficial role in cerebral ischemic injury. Previous reports have demonstrated that transcutaneous electrical acupoint stimulation (TEAS) exerts a beneficial effect on ischemic stroke; however, whether SIRT1 participates in the underlying mechanism for the neuroprotective effects of TEAS against ischemic brain damage has not been confirmed. METHODS: The rat models of middle cerebral artery occlusion/reperfusion (MCAO/R) were utilized in the current experiment. After MCAO/R surgery, rats in TEAS, EC and EX group received TEAS intervention with or without the injection of EX527, the SIRT1 inhibitor. Neurological deficit scores, infarct volume, hematoxylin eosin (HE) staining and apoptotic cell number were measured. The results of RNA sequencing were analyzed to determine the differential expression changes of genes among sham, MCAO and TEAS groups, in order to investigate the possible pathological processes involved in cerebral ischemia and explore the protective mechanisms of TEAS. Moreover, oxidative stress markers including MDA, SOD, GSH and GSH-Px were measured with assay kits. The levels of the proinflammatory cytokines, such as IL-6, IL-1ß and TNF-α, were detected by ELISA assay, and Iba-1 (the microglia marker protein) positive cells was measured by immunofluorescence (IF). Western blot and IF were utilized to examine the levels of key molecules in SIRT1/FOXO3a and SIRT1/BRCC3/NLRP3 signaling pathways. RESULTS: TEAS significantly decreased brain infarcted size and apoptotic neuronal number, and alleviated neurological deficit scores and morphological injury by activating SIRT1. The results of RNA-seq and bioinformatic analysis revealed that oxidative stress and inflammation were the key pathological mechanisms, and TEAS alleviated oxidative injury and inflammatory reactions following ischemic stroke. Then, further investigation indicated that TEAS notably attenuated neuronal apoptosis, neuroinflammation and oxidative stress damage in the hippocampus of rats with MCAO/R surgery. Moreover, TEAS intervention in the MCAO/R model significantly elevated the expressions of SIRT1, FOXO3a, CAT, BRCC3, NLRP3 in the hippocampus. Furthermore, EX527, as the inhibitor of SIRT1, obviously abolished the anti-oxidative stress and anti-neuroinflammatory roles of TEAS, as well as reversed the TEAS-mediated elevation of SIRT1, FOXO3a, CAT and reduction of BRCC3 and NLRP3 mediated by following MCAO/R surgery. CONCLUSIONS: In summary, these findings clearly suggested that TEAS attenuated brain damage by suppressing apoptosis, oxidative stress and neuroinflammation through modulating SIRT1/FOXO3a and SIRT1/BRCC3/NLRP3 signaling pathways following ischemic stroke, which can be a promising treatment for stroke patients.

Neurovascular Correlates of Cobalamin, Folate, and Homocysteine in Dementia.

BACKGROUND: Cobalamin (Cbl) and folate are common supplements clinicians prescribe as an adjuvant therapy for dementia patients, on the presumption of their neurotrophic and/or homocysteine (Hcy) lowering effect. However, the treatment efficacy has been found mixed and the effects of Cbl/folate/Hcy on the human brain remain to be elucidated. OBJECTIVE: To explore the neurovascular correlates of Cbl/folate/Hcy in Alzheimer's disease (AD) and subcortical ischemic vascular dementia (SIVD). METHODS: Sixty-seven AD patients and 57 SIVD patients were prospectively and consecutively recruited from an outpatient clinic. Multimodal 3-Tesla magnetic resonance imaging was performed to quantitatively evaluate cerebral blood flow (CBF) and white matter integrity. The relationship between neuroimaging metrics and the serum levels of Cbl/folate/Hcy was examined by using the Kruskal-Wallis test, partial correlation analysis, and moderation analysis, at a significance level of 0.05. RESULTS: As a whole, CBF mainly associated with Cbl/folate while white matter hyperintensities exclusively associated with Hcy. As compared with AD, SIVD exhibited more noticeable CBF correlates (spatially widespread with Cbl and focal with folate). In SIVD, a bilateral Cbl-moderated CBF coupling was found between medial prefrontal cortex and ipsilateral basal ganglia, while in the fronto-subcortical white matter tracts, elevated Hcy was associated with imaging metrics indicative of increased injury in both axon and myelin sheath. CONCLUSIONS: We identified the neurovascular correlates of previously reported neurotrophic effect of Cbl/folate and neurotoxic effect of Hcy in dementia. The correlates exhibited distinct patterns in AD and SIVD. The findings may help improving the formulation of supplemental Cbl/folate treatment for dementia.

Active fraction of Polyrhachis vicina (Roger) alleviated cerebral ischemia/reperfusion injury by targeting SIRT3-mediated mitophagy and angiogenesis.

BACKGROUND: Damaged mitophagy and impaired angiogenesis involve in the pathogenic development of ischemic stroke. Active fraction of Polyrhachis vicina (Roger) (AFPR) showed great potential on neurological disease with it's remarkable anti-inflammatory and anti-oxidative effects. PURPOSE: This study designed to clarify the correlation between Pink1/Parkin-mediated mitophagy and angiogenesis after stroke, and to elucidate the role of SIRT3 in regulating mitophagy and angiogenesis, and to address the mechanism of AFPR on promoting mitophagy and angiogenesis in microvessels endothelium of ischemic brain. STUDY DESIGN: A cerebral ischemia/reperfusion (CIR) rat model was developed by middle cerebral artery occlusion procedure. bEnd.3 cells were exposed to oxygen-glucose deprivation/reoxygenation (OGD/R) to mimic CIR process. Neurological function, mitophagy and angiogenesis related indicators were measured. SIRT3 siRNA and 3-MA were used to verify the interaction between SIRT3-mediated mitophagy and angiogenesis. METHODS: CIR rats were orally treated with AFPR (8 and 4 g raw drug /kg) and Nimodipine (10.8 mg/kg) for 12 days to mimic the recovery phase post-stroke. The neurological function assessment, TTC staining, HE staining, TUNEL staining and Nissl staining were performed to assess neuroprotective effects of AFPR against CIR. Then CD31-labeled microvessel density in brain was visualized and quantified by immunofluorescence staining. Mitochondrial ultrastructure was assessed by transmission electron microscope scanning. Expressions of relative proteins,e.g. SIRT3, Pink1, Parkin, LC3-II, p62, VEGFA, involving in mitophagy and angiogenesis, were detected by Western blotting analysis. In vitro, bEnd.3 cells were cultured with AFPR or in combination of autophagy inhibitor 3-MA during the reoxygenation. Then cell viability, and LDH releasing were measured. Angiogenic indicators,such as migration and tube formation activity, VEGFA level were determined. To assess effects of AFPR on mitophagy, mitophagy-related proteins were detected, as well as the autophagosome engulfment and lysosome degradation of mitochondria. To address the role of SIRT3, deacetylation activity of SIRT3 was validated by detecting acetylated FOXO3A level with co-immunoprecipitation (Co-IP) assay. Pre-treatment of siRNA or combination use of 3-MA were used to verify the detailed mechanism. RESULTS: AFPR remarkably reduced neurological scores and infarct size, alleviated neuron apoptosis in cortex, and increased Nissl density in hippocampus of CIR rats. In addition, AFPR significantly promoted angiogenesis by increasing microvessels density and VEGFA expressions, increased SIRT3 expression, and activated Pink1/Parkin mediated mitophagy. In bEnd.3 cells, the combination use of 3-MA and AFPR further demonstrated that AFPR might promote angiogenesis after OGD/R injury through activating Pink1/Parkin mediated mitophagy. Co-IP assay suggested AFPR reduced acetylated FOXO3A level. This might be correlated with an elevation of SIRT3 expression and it's deacetylation activity. SIRT3 siRNA pretreatment significantly abolished the activation of mitophagy through Pink1/Parkin axis, eventually inhibited angiogenesis. CONCLUSION: AFPR promoted angiogenesis through activating mitophagy after cerebral ischemia reperfusion, which might partially involved in the amelioration of SIRT3-mediated regulation on Pink1/Parkin axis. Our study will shed new light on the role of SIRT3 in ischemic brain, especially in regulating mitophagy and angiogenesis after stroke.

Neuroprotective effect of Potentilla reptans L. root in the rat brain global ischemia/reperfusion model.

Stroke is the most common cause of death among neurological diseases. The protective effects of Potentilla reptans L. include antioxidative, anti-inflammatory, and antiapoptotic effects. In this study, the brain protection and beta-amyloid effects of P. reptans root extract were investigated in the rat brain ischemia/reperfusion (IR) model. Forty male Wistar rats were randomly divided into five groups (n = 8), including IR, sham, and three groups receiving P. reptans with concentrations of 0.025, 0.05, and 0.1 (g/kg/b.w.), which were injected daily for 7 days. For the IR model, the common carotid artery was occluded bilaterally for 8 min. All injections were intraperitoneal (IP). The shuttle box test was used to measure passive avoidance memory. Then the brain tissue was extracted for the histological examination of neuron counts and ß-amyloid plaques using a morphometric technique, and finally, Statistical Package for the Social Sciences software was used for statistical analysis of the data. Pretreatment with P. reptans improved memory impairment. Also, by examining the tissues of the CA1, CA3, and dentate gyrus areas of the hippocampus, it was observed that the number of plaques in the groups receiving P. reptans extract was reduced compared to the IR group, especially at the concentration of 0.05 g/kg/b.w. Also, P. reptans improved the number of neurons at all concentrations, in which the concentration of 0.05 g/kg/b.w. showed more effective therapeutic results. Taken together, we found that P. reptans root extract has beneficial effects on memory impairment, neuronal loss, and ß-amyloid accumulation.

Protective effect of Xingnaojing injection on ferroptosis after cerebral ischemia injury in MCAO rats and SH-SY5Y cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Xingnaojing（XNJ）injection is a traditional Chinese medicine injection with neuroprotective effect, which has been widely used in the treatment of stroke for many years. AIM OF THE STUDY: This study aimed to explore the potential mechanism of XNJ in cerebral ischemia mediated by ferroptosis using proteomics and in vivo and in vitro experiments. MATERIALS AND METHODS: After the rat model of middle cerebral artery occlusion (MCAO) was successfully established, they were randomly divided into model, XNJ, and deferoxamine (DFO) group. Triphenyl tetrazolium chloride (TTC) staining, Hematoxylin and eosin (H&E), and Nissl staining were used to observe the infarct area, pathological changes and the degree of neuronal apoptosis of rat brain. Proteins extracted from rat brain tissues were analyzed by quantitative proteomics using tandem mass tags (TMT). Western blotting and immunohistochemical assessment were used to measure the expression of ferroptosis-related proteins. In vitro, the SH-SY5Y cells were subjected to hypoxia (37°C/5% CO2/1% O2) for 24 h to observe the survival rate, and detect the reactive oxygen species (ROS) content and ferroptosis-related proteins. RESULTS: In TTC and H&E experiments, we found that XNJ drug treatment reduced the infarct volume and brain tissue damage in MCAO rats. Nissl staining also showed that compared with MCAO group rats, the Nissl bodies of brain tissue after XNJ drug intervention were clear with a 3.54-fold increased times, suggesting that XNJ improved cerebral infraction, and neurological deficits in MCAO rats. Proteomics identified 101 intersected differentially expressed proteins (DEPs). According to the bioinformatics analysis, these DEPs were closely related to ferroptosis. Further research indicated that MCAO-induced cerebral ischemia was alleviated by upregulating recombinant glutathione peroxidase 4 (GPX4), ferroportin (FPN) expression, Heme oxygenase-1 (HO-1) expression, and downregulating cyclooxygenase-2 (COX-2), transferring receptor (TFR) and divalent metal transporter-1 (DMT1) expression after XNJ treatment. In addition, in vitro experiment indicated that XNJ improved the survival rate of hypoxia-damaged SH-SY5Y cells. XNJ increased the level of GPX4 and inhibited the protein expression of COX-2 and TFR after cell hypoxia. Moreover, different concentrations of XNJ (0.25%, 0.5%, 1%) reduced the ROS content of hypoxic cells, suggesting that XNJ could inhibit hypoxia-induced cell damage by regulating the expression of ferroptosis-related proteins and decreasing the production of ROS. CONCLUSIONS: XNJ could promote the recovery of neurological function in MCAO rats and hypoxia SH-SY5Y cells by regulating ferroptosis.

[Transient changes in food preference in a patient with cerebellar infarction].

A 44-year-old woman was admitted to our hospital due to dizziness and ataxia of the trunk and right upper limb. Brain MRI revealed an acute infarct lesion in the right posterior inferior cerebellar artery territory. In addition to the cognitive deterioration observed in the subacute phase, a change was noted in her food preference-from light-tasting, low-caloric Japanese cuisine, sugarless coffee, and hot drinks to strong-tasting, high-caloric Western cuisine, sugar-rich coffee, and iced drinks. Single-photon emission computed tomography showed hypoperfusion in the bilateral frontal lobes and right cerebellum. These cognitive and food preference-related changes were gradually restored over six months after the onset. The reduced cerebral blood flow in the bilateral frontal lobes also restored along with the clinical improvement, with the maximal changes in the bilateral subcallosal areas. This case suggests that changes in food preference can occur as a symptom of cerebellar infarction, possibly by the mechanism similar to cerebellar cognitive affective syndrome.

Scutellarin acts on the AR-NOX axis to remediate oxidative stress injury in a mouse model of cerebral ischemia/reperfusion injury.

BACKGROUND: Oxidative stress plays an important role in the pathology of ischemic stroke. Studies have confirmedthat scutellarin has antioxidant effects against ischemic injury, and we also reported that the involvement of Aldose reductase (AR) in oxidative stress and cerebral ischemic injury, in this study we furtherly explicit whether the antioxidant effect of scutellarin on cerebral ischemia injury is related to AR gene regulation and its specific mechanism. METHODS: C57BL/6N mice (Wild-type, WT) and AR knockout (AR-/-) mice suffered from transient middle cerebral artery occlusion (tMCAO) injury (1 h occlusion followed by 3 days reperfusion), and scutellarin was administered from 2 h before surgery to 3 days after surgery. Subsequently, neurological function was assessed by the modified Longa score method, the histopathological morphology observed with 2,3,5-triphenyltetrazolium chloride (TTC) and hematoxylin-eosin (HE) staining. Enzyme-linked immunosorbent assay (Elisa) was used to detect the levels of ROS, 4-hydroxynonenal (4-HNE), 8-hydroxydeoxyguanosine (8-OHDG), Neurotrophin-3 (NT-3), poly ADP-ribose polymerase-1 (PARP1) and 3-nitrotyrosine (3-NT) in the ischemic penumbra regions. Quantitative proteomics profiling using quantitative nano-HPLC-MS/MS were performed to compare the protein expression difference between AR-/- and WT mice with or without tMCAO injury. The expression of AR, nicotinamide adenine dinucleotide phosphate oxidases (NOX1, NOX2 and NOX4) in the ipsilateral side of ischemic brain were detected by qRT-PCR, Western blot and immunofluorescence co-staining with NeuN. RESULTS: Scutellarin treatment alleviated brain damage in tMCAO stroke model such as improved neurological function deficit, brain infarct area and neuronal injury and reduced the expression of oxidation-related products, moreover, also down-regulated tMCAO induced AR mRNA and protein expression. In addition, the therapeutic effect of scutellarin on the reduction of cerebral infarction area and neurological function deficits abolished in AR-/- mice under ischemia cerebral injury, which indicated that the effect of scutellarin treatment on tMCAO injury is through regulating AR gene. Proteomic analysis of AR-/- and WT mice indicated AR knockout would affect oxidation reaction even as NADPH related process and activity in mice under cerebral ischemia conditions. Moreover, NOX isoforms (NOX1, NOX2 and NOX4) mRNA and protein expression were significant decreased in neurons of penumbra region in AR-/- mice compared with that in WT mice at 3d after tMCAO injury, which indicated that AR should be the upstream protein regulating NOX after cerebral ischemia. CONCLUSIONS: We first reported that AR directly regulates NOX subtypes (not only NOX2 but also NOX1 and NOX4) after cerebral ischaemic injury. Scutellarin specifically targets the AR-NOX axis and has antioxidant effects in mice with cerebral ischaemic injury, providing a theoretical basis and accurate molecular targets for the clinical application of scutellarin.

Berberine protects against chronic cerebral hypoperfusion-induced cognitive impairment and hippocampal damage via regulation of the ERK/Nrf2 pathway.

Vascular cognitive impairment caused by chronic cerebral hypoperfusion (CCH) seriously affects the quality of life of elderly patients and places a great burden on society and family. With the development of traditional Chinese medicine (TCM), TCM approaches to the prevention and treatment of senile ischemic cerebrovascular disease has received increasing attention. In this study, rats with bilateral common carotid artery occlusion (BCCAO) were treated with berberine (BBR). Their learning and memory function, neuronal injury and repair, the extracellular regulatory protein kinase (ERK)/nuclear factor-E2-related factor 2 (Nrf2) signaling pathway, and impairment and improvement of the blood-brain barrier (BBB) were evaluated. This study found that BBR can alleviate the pathological injury to the brain, reduce neuronal loss and promote neuronal cell survival after CCH by interfering with the ERK/Nrf2 signaling pathway. BBR can reduce BBB injury in CCH rats by inhibiting the expression of VEGF-A and MMP-9 in plasma, which reveals a protective effect of BBR on vascular cognitive impairment. This study provides a new research direction for BBR in the treatment of ischemic cerebrovascular disease.

Hydroxysafflor Yellow A Alleviates Ischemic Stroke in Rats via HIF-1[Formula: see text], BNIP3, and Notch1-Mediated Inhibition of Autophagy.

Stroke has become a major cause of death and disability worldwide. The cellular recycling pathway autophagy has been implicated in ischemia-induced neuronal changes, but whether autophagy plays a beneficial or detrimental role is controversial. Hydroxysafflor Yellow A (HSYA), a popular herbal medicine, is an extract of Carthamus tinctorius and is used to treat ischemic stroke (IS) in China. HSYA has been shown to prevent cardiovascular and cerebral ischemia/reperfusion injury in animal models. However, the specific active ingredients and molecular mechanisms of HSYA in IS remain unclear. Here, we investigated the effect of HSYA treatment on autophagy in a rat model of IS. IS was induced in rats by middle cerebral artery occlusion. Rats were treated once daily for 3 days with saline, HYSA, or the neuroprotective agent Edaravone. Neurobehavioral testing was performed on days 1, 2, and 3 post-surgery. Brains were removed on day 3 post-surgery for histological evaluation of infarct area, morphology, and for qRT-PCR and western blot analysis of the expression of the autophagy factor LC3 and the signaling molecules HIF-1[Formula: see text], BNIP3, and Notch1. Molecular docking studies were performed in silico to predict potential interactions between HSYA and LC3, HIF-1[Formula: see text], BNIP3, and Notch1 proteins. The result showed that HSYA treatment markedly alleviated IS-induced neurobehavioral deficits and reduced brain infarct area and tissue damage. HSYA also significantly reduced hippocampal expression levels of LC3, HIF-1[Formula: see text], BNIP3, and Notch1. The beneficial effect of HSYA was generally superior to that of Edaravone. Molecular modeling suggested that HSYA may bind strongly to HIF-1[Formula: see text], BNIP3, and Notch1 but weakly to LC3. In conclusion, HSYA inhibits post-IS autophagy induction in the brain, possibly by suppressing HIF-1[Formula: see text], BNIP3 and Notch1. HSYA may have utility as a post-IS neuroprotective agent.

Do Concentration or Activity of Selenoproteins Change in Acute Stroke Patients? A Systematic Review and Meta-Analyses.

INTRODUCTION: Stroke is characterized by deleterious oxidative stress. Selenoprotein enzymes are essential endogenous antioxidants, and detailed insight into their role after stroke could define new therapeutic treatments. This systematic review aimed to elucidate how blood selenoprotein concentration and activity change in the acute phase of stroke. METHODS: We searched PubMed, EMBASE, and Medline databases for studies measuring serial blood selenoprotein concentration or activity in acute stroke patients or in stroke patients compared to non-stroke controls. Meta-analyses of studies stratified by the type of stroke, blood compartment, and type of selenoprotein measurement were conducted. RESULTS: Eighteen studies and data from 941 stroke patients and 708 non-stroke controls were included in this review. Glutathione peroxidase (GPx) was the only identified selenoprotein, and its activity was most frequently measured. Results from 12 studies and 693 patients showed that compared to non-stroke controls in acute ischaemic stroke patients, the GPx activity increased in haemolysate (standardized mean difference [SMD]: 0.27, 95% CI: 0.07-0.47) but decreased in plasma (mean difference [MD]: -1.08 U/L, 95% CI: -1.94 to -0.22) and serum (SMD: -0.54, 95% CI: -0.91 to -0.17). From 4 identified studies in 106 acute haemorrhagic stroke patients, the GPx activity decreased in haemolysate (SMD: -0.40, 95% CI: -0.68 to -0.13) and remained unchanged in plasma (MD: -0.10 U/L, 95% CI: -0.81 to 0.61) and serum (MD: -5.00 U/mL, 95% CI: -36.17 to 26.17) compared to non-stroke controls. Results from studies assessing the GPx activity in the haemolysate compartment were inconsistent and characterized by high heterogeneity. CONCLUSIONS: Our results suggest a reduction of the blood GPx activity in acute ischaemic stroke patients, a lack of evidence regarding a role for GPx in haemorrhagic stroke patients, and insufficient evidence for other selenoproteins.

Salvianolic acid A relieves cognitive disorder after chronic cerebral ischemia: Involvement of Drd2/Cryab/NF-κB pathway.

Chronic cerebral ischemia (CCI) refers to long-term hypoperfusion of cerebral blood flow with the main clinical manifestations of progressive cognitive impairment. The pathological mechanism of CCI is complex, and there is a lack of effective treatments. Salvianolic acid A (SalA) is a neuroprotective extract of Salvia miltiorrhiza with the effects of anti-inflammation and anti-apoptosis. In this study, the effect of SalA on cognitive function and Drd2/Cryab/NF-κB signaling pathway in rats with CCI was investigated. Morris water maze and open field test were used to observe the effects of SalA on the cognitive function of CCI rats. The pathological changes in the brain were observed by HE, Nissl, and LFB staining. TUNEL staining, enzyme-linked immunosorbent assay, and western blot analysis were used to detect the inflammatory and apoptosis in the cortex and hippocampus. The expression of Drd2/Cryab/NF-κB pathway-related molecules and Drd2 localization were detected by western blotting and dual immunofluorescence, respectively. SH-SY5Y cells were exposed to chronic hypoglycemic and hypoxic injury in vitro, and Drd2 inhibitor haloperidol was used to verify the involved pathway. The results showed that SalA could improve the cognitive function of CCI rats, reduce pathological damage of cortex and hippocampus, inhibit neuroinflammation and apoptosis, and suppress the activation of NF-κB by regulating Drd2/Cryab pathway. And SalA inhibited NF-κB activation and nuclear translocation in SH-SY5Y cells by upregulating Drd2/Cryab pathway, which was reversed by haloperidol interference. In conclusion, SalA could relieve CCI-induced cognitive impairment in rats, at least partly through the Drd2/Cryab/NF-κB pathway.

Electroacupuncture Promotes the Survival of the Grafted Human MGE Neural Progenitors in Rats with Cerebral Ischemia by Promoting Angiogenesis and Inhibiting Inflammation.

Stem cells have the potential as a regenerative therapy for cerebral ischemia by improving functional outcomes. However, cell transplantation has some limitations, including a low rate of the grafted cell survival. There is still a major challenge of promoting the harmonious symbiosis between grafted cells and the host. Acupuncture can effectively improve the functional outcome after cerebral ischemia. The present study evaluated the therapeutic effects and explored the mechanism of combined medial ganglionic eminence (MGE) neural progenitors differentiated from human embryonic stem cells (hESCs) with electroacupuncture (EA) in a bilateral common carotid artery occlusion (2VO) rat model. The results showed that EA could promote the survival of the grafted MGE neural progenitors differentiated from hESCs and alleviate learning and memory impairment in rats with cerebral ischemia. This may have partially resulted from inhibited expression of TNF-α and IL-1ß and increased vascular endothelial growth factor (VEGF) expression and blood vessel density in the hippocampus. Our findings indicated that EA could promote the survival of the grafted MGE neural progenitors and enhance transplantation therapy's efficacy by promoting angiogenesis and inhibiting inflammation.

Sex-Specific Differences in Autophagic Responses to Experimental Ischemic Stroke.

Ischemic stroke triggers a series of complex pathophysiological processes including autophagy. Differential activation of autophagy occurs in neurons derived from males versus females after stressors such as nutrient deprivation. Whether autophagy displays sexual dimorphism after ischemic stroke is unknown. We used a cerebral ischemia mouse model (middle cerebral artery occlusion, MCAO) to evaluate the effects of inhibiting autophagy in ischemic brain pathology. We observed that inhibiting autophagy reduced infarct volume in males and ovariectomized females. However, autophagy inhibition enhanced infarct size in females and in ovariectomized females supplemented with estrogen compared to control mice. We also observed that males had increased levels of Beclin1 and LC3 and decreased levels of pULK1 and p62 at 24 h, while females had decreased levels of Beclin1 and increased levels of ATG7. Furthermore, the levels of autophagy markers were increased under basal conditions and after oxygen and glucose deprivation in male neurons compared with female neurons in vitro. E2 supplementation significantly inhibited autophagy only in male neurons, and was beneficial for cell survival only in female neurons. This study shows that autophagy in the ischemic brain differs between the sexes, and that autophagy regulators have different effects in a sex-dependent manner in neurons.

Danhong injection enhances the therapeutic effect of mannitol on hemispheric ischemic stroke by ameliorating blood-brain barrier disruption.

Mannitol, a representative of hyperosmolar therapy, is indispensable for the treatment of malignant cerebral infarction, but its therapeutic effect is limited by its exacerbation of blood-brain barrier (BBB) disruption. This study was to explore whether Danhong injection (DHI), a standardized product extracted from Salvia miltiorrhiza Bunge and Carthamus tinctorius L., inhibits the destructive effect of mannitol on BBB and thus enhancing the treatment of hemispheric ischemic stroke. SD rats were subjected to pMCAO followed by intravenous bolus injections of mannitol with/without DHI intervention. Neurological deficit score, brain edema, infarct volume at 24 h after MCAO and histopathology, microvascular ultrastructure, immunohistochemistry and immunofluorescence staining of endothelial cell junctions, energy metabolism in the ischemic penumbra were assessed. Intravenous mannitol after MCAO resulted in a decrease in 24 h mortality and cerebral edema, whereas no significant benefit on neurological deficits, infarct volume and microvascular ultrastructure. Moreover, mannitol led to the loss of endothelial integrity, manifested by the decreased expression of occludin, junctional adhesion molecule-1 (JAM-1) and zonula occluden-1 (ZO-1) and the discontinuity of occludin staining around the periphery of endothelial cells. Meanwhile, after mannitol treatment, energy-dependent vimentin and F-actin, ATP content, and ATP5D expression were down-regulated, while MMP2 and MMP9 expression increased in the ischemic penumbra. All the insults after mannitol treatment were attenuated by addition of intravenous DHI. The results suggest DHI as a potential remedy to attenuate mannitol-related BBB disruption, and the potential of DHI to upregulate energy metabolism and inhibit the activity of MMPs is likely attributable to its effects observed.

Neuroprotective and Anti-Inflammatory Effects of Pinus densiflora Bark Extract in Gerbil Hippocampus Following Transient Forebrain Ischemia.

Korean red pine (Pinus densiflora) belongs to the Genus Pinus, and its bark contains a great amount of naturally occurring phenolic compounds. Until now, few studies have been conducted to assess the neuroprotective effects of Pinus densiflora bark extract against brain ischemic injury. The aim of this study was to investigate the neuroprotective effects of pre-treatment with the extract in the hippocampus following 5-min transient forebrain ischemia in gerbils. Furthermore, this study examined the anti-inflammatory effect as a neuroprotective mechanism of the extract. Pinus densiflora bark was extracted by pure water (100 °C), and this extract was quantitatively analyzed and contained abundant polyphenols, flavonoids, and proanthocyanidins. The extract (25, 50, and 100 mg/kg) was orally administered once a day for seven days before the ischemia. In the gerbil hippocampus, death of the pyramidal neurons was found in the subfield cornu ammonis 1 (CA1) five days after the ischemia. This death was significantly attenuated by pre-treatment with 100 mg/kg, not 25 or 50 mg/kg, of the extract. The treatment with 100 mg/kg of the extract markedly inhibited the activation of microglia (microgliosis) and significantly decreased the expression of pro-inflammatory cytokines (interleukin 1ß and tumor necrosis factor α). In addition, the treatment significantly increased anti-inflammatory cytokines (interleukin 4 and interleukin 13). Taken together, this study clearly indicates that pre-treatment with 100 mg/kg of Pinus densiflora bark extract in gerbils can exert neuroprotection against brain ischemic injury by the attenuation of neuroinflammatory responses.