OBJECTIVES: To investigate the neuroprotective effects of electroacupuncture (EA) preconditioning against cerebral ischemia-reperfusion injury (CIRI) mediated by gut microbiota modulation, Nrf2/HO-1 pathway activation, and ferroptosis suppression. METHODS: Adult male SD rats were divided into sham operation group, CIRI model group, and EA preconditioning group. In the latter two groups, rat models of CIRI were established by middle cerebral artery occlusion (MCAO), and in EA preconditioning group, EA was applied at Baihui (DU20) and Zusanli (ST36) for 3 days before modeling. Neurological deficits, cerebral infarction, and hippocampal pathology of the rats were evaluated using behavioral tests, TTC staining, and Nissl and HE staining, and the oxidative stress markers (MDA, ROS, and SOD), apoptosis/ferroptosis-related proteins (Bax, Bcl-2, GPX4, and SLC7A11), and changes in gut microbiota were analyzed. RESULTS: EA preconditioning significantly reduced neurological deficits, decreased infarct volume, promoted hippocampal neuronal survival, and improved structural integrity of the hippocampal neurons in MCAO rats. EA preconditioning also significantly lowered MDA and ROS and increased SOD levels, upregulated Bcl-2, GPX4, and SLC7A11 expressions, and downregulated Bax expression in the hippocampal tissue of the rats, causing also activation of Nrf2/HO-1 signaling and improvement of gut microbiota composition. CONCLUSIONS EA preconditioning alleviates CIRI in rats by suppressing ferroptosis and apoptosis, enhancing antioxidant defenses via activating Nrf2/HO-1 signaling, and regulating the gut-brain axis.
Animals ; Electroacupuncture ; NF-E2-Related Factor 2/metabolism* ; Rats, Sprague-Dawley ; Signal Transduction ; Reperfusion Injury/therapy* ; Ferroptosis ; Male ; Rats ; Brain Ischemia ; Gastrointestinal Microbiome ; Heme Oxygenase (Decyclizing)/metabolism* ; Brain/metabolism* ; Oxidative Stress ; Heme Oxygenase-1/metabolism* ; Apoptosis

OBJECTIVES: To evaluate the effect of eye acupuncture on neural function and angiogenesis of ischemic cerebral tissue in rats, and explore the roles of METTL3-mediated m⁶A methylation and the HIF-1α/VEGF-A signal axis in mediating this effect. METHODS: Fifty SD rats were randomized into normal control group, sham-operated group, model group, eye acupuncture group and DMOG (a HIF-1α agonist) group. Rat models of cerebral ischemia/reperfusion injury (CIRI) were established using a modified thread thrombus method, and the changes in neurological deficits of the rats after interventions were evaluated. TTC and Nissl staining were used to examine the changes in infarction size and neuronal injury, and cerebral angiogenesis was detected by double-immunofluorescence staining. m⁶A methylation modification level in the brain tissue was detected by ELISA, and RT-qPCR and Western blotting were used to detect the mRNA and protein expressions of METTL3 and HIF-1α/VEGF-A. RESULTS: Compared with the control and sham-operated rats, the CIRI rats had significantly higher neurological deficit scores with larger cerebral infarction area, a greater number of CD31- and EDU-positive new vessels, higher expression levels of HIF-1α and VEGF-A, reduced number of Nissl bodies and m6A methylation level, and lowered METTL3 protein and mRNA expressions. All these changes were significantly improved by interventions with eye acupuncture after modeling or intraperitoneal injections of DMOG for 7 consecutive days prior to modeling, and the effects of the two interventions were similar. CONCLUSIONS Eye acupuncture can improve neurological deficits in CIRI rat models possibly by promoting cortical angiogenesis via upregulating METTL3-mediated m⁶A methylation and regulating the HIF-1α/VEGF-A signal axis.
Animals ; Rats, Sprague-Dawley ; Methyltransferases/metabolism* ; Reperfusion Injury/physiopathology* ; Methylation ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism* ; Rats ; Vascular Endothelial Growth Factor A/metabolism* ; Brain Ischemia/metabolism* ; Acupuncture Therapy ; Male ; Up-Regulation ; Neovascularization, Physiologic ; Angiogenesis ; Adenosine/analogs & derivatives*

OBJECTIVES: To evaluate the therapeutic effect of gastrodin against hypoxic-ischemic brain damage (HIBD) in neonatal mice and explore the role of GPX4/SLC7A11/FTH1 signaling in mediating its effect. METHODS: Twenty-four 9- to 11-day-old C57BL/6J mice were randomized equally into 4 groups for sham operation, HIBD modeling by right common carotid artery ligation and subsequent exposure to hypoxia for 1 h, or gastrodin treatment at 100 or 200 mg/kg before and at 1 and 2 days after modeling. The mice then underwent neurological assessment (Zea-Longa scores), and the cerebral cortical penumbra tissue were collected for HE and Nissl staining, detection of ferroptosis biomarkers and protein expressions of GPX4, SLC7A11, and FTH1 with Western blotting and immunofluorescence co-localization, and observation of mitochondrial ultrastructure with electron microscopy. In cultured HT22 neuronal cells with oxygen-glucose deprivation (OGD) for 2 h, the effects of pretreatments with 0.5 mmol/L gastrodin, 10 μmol/L RSL3 (a GPX4 inhibitor), alone or in combination, were analyzed on expressions of ferroptosis-related proteins, cellular Fe²⁺, ROS, lipid peroxidation, MDA, and GSH levels, mitochondrial membrane potential (JC-1), and cell viability. RESULTS: Gastrodin treatment at the two doses both significantly ameliorated HIBD and neurological deficits of the mice, reduced mitochondrial damage and Fe²⁺, MDA and ROS levels, increased GSH level, and upregulated GPX4, SLC7A11, and FTH1 protein expressions. In HT22 cells, gastrodin pretreatment obviously attenuated OGD-induced ferroptosis and improved cell viability and mitochondrial function. Co-treatment with RSL3 potently abrogated the inhibitory effects of gastrodin on Fe²⁺, ROS, BODIPY-C11, and MDA levels and attenuated its protective effects on GSH level, cell viability, and mitochondrial membrane potential. CONCLUSIONS Gastrodin provides neuroprotective effects in neonatal mice with HIBD by suppressing neuronal ferroptosis via upregulating the GPX4/SLC7A11/FTH1 signaling pathway.
Animals ; Ferroptosis/drug effects* ; Hypoxia-Ischemia, Brain/drug therapy* ; Mice ; Mice, Inbred C57BL ; Signal Transduction/drug effects* ; Phospholipid Hydroperoxide Glutathione Peroxidase ; Glucosides/pharmacology* ; Animals, Newborn ; Benzyl Alcohols/pharmacology* ; Amino Acid Transport System y+/metabolism*

Hypoxic-ischemic (HI) brain damage poses a high risk of death or lifelong disability, yet effective treatments remain elusive. Here, we demonstrated that miR-100-5p levels in the lesioned cortex increased after HI insult in neonatal mice. Knockdown of miR-100-5p expression in the brain attenuated brain injury and promoted functional recovery, through inhibiting the cleaved-caspase-3 level, microglia activation, and the release of proinflammation cytokines following HI injury. Engineered extracellular vesicles (EVs) containing neuron-targeting rabies virus glycoprotein (RVG) and miR-100-5p antagonists (RVG-EVs-Antagomir) selectively targeted brain lesions and reduced miR-100-5p levels after intranasal delivery. Both pre- and post-HI administration showed therapeutic benefits. Mechanistically, we identified protein phosphatase 3 catalytic subunit alpha (Ppp3ca) as a novel candidate target gene of miR-100-5p, inhibiting c-Fos expression and neuronal apoptosis following HI insult. In conclusion, our non-invasive method using engineered EVs to deliver miR-100-5p antagomirs to the brain significantly improves functional recovery after HI injury by targeting Ppp3ca to suppress neuronal apoptosis.
Animals ; MicroRNAs/metabolism* ; Extracellular Vesicles/metabolism* ; Mice ; Recovery of Function/physiology* ; Hypoxia-Ischemia, Brain/therapy* ; Mice, Inbred C57BL ; Antagomirs/administration & dosage* ; Male ; Animals, Newborn ; Apoptosis/drug effects* ; Brain Injuries/metabolism* ; Glycoproteins ; Peptide Fragments ; Viral Proteins

OBJECTIVE: To observe the neuroprotective effect of 6-shogaol (6-SH) in global cerebral ischemia/reperfusion injury (CIRI) following cardiac arrest (CA) and cardiopulmonary resuscitation (CPR) in rats. METHODS: Computer-aided molecular docking was used to determine whether 6-SH could spontaneously bind to death-associated protein kinase 1 (DAPK1). SPF-grade male SD rats were randomly divided into a sham group (n = 5), a CPR group (n = 7), and a CPR+6-SH group (n = 7). The CPR group and CPR+6-SH group were further divided into 12-, 24-, and 48-hour subgroups based on observation time points. A rat model of global CIRI after CA-CPR was established by asphyxiation. In the sham group, only tracheal and vascular intubation was performed without asphyxia and CPR induction. The CPR group was intraperitoneally injected with 1 mL of normal saline immediately after successful modeling. The CPR+6-SH group received an intraperitoneal injection of 20 mg/kg 6-SH (1 mL) immediately after successful modeling, followed by administration every 12 hours until the endpoint. Neurological Deficit Score (NDS) was recorded at each time point after modeling. After completion of observation at each time point, rats were anesthetized and sacrificed, and brain tissue specimens were collected. Histopathological changes of neurons were observed under light microscopy after hematoxylin-eosin (HE) staining. Ultrastructural changes of hippocampal neurons and autophagy were observed by transmission electron microscopy (TEM). Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect mRNA expression levels of DAPK1, vacuolar protein sorting 34 (VPS34), Beclin1, and microtubule-associated protein 1 light chain 3 (LC3) in brain tissues. Western blotting was used to detect protein expression levels of DAPK1, phosphorylated DAPK1 at serine 308 (p-DAPK1 ser308), VPS34, Beclin1, and LC3. Immunofluorescence was used to observe Beclin1 and LC3 expression in brain tissues under a fluorescence microscope. RESULTS: Molecular docking results indicated that 6-SH could spontaneously bind to DAPK1. Compared with the sham group, the NDS scores of the CPR group rats were significantly increased at all modeling time points; under light microscopy, disordered cell arrangement, widened intercellular spaces, and edema were observed in brain tissues, with pyknotic and necrotic nuclei in some areas; under TEM, mitochondria were markedly swollen with intact membranes, dissolved matrix, reduced or disappeared cristae, vacuolization, and increased autophagosomes. Compared with the CPR group, the NDS scores of the CPR+6-SH group rats were significantly decreased at all modeling time points; under light microscopy, local neuronal edema and widened perinuclear space were observed; under TEM, mitochondria were mostly mildly swollen with intact membranes, fewer autophagosomes, and alleviated injury. RT-qPCR results showed that compared with the sham group, mRNA expression levels of DAPK1, VPS34, Beclin1, and LC3 in brain tissues were significantly upregulated in all CPR subgroups, with the most pronounced changes at 24 hours. Compared with the CPR group, the CPR+6-SH group showed significantly lower mRNA expression of the above indicators at each time point [24 hours post-modeling (relative expression): DAPK1 mRNA: 3.41±0.68 vs. 4.48±0.62; VPS34 mRNA: 3.63±0.49 vs. 4.66±1.18; Beclin1 mRNA: 3.08±0.49 vs. 4.04±0.22; LC3 mRNA: 2.60±0.36 vs. 3.67±0.62; all P < 0.05]. Western blotting results showed that compared with the sham group, the protein expression levels of DAPK1, VPS34, Beclin1, and LC3 in all CPR subgroups were significantly increased, while the expression of p-DAPK1 ser308 was significantly decreased, with the most pronounced changes observed in the CPR 24-hour subgroup. Compared with the CPR group, the CPR+6-SH subgroups exhibited significantly reduced protein expression of DAPK1, VPS34, Beclin1, and LC3 [24-hour post-modeling: DAPK1/β-actin: 1.88±0.22 vs. 2.47±0.22; VPS34/β-actin: 2.55±0.06 vs. 3.46±0.05; Beclin1/β-actin: 2.12±0.03 vs. 2.87±0.03; LC3/β-actin: 2.03±0.24 vs. 3.17±0.23; all P < 0.05]. Conversely, the expression of p-DAPK1 ser308 was significantly upregulated in the CPR+6-SH group compared to the CPR group [24-hour post-modeling: p-DAPK1 ser308/β-actin: 0.40±0.02 vs. 0.20±0.07, P < 0.05]. Under the fluorescence microscope, fluorescence intensities of Beclin1 and LC3 in the CPR 24-hour group were significantly higher than those in the sham 24-hour group; compared with the CPR 24-hour group, the CPR+6-SH 24-hour group showed significantly reduced fluorescence intensities of Beclin1 and LC3. CONCLUSION 6-SH inhibited the expression of DAPK1, alleviated excessive autophagy after global CIRI following CA-CPR in rats, and exerted neuroprotective effects. The mechanism may be related to phosphorylation at the DAPK1 ser308 site.
Animals ; Rats, Sprague-Dawley ; Male ; Rats ; Cardiopulmonary Resuscitation ; Autophagy/drug effects* ; Heart Arrest/therapy* ; Death-Associated Protein Kinases/metabolism* ; Reperfusion Injury/metabolism* ; Disease Models, Animal ; Neuroprotective Agents/pharmacology* ; Brain Ischemia/metabolism*

Microglia, the resident immune cells in the central nervous system (CNS), rapidly transition from a resting to an active state in the acute phase of ischemic brain injury. This active state mediates a pro-inflammatory response that can exacerbate the injury. Targeting the pro-inflammatory response of microglia in the semi-dark band during this acute phase may effectively reduce brain injury. Shionone (SH), an active ingredient extracted from the dried roots and rhizomes of the genus Aster (Asteraceae), has been reported to regulate the inflammatory response of macrophages in sepsis-induced acute lung injury. However, its function in post-stroke neuroinflammation, particularly microglia-mediated neuroinflammation, remains uninvestigated. This study found that SH significantly inhibited lipopolysaccharide (LPS)-induced elevation of inflammatory cytokines, including interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), and inducible nitric oxide synthase (iNOS), in microglia in vitro. Furthermore, the results demonstrated that SH alleviated infarct volume and improved behavioral performance in middle cerebral artery occlusion (MCAO) mice, which may be attributed to the inhibition of the microglial inflammatory response induced by SH treatment. Mechanistically, SH potently inhibited the phosphorylation of serine-threonine protein kinase B (AKT), mammalian target of rapamycin (mTOR), and signal transducer and activator of transcription 3 (STAT3). These findings suggest that SH may be a potential therapeutic agent for relieving ischemic stroke (IS) by alleviating microglia-associated neuroinflammation.
Animals ; Microglia/immunology* ; Mice ; Male ; Mice, Inbred C57BL ; Brain Ischemia/immunology* ; Neuroinflammatory Diseases/drug therapy* ; Neuroprotective Agents/administration & dosage* ; Interleukin-1beta/genetics* ; STAT3 Transcription Factor/genetics* ; TOR Serine-Threonine Kinases/genetics* ; Tumor Necrosis Factor-alpha/genetics* ; Proto-Oncogene Proteins c-akt/immunology* ; Nitric Oxide Synthase Type II/genetics* ; Lipopolysaccharides

Endovascular treatment has become the primary treatment for patients with acute ischemic stroke (AIS) due to large vessel occlusion. Its development and clinical application have expanded significantly in recent years. Imaging assessment plays a pivotal role in guiding both preoperative screening and postoperative management in AIS patients undergoing such interventions. However, updated expert consensus and standardized guidelines remain lacking. To address this gap, the Professional Committee of Interventional Neurology of the Chinese Research Hospital Association convened leading cerebrovascular disease experts to formulate consensus recommendations based on domestic clinical practice, aiming to provide authoritative guidance for imaging evaluation in this patient population.
Humans ; Endovascular Procedures ; Ischemic Stroke/therapy* ; Consensus ; Stroke/therapy* ; Brain Ischemia/therapy*

OBJECTIVE: Emerging evidence suggests that exposure to ultrafine particulate matter (UPM, aerodynamic diameter < 0.1 µm) is associated with adverse cardiovascular events. Previous studies have found that Shenlian (SL) extract possesses anti-inflammatory and antiapoptotic properties and has a promising protective effect at all stages of the atherosclerotic disease process. In this study, we aimed to investigated whether SL improves UPM-aggravated myocardial ischemic injury by inhibiting inflammation and cell apoptosis. METHODS: We established a mouse model of MI+UPM. Echocardiographic measurement, measurement of myocardialinfarct size, biochemical analysis, enzyme-linked immunosorbent assay (ELISA), histopathological analysis, Transferase dUTP Nick End Labeling (TUNEL), Western blotting (WB), Polymerase Chain Reaction (PCR) and so on were used to explore the anti-inflammatory and anti-apoptotic effects of SL in vivo and in vitro. RESULTS: SL treatment can attenuate UPM-induced cardiac dysfunction by improving left ventricular ejection fraction, fractional shortening, and decreasing cardiac infarction area. SL significantly reduced the levels of myocardial enzymes and attenuated UPM-induced morphological alterations. Moreover, SL significantly reduced expression levels of the inflammatory cytokines IL-6, TNF-α, and MCP-1. UPM further increased the infiltration of macrophages in myocardial tissue, whereas SL intervention reversed this phenomenon. UPM also triggered myocardial apoptosis, which was markedly attenuated by SL treatment. The results of in vitro experiments revealed that SL prevented cell damage caused by exposure to UPM combined with hypoxia by reducing the expression of the inflammatory factor NF-κB and inhibiting apoptosis in H9c2 cells. CONCLUSION Overall, both in vivo and in vitro experiments demonstrated that SL attenuated UPM-aggravated myocardial ischemic injury by inhibiting inflammation and cell apoptosis. The mechanisms were related to the downregulation of macrophages infiltrating heart tissues.
Animals ; Apoptosis/drug effects* ; Particulate Matter/adverse effects* ; Mice ; Male ; Inflammation/drug therapy* ; Drugs, Chinese Herbal/therapeutic use* ; Mice, Inbred C57BL ; Myocardial Ischemia/drug therapy* ; Cell Line

INTRODUCTION: Achieving low-density lipoprotein cholesterol (LDL-C) levels is key to preventing atherosclerotic cardiovascular events. However, many high-risk cardiovascular patients still experience poor LDL-C goal attainment and receive suboptimal lipid-lowering therapy (LLT) prescriptions. Herein, we evaluated LLT prescription patterns, LDL-C goal attainment and cardiovascular mortality among this population group in Singapore. METHODS: This prospective observational cohort study included 555 patients with ischaemic heart disease (IHD) admitted to the hospital in 2020. The LLT prescriptions, corresponding LDL-C levels and cardiovascular outcomes were assessed over a 24-month period. RESULTS: Most participants were male (82.3%), with 48.5% identified as Chinese. High-intensity statin prescriptions increased from 45.4% at hospital admission to 87.1% at discharge and remained stable at approximately 80% at 6, 12, and 24 months post-discharge. Combination LLT prescriptions increased from 12.3% at discharge to 33.8% by 24 months. Ezetimibe was the most commonly prescribed second-line LLT (40.8%), followed by inclisiran (1.09%) and anti-proprotein convertase subtilisin/kexin type 9 monoclonal antibody therapies (0.87%). Over 24 months, LDL-C goal attainment rates were 22.1% for LDL-C < 1.4 mmol/L and 47.2% for LDL-C < 1.8 mmol/L. Multivariable Cox proportional hazards regression indicated that achieving LDL-C < 1.8 mmol/L goal was associated with a reduction in all-cause mortality at 24 months (hazard ratio 0.53, 95% confidence interval 0.30-0.94, P = 0.030). CONCLUSION Treatment gaps in lipid management persist in 80% of the study population, indicating that statin monotherapy alone is insufficient to achieve LDL-C goals. Greater efforts to improve LDL-C goal attainment rates in high-risk cardiovascular patients are imperative.
Humans ; Male ; Cholesterol, LDL/blood* ; Female ; Myocardial Ischemia/drug therapy* ; Middle Aged ; Prospective Studies ; Aged ; Singapore/epidemiology* ; Hydroxymethylglutaryl-CoA Reductase Inhibitors/therapeutic use* ; Ezetimibe/therapeutic use* ; Anticholesteremic Agents/therapeutic use* ; Treatment Outcome

OBJECTIVE: To observe the effects of Xingnao Kaiqiao acupuncture technique (for regaining consciousness and opening orifice) on methylation of N6-methyladenosine (m6A), and key methyltransferases and demethylases, so as to clarify the mechanism of acupuncture on cerebral ischemia-reperfusion injury (CIRI). METHODS: Of 68 male Sprague-Dawley rats of SPF grade, 15 rats were randomly selected as a sham-operation group, and the remaining rats were subjected to the model of middle cerebral artery occlusion using the suture ligation. CIRI was induced by ischemia for 2 h followed by reperfusion. Rats that failed to modeling or died were excluded. The rest 45 rats were randomly divided into three groups, i.e. model group, acupuncture group, and non-point acupuncture group, with 15 rats in each group. The rats in the acupuncture group were treated with acupuncture at bilateral "Neiguan" (PC6) and "Shuigou" (GV26). In the non-point acupuncture group, acupuncture was delivered at three non-points, located 3 mm below the bilateral midaxillary line and 3 mm lateral to the tip of the coccyx. One intervention was operated in these two acupuncture groups and the needles were retained for 30 min. Before modeling start and 2 h after ischemia, a laser speckle flowmeter was used to monitor the cerebral blood perfusion. In 2 h of ischemia and 24 h of reperfusion, the neurological behavioral score was evaluated. The volume of rat cerebral infarction was determined by triphenyltetrazolium chloride (TTC) staining, and the level of m6A methylation in ischemic cortical area was detected by Dot blot, and the protein and mRNA expression of the demethylase i.e. fat mass and obesity-associated protein (FTO), AlkB homolog 5 (ALKBH5) and key methyltransferases, i.e. methyltransferase-like 3 (METTL3), methyltransferase-like 14 (METTL14), and Wilms' tumor 1-associated protein (WTAP) in ischemic cortical area were detected by Western blot and real-time PCR. RESULTS: Cerebral blood perfusion decreased by>70% after 2 h ischemia. Compared with the sham-operation group, the neurobehavioral score and the percentage of cerebral infarction volume increased in the model group (P<0.01); the level of m6A methylation in the ischemic cortical area increased (P<0.01), the protein and mRNA expression of FTO decreased (P<0.01), and that of ALKBH5, METTL3, and METTL14 increased (P<0.01, P<0.05) in the model group. When compared with the model group and the non-point acupuncture group, the acupuncture group showed a decrease in the neurobehavioral score and the percentage of cerebral infarction volume (P<0.01), the level of m6A methylation in the ischemic cortical area was reduced (P<0.01, P<0.05), and the protein and mRNA expression of FTO was elevated (P<0.01). CONCLUSION Xingnao Kaiqiao acupuncture technique presents its protective effect on the brain in the rats with CIRI, which is related to up-regulating the expression of FTO and modulating m6A methylation.
Animals ; Rats, Sprague-Dawley ; Male ; Acupuncture Therapy ; Reperfusion Injury/genetics* ; Rats ; Brain Ischemia/genetics* ; Humans ; Adenosine/metabolism* ; Methylation ; Acupuncture Points ; Cerebral Cortex/metabolism*