This study aims to explore whether Naoxintong Capsules improve multiple cerebral infarctions and myocardial injury via promoting angiogenesis, thereby exerting a simultaneous treatment effect on both the brain and heart. Male SD rats were randomly divided into six groups: sham-operated group, model group, high-dose, medium-dose, and low-dose groups of Naoxintong Capsules(440, 220, and 110 mg·kg~(-1)), and nimodipine group(10.8 mg·kg~(-1)). Rat models of multiple cerebral infarctions were established by injecting autologous thrombus, and samples were collected and tested seven days after modeling. Evaluations included multiple cerebral infarction model assessments, neurological function scores, grip strength tests, and rotarod tests, so as to evaluate neuromotor functions. Morphological structures of brain and heart tissue were observed using hematoxylin-eosin(HE) staining, Nissl staining, and Masson staining. Network pharmacology was employed to screen the mechanisms of Naoxintong Capsules in improving multiple cerebral infarctions and myocardial injury. Neuronal and myocardial cell ultrastructures were observed using transmission electron microscopy. Apoptosis rate in brain neuronal cells was detected by TdT-mediated dUTP nick end labeling(TUNEL) staining, and reactive oxygen species(ROS) levels in myocardial cells were measured. Immunofluorescence was used to detect the expression of platelet endothelial cell adhesion molecule-1(CD31), antigen identified by monoclonal antibody Ki67(Ki67), hematopoietic progenitor cell antigen CD34(CD34), and hypoxia inducible factor-1α(HIF-1α) in brain and myocardial tissue. Western blot, and real-time quantitative polymerase chain reaction(RT-qPCR) were used to detect the expression of HIF-1α, vascular endothelial growth factor(VEGF), vascular endothelial growth factor receptor 2(VEGFR2), sarcoma(Src), basic fibroblast growth factor(bFGF), angiopoietin-1(Ang-1), and TEK receptor tyrosine kinase(Tie-2). Compared with the model group, the medium-dose group of Naoxintong Capsules showed significantly lower neurological function scores, increased grip strength, and prolonged time on the rotarod. Pathological damage in brain and heart tissue was reduced, with increased and more orderly arranged mitochondria in neurons and cardiomyocytes. Apoptosis in brain neuronal cells was decreased, and ROS levels in cardiomyocytes were reduced. The microvascular density and endothelial cells of new blood vessels in brain and heart tissue increased, with increased overlapping regions of CD31 and Ki67 expression. The relative protein and mRNA expression levels of HIF-1α, VEGF, VEGFR2, Src, Ang-1, Tie-2, and bFGF were elevated in brain tissue and myocardial tissue. Naoxintong Capsules may improve multiple cerebral infarctions and myocardial injury by mediating HIF-1α/VEGF expression to promote angiogenesis.
Animals ; Male ; Drugs, Chinese Herbal/administration & dosage* ; Rats, Sprague-Dawley ; Rats ; Cerebral Infarction/genetics* ; Hypoxia-Inducible Factor 1, alpha Subunit/genetics* ; Vascular Endothelial Growth Factor A/genetics* ; Capsules ; Signal Transduction/drug effects* ; Humans ; Brain/metabolism* ; Myocardium/metabolism* ; Apoptosis/drug effects*

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 investigate the effects of Naoluo Xintong Decoction (NLXTD) on proliferation of rat brain microvascular endothelial cells (BMECs) after oxygen-glucose deprivation/reoxygenation (OGD/R) injury and role of the HIF-1α/VEGF pathway in mediating its effect. METHODS: Using a BMEC model of OGD/R, we tested the effects of 10% NLXTD-medicated rat serum, alone or in combination with 2ME2 or 10% NAKL, on cell proliferation, migration, tube-forming ability and permeability using CCK-8 assay, Transwell chamber assay, tube formation assay and permeability assay. Cellular expressions of VEGF and Notch were detected using ELISA and laser confocal immunofluorescence analysis, and the expressions of HIF-1α, VEGFR2, Notch1, ERK and P-ERK1/2 proteins were detected with Western blotting. RESULTS: OGD/R injury significantly decreased viability of BMECs. NLXTD treatment of the cells with OGD/R could significantly promoted cell proliferation, migration and tube formation ability, but these effects were strongly attenuated by application of 2ME2. NLXTD treatment also significantly increased the percentages of VEGF- and Notch-positive cells in the cell models and obviously enhanced the expression levels of HIF-1α, VEGFR2, Notch1 and P-ERK1/2. CONCLUSIONS NLXTD promotes proliferation, migration, and tube formation of rat BMECs after OGD/R injury possibly by activating the HIF-1α/VEGF signaling pathway.
Animals ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism* ; Drugs, Chinese Herbal/pharmacology* ; Vascular Endothelial Growth Factor A/metabolism* ; Endothelial Cells/metabolism* ; Rats ; Cell Proliferation/drug effects* ; Signal Transduction/drug effects* ; Glucose ; Brain/blood supply* ; Cells, Cultured ; Rats, Sprague-Dawley ; Vascular Endothelial Growth Factor Receptor-2/metabolism* ; Oxygen/metabolism* ; Cell Hypoxia

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

Hypoxic injury (HI) in the prenatal period often causes neonatal neurological disabilities. Due to the difficulty in obtaining clinical samples, the molecular and cellular mechanisms remain unclear. Here we use vascularized cerebral organoids to investigate the hypoxic injury phenotype and explore the intercellular interactions between vascular and neural tissues under hypoxic conditions. Our results indicate that fused vascularized cerebral organoids exhibit broader hypoxic responses and larger decreases in panels of neural development-related genes when exposed to low oxygen levels compared to single cerebral organoids. Interestingly, vessels also exhibit neural protective effects on T-box brain protein 2⁺ intermediate progenitors (IPs), which are markedly lost in HI cerebral organoids. Furthermore, we identify the role of bone morphogenic protein signaling in protecting IPs. Thus, this study has established an in vitro organoid system that can be used to study the contribution of vessels to brain injury under hypoxic conditions and provides a strategy for the identification of intervention targets.
Organoids/pathology* ; Animals ; Mice ; Hypoxia, Brain/metabolism* ; Brain/blood supply* ; Neurons/metabolism*

The discovery of neuroglobin (Ngb), a brain- or neuron-specific member of the hemoglobin family, has revolutionized our understanding of brain oxygen metabolism. Currently, how Ngb plays such a role remains far from clear. Here, we report a novel mechanism by which Ngb might facilitate neuronal oxygenation upon hypoxia or anemia. We found that Ngb was present in, co-localized to, and co-migrated with mitochondria in the cell body and neurites of neurons. Hypoxia induced a sudden and prominent migration of Ngb towards the cytoplasmic membrane (CM) or cell surface in living neurons, and this was accompanied by the mitochondria. In vivo, hypotonic and anemic hypoxia induced a reversible Ngb migration toward the CM in cerebral cortical neurons in rat brains but did not alter the expression level of Ngb or its cytoplasm/mitochondria ratio. Knock-down of Ngb by RNA interference significantly diminished respiratory succinate dehydrogenase (SDH) and ATPase activity in neuronal N2a cells. Over-expression of Ngb enhanced SDH activity in N2a cells upon hypoxia. Mutation of Ngb at its oxygen-binding site (His⁶⁴) significantly increased SDH activity and reduced ATPase activity in N2a cells. Taken together, Ngb was physically and functionally linked to mitochondria. In response to an insufficient oxygen supply, Ngb migrated towards the source of oxygen to facilitate neuronal oxygenation. This novel mechanism of neuronal respiration provides new insights into the understanding and treatment of neurological diseases such as stroke and Alzheimer's disease and diseases that cause hypoxia in the brain such as anemia.
Rats ; Animals ; Neuroglobin/metabolism* ; Globins/metabolism* ; Nerve Tissue Proteins/metabolism* ; Neurons/metabolism* ; Hypoxia/metabolism* ; Brain/metabolism* ; Oxygen ; Anemia/metabolism* ; Adenosine Triphosphatases/metabolism*

Hypoxia, as an important hallmark of the tumor microenvironment, is a major cause of oxidative stress and plays a central role in various malignant tumors, including glioblastoma. Elevated reactive oxygen species (ROS) in a hypoxic microenvironment promote glioblastoma progression; however, the underlying mechanism has not been clarified. Herein, we found that hypoxia promoted ROS production, and the proliferation, migration, and invasion of glioblastoma cells, while this promotion was restrained by ROS scavengers N-acetyl-L-cysteine (NAC) and diphenyleneiodonium chloride (DPI). Hypoxia-induced ROS activated hypoxia-inducible factor-1α (HIF-1α) signaling, which enhanced cell migration and invasion by epithelial-mesenchymal transition (EMT). Furthermore, the induction of serine protease inhibitor family E member 1 (SERPINE1) was ROS-dependent under hypoxia, and HIF-1α mediated SERPINE1 increase induced by ROS via binding to the SERPINE1 promoter region, thereby facilitating glioblastoma migration and invasion. Taken together, our data revealed that hypoxia-induced ROS reinforce the hypoxic adaptation of glioblastoma by driving the HIF-1α-SERPINE1 signaling pathway, and that targeting ROS may be a promising therapeutic strategy for glioblastoma.
Humans ; Cell Hypoxia ; Cell Line, Tumor ; Glioblastoma/pathology* ; Hypoxia-Inducible Factor 1, alpha Subunit/metabolism* ; Plasminogen Activator Inhibitor 1/metabolism* ; Reactive Oxygen Species/metabolism* ; Signal Transduction ; Tumor Microenvironment ; Brain Neoplasms/pathology*

Objective To investigate the protective effect of artesunate on hypoxic-ischemic brain damage (HIBD) and its mechanism in neonatal rats. Methods 7-day-old neonatal SD rats were randomly divided into sham operation group, model group, artesunate 5 mg/kg group, artesunate 10 mg/kg group, artesunate 20 mg/kg group and dexamethasone 6 mg/kg group, with 18 rats in each group. HIBD models were established in groups except for the sham operation group. The sham operation group only needed to separate the left common carotid artery without ligation and nitrogen-oxygen mixed gas ventilation. Each group was injected with drug intraperitoneally right after surgery and the rats in the sham operation group and the model group were injected with an equal volume of normal saline (once a day for a total of 5 times). One hour after the last injection, the rats in each group were scored for neurological defects. After the rats were sacrificed, the brain water content was measured and the pathological changes of the brain tissues of rats were observed. Terminal-deoxynucleotidyl transferase mediated nick end labeling (TUNEL) was used to detect the neuronal cell apoptosis, and ELISA was applied to detect the levels of IL-1β, IL-6 and TNF-α in brain tissues and peripheral blood of each group of rats. Western blot analysis was adopted to detect the protein expression levels of NLR family pyrin domain containing 3 (NLRP3), apoptosis-associated speck-like protein containing CARD (ASC) and caspase-1 in the rats brain tissues of each group. Results Compared with the model group, the neurological deficit score was decreased; the pathological damage of brain tissues was relieved; the brain water content was significantly reduced; the apoptosis number of hippocampal neurons was decreased significantly; the levels of IL-1β, IL-6 and TNF-α in brain tissues and peripheral blood were significantly reduced; the protein expression levels of NLRP3, ASC and caspase-1 were significantly lowered in the middle-dose and high-dose artesunate groups and the dexamethasone group. Conclusion Artesunate can improve the neurological function, relieve the brain damage, and alleviate the brain edema in neonatal rats with HIBD. It can protect the HIBD, which may be related to the inhibition of NLRP3 inflammasome activation and reduction of inflammatory cytokine secretion.
Animals ; Rats ; Animals, Newborn ; Artesunate/pharmacology* ; Brain/metabolism* ; Caspases/metabolism* ; Dexamethasone ; Hypoxia-Ischemia, Brain/pathology* ; Inflammasomes ; Interleukin-6/metabolism* ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Rats, Sprague-Dawley ; Tumor Necrosis Factor-alpha/metabolism* ; Water/metabolism*

OBJECTIVES: To study the effect of ligustrazine injection on mitophagy in neonatal rats with hypoxic-ischemic encephalopathy (HIE) and its molecular mechanism. METHODS: Neonatal Sprague-Dawley rats, aged 7 days, were randomly divided into a sham-operation group with 8 rats, a model group with 12 rats, and a ligustrazine group with 12 rats. The rats in the model group and the ligustrazine group were used to establish a neonatal rat model of HIE by ligation of the left common carotid artery followed by hypoxia treatment, and blood vessels were exposed without any other treatment for the rats in the sham-operation group. The rats in the ligustrazine group were intraperitoneally injected with ligustrazine (20 mg/kg) daily after hypoxia-ischemia, and those in the sham-operation group and the model group were intraperitoneally injected with an equal volume of normal saline daily. Samples were collected after 7 days of treatment. Hematoxylin and eosin staining and Nissl staining were used to observe the pathological changes of neurons in brain tissue; immunohistochemical staining was used to observe the positive expression of PINK1 and Parkin in the hippocampus and cortex; TUNEL staining was used to measure neuronal apoptosis; Western blotting was used to measure the expression levels of the mitophagy pathway proteins PINK1 and Parkin and the autophagy-related proteins Beclin-1, microtubule-associated protein 1 light chain 3 (LC3), and ubiquitin-binding protein (P62). RESULTS: Compared with the sham-operation group, the model group had a significant reduction in the number of neurons, an increase in intercellular space, loose arrangement, lipid vacuolization, and a reduction in Nissl bodies. The increased positive expression of PINK1 and Parkin, apoptosis rate of neurons, and protein expression levels of PINK1, Parkin, Beclin1 and LC3 (P<0.05) and the decreased protein expression level of P62 in the hippocampus were also observed in the model group (P<0.05). Compared with the model group, the ligustrazine group had a significant increase in the number of neurons with ordered arrangement and an increase in Nissl bodies, significant reductions in the positive expression of PINK1 and Parkin, the apoptosis rate of neurons, and the protein expression levels of PINK1, Parkin, Beclin1, and LC3 (P<0.05), and a significant increase in the protein expression level of P62 (P<0.05). CONCLUSIONS Ligustrazine can alleviate hypoxic-ischemic brain damage and inhibit neuronal apoptosis in neonatal rats to a certain extent, possibly by inhibiting PINK1/Parkin-mediated autophagy.
Rats ; Animals ; Hypoxia-Ischemia, Brain/metabolism* ; Animals, Newborn ; Rats, Sprague-Dawley ; Beclin-1 ; Autophagy ; Ubiquitin-Protein Ligases/metabolism* ; Protein Kinases/metabolism*