Brain Protein Expression Profile Confirms the Protective Effect of the ACTH(4–7)PGP Peptide (Semax) in a Rat Model of Cerebral Ischemia–Reperfusion

The Semax (Met-Glu-His-Phe-Pro-Gly-Pro) peptide is a synthetic melanocortin derivative that is used in the treatment of ischemic stroke. Previously, studies of the molecular mechanisms underlying the actions of Semax using models of cerebral ischemia in rats showed that the peptide enhanced the transcription of neurotrophins and their receptors and modulated the expression of genes involved in the immune response. A genome-wide RNA-Seq analysis revealed that, in the rat transient middle cerebral artery occlusion (tMCAO) model, Semax suppressed the expression of inflammatory genes and activated the expression of neurotransmitter genes. Here, we aimed to evaluate the effect of Semax in this model via the brain expression profiling of key proteins involved in inflammation and cell death processes (MMP-9, c-Fos, and JNK), as well as neuroprotection and recovery (CREB) in stroke. At 24 h after tMCAO, we observed the upregulation of active CREB in subcortical structures, including the focus of the ischemic damage; downregulation of MMP-9 and c-Fos in the adjacent frontoparietal cortex; and downregulation of active JNK in both tissues under the action of Semax. Moreover, a regulatory network was constructed. In conclusion, the suppression of inflammatory and cell death processes and the activation of recovery may contribute to the neuroprotective action of Semax at both the transcriptome and protein levels.

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Cerebral Ischemia Causes Dysregulation of Synaptic Adhesion in Mouse Synaptosomes

Journal of Cerebral Blood Flow & Metabolism ◽

10.1038/sj.jcbfm.9600510 ◽

2007 ◽

Vol 28 (1) ◽

pp. 99-110 ◽

Cited By ~ 15

Author(s):

Willard J Costain ◽

Ingrid Rasquinha ◽

Jagdeep K Sandhu ◽

Peter Rippstein ◽

Bogdan Zurakowski ◽

...

Keyword(s):

Cell Death ◽

Cerebral Ischemia ◽

Adhesion Molecules ◽

Neuronal Cell Death ◽

Neuronal Cell ◽

Artery Occlusion ◽

Synaptic Proteins ◽

Mrna Levels ◽

Neuronal Nuclei ◽

Protein Levels

Synaptic pathology is observed during hypoxic events in the central nervous system in the form of altered dendrite structure and conductance changes. These alterations are rapidly reversible, on the return of normoxia, but are thought to initiate subsequent neuronal cell death. To characterize the effects of hypoxia on regulators of synaptic stability, we examined the temporal expression of cell adhesion molecules (CAMs) in synaptosomes after transient middle cerebral artery occlusion (MCAO) in mice. We focused on events preceding the onset of ischemic neuronal cell death (< 48 h). Synaptosome preparations were enriched in synaptically localized proteins and were free of endoplasmic reticulum and nuclear contamination. Electron microscopy showed that the synaptosome preparation was enriched in spheres (≈650 nm in diameter) containing secretory vesicles and postsynaptic densities. Forebrain mRNA levels of synaptically located CAMs was unaffected at 3 h after MCAO. This is contrasted by the observation of consistent downregulation of synaptic CAMs at 20 h after MCAO. Examination of synaptosomal CAM protein content indicated that certain adhesion molecules were decreased as early as 3 h after MCAO. For comparison, synaptosomal Agrn protein levels were unaffected by cerebral ischemia. Furthermore, a marked increase in the levels of p-Ctnnb1 in ischemic synaptosomes was observed. p-Ctnnb1 was detected in hippocampal fiber tracts and in cornu ammonis 1 neuronal nuclei. These results indicate that ischemia induces a dysregulation of a subset of synaptic proteins that are important regulators of synaptic plasticity before the onset of ischemic neuronal cell death.

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Surface-modified engineered exosomes attenuated cerebral ischemia/reperfusion injury by targeting the delivery of quercetin towards impaired neurons

Journal of Nanobiotechnology ◽

10.1186/s12951-021-00879-4 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Lin Guo ◽

Zhixuan Huang ◽

Lijuan Huang ◽

Jia Liang ◽

Peng Wang ◽

...

Keyword(s):

Cerebral Ischemia ◽

Reperfusion Injury ◽

Targeted Delivery ◽

Ischemia Reperfusion Injury ◽

Infarct Volume ◽

Ischemia Reperfusion ◽

Artery Occlusion ◽

Therapeutic Drug ◽

Cerebral Ischemia Reperfusion ◽

Cerebral Ischemia Reperfusion Injury

Abstract Background The incidence of ischemic stroke in the context of vascular disease is high, and the expression of growth-associated protein-43 (GAP43) increases when neurons are damaged or stimulated, especially in a rat model of middle cerebral artery occlusion/reperfusion (MCAO/R). Experimental design We bioengineered neuron-targeting exosomes (Exo) conjugated to a monoclonal antibody against GAP43 (mAb GAP43) to promote the targeted delivery of quercetin (Que) to ischemic neurons with high GAP43 expression and investigated the ability of Exo to treat cerebral ischemia by scavenging reactive oxygen species (ROS). Results Our results suggested that Que loaded mAb GAP43 conjugated exosomes (Que/mAb GAP43-Exo) can specifically target damaged neurons through the interaction between Exo-delivered mAb GAP43 and GAP43 expressed in damaged neurons and improve survival of neurons by inhibiting ROS production through the activation of the Nrf2/HO-1 pathway. The brain infarct volume is smaller, and neurological recovery is more markedly improved following Que/mAb GAP43-Exo treatment than following free Que or Que-carrying exosome (Que-Exo) treatment in a rat induced by MCAO/R. Conclusions Que/mAb GAP43-Exo may serve a promising dual targeting and therapeutic drug delivery system for alleviating cerebral ischemia/reperfusion injury.

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LINGO-1 shRNA protects the brain against ischemia/reperfusion injury by inhibiting the activation of NF-κB and JAK2/STAT3

Human Cell ◽

10.1007/s13577-021-00527-x ◽

2021 ◽

Author(s):

Jiaying Zhu ◽

Zhu Zhu ◽

Yipin Ren ◽

Yukang Dong ◽

Yaqi Li ◽

...

Keyword(s):

Cerebral Ischemia ◽

Nuclear Translocation ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Glucose Deprivation ◽

Artery Occlusion ◽

Mice Model ◽

Down Regulation

AbstractLINGO-1 may be involved in the pathogenesis of cerebral ischemia. However, its biological function and underlying molecular mechanism in cerebral ischemia remain to be further defined. In our study, middle cerebral artery occlusion/reperfusion (MACO/R) mice model and HT22 cell oxygen–glucose deprivation/reperfusion (OGD/R) were established to simulate the pathological process of cerebral ischemia in vivo and in vitro and to detect the relevant mechanism. We found that LINGO-1 mRNA and protein were upregulated in mice and cell models. Down-regulation LINGO-1 improved the neurological symptoms and reduced pathological changes and the infarct size of the mice after MACO/R. In addition, LINGO-1 interference alleviated apoptosis and promoted cell proliferation in HT22 of OGD/R. Moreover, down-regulation of LINGO-1 proved to inhibit nuclear translocation of p-NF-κB and reduce the expression level of p-JAK2 and p-STAT3. In conclusion, our data suggest that shLINGO-1 attenuated ischemic injury by negatively regulating NF-KB and JAK2/STAT3 pathways, highlighting a novel therapeutic target for ischemic stroke.

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miR-380-5p facilitates NRF2 and attenuates cerebral ischemia/reperfusion injury-induced neuronal cell death by directly targeting BACH1

Translational Neuroscience ◽

10.1515/tnsci-2020-0172 ◽

2021 ◽

Vol 12 (1) ◽

pp. 210-217

Author(s):

Yibiao Wang ◽

Min Xu

Keyword(s):

Cell Death ◽

Cerebral Ischemia ◽

Reperfusion Injury ◽

Ischemia Reperfusion Injury ◽

Ischemia Reperfusion ◽

Neuronal Cell Death ◽

Neuronal Cell ◽

Luciferase Assay ◽

Neuroprotective Effects ◽

Cerebral Ischemia Reperfusion

Abstract Background This study aimed to explore the role of miR-380-5p in cerebral ischemia/reperfusion (CIR) injury-induced neuronal cell death and the potential signaling pathway involved. Methodology Human neuroblastoma cell line SH-SY5Y cells were used in this study. Oxygen and glucose deprivation/reperfusion (OGD/R) model was used to mimic ischemia/reperfusion injury. CCK-8 assay and flow cytometry were used to examine cell survival. Quantitative real time PCR (RT-qPCR) assay and Western blotting were used to measure the change of RNA and protein expression, respectively. TargetScan and Luciferase assay was used to confirm the target of miR-380-5p. Malondialdehyde (MDA) superoxide dismutase (SOD) and glutathione peroxidase (GSHPx) were measured using commercial kits. Results miR-380-5p was downregulated in SH-SY5Y cells after OGD/R. Cell viability was increased by miR-380-5p, while cell apoptosis was reduced by miR-380-5p mimics. MDA was reduced by miR-380-5p mimics, while SOD and GSHPx were increased by miR-380-5p. Results of TargetScan and luciferase assay have showed that BACH1 is the direct target of miR-380-5p. Expression of NRF2 was upregulated after OGD/R, but was not affected by miR-380-5p. mRNA expression of HO-1 and NQO1 and ARE activity were increased by miR-380-5p. Overexpression of BACH1 reversed the antioxidant and neuroprotective effects of miR-380-5p. Conclusion miR-380-5p inhibited cell death induced by CIR injury through target BACH1 which also facilitated the activation of NRF2, indicating the antioxidant and neuroprotective effects of miR-380-5p.

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miR-182-5p and miR-378a-3p regulate ferroptosis in I/R-induced renal injury

Cell Death and Disease ◽

10.1038/s41419-020-03135-z ◽

2020 ◽

Vol 11 (10) ◽

Author(s):

Chenguang Ding ◽

Xiaoming Ding ◽

Jin Zheng ◽

Bo Wang ◽

Yang Li ◽

...

Keyword(s):

Cell Death ◽

Renal Injury ◽

Molecular Mechanisms ◽

Ischemia Reperfusion ◽

Kidney Injury ◽

Luciferase Reporter ◽

Renal Tubular Cell ◽

In Cells

Abstract Renal tubular cell death is the key factor of the pathogenesis of ischemia/reperfusion (I/R) kidney injury. Ferroptosis is a type of regulated cell death (RCD) found in various diseases. However, the underlying molecular mechanisms related to ferroptosis in renal I/R injury remain unclear. In the present study, we investigated the regulatory role of microRNAs on ferroptosis in I/R-induced renal injury. We established the I/R-induced renal injury model in rats, and H/R induced HK-2 cells injury in vitro. CCK-8 was used to measure cell viability. Fe2+ and ROS levels were assayed to evaluate the activation of ferroptosis. We performed RNA sequencing to profile the miRNAs expression in H/R-induced injury and ferroptosis. Western blot analysis was used to detect the protein expression. qRT-PCR was used to detect the mRNA and miRNA levels in cells and tissues. We further used luciferase reporter assay to verify the direct targeting effect of miRNA. We found that ischemia/reperfusion-induced ferroptosis in rat’s kidney. We identified that miR-182-5p and miR-378a-3p were upregulated in the ferroptosis and H/R-induced injury, and correlates reversely with glutathione peroxidases 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11) expression in renal I/R injury tissues, respectively. In vitro studies showed that miR-182-5p and miR-378a-3p induced ferroptosis in cells. We further found that miR-182-5p and miR-378a-3p regulated the expression of GPX4 and SLC7A11 negatively by directly binding to the 3′UTR of GPX4 and SLC7A11 mRNA. In vivo study showed that silencing miR-182-5p and miR-378a-3p alleviated the I/R-induced renal injury in rats. In conclusion, we demonstrated that I/R induced upregulation of miR-182-5p and miR-378a-3p, leading to activation of ferroptosis in renal injury through downregulation of GPX4 and SLC7A11.

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Zuogui Pill Attenuates Neuroinflammation and Improves Cognitive Function in Cerebral Ischemia Reperfusion-Injured Rats

NeuroImmunoModulation ◽

10.1159/000519010 ◽

2021 ◽

pp. 1-8

Author(s):

Hong Liu ◽

Qiaomei Dai ◽

Jing Yang ◽

Yuwei Zhang ◽

Bo Zhang ◽

...

Keyword(s):

Cognitive Function ◽

Cerebral Ischemia ◽

Cell Viability ◽

Ischemia Reperfusion ◽

Glucose Deprivation ◽

Artery Occlusion ◽

Oxygen And Glucose Deprivation ◽

Infarct Area ◽

Zuogui Pill ◽

Adverse Influence

Introduction: Cerebral ischemia and reperfusion (CI/R) injury is a devasting cerebrovascular disease, accompanied with ischemia stroke, cerebral infarction. Zuogui Pill (ZGP), as a Chinese traditional medicine, is proved to be effective in many diseases and cancers. Our study aimed to detect the roles of ZGP in CI/R injury. Methods: Neural stem cells were isolated from rats and induced by oxygen and glucose deprivation and recovery. CCK-8 and flow cytometry were applied to assess the function of ZGP on cell viability and apoptosis. Rat CI/R injury models were established by the middle cerebral artery occlusion and reperfusion. The function of ZGP on CI/R injury was identified via evaluating modified neurological severity score, infarct area, and cognitive impairment. Results: Compared to the control, the cell viability was obviously decreased in the oxygen and glucose deprivation and recovery (OGD/R) group, while the adverse influence on cells was reversed by cultured plus 10% ZGP serum. Consistently, ZGP attenuated the influence of OGD/R on cell apoptosis. More importantly, ZGP could alleviate CI/R injury of rats by reducing neurological damage and infarct area and promoting cognitive function. Conclusion: This study provided protective roles of ZGP on cell viability and apoptosis induced by OGD/R. In addition, ZGP played protective roles on neuroinflammation and cognitive function in rats.

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Gossypol Acetic Acid Attenuates Cardiac Ischemia/Reperfusion Injury in Rats via an Antiferroptotic Mechanism

Biomolecules ◽

10.3390/biom11111667 ◽

2021 ◽

Vol 11 (11) ◽

pp. 1667

Author(s):

Jian-Hong Lin ◽

Kun-Ta Yang ◽

Pei-Ching Ting ◽

Yu-Po Luo ◽

Ding-Jyun Lin ◽

...

Keyword(s):

Lipid Peroxidation ◽

Cell Death ◽

Acetic Acid ◽

Ischemia Reperfusion ◽

Neonatal Rat ◽

Mrna Levels ◽

H9c2 Cells ◽

Rat Cardiomyocytes ◽

Protein Levels ◽

Gossypol Acetic Acid

Myocardial ischemia/reperfusion (I/R) injury has been associated with ferroptosis, which is characterized by an iron-dependent accumulation of lipid peroxide to lethal levels. Gossypol acetic acid (GAA), a natural product taken from the seeds of cotton plants, prevents oxidative stress. However, the effects of GAA on myocardial I/R-induced ferroptosis remain unclear. This study investigated the ability of GAA to attenuate I/R-induced ferroptosis in cardiomyocytes along with the underlying mechanisms in a well-established rat model of myocardial I/R and isolated neonatal rat cardiomyocytes. H9c2 cells and cardiomyocytes were treated with the ferroptosis inducers erastin, RSL3, and Fe-SP. GAA could protect H9c2 cells against ferroptotic cell death caused by these ferroptosis inducers by decreasing the production of malondialdehyde and reactive oxygen species, chelating iron content, and downregulating mRNA levels of Ptgs2. GAA could prevent oxygen-glucose deprivation/reperfusion-induced cell death and lipid peroxidation in the cardiomyocytes. Moreover, GAA significantly attenuated myocardial infarct size, reduced lipid peroxidation, decreased the mRNA levels of the ferroptosis markers Ptgs2 and Acsl4, decreased the protein levels of ACSL4 and NRF2, and increased the protein levels of GPX4 in I/R-induced ex vivo rat hearts. Thus, GAA may play a cytoprotectant role in ferroptosis-induced cardiomyocyte death and myocardial I/R-induced ferroptotic cell death.

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Up-regulation of miR-499a-5p decreases cerebral ischemia/reperfusion injury by targeting PDCD4

10.21203/rs.3.rs-297082/v1 ◽

2021 ◽

Author(s):

Weifeng Shan ◽

Huifeng Ge ◽

Bingquan Chen ◽

Linger Huang ◽

Shaojun Zhu ◽

...

Keyword(s):

Ischemic Stroke ◽

Cerebral Ischemia ◽

Ischemia Reperfusion Injury ◽

Cell Model ◽

Ischemia Reperfusion ◽

Glucose Deprivation ◽

Artery Occlusion ◽

Tunel Staining ◽

Cerebral Ischemia Reperfusion

Abstract MiR-499a-5p was significantly down-regulated in degenerative tissues and correlated with apoptosis. Nonetheless, the biological function of miR-499a-5p in acute ischemic stroke has been still unclear. In this study, we found the plasma levels of miR-499a-5p were significantly down-regulated in 64 ischemic stroke patients and negatively correlated with the National Institutes of Health Stroke Scale score. Then, we constructed cerebral ischemia/reperfusion (I/R) injury in rats after middle cerebral artery occlusion and subsequent reperfusion and oxygen-glucose deprivation and reoxygenation (OGD/R) treated SH-SY5Y cell model. Transfection with miR-499a-5p mimic was accomplished by intracerebroventricular injection in the in vivo I/R injury model. We further found miR-499a-5p overexpression decreased infarct volumes and cell apoptosis in the in vivo I/R stroke model using TTC and TUNEL staining. PDCD4 was a direct target of miR-499a-5p by luciferase report assay and western blotting. Knockdown of PDCD4 reduced the infarct damage and cortical neuron apoptosis caused by I/R injury. MiR-499a-5p exerted neuroprotective roles mainly through inhibiting PDCD4-mediated apoptosis by CCK-8 assay, LDH release assay and flow cytometry analysis. These findings suggest that miR-499a-5p might represent a novel target that regulates brain injury by inhibiting PDCD4-mediating apoptosis.

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The RIG-I Signal Pathway Mediated Panax notoginseng Saponin Anti-Inflammatory Effect in Ischemia Stroke

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2021/8878428 ◽

2021 ◽

Vol 2021 ◽

pp. 1-14

Author(s):

Chujun Zhang ◽

Sai Zhang ◽

Lanxiang Wang ◽

Soyeon Kang ◽

Jiabao Ma ◽

...

Keyword(s):

Cerebral Ischemia ◽

Molecular Mechanisms ◽

Chinese Herb ◽

Inflammatory Cells ◽

Panax Notoginseng ◽

Artery Occlusion ◽

Blue Staining ◽

Bioactive Constituents ◽

Anti Inflammatory ◽

Cerebral Artery Occlusion

Panax notoginseng saponins (PNS), the main bioactive constituents of a traditional Chinese herb Panax notoginseng, were commonly used for ischemic stroke in China. However, the associated cellular and molecular mechanisms of PNS have not been well examined. This study aimed to decipher the underlying molecular target of PNS in the treatment of cerebral ischemia. The oxygen-glucose-deprived (OGD) model of rat brain microvascular endothelial cells (BMECs) was used in this study. The alteration of gene expression in rat BMECs after PNS treatment was measured by microarray and indicated that there were 38 signaling pathways regulated by PNS. Among them, RIG-I receptor and related signaling molecules TNF receptor-associated factor 2 (Traf2) and nuclear factor-kappa B (NF-κB) were significantly suppressed by PNS, which was verified again in OGD-induced BMECs measured by FQ-PCR and western blotting and in middle cerebral artery occlusion (MCAO) rats measured by immunohistochemistry. The levels of TNF-α, IL-8, and the downstream cytokines regulated by RIG-I receptor pathway were also decreased by PNS. Meanwhile, the neurological evaluation, hematoxylin and eosin (HE) staining, and Evans blue staining were conducted to evaluate the effect of PNS in MCAO rats. Results showed PNS significantly improved functional outcome and cerebral vascular leakage. Flow cytometry showed the number of the inflammatory cells infiltrated in brain tissue was decreased in PNS treatment. Our results identified that RIG-I signaling pathway mediated anti-inflammatory properties of PNS in cerebral ischemia, which provided the novel insights of PNS application in clinics.

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Leukocyte-Endothelium Interactions during Permanent Focal Cerebral Ischemia in Mice

Journal of Cerebral Blood Flow & Metabolism ◽

10.1097/01.wcb.0000117812.35136.5b ◽

2004 ◽

Vol 24 (6) ◽

pp. 668-676 ◽

Cited By ~ 41

Author(s):

Hiroharu Kataoka ◽

Seong-Woong Kim ◽

Nikolaus Plesnila

Keyword(s):

Cerebral Ischemia ◽

Brain Damage ◽

Molecular Mechanisms ◽

Focal Cerebral Ischemia ◽

Fluorescent Microscopy ◽

Capillary Density ◽

Artery Occlusion ◽

Cerebral Artery Occlusion ◽

Adherent Leukocytes

The contribution of leukocyte infiltration to brain damage after permanent focal cerebral ischemia and the underlying molecular mechanisms are still unclear. Therefore, the aim of this study was to establish a mouse model for the visualization of leukocytes in the cerebral microcirculation in vivo and to investigate leukocyte-endothelial interaction (LEI) after permanent middle cerebral artery occlusion (MCAO). Sham-operated 129/Sv mice showed physiologic LEI in pial venules as observed by intravital fluorescent microscopy. Permanent focal cerebral ischemia induced a significant increase of LEI predominantly in pial venules. The number of rolling and adherent leukocytes reached 36.5 ± 13.2/100 μm × min and 22.5 ± 7.9/100 μm × min, respectively at 120 minutes after MCAO ( P = 0.016 vs. control). Of note, rolling and adherent leukocytes were also observed in arterioles of ischemic animals (7.3 ± 3.0/100 μm × min rolling and 3.0 ± 3.6/100 μm × min adherent). Capillary density was not different between groups. These results demonstrate that leukocytes accumulate in the brain not only after transient but also after permanent focal cerebral ischemia and may therefore contribute to brain damage after stroke without reperfusion.

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