Pathogenesis Of Cerebral Ischemia-reperfusion Injury Research Articles

DJ-1 regulates astrocyte activation through miR-155/SHP-1 signaling in cerebral ischemia/reperfusion injury.

Reactive astrocyte activation in the context of cerebral ischemia/reperfusion (I/R) injury gives rise to two distinct subtypes: the neurotoxic A1 type and the neuroprotective A2 type. DJ-1 (Parkinson disease protein 7, PARK7), originally identified as a Parkinson's disease-associated protein, is a multifunctional anti-oxidative stress protein with molecular chaperone and signaling functions. SHP-1 (Src homology 2 domain-containing phosphatase-1) is a protein tyrosine phosphatase closely associated with cellular signal transduction. miR-155 is a microRNA that participates in cellular functions by regulating gene expression. Recent studies have uncovered the relationship between DJ-1 and astrocyte-mediated neuroprotection, which may be related to its antioxidant properties and regulation of signaling molecules such as SHP-1. Furthermore, miR-155 may exert its effects by influencing SHP-1, providing a potential perspective for understanding the molecular mechanisms of stroke. A middle cerebral artery occlusion/reperfusion (MCAO/R) model and an oxygen-glucose deprivation/reperfusion (OGD/R) model were established to simulate focal cerebral I/R injury invivo and invitro, respectively. The invivo interaction between DJ-1 and SHP-1 has been experimentally validated through immunoprecipitation. Overexpression of DJ-1 attenuates I/R injury and suppresses miR-155 expression. In addition, inhibition of miR-155 upregulates SHP-1 expression and modulates astrocyte activation phenotype. These findings suggest that DJ-1 mediates astrocyte activation via the miR-155/SHP-1 pathway, playing a pivotal role in the pathogenesis of cerebral ischemia-reperfusion injury. Our results provide a potential way for exploring the pathogenesis of ischemic stroke and present promising targets for pharmacological intervention.

Application and mechanisms of Sanhua Decoction in the treatment of cerebral ischemia-reperfusion injury.

Cerebral ischemia-reperfusion is a process in which the blood supply to the brain is temporarily interrupted and subsequently restored. However, it is highly likely to lead to further aggravation of pathological damage to ischemic tissues or the nervous system., and has accordingly been a focus of extensive clinical research. As a traditional Chinese medicinal formulation, Sanhua Decoction has gradually gained importance in the treatment of cerebrovascular diseases. Its main constituents include Citrus aurantium, Magnolia officinalis, rhubarb, and Qiangwu, which are primarily used to regulate qi. In the treatment of neurological diseases, the therapeutic effects of the Sanhua Decoction are mediated via different pathways, including antioxidant, anti-inflammatory, and neurotransmitter regulatory pathways, as well as through the protection of nerve cells and a reduction in cerebral edema. Among the studies conducted to date, many have found that the application of Sanhua Decoction in the treatment of neurological diseases has clear therapeutic effects. In addition, as a natural treatment, the Sanhua Decoction has received widespread attention, given that it is safer and more effective than traditional Western medicines. Consequently, research on the mechanisms of action and efficacy of the Sanhua Decoctions in the treatment of cerebral ischemia-reperfusion injury is of considerable significance. In this paper, we describe the pathogenesis of cerebral ischemia-reperfusion injury and review the current status of its treatment to examine the therapeutic mechanisms of action of the Sanhua Decoction. We hope that the findings of the research presented herein will contribute to a better understanding of the efficacy of this formulation in the treatment of cerebral ischemia-reperfusion, and provide a scientific basis for its application in clinical practice.

Targeting Microglia/Macrophages Notch1 Protects Neurons from Pyroptosis in Ischemic Stroke.

The immune-inflammatory cascade and pyroptosis play an important role in the pathogenesis of cerebral ischemia-reperfusion injury (CIRI). The maintenance of immune homeostasis is inextricably linked to the Notch signaling pathway, but whether myeloid Notch1 affects microglia polarization as well as neuronal pyroptosis in CIRI is not fully understood. This study was designed to clarify the role of myeloid Notch1 in CIRI, providing new therapeutic strategies for ischemic stroke. Myeloid-specific Notch1 knockout (Notch1M-KO) mice and the floxed Notch1 (Notch1FL/FL) mice were subjected to middle cerebral artery occlusion (MCAO). After 3 days of CIRI, we evaluated the neurological deficit score and cerebral infarction volume. Immunofluorescence staining was used to detect the expression of Notch1 and microglial subtype markers. Cerebral infiltrating macrophages were detected by flow cytometry. RT-qPCR was used to detect pro-inflammatory cytokines. Western blot was used to detect the expression of pyroptosis related proteins. The Notch1-siRNA transfected BV2 cells were co-cultured with HT22 cells to investigate the potential mechanisms by which microglial Notch1 affects neuronal pyroptosis induced by anoxia/reoxygenation in vitro. We found that Notch1 was activated in cerebral microglia/macrophages after CIRI. Myeloid Notch1 deficiency decreased the cerebral infarct volume (24.17 ± 3.29 vs. 36.17 ± 2.27, p < 0.001), neurological function scores (2.33 ± 0.47 vs. 3.17 ± 0.37, p < 0.001) and the infiltration of peripheral monocytes/macrophages (3.26 ± 0.53 vs. 5.67 ± 0.57, p < 0.01). Strikingly, myeloid-specific Notch1 knockout alleviated pyroptosis. Compared with microglia M1, increased microglia M2 were detected in the ischemic penumbra. In parallel in vitro co-culture experiments, we found that Notch1 knockdown in microglial BV2 cells inhibited anoxia/reoxygenation-induced JAK2/STAT3 activation and pyroptosis in hippocampal neuron HT22 cells. Our findings elucidate the underlying mechanism of the myeloid Notch1 signaling pathway in regulating neuronal pyroptosis in CIRI, suggesting that targeting myeloid-specific Notch1 is an effective strategy for the treatment of ischemic stroke.

Hyperbaric oxygen improves cerebral ischemia-reperfusion injury in rats via inhibition of ferroptosis

Background: Our previous study found that hyperbaric oxygen (HBO) attenuated cognitive impairment in mice induced by cerebral ischemia-reperfusion injury (CIRI). However, its mechanism of action is not fully understood. In this study, we aimed to establish a rat model of cerebral ischemia-reperfusion, explore the possible role of ferroptosis in the pathogenesis of CIRI, and observe the effect of HBO on ferroptosis-mediated CIRI. Methods: Sprague Dawley (SD) rats were randomly divided into control, model, Ferrostatin-1 (Fer-1), HBO and Fer-1+ HBO groups. Morris water maze, myelin basic protein (MBP) and β-tubulin immunoreactivity were assessed to evaluate the neuroprotective effects of HBO on cerebral ischemia reperfusion injury. Ferroptosis were examined to investigate the mechanism underlying the effects of HBO. Results: Our result showed that Fer-1 and HBO improved learning and memory ability in the navigation trail and probe trail of the Morris water maze and increased MBP and β-tubulin immunoreactivity of the cortex in the model rats. The levels of ferritin, malondialdehyde (MDA) and glutathione (GSH) in the serum were also reversed by Fer-1 and HBO treatment. Mitochondrial cristae dissolution and vacuolization were observed in the model group by transmission electron microscopy and these conditions were improved in the Fer-1 and HBO groups. Furthermore, Fer-1 and HBO treatment reversed Prostaglandin-Endoperoxide Synthase 2 (PTGS2), Iron Responsive Element Binding Protein 2 (IREB2), acyl-CoA synthetase long chain family member 4 (ACSL4) and Solute Carrier Family 7 Member 11 (SLC7A11) mRNA levels and Transferrin Receptor 1 (TFR1), ferritin light chain (FTL), ferritin heavy chain 1 (FTH1), glutathione peroxidase 4 (GPX4), Nuclear factor E2-related factor 2 (Nrf2), lysophosphatidylcholine acyltransferase 3 (LPCAT3), c-Jun N-terminal kinase (JNK), phosphorylated c-Jun N-terminal kinase (P-JNK) phosphorylated Extracellular signal-regulated protein kinase (P-ERK) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MEK) protein levels. The above changes were more pronounced in Fer-1+ HBOGroup. Discussion: The results of the present study indicated that HBO improves cerebral ischemia-reperfusion injury in rats, which may be related to inhibition of ferroptosis. This also means that ferroptosis may become a new target of HBO against CIRI.

Neutral polysaccharide from Gastrodia elata alleviates cerebral ischemia-reperfusion injury by inhibiting ferroptosis-mediated neuroinflammation via the NRF2/HO-1 signaling pathway.

The crosstalk between ferroptosis and neuroinflammation considerably impacts the pathogenesis of cerebral ischemia-reperfusion injury (CIRI). Neutral polysaccharide from Gastrodia elata (NPGE) has shown significant effects against oxidative stress and inflammation. This study investigated the potential effects of NPGE on CIRI neuropathology. The effects of NPGE were studied in a mouse model of ischemic stroke (IS) and in oxygen-glucose deprivation/reperfusion (OGD/R)-induced HT22 cells. NPGE treatment decreased neurological deficits, reduced infarct volume, and alleviated cerebral edema in IS mice, and promoted the survival of OGD/R-induced HT22 cells. Mechanistically, NPGE treatment alleviated neuronal ferroptosis by upregulating GPX4 levels, lowering reactive oxygen species (ROS), malondialdehyde (MDA), and Fe2+ excessive hoarding, and meliorating GSH levels and SOD activity. Additionally, it inhibited neuroinflammation by down-regulating the level of IL-1β, IL-6, TNF-α, NLRP3, and HMGB1. Meanwhile, NPGE treatment alleviated ferroptosis and inflammation in erastin-stimulated HT22 cells. Furthermore, NPGE up-regulated the expression of NRF2 and HO-1 and promoted the translocation of NRF2 into the nucleus. Using the NRF2 inhibitor brusatol, we verified that NRF2/HO-1 signaling mediated the anti-ferroptotic and anti-inflammatory properties of NPGE. Collectively, our results demonstrate the protective effects of NPGE and highlight its therapeutic potential as a drug component for CIRI treatment.

Neuroprotective Effects of Activin A against Cerebral Ischemia/Reperfusion Injury in Mice by Enhancing Nrf2 Expression to Attenuate Neuronal Ferroptosis.

Activin A (Act A) is a member of the transforming growth factor-β (TGF-β) superfamily and can protect against ischemic cerebral injury. Ferroptosis, a newly discovered type of programmed cell death, contributes to the pathogenesis of cerebral ischemia-reperfusion injury (CIRI). However, little is known on whether Act A can modulate neuronal ferroptosis to protect against CIRI in a mouse model of middle cerebral artery occlusion (MCAO) and an HT22 cell model of oxygen-glucose deprivation/reoxygenation (OGD/R). The results indicated that Act A treatment relieved CIRI by improving neurological deficits and reducing the infarct volume in mice. MCAO stimulated iron accumulation and malondialdehyde formation and upregulated ACSL4 expression but downregulated GPX4 expression, a hallmark of ferroptosis in the brain of mice. Treatment with Act A significantly mitigated MCAO-triggered ferroptosis in the brain of mice. Furthermore, Act A treatment enhanced the MCAO-upregulated nuclear factor erythroid-2-related factor 2 (Nrf2) expression in the brains of mice. Similar results were observed in HT22 cells following OGD/R and pretreatment with Act A. The neuronal protective effect of Act A in HT22 cells was attenuated by treatment with ML385, an Nrf2 inhibitor. To conclude, Act A attenuated CIRI by enhancing Nrf2 expression and inhibiting neuronal ferroptosis.

CTRP1 Attenuates Cerebral Ischemia/Reperfusion Injury via the PERK Signaling Pathway.

Cerebral ischemic stroke is one of the leading causes of death worldwide. Previous studies have shown that circulating levels of CTRP1 are upregulated in patients with acute ischemic stroke. However, the function of CTRP1 in neurons remains unclear. The purpose of this study was to explore the role of CTRP1 in cerebral ischemia reperfusion injury (CIRI) and to elucidate the underlying mechanism. Middle cerebral artery occlusion/reperfusion (MCAO/R) and oxygen–glucose deprivation/reoxygenation (OGD/R) models were used to simulate cerebral ischemic stroke in vivo and in vitro, respectively. CTRP1 overexpression lentivirus and CTRP1 siRNA were used to observe the effect of CTRP1 expression, and the PERK selective activator CCT020312 was used to activate the PERK signaling pathway. We found the decreased expression of CTRP1 in the cortex of MCAO/R-treated rats and OGD/R-treated primary cortical neurons. CTRP1 overexpression attenuated CIRI, accompanied by the reduction of apoptosis and suppression of the PERK signaling pathway. Interference with CTRP1 expression in vitro aggravated apoptotic activity and increased the expression of proteins involved in the PERK signaling pathway. Moreover, activating the PERK signaling pathway abolished the protective effects of CTRP1 on neuron injury induced by CIRI in vivo and in vitro. In conclusion, CTRP1 protects against CIRI by reducing apoptosis and endoplasmic reticulum stress (ERS) through inhibiting the PERK-dependent signaling pathway, suggesting that CTRP1 plays a crucial role in the pathogenesis of CIRI.

Metformin Protects against Oxidative Stress Injury Induced by Ischemia/Reperfusion via Regulation of the lncRNA-H19/miR-148a-3p/Rock2 Axis.

Previous studies have shown that metformin not only is a hypoglycemic agent but also has neuroprotective effects. However, the mechanism of action of metformin in ischemic stroke is unclear. Oxidative stress is an important factor in the pathogenesis of cerebral ischemia-reperfusion injury. It has been reported that metformin is associated with stroke risk in the clinical population. This study is aimed at investigating the effect and mechanism of metformin in an experimental model of oxidative stress induced by ischemia/reperfusion (I/R) in vivo and oxygen glucose deprivation/reperfusion (OGD/R) in vitro. Metformin (100, 200, and 300 mg/kg) was administered intraperitoneally immediately after induction of cerebral ischemia. The indicators of oxidative stress selected were antioxidant enzyme activities of catalase, malondialdehyde (MDA), nitric oxide (NO), superoxide dismutase (SOD), and glutathione peroxidation enzyme (GSHPx). First, we demonstrated that metformin can significantly alleviate acute and chronic cerebral I/R injury and it has a strong regulatory effect on stroke-induced oxidative stress. It can reduce the elevated activities of MDA and NO and increase the levels of GSHPx and SOD in the cerebrum of mice and N2a cells exposed to I/R. Furthermore, real-time PCR and western blot were used to detect the expression of long noncoding RNA H19 (lncRNA-H19), microRNA-148a-3p (miR-148a-3p), and Rho-associated protein kinase 2 (Rock2). The direct interaction of lncRNA-H19, miR-148a-3p, and Rock2 was tested using a dual luciferase reporter assay. lncRNA-H19 altered OGD/R-induced oxidative stress by modulating miR-148a-3p to increase Rock2 expression. The expression of lncRNA-H19 and Rock2 could be downregulated with metformin in vivo and in vitro. In conclusion, our study confirmed that metformin exerts neuroprotective effects by regulating ischemic stroke-induced oxidative stress injury via the lncRNA-H19/miR-148a-3p/Rock2 axis. These results provide new evidence that metformin may represent a potential treatment for stroke-related brain injury.

Nur77 promotes cerebral ischemia-reperfusion injury via activating INF2-mediated mitochondrial fragmentation.

Mitochondrial fragmentation drastically regulates mitochondrial homeostasis in brain illness. However, the role of mitochondrial fragmentation in cerebral ischemia-reperfusion (IR) injury remains unclear. Nur77, a regulator of mitochondrial homeostasis, is associated with heart and liver IR injury, but its effects on mitochondrial function in cerebral IR injury has not been studied intensively. The aim of our study is to explore whether cerebral IR injury is modulated by Nur77 via modification of mitochondrial homeostasis. Our results indicated that Nur77 was upregulated in reperfused brain tissues. Genetic ablation of Nur77 reduced infarction area and promoted neuron survival under IR burden. Biochemical analysis demonstrated that Nur77 deletion protected mitochondrial function, attenuated mitochondrial oxidative stress, preserved mitochondrial potential, and blocked mitochondria-related cell apoptosis. In addition, we illustrated that Nur77 mediated mitochondrial damage via evoking mitochondrial fragmentation that occurred through increased mitochondrial fission and decreased fusion. Besides, our results also demonstrated that Nur77 controlled mitochondrial fragmentation via upregulating INF2 in a manner dependent on the Wnt/β-catenin pathway; inhibition of the Wnt pathway abrogated the protective effect of Nur77 deletion on reperfused-mediated neurons. Altogether, our study highlights that the pathogenesis of cerebral IR injury is associated with Nur77 activation followed by augmented mitochondrial fragmentation via an abnormal Wnt/β-catenin/INF2 pathway. Accordingly, Nur77-dependent mitochondrial fragmentation and the Wnt/β-catenin/INF2 axis may represent novel therapeutic targets to reduce cerebral IR injury.

Nurr1 exacerbates cerebral ischemia-reperfusion injury via modulating YAP-INF2-mitochondrial fission pathways

Nurr1, a nuclear transcription factor, has been linked to ischemia-reperfusion injury (IRI) in heart and kidney via modulating mitochondrial homeostasis. However, its role in cerebral ischemia-reperfusion has not been defined. In the present study, we found that cerebral IRI significantly increased the expression of Nurr1 and genetic ablation of Nurr1 attenuated the infarction area and reduced the neuron apoptosis under brain IRI burden. Functional studies have demonstrated that Nurr1 induced neuron death via activating mitochondrial fission. Aberrant mitochondrial fission promoted mitochondrial membrane potential reduction, evoked cellular oxidative stress and activated caspase-9-dependent mitochondrial apoptotic pathway. Interestingly, Nurr1 deletion alleviated fission-mediated mitochondrial damage, sustaining mitochondrial homeostasis and favoring neuron survival. Further, we found that Nurr1 deletion modulated mitochondrial fission via preventing INF2 upregulation in a manner dependent on YAP pathways. Either pharmacological blockade of YAP pathway or overexpression of INF2 abrogated the inhibitory effect of Nurr1 deletion on mitochondrial fission, leading to neuron death via mitochondrial apoptosis. Altogether, our results report that the pathogenesis of cerebral ischemia-reperfusion injury is associated with Nurr1 upregulation followed by augmented mitochondrial fission via an abnormal YAP-INF2 pathways.

NR4A1 Promotes Cerebral Ischemia Reperfusion Injury by Repressing Mfn2-Mediated Mitophagy and Inactivating the MAPK-ERK-CREB Signaling Pathway.

Mitochondrial dysfunction has been acknowledged as the key pathogenic mechanism in cerebral ischemia-reperfusion (IR) injury. Mitophagy is the protective system used to sustain mitochondrial homeostasis. However, the upstream regulator of mitophagy in response to brain IR injury is not completely understood. Nuclear receptor subfamily 4 group A member 1 (NR4A1) has been found to be associated with mitochondrial protection in a number of diseases. The aim of our study is to explore the functional role of NR4A1 in cerebral IR injury, with a particular focus on its influence on mitophagy. Wild-type mice and NR4A1-knockout mice were used to generate cerebral IR injury in vivo. Mitochondrial function and mitophagy were detected via immunofluorescence assays and western blotting. Cellular apoptosis was determined via MTT assays, caspase-3 activity and western blotting. Our data revealed that NR4A1 was significantly increased in the reperfused brain tissues. Genetic ablation of NR4A1 reduced the cerebral infarction area and repressed neuronal apoptosis. The functional study demonstrated that NR4A1 modulated cerebral IR injury by inducing mitochondrial damage. Higher NR4A1 promoted mitochondrial potential reduction, evoked cellular oxidative stress, interrupted ATP generation, and initiated caspase-9-dependent apoptosis. Mechanistically, NR4A1 induced mitochondrial damage by disrupting Mfn2-mediated mitophagy. Knockdown of NR4A1 elevated Mfn2 expression and therefore reversed mitophagic activity, sending a prosurvival signal for mitochondria in the setting of cerebral IR injury. Further, we demonstrated that NR4A1 modulated Mfn2 expression via the MAPK-ERK-CREB signaling pathway. Blockade of the ERK pathway could abrogate the permissive effect of NR4A1 deletion on mitophagic activation, contributing to neuronal mitochondrial apoptosis. Overall, our results demonstrate that the pathogenesis of cerebral IR injury is closely associated with a drop in protective mitophagy due to increased NR4A1 through the MAPK-ERK-CREB signaling pathway.

Levo-tetrahydropalmatine Attenuates Neuron Apoptosis Induced by Cerebral Ischemia-Reperfusion Injury: Involvement of c-Abl Activation.

Ischemic stroke is one of the most dangerous acute diseases which causes death or deformity. Apoptosis has been shown to play an important role in the development and pathogenesis of cerebral ischemia-reperfusion injury (I/R injury), but the related mechanism is unclear. Levo-tetrahydropalmatine (L-THP), a bioactive ingredient extracted from the Chinese herb Corydalis, can penetrate the blood-brain barrier and exert various pharmacological effects on neural tissues. The present study examined the neuroprotective effect of L-THP on neuronal apoptosis induced by cerebral I/R injury. Results showed that pretreatment with L-THP (12.5, 25, and 50mg/kg) improved neurological outcomes and reduced infarct volume and cerebral edema in comparison with the brains of the middle cerebral artery occlusion (MCAO) group. These findings provided evidence for the neuroprotective effects of L-THP against cerebral I/R injury. Furthermore, administration of L-THP enhanced the expression of Bcl-2 and attenuated the content of Bax, cleaved caspase-3, and PARP. L-THP could improve the reduction of NeuN-positive cells induced by I/R injury. These results suggested that L-THP could inhibit neuroapoptosis in cerebral ischemic rats. c-Abl was discovered as the critical protein responsible for neurocyte apoptosis; however, few data have been published on the relation between ischemic stroke and the expression of c-Abl. We found that both c-Abl expression and neuronal apoptosis were significantly increased in the MCAO group, while pretreatment with L-THP could ameliorate this effect. Therefore, we deduced that reduced c-Abl overexpression may play a role in the anti-apoptosis effect of L-THP after cerebral I/R injury. Thus, L-THP may provide a potential therapeutic approach for the treatment of ischemic stroke. Graphical Abstract ᅟ.

CFTR prevents neuronal apoptosis following cerebral ischemia reperfusion via regulating mitochondrial oxidative stress.

The cystic fibrosis transmembrane conductance regulator (CFTR) is linked to cell apoptosis and abundantly expressed in brain tissue. Mitochondrial oxidative stress plays a key role in activating apoptotic pathway following cerebral ischemia reperfusion (IR) injury. Reduced glutathione (GSH) is exclusively synthesized in cytosol but distributed in mitochondria. In the present study, we investigated whether CFTR affected mitochondrial oxidative stress via regulating GSH and thereby protected neurons against apoptosis following cerebral IR. Brains were subjected to global IR by four-vessel occlusion and CFTR activator forskolin (FSK) was used in vivo. CFTR silence was performed in vitro for neurons by RNA interference. We found that FSK suppressed neuronal apoptosis whereas CFTR silence enhanced neuronal apoptosis. FSK prevented the elevations in reactive oxygen species (ROS) and caspase activities while FSK inhibited the reductions in complex I activity and mitochondrial GSH level following IR. FSK decreased mitochondrial oxidative stress partially and preserved mitochondrial function. On the contrary, CFTR silence exaggerated mitochondrial dysfunction. CFTR loss increased hydrogen peroxide (H2O2) level and decreased GSH level in mitochondria. Importantly, we showed that CFTR was located on mitochondrial membrane. GSH transport assay suggested that GSH decrease may be a consequence not a reason for mitochondrial oxidative stress mediated by CFTR disruption. Our results highlight the central role of CFTR in the pathogenesis of cerebral IR injury. CFTR regulates neuronal apoptosis following cerebral IR via mitochondrial oxidative stress-dependent pathway. The mechanism of CFTR-mediated mitochondrial oxidative stress needs further studies. KEY MESSAGES: CFTR activation protects brain tissue against IR-induced apoptosis and oxidative stress. CFTR disruption enhances H2O2-induced neuronal apoptosis and CFTR loss leads to mitochondrial oxidative stress. CFTR regulates IR-induced neuronal apoptosis via mitochondrial oxidative stress. CFTR may be a potential therapeutic target to cerebral IR damage.

Circular RNA expression alterations are involved in OGD/R-induced neuron injury

Cerebral ischemia-reperfusion injury (IRI) is a common clinical pathological process, and it is a key step in causing further ischemic organ damage. The mechanism of cerebral IRI is still not fully understood, leading to a lack of effective treatment. It has been demonstrated that circular RNAs (circRNAs) can act as miRNA sponges and play an important role in regulating gene expression through a circRNA-miRNA-gene pathway. The specific role of circRNAs in the pathogenesis of cerebral IRI, however, is still unclear. Thus, in the present study, we investigated circRNA expression differences in HT22 cells with oxygen–glucose deprivation/reoxygenation (OGD/R) versus normal controls. The results from circRNA microarrays revealed that 15 circRNAs were significantly altered in the OGD/R model (p < 0.05) compared with the control group. Among them, 3 were significantly up-regulated, and the other 12 were down-regulated. Furthermore, the up-regulated expression of mmu-circRNA-015947 was verified using quantitative real-time polymerase chain reaction (qRT-PCR). Bioinformatics analysis revealed that up-regulated expression of mmu-circRNA-015947 could interact with miRNAs (mmu-miR-188-3p, mmu-miR-329-5p, mmu-miR-3057-3p, mmu-miR-5098 and mmu-miR-683) and thereby enhance target gene expression. KEGG pathway analysis predicted that mmu-circRNA-015947 may participate in apoptosis-related, metabolism-related and immune-related pathways, which are known to be involved in the pathogenesis of IRI. This research suggests that the overlapping expression of mmu-circRNA-015947 might be involved in the process of cerebral IRI and presents a novel molecular target for clinical therapy.

The Possible Damaged Mechanism and the Preventive Effect of Monosialotetrahexosylganglioside in a Rat Model of Cerebral Ischemia-Reperfusion Injury

The pathogenesis of cerebral ischemia-reperfusion injury (CIRI) is not completely clear and therapies are limited now. Therefore, our study aimed to investigate the possible pathogenesis and preventive approach of CIRI through analyzing changes of aspartate (Asp), glutamate (Glu), mitochondrial calcium (MCa), calmodulin (CaM), and malondialdehyde (MDA) contents and ultramicropathology in hippocampus and cerebral cortex of ischemic susceptible injured regions and the effect of monosialotetrahexosylganglioside (GM1) in the rat model of CIRI. Contents of Asp, Glu, MCa, CaM, and MDA in hippocampus and cerebral cortex tissues were measured by a high-performance liquid chromatography, atomic absorption spectrophotometer, and ordinary spectrophotometer, respectively, changes of ultramicrostructure in neurons of the hippocampus CA1 region and frontal cerebral cortex were observed by a transmission electron microscope. Contents of Asp and Glu in hippocampus and cerebral cortex tissues of CIRI groups significantly decreased and contents of MCa, CaM, and MDA significantly increased than those in control groups, and the ultramicrostructure in neurons of the hippocampus CA1 region and frontal cerebral cortex revealed a significant damaged change, and GM1 significantly ameliorated changes of Asp, Glu, MCa, CaM, and MDA contents in hippocampus and ultrastructural changes in neurons of the hippocampus CA1 region and frontal cerebral cortex. Our findings further support that the abnormal release and/or reuptake of excitatory amino acid neurotransmitters, the disordered calcium homeostasis and the excessive production and/or reduced elimination of reactive oxygen species contribute to the pathogenesis of CIRI, and GM1 can partially prevent these pathogenesises to exert the protective effect on CIRI.

Abstract TP268: Huwe1 Is Neuroprotective Against Monkey Cerebral Ischemia-reperfusion Injury By Regulating P53- And Mcl1- Dependent Apoptotic Pathways

Background: Neural cell apoptosis is one of the dominated mechanisms of cerebral ischemia-reperfusion injury (IRI). Anti-apotosis factor Mcl1 and apotosis factor p53 have been confirmed to play crucial roles in the apoptotic regulation previously. Mcl1 and p53 are the substrates of the ubiquitin ligases Huwe1, and which has been suggested as a potent therapeutic target in regulating cell apoptosis. Our aim is to explore whether Huwe1 modulates neural apoptosis by degrading Mcl1, p53 in cerebral ischemia-reperfusion injury. Methods: Eight health male adult rhesus monkeys were recruited in our study and were randomly devided into four groups: sham, PBS, siRNA silencing Huwe1 lentiviral vector and empty vector group. Except the sham group, the other groups were received corespondant intracranial injections and transient right middle cerebral occlusion (MCAO). A series of neurological evaluation and MRI were ulitized to evaluate animals. GFP flurescence was used to test transfection efficiency and apoptosis was detected by TUNEL staining. The expression level of related factors in causatum, putamen, temporal lobe and hippocampus were analyzed with western blot, QPCR, and immunohistochemistry. Results: The neurological deficit was much more serious in the silencing Huwe1 group than both empty vector and PBS group. The expression of Huwe1, Mcl1 and P53 were appeared a tendency to upregulate in the right caudatum and putament in model group than sham group in both protein and nucleotides level. Moreover, the number of apoptosis cells tested by TUNEL was the highest in the silencing Huwe 1 group, induced by decreasing expression of Huwe1 and obviously upragulating P53 expression. However, none of them has significant difference in other remote positions. Conclusion: Our study provides a potential novel mechnism that Huwe1 palys an important role in regulating neural apoptosis by regadading Mcl1 and P53 in cerebral IRI. In addition, for the first time, we confirm that the anti-apoptosis factor Mcl1 engages in the pathogenesis of cerebral IRI. It is possible that Huwe1 can be a potential therapeutic target for cerebral ischemia.

The Alternative Complement Pathway Propagates Inflammation and Injury in Murine Ischemic Stroke

There is mounting evidence indicating an important role for complement in the pathogenesis of cerebral ischemia-reperfusion injury, or ischemic stroke. The role of the alternative complement pathway in ischemic stroke has not been investigated, and there is conflicting data on the role of the terminal pathway. In this study, we show that compared with wild-type mice, mice deficient in the alternative pathway protein factor B or mice treated with the alternative pathway inhibitor CR2-fH have improved outcomes after 60-min middle cerebral artery occlusion and 24-h reperfusion. Factor B-deficient or CR2-fH-treated mice were protected in terms of improved neurologic function and reduced cerebral infarct, demyelination, P-selectin expression, neutrophil infiltration, and microthrombi formation. Mice deficient in both the classical and lectin pathways (C1q/MBL deficient) were also protected from cerebral ischemia-reperfusion injury, and there was no detectable C3d deposition in the ipsilateral brain of these mice. These data demonstrate that the alternative pathway is not alone sufficient to initiate complement activation and indicate that the alternative pathway propagates cerebral injury via amplification of the cascade. Deficiency of C6, a component of the terminal cytolytic membrane attack complex, had no effect on outcome after ischemic stroke, indicating that the membrane attack complex is not involved in mediating injury in this model. We additionally show that the protective effect of factor B deficiency and CR2-fH treatment is sustained in the subacute stage of infarct development, adding to the clinical relevance of these findings.