Effects of extracellular vesicles from mesenchymal stem cells on oxygen-glucose deprivation/reperfusioninduced neuronal injury

BACKGROUND: Small extracellular vesicles (sEVs) from bone marrow mesenchymal stemcells (BMSCs) have shown therapeutic potential for cerebral ischemic diseases. However, themechanisms by which BMSC-derived sEVs (BMSC-sEVs) protect neurons against cerebral ischemia/reperfusion (I/R) injury remain unclear. In this study, we explored the neuroprotective effects ofBMSC-sEVs in the primary culture of rat cortical neurons exposed to oxygen-glucose deprivation andreperfusion (OGD/R) injury.METHODS: The primary cortical neuron OGD/R model was established to simulate the processof cerebral I/R in vitro. Based on this model, we examined whether the mechanism through whichBMSC-sEVs could rescue OGD/R-induced neuronal injury.RESULTS: BMSC-sEVs (20 μg/mL, 40 μg/mL) significantly decreased the reactive oxygenspecies (ROS) productions, and increased the activities of superoxide dismutase (SOD) and glutathioneperoxidase (GPx). Additionally, BMSC-sEVs prevented OGD/R-induced neuronal apoptosis in vivo, asindicated by increased cell viability, reduced lactate dehydrogenase (LDH) leakage, decreased terminaldeoxynucleotidyl transferase (TdT)-mediated dUTP nick end labeling (TUNEL) staining-positivecells, down-regulated cleaved caspase-3, and up-regulated Bcl-2/Bax ratio. Furthermore, Westernblot and flow cytometry analysis indicated that BMSC-sEV treatment decreased the expression ofphosphorylated calcium/calmodulin-dependent kinase II (p-CaMK II)/CaMK II, suppressed the increaseof intracellular calcium concentration ([Ca2+]i) caused by OGD/R in neurons.CONCLUSIONS: These results demonstrate that BMSC-sEVs have signifi cant neuroprotectiveeff ects against OGD/R-induced cell injury by suppressing oxidative stress and apoptosis, and Ca2+/CaMK II signaling pathways may be involved in this process.

Protective effect of mesenchymal stem cell-derived exosomal treatment of hippocampal neurons against oxygen-glucose deprivation/reperfusion-induced injury

BACKGROUND:Individuals who survive a cardiac arrest often sustain cognitive impairments due to ischemia-reperfusion injury.Mesenchymal stem cell(MSC)transplantation is used to reduce tissue damage,but exosomes are more stable and highly conserved than MSCs.This study was conducted to investigate the therapeutic effects of MSC-derived exosomes(MSC-Exo)on cerebral ischemia-reperfusion injury in an in vitro model of oxygen-glucose deprivation/reperfusion(OGD/R),and to explore the underlying mechanisms.METHODS:Primary hippocampal neurons obtained from 18-day Sprague-Dawley rat embryos were subjected to OGD/R treatment,with or without MSC-Exo treatment.Exosomal integration,cell viability,mitochondrial membrane potential,and generation of reactive oxygen species(ROS)were examined.Terminal deoxynucleotidyl transferase-mediated 2’-deoxyuridine 5’-triphosphate nickend labeling(TUNEL)staining was performed to detect neuronal apoptosis.Moreover,mitochondrial function-associated gene expression,Nrf2 translocation,and expression of downstream antioxidant proteins were determined.RESULTS:MSC-Exo attenuated OGD/R-induced neuronal apoptosis and decreased ROS generation(P<0.05).The exosomes reduced OGD/R-induced Nrf2 translocation into the nucleus(2.14±0.65 vs.5.48±1.09,P<0.01)and increased the intracellular expression of antioxidative proteins,including superoxide dismutase and glutathione peroxidase(17.18±0.97 vs.14.40±0.62,and 20.65±2.23 vs.16.44±2.05,respectively;P<0.05 for both).OGD/R significantly impaired the mitochondrial membrane potential and modulated the expression of mitochondrial functionassociated genes,such as PINK,DJ1,LRRK2,Mfn-1,Mfn-2,and OPA1.The abovementioned changes were partially reversed by exosomal treatment of the hippocampal neurons.CONCLUSIONS:MSC-Exo treatment can alleviate OGD/R-induced oxidative stress and dysregulation of mitochondrial function-associated genes in hippocampal neurons.Therefore,MSCExo might be a potential therapeutic strategy to prevent OGD/R-induced neuronal injury.

Neuroprotective effect and mechanism of daidzein in oxygen-glucose deprivation/reperfusion injury based on experimental approaches and network pharmacology

Background:Daidzein,phytoestrogens derived from the Pueraria lobata(Willd.)Ohwi root used in traditional Chinese medicine,has a wide range of biological activities,including antioxidant,anti-inflammatory,and neuroprotection.However,the neuroprotective role of daidzein in oxygen-glucose deprivation/reperfusion injury and its underlying mechanism are still unknown.Methods:In this study,we used pheochromocytoma cells induced by oxygen-glucose deprivation and reperfusion to study the potential effect in the protection of the nerve cells.Then,we used molecular docking simulation and network pharmacology to predict the possible targets and pharmacological pathways of daidzein.Western blot was used to verify the expression of target proteins with or without adding the inhibitors.Results:After daidzein treatment,cell vitality had an upward trend(P<0.05)and the release of lactate dehydrogenase had a downward trend(P<0.01)in dose-dependent compared with the model group by exposure to oxygen-glucose deprivation and reperfusion.Several core targets were analyzed through network pharmacology and molecular docking including catalase,peroxisome proliferator-activated receptor gamma,vascular endothelial growth factor A,interleukin-6,tumor necrosis factor,nitric oxide synthase 3,prostaglandin-endoperoxide synthase 2,and RAC-alpha serine/threonine kinase 1.These results suggest that catalase may be a first-ranked target for the neuroprotective role of daidzein.Gene Ontology enrichment analysis indicated the pathways mainly contained molecule metabolic process,while Kyoto Encyclopedia of Genes and Genomes enrichment analysis focus on pathways in terms of inflammation such as tumor necrosis factor signal pathway.Then,Western blot results showed that daidzein had a significant increase on the expression of protein catalase(P<0.01).Daidzein reversed catalase level alterations after oxygen-glucose deprivation reperfusion injury in a dose-dependent manner which was consistent with the catalase antagonists-based experiments.Conclusion:These outcomes provide new insights into the neuroprotective effect and mechanism of daidzein in oxygen-glucose deprivation/reperfusion injury.

Shuxuetong injection protects cerebral microvascular endothelial cells against oxygen-glucose deprivation reperfusion

Shuxuetong injection composed of leech(Hirudo nipponica Whitman) and earthworm(Pheretima aspergillum) has been used for the clinical treatment of acute stroke for many years in China. However, the precise neuroprotective mechanism of Shuxuetong injection remains poorly understood. Here, cerebral microvascular endothelial cells(bEnd.3) were incubated in glucose-free Dulbecco's modified Eagle's medium containing 95% N_2/5% CO_2 for 6 hours, followed by high-glucose medium containing 95% O_2 and 5% CO_2 for 18 hours to establish an oxygen-glucose deprivation/reperfusion model. This in vitro cell model was administered Shuxuetong injection at 1/32, 1/64, and 1/128 concentrations(diluted 32-, 64-, and 128-times). Cell Counting Kit-8 assay was used to evaluate cell viability. A fluorescence method was used to measure lactate dehydrogenase, and a fluorescence microplate reader used to detect intracellular reactive oxygen species. A fluorescent probe was also used to measure mitochondrial superoxide production. A cell resistance meter was used to measure transepithelial resistance and examine integrity of monolayer cells. The fluorescein isothiocyanate-dextran test was performed to examine blood-brain barrier permeability. Real-time reverse transcription polymerase chain reaction was performed to analyze mRNA expression levels of tumor necrosis factor alpha, interleukin-1β, interleukin-6, and inducible nitric oxide synthase. Western blot assay was performed to analyze expression of caspase-3, intercellular adhesion molecule 1, vascular cell adhesion molecule 1, occludin, vascular endothelial growth factor, cleaved caspase-3, B-cell lymphoma 2, phosphorylated extracellular signal-regulated protein kinase, extracellular signal-regulated protein kinase, nuclear factor-κB p65, I kappa B alpha, phosphorylated I kappa B alpha, I kappa B kinase, phosphorylated I kappa B kinase, claudin-5, and zonula occludens-1. Our results show that Shuxuetong injection increases bEnd.3 cell viability and B-cell lymphoma 2 expression, reduces cleaved caspase-3 expression, inhibits production of reactive oxygen species and mitochondrial superoxide, suppresses expression of tumor necrosis factor alpha, interleukin-1β, interleukin-6, inducible nitric oxide synthase mRNA, intercellular adhesion molecule-1, and vascular cell adhesion molecule-1, markedly increases transepithelial resistance, decreases blood-brain barrier permeability, upregulates claudin-5, occludin, and zonula occludens-1 expression, reduces nuclear factor-κB p65 and vascular endothelial growth factor expression, and reduces I kappa B alpha, extracellular signal-regulated protein kinase 1/2, and I kappa B kinase phosphorylation levels. Overall, these findings suggest that Shuxuetong injection has protective effects on brain microvascular endothelial cells after oxygen-glucose deprivation/reperfusion. Moreover, its protective effect is associated with reduction of mitochondrial superoxide production, inhibition of the inflammatory response, and inhibition of vascular endothelial growth factor, extracellular signal-regulated protein kinase 1/2, and the nuclear factor-κB p65 signaling pathway.

Silencing Huwe1 reduces apoptosis of cortical neurons exposed to oxygen-glucose deprivation and reperfusion

HECT, UBA and WWE domain-containing 1(Huwe1), an E3 ubiquitin ligase involved in the ubiquitin-proteasome system, is widely expressed in brain tissue. Huwe1 is involved in the turnover of numerous substrates, including p53, Mcl-1, Cdc6 and N-myc, thereby playing a critical role in apoptosis and neurogenesis. However, the role of Huwe1 in brain ischemia and reperfusion injury remains unclear. Therefore, in this study, we investigated the role of Huwe1 in an in vitro model of ischemia and reperfusion injury. At 3 days in vitro, primary cortical neurons were transduced with a control or shRNA-Huwe1 lentiviral vector to silence expression of Huwe1. At 7 days in vitro, the cells were exposed to oxygen-glucose deprivation for 3 hours and reperfusion for 24 hours. To examine the role of the c-Jun N-terminal kinase(JNK)/p38 pathway, cortical neurons were pretreated with a JNK inhibitor(SP600125) or a p38 MAPK inhibitor(SB203508) for 30 minutes at 7 days in vitro, followed by ischemia and reperfusion. Neuronal apoptosis was assessed by TUNEL assay. Protein expression levels of JNK and p38 MAPK and of apoptosis-related proteins(p53, Gadd45 a, cleaved caspase-3, Bax and Bcl-2) were measured by western blot assay. Immunofluorescence labeling for cleaved caspase-3 was performed. We observed a significant increase in neuronal apoptosis and Huwe1 expression after ischemia and reperfusion. Treatment with the shRNA-Huwe1 lentiviral vector markedly decreased Huwe1 levels, and significantly decreased the number of TUNEL-positive cells after ischemia and reperfusion. The silencing vector also downregulated the pro-apoptotic proteins Bax and cleaved caspase-3, and upregulated the anti-apoptotic proteins Gadd45 a and Bcl-2. Silencing Huwe1 also significantly reduced p-JNK levels and increased p-p38 levels. Our findings show that downregulating Huwe1 affects the JNK and p38 MAPK signaling pathways as well as the expression of apoptosis-related genes to provide neuroprotection during ischemia and reperfusion. All animal experiments and procedures were approved by the Animal Ethics Committee of Sichuan University, China in January 2018(approval No. 2018013).

Dynamic changes in proprotein convertase 2 activity in cortical neurons after ischemia/reperfusion and oxygen-glucose deprivation

In this study, a rat model of transient focal cerebral ischemia was established by performing 100 minutes of middle cerebral artery occlusion, and an in vitro model of experimental oxygen-glucose deprivation using cultured rat cortical neurons was established. Proprotein convertase 2 activity gradually decreased in the ischemic cortex with increasing duration of reperfusion. In cultured rat cortical neurons, the number of terminal deoxynucleotidyl transferase-mediated 2＇-deoxyuridine 5＇-triphosphate-biotin nick end labeling-positive neurons significantly increased and proprotein convertase 2 activity also decreased gradually with increasing duration of oxygen-glucose deprivation. These experimental findings indicate that proprotein convertase 2 activity decreases in ischemic rat cortex after reperfusion, as well as in cultured rat cortical neurons after oxygen-glucose deprivation. These changes in enzyme activity may play an important pathological role in brain injury.

Intervention Timing and Effect of PJ34 on Astrocytes During Oxygen-glucose Deprivation/reperfusion and Cell Death Pathways

Poly （ADP-ribose） polymerase-I （PARP-1） plays as a double edged sword in cerebral ischemia-reperfusion, hinging on its effect on the intracellular energy storage and injury severity, and the prognosis has relationship with intervention timing. During ischemia injury, apoptosis and oncosis are the two main cell death pathway sin the ischemic core. The participation of astrocytes in ische- mia-reperfusion induced cell death has triggered more and more attention. Here, we examined the pro- tective effects and intervention timing of the PARP-1 inhibitor PJ34, by using a mixed oxygen-glucose deprivation/reperfusion （OGDR） model of primary rat astrocytes in vitro, which could mimic the ische- mia-reperfusion damage in the ＂ischemic core＂. Meanwhile, cell death pathways of various P J34 treated astrocytes were also investigated. Our results showed that P J34 incubation （10 μmol/L） did not affect release of lactate dehydrogenase （LDH） from astrocytes and cell viability or survival 1 h after OGDR. Interestingly, after 3 or 5 h OGDR, P J34 significantly reduced LDH release and percentage of PI-positive cells and increased cell viability, and simultaneously increased the caspase-dependent apop- totic rate. The intervention timing study demonstrated that an earlier and longer P J34 intervention dur- ing reperfusion was associated with more apparent protective effects. In conclusion, earlier and longer PJ34 intervention provides remarkable protective effects for astrocytes in the ＂ischaemic core＂ mainly by reducing oncosis of the astrocytes, especially following serious OGDR damage.

Establishment of oxygen glucose deprivation reperfusion model of senescent SH-SY5Y cells

Obejective:To explore the establishment of an oxygen glucose deprivation/reperfusion model of senescent SH-SY5Y cells.Methods:SH-SY5Y cells were randomly divided into control(D-galactose 0 mmol/L group),D-galactose(25 mmol/L,50 mmol/L,100 mmol/L,200 mmol/L,400 mmol/L)groups,and treated with corresponding concentrations of D-galactose for 48 h.The changes of cell morphology,β-galactosidase,the cell morphology,β-galactosidase activity by microscopic observation,cell proliferation rate by EdU kit and cell survival rate by CCK-8 assay were used to determine the decaying concentration of D-galactose and to establish the senescence model.The senescent SH-SY5Y cells were randomly divided into control group(oxygen glucose deprivation without treatment group),oxygen glucose deprivation treatment(0.5 h,1 h,1.5 h,2 h)group,followed by re-glucose reoxygenation for 24 h,and CCK-8 assay for the survival rate of senescent SH-SY5Y cells.Results:There were no significant changes in cell morphology and β-gal activity in the 25 mmol/L and 50 mmol/L groups compared with the control group(P>0.05),cytosolic hypertrophy was seen in the cells of the 100 mmol/L group,chromatin fixation in the cells of the 200 mmol/L group,and massive vacuolization in the cells of the 400 mmol/L group;the positive rate ofβ-galactosidase staining in the cells of the(100-400 mmol/L)group was significantly higher compared with the control group(P<0.05),with little difference between the 100 mmol/L and 200 mmol/L groups(P>0.05);the cell proliferation ability of the(100-400 mmol/L)group was significantly decreased in a concentration-dependent manner(P<0.05);the cell survival rate was decreased in a concentration-dependent manner(P<0.05),with IC_(50) between 100 mmol/L and 200 mmol/L.The survival of senescent SH-SY5Y cells showed a time-dependent decrease in oxygen-glucose deprivation(P<0.05),with an IC_(50) close to 1 h.Conclusion:D-gal concentration of 100 mmoL/L and 48 h of cell action could establish a survival rate of about 50%of senescent SH-SY5Y cells,and oxygen glucose deprivation of senescent SH-SY5Y cells for 1 h and reperfusion for 24 h could establish an oxygen glucose deprivation/reperfusion model of senescent SH-SY5Y cells with a survival rate close to 50%.

IcarisideⅡ alleviates oxygen-glucose deprivation and reoxygenation-induced PC12 celloxidative injury by activating Nrf2 / SIRT3signaling pathway

OBJECTIVE To investigate icariside(ICS)Ⅱ protects against PC12 cel damage induced by oxygen-glucose deprivation and reoxygenation and explore its mechanism.METHODS The oxidative stress injury model was induced by oxygen-glucose deprivation/reoxygenation(OGD/R) 2 h/24 h in PC12 cells.N-acetyl-lcysteine(NAC),a classical anti-oxidant,was used as positive control.Pharmacodynamic experimental study groups as follows:control,control+ICS Ⅱ50 μmol·L^(-1),OGD/R,OGD/R+ICSⅡ 12.5 μmol·L^(-1),OGD/R + ICS Ⅱ 25 μmol·L^(-1),OGD/R + ICS Ⅱ50 μmol·L^(-1),and OGD/R+NAC 100 μmol·L^(-1) groups.Cell viability and lactate dehydrogenase(LDH) leakage rate were measured by MTT assay and LDH ELISA kit,respectively.Moreover,reactive oxygen species(ROS) ELISA kit was used for detection of intracellular ROS generation,Mito-SOX fluorescence staining was used for detecting production of ROS in mitochondria and mitochondrial membrane potential(MMP)was detected by rhodamine 123 dye.In addition,PC12 cells apoptosis was detected by one-step TUNEL assay.Furthermore,the expressions of nuclear factor erythroid 2-related factors(Nrf2),Keap1,HO^(-1),NQO^(-1),silent information regulator 3(SIRT3),IDH2,Bax,Bcl-2 and caspase 3 were detected by Western blotting analysis.RESULTS The results of MTT and LDH assay showed that OGD/R reduced the cell viability and improved LDH release compared with the control or ICSⅡ 50 μmol·L^(-1) alone(P<0.01).Meanwhile,OGD/R not only increased intracellular and mitochondrial ROS generation,but also elevated the fluorescence intensity of TUNEL staining,at the same time,the MMP was declined when challenged by OGD/R.Furthermore,the Western blotting results showed that OGD/R induced the increase in the expression of cytoplasm-Nrf2,Keap1,Bax and cleaved-caspase 3 level,while the decrease in the expression of nucleus-Nrf2,HO^(-1),NQO^(-1),SIRT3,IDH2 and Bcl-2(P<0.05).However,ICS Ⅱ significantly increased the viability of PC12 cells and reduced LDH leakage(P<0.01).Notably,ICS Ⅱ also suppressed ROS generation both in the intracellular and mitochondria,as well as restored MMP.It was also worthy to note that ICS Ⅱ decreased the expressions of cytoplasmNrf2,Keap1,Bax and the level of cleaved-caspase3,whereas,it increased the expressions of nucleus-Nrf2,HO^(-1),NQO^(-1),SIRT3,IDH2 and Bcl-2(P<0.05).CONCLUSION ICSⅡ reduced OGD/Rinduced oxidative damage in PC12 cells under the laboratory conditions,and its underlying mechanism may be related to the regulation of Nrf2/SIRT3 signaling pathway.

Protective mechanisms of micro RNA-27a against oxygen-glucose deprivation-induced injuries in hippocampal neurons

Hypoxic injuries during fetal distress have been shown to cause reduced expression of micro RNA-27a（mi R-27a）,which regulates sensitivity of cortical neurons to apoptosis.We hypothesized that miR-27 a overexpression attenuates hypoxia- and ischemia-induced neuronal apoptosis by regulating FOXO1,an important transcription factor for regulating the oxidative stress response.miR-27 a mimic was transfected into hippocampal neurons to overexpress miR-27 a.Results showed increased hippocampal neuronal viability and decreased caspase-3 expression.The luciferase reporter gene system demonstrated that mi R-27 a directly binded to FOXO1 3′UTR in hippocampal neurons and inhibited FOXO1 expression,suggesting that FOXO1 was the target gene for mi R-27 a.These findings confirm that mi R-27 a protects hippocampal neurons against oxygen-glucose deprivation-induced injuries.The mechanism might be mediated by modulation of FOXO1 and apoptosis-related gene caspase-3 expression.

Rac1 relieves neuronal injury induced by oxygen-glucose deprivation and re-oxygenation via regulation of mitochondrial biogenesis and function

Certain microRNAs(miRNAs)can function as neuroprotective factors after reperfusion/ischemia brain injury.miRNA-142-3p can participate in the occurrence and development of tumors and myocardial ischemic injury by negatively regulating the activity of Rac1,but it remains unclear whether miRNA-142-3p also participates in cerebral ischemia/reperfusion injury.In this study,a model of oxygen-glucose deprivation/re-oxygenation in primary cortical neurons was established and the neurons were transfected with miR-142-3p agomirs or miR-142-3p antagomirs.miR-142-3p expression was down-regulated in neurons when exposed to oxygen-glucose deprivation/re-oxygenation.Over-expression of miR-142-3p using its agomir remarkably promoted cell death and apoptosis induced by oxygen-glucose deprivation/re-oxygenation and improved mitochondrial biogenesis and function,including the expression of peroxisome proliferator-activated receptor-γcoactivator-1α,mitochondrial transcription factor A,and nuclear respiratory factor 1.However,the opposite effects were produced if miR-142-3p was inhibited.Luciferase reporter assays verified that Rac Family Small GTPase 1(Rac1)was a target gene of miR-142-3p.Over-expressed miR-142-3p inhibited NOX2 activity and expression of Rac1 and Rac1-GTPase(its activated form).miR-142-3p antagomirs had opposite effects after oxygen-glucose deprivation/re-oxygenation.Our results indicate that miR-142-3p down-regulates the expression and activation of Rac1,regulates mitochondrial biogenesis and function,and inhibits oxygen-glucose deprivation damage,thus exerting a neuroprotective effect.The experiments were approved by the Committee of Experimental Animal Use and Care of Central South University,China(approval No.201703346)on March 7,2017.

Vav1 promotes inflammation and neuronal apoptosis in cerebral ischemia/reperfusion injury by upregulating microglial and NLRP3 inflammasome activation

Microglia,which are the resident macrophages of the central nervous system,are an important part of the inflammatory response that occurs after cerebral ischemia.Vav guanine nucleotide exchange factor 1(Vav1) is a guanine nucleotide exchange factor that is related to microglial activation.However,how Vav1 participates in the inflammato ry response after cerebral ischemia/reperfusion inj ury remains unclea r.In this study,we subjected rats to occlusion and repe rfusion of the middle cerebral artery and subjected the BV-2 mic roglia cell line to oxygen-glucose deprivatio n/reoxygenation to mimic cerebral ischemia/repe rfusion in vivo and in vitro,respectively.We found that Vav1 levels were increased in the brain tissue of rats subjected to occlusion and reperfusion of the middle cerebral arte ry and in BV-2 cells subjected to oxygen-glucose deprivation/reoxygenation.Silencing Vav1 reduced the cerebral infarct volume and brain water content,inhibited neuronal loss and apoptosis in the ischemic penumbra,and im p roved neurological function in rats subjected to occlusion and repe rfusion of the middle cerebral artery.Further analysis showed that Vav1 was almost exclusively localized to microglia and that Vav1 downregulation inhibited microglial activation and the NOD-like receptor pyrin 3(NLRP3) inflammasome in the ischemic penumbra,as well as the expression of inflammato ry facto rs.In addition,Vov1 knoc kdown decreased the inflammatory response exhibited by BV-2 cells after oxygen-glucose deprivation/reoxyge nation.Taken together,these findings show that silencing Vav1 attenuates inflammation and neuronal apoptosis in rats subjected to cerebral ischemia/repe rfusion through inhibiting the activation of mic roglia and NLRP3 inflammasome.

Upregulation of CDGSH iron sulfur domain 2 attenuates cerebral ischemia/reperfusion injury

CDGSH iron sulfur domain 2 can inhibit ferroptosis,which has been associated with cerebral ischemia/reperfusion,in individuals with head and neck cancer.Therefore,CDGSH iron sulfur domain 2 may be implicated in cerebral ischemia/reperfusion injury.To validate this hypothesis in the present study,we established mouse models of occlusion of the middle cerebral artery and HT22 cell models of oxygen-glucose deprivation and reoxygenation to mimic cerebral ischemia/reperfusion injury in vivo and in vitro,respectively.We found remarkably decreased CDGSH iron sulfur domain 2 expression in the mouse brain tissue and HT22 cells.When we used adeno-associated virus and plasmid to up-regulate CDGSH iron sulfur domain 2 expression in the brain tissue and HT22 cell models separately,mouse neurological dysfunction was greatly improved;the cerebral infarct volume was reduced;the survival rate of HT22 cells was increased;HT22 cell injury was alleviated;the expression of ferroptosis-related glutathione peroxidase 4,cystine-glutamate antiporter,and glutathione was increased;the levels of malondialdehyde,iron ions,and the expression of transferrin receptor 1 were decreased;and the expression of nuclear-factor E2-related factor 2/heme oxygenase 1 was increased.Inhibition of CDGSH iron sulfur domain 2 upregulation via the nuclear-factor E2-related factor 2 inhibitor ML385 in oxygen-glucose deprived and reoxygenated HT22 cells blocked the neuroprotective effects of CDGSH iron sulfur domain 2 up-regulation and the activation of the nuclear-factor E2-related factor 2/heme oxygenase 1 pathway.Our data indicate that the up-regulation of CDGSH iron sulfur domain 2 can attenuate cerebral ischemia/reperfusion injury,thus providing theoretical support from the perspectives of cytology and experimental zoology for the use of this protein as a therapeutic target in patients with cerebral ischemia/reperfusion injury.

Curcumin pretreatment and post-treatment both improve the antioxidative ability of neurons with oxygen-glucose deprivation

Recent studies have shown that induced expression of endogenous antioxidative enzymes thr- ough activation of the antioxidant response element/nuclear factor erythroid 2-related factor 2 （Nrf2） pathway may be a neuroprotective strategy. In this study, rat cerebral cortical neurons cultured in vitro were pretreated with 10 ktM curcumin or post-treated with 5 pM curcumin, respectively before or after being subjected to oxygen-glucose deprivation and reoxygenation for 24 hours. Both pretreatment and post-treatment resulted in a significant decrease of cell injury as indicated by propidium iodide/Hoechst 33258 staining, a prominent increase of Nrf2 protein expression as indicated by western blot analysis, and a remarkable increase of protein expression and enzyme activity in whole cell lysates of thioredoxin before ischemia, after ischemia, and after reoxygenation. In addition, post-treatment with curcumin inhibited early DNA/RNA oxidation as indicated by immunocytochemistry and increased nuclear Nrf2 protein by inducing nuclear accumulation of Nrf2. These findings suggest that curcumin activates the expression of thi- oredoxin, an antioxidant protein in the Nrf2 pathway, and protects neurons from death caused by oxygen-glucose deprivation in an in vitro model of ischemia/reperfusion. We speculate that pharmacologic stimulation of antioxidant gene expression may be a promising approach to neu- roprotection after cerebral ischemia.

Activin A prevents neuron-like PC12 cell apoptosis after oxygen-glucose deprivation

In this study, PC12 cells were induced to differentiate into neuron-like cells using nerve growth factor, and were subjected to oxygen-glucose deprivation. Cells were treated with 0, 10, 20, 30, 50, 100 ng/mL exogenous Activin A. The 3-（4,5-dimethyl-2-thiazolyl）-2,5-diphenyl tetrazolium bromide assay and Hoechst 33324 staining showed that the survival percentage of PC12 cells significantly decreased and the rate of apoptosis significantly increased after oxygen-glucose deprivation. Exogenous Activin A significantly increased the survival percentage of PC12 cells in a dose-dependent manner. Reverse transcription-PCR results revealed a significant increase in Activin receptor IIA, Smad3 and Smad4 mRNA levels, which are key sites in the Activin A/Smads signaling pathway, in neuron-like cells subjected to oxygen-glucose deprivation, while mRNA expression of the apoptosis-regulation gene caspase-3 decreased. Our experimental findings indicate that exogenous Activin A plays an anti-apoptotic role and protects neurons by means of activating the Activin A/Smads signaling pathway.

Expression of hypoxia-inducible factor 1 alpha and oligodendrocyte lineage gene-1 in cultured brain slices after oxygen-glucose deprivation

Oligodendrocyte lineage gene-1 expressed in oligodendrocytes may trigger the repair of neuronal myelin impairment, and play a crucial role in myelin repair. Hypoxia-inducible factor la, a transcription factor, is of great significance in premature infants with hypoxic-ischemic brain damage There is little evidence of direct regulatory effects of hypoxia-inducible factor le on oligodendrocyte lineage gene-l. In this study, brain slices of Sprague-Dawley rats were cultured and subjected to oxygen-glucose deprivation. Then, slices were transfected with hypoxia-inducible factor la or oligodendrocyte lineage gene-1. The expression levels of hypoxia-inducible factor la and oligodendrocyte lineage gene-1 were significantly up-regulated in rat brains prior to transfection, as detected by immunohistochemical staining. Eight hours after transfection of slices with hypoxia-inducible factor la, oligodendrocyte lineage gene-1 expression was upregulated, and reached a peak 24 hours after transfection. Oligodendrocyte lineage gene-1 transfection induced no significant differences in hypoxia-inducible factor la levels in rat brain tissues with oxygen-glucose deprivation. These experimental findings indicate that hypoxia-inducible factor la can regulate oligodendrocyte lineage gene-1 expression in hypoxic brain tissue, thus repairing the neural impairment.

Neuroprotective effect of penehyclidine hydrochloride on focal cerebral ischemia-reperfusion injury

Penehyclidine hydrochloride can promote microcirculation and reduce vascular permeability. However, the role of penehyclidine hydrochlodde in cerebral ischemia-reperfusion injury remains unclear. In this study, in vivo middle cerebral artery occlusion models were established in experimental rats, and penehyclidine hydrochloride pretreatment was given via intravenous injection prior to model establishment. Tetrazolium chloride, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling and immunohistochemical staining showed that, penehyclidine hydrochloride pretreatment markedly attenuated neuronal histopathological changes in the cortex, hippocampus and striatum, reduced infarction size, increased the expression level of BcI-2, decreased the expression level of caspase-3, and inhibited neuronal apoptosis in rats with cerebral ischemia-reperfusion injury. Xanthine oxidase and thiobarbituric acid chromogenic results showed that penehyclidine hydrochloride upregulated the activity of superoxide dismutase and downregulated the concentration of malondialdehyde in the ischemic cerebral cortex and hippocampus, as well as reduced the concentration of extracellular excitatory amino acids in rats with cerebral ischemia-reperfusion injury. In addition, penehyclidine hydrochloride inhibited the expression level of the NR1 subunit in hippocampal nerve cells in vitro following oxygen-glucose deprivation, as detected by PCR. Experimental findings indicate that penehyclidine hydrochloride attenuates neuronal apoptosis and oxidative stress injury after focal cerebral ischemia-reperfusion, thus exerting a neuroprotective effect.

The pathways by which mild hypothermia inhibits neuronal apoptosis following ischemia/reperfusion injury

Several studies have demonstrated that mild hypothermia exhibits a neuroprotective role and it can inhibit endothelial cell apoptosis following ischemia/reperfusion injury by decreasing casp- ase-3 expression, It is hypothesized that mild hypothermia exhibits neuroprotective effects on neurons exposed to ischemia/reperfusion condition produced by oxygen-glucose deprivation. Mild hypothermia significantly reduced the number of apoptotic neurons, decreased the expres- sion of pro-apoptotic protein Bax and increased mitochondrial membrane potential, with the peak of anti-apoptotic effect appearing between 6 and 12 hours after the injury. These findings indicate that mild hypothermia inhibits neuronal apoptosis following ischemia/reperfusion injury by protecting the mitochondria and that the effective time window is 6-12 hours after ischemia/reperfusion injury.

Long non-coding RNA MEG3 regulates autophagy after cerebral ischemia/reperfusion injury

Severe cerebral ischemia/reperfusion injury has been shown to induce high-level autophagy and neuronal death.Therefore,it is extremely important to search for a target that inhibits autophagy activation.Long non-coding RNA MEG3 participates in autophagy.However,it remains unclear whether it can be targeted to regulate cerebral ischemia/reperfusion injury.Our results revealed that in oxygen and glucose deprivation/reoxygenation-treated HT22 cells,MEG3 expression was obviously upregulated,and autophagy was increased,while knockdown of MEG3 expression greatly reduced autophagy.Furthermore,MEG3 bound mi R-181 c-5 p and inhibited its expression,while mi R-181 c-5 p bound to autophagy-related gene ATG7 and inhibited its expression.Further experiments revealed that mir-181 c-5 p overexpression reversed the effect of MEG3 on autophagy and ATG7 expression in HT22 cells subjected to oxygen and glucose deprivation/reoxygenation.In vivo experiments revealed that MEG3 knockdown suppressed autophagy,infarct volume and behavioral deficits in cerebral ischemia/reperfusion mice.These findings suggest that MEG3 knockdown inhibited autophagy and alleviated cerebral ischemia/reperfusion injury through the mi R-181 c-5 p/ATG7 signaling pathway.Therefore,MEG3 can be considered as an intervention target for the treatment of cerebral ischemia/reperfusion injury.This study was approved by the Animal Ethics Committee of the First Affiliated Hospital of Zhengzhou University,China(approval No.XF20190538)on January 4,2019.

Icariside Ⅱ, a PDE5 inhibitor, attenuates cerebral ischemia/reperfusion injury through activating BDNF/TrkB/CREB signaling pathway

OBJECTIVE To explore the effects and mechanism of icariside Ⅱ(ICS Ⅱ),a pharmacologically active compound derived from herbal Epimedii with previous study-proved phosphodiesterase 5(PDE5) inhibitors,was investigated in vivo using a middle cerebral artery occlusion/reperfusion(MCAO/R) model in rats and in vitro using an oxygen-glucose deprivation/reperfusion(OGD/R) model in primary hippocampal neurons.METHODS Laser Doppler flowmeter was introduced to examine the cerebral blood flow of MCAO/R rats.The neurological deficits scores,brain water content and infarction volume were assessed after MCAO/R.OGD/R-induced primary hippocampal neuronal injury and apoptosis were examined by MTT,lactate dehydrogenase(LDH) release,TUNEL staining and flow cytometry,respectively.Expressions of PDE5 A and memory-related signaling pathways were measured using Western blotting analysis.The direct interaction between ICS Ⅱand PDE5 was further evaluated by molecular docking.RESULTS ICS Ⅱ significantly decreased the infraction volume in MCAO/R rats.Furthermore,ICS Ⅱ significantly abrogated OGD/R-induced hippocampal neuronal death.Moreover,ICSⅡ not only effectively restored the 3′ 5′-cyclic guanosine monophosphate(cGMP) level and protein kinase G(PKG) activity both in vivo and in vitro,but also increased brain-derived neurotrophic factor(BDNF),tyrosine protein kinase B(TrkB) and cAMP response element-binding protein(CREB) expressions,thereby inhibited hippocampal neuronal apoptosis.Mechanistically,the beneficial effects of ICS Ⅱ was attributed to its activation of the PKG/TrkB/BDNF via increasing BDNF expression,evidenced by that the inhibition effects of ICSⅡ was abrogated by Rp-8-BrcGMPS,a PKG inhibitor,or ANA-12,a TrkB inhibitor.ICSⅡ also decreased both protein level and activity of PDE5.Notably,ICSⅡ might effectively bind and inhibite PDE5 as demonstrated by relatively high binding score.CONCLUSION ICSⅡ significantly protect against cerebral ischemia/reperfusion injury in rats and rescues OGD/Rinduced hippocampal neuronal injury,and the underling mechanisms are,at least partly,due to inhibition of PDE5 and activation of BDNF/TrkB/CREB signaling pathway.Hence ICS Ⅱ may be an effective agent for combating cerebral ischemia/reperfusion injury.