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%.

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).

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.

Salidroside attenuates oxygen and glucose deprivation-induced neuronal injury by inhibiting ferroptosis

Objective: To evaluate the effect of salidroside on oxygen and glucose deprivation(OGD)-treated NT2 cells and its underlying mechanisms of action.Methods: Retinoic acid was used to induce the differentiation of NT2 cells into neurons. The effects of salidroside on survival, apoptosis, inflammatory response, and oxidative stress of neurons undergoing OGD were evaluated. Using precursor cells as controls, the effect of salidroside on the differentiation progression of OGDtreated cells was evaluated. In addition, the effect of erastin, a ferroptosis inducer, on NT2 cells was examined to investigate the underlying mechanisms of neuroprotective action of salidroside.Results: Salidroside alleviated the effects of OGD on neuronal survival, apoptosis, inflammation, and oxidative stress, and promoted NT2 cell differentiation. Moreover, salidroside prevented ferroptosis of OGD-treated cells, which was abolished following erastin treatment, indicating that ferroptosis mediated the regulatory pathway of salidroside.Conclusions: Salidroside attenuates OGD-induced neuronal injury by inhibiting ferroptosis and promotes neuronal differentiation.

Dehydrocostuslactone protects against oxygen-glucose deprivation/reperfusion-induced injury by inhibiting autophagy and apoptosis in PC12 cells

OBJECTIVE TO investigate the neural protection of dehydrocostus lactone(DHL)against neuronal injury induced by oxygen and glucose deprivation/reperfusion(OGD/R)in differentiated PC12 cells.METHODS We used a cellular model of 2 h of OGD and 24 h of reperfusion to mimic cerebral ischemia-reperfusion injury.Cell viability was used to reflect the degree of OGD/R-induced injury.Cells were treated with DHL during the reperfusion phase.Cell Counting Kit(CCK-8)and LDH assays were performed to determine the optimal dose of DHL and cell viability.Flow cytometry analysis and Monodansylcadaverine(MDC)staining were then conducted to detect apoptosis rate and autophagosome formation after OGD/R in PC12 cells.Immunofluorescence and Western blotting analyses were used to detect the expres⁃sion of proteins associated with autophagy and apoptosis.RESULTS OGD/R significantly decreased cell viability and increased apoptosis rate.The expression levels of autophagy-related proteins,namely,LC3 and Beclin-1,and apoptosisrelated proteins,namely,Bax and caspase-3 increased,but that of the anti-apoptosis Bcl-2 protein decreased.However,DHL attenuated OGD/R-induced neuronal injury through inhibition of apoptosis and autophagy properties by modulating au⁃tophagy-associated proteins(LC3 and Beclin-1)and apoptosis-modulating proteins(caspase-3 and Bcl-2/Bax).CONCLU⁃SION Our data provide an evidence for the neuroprotective effect of DHL against ischemic neuronal injury.Hence,DHL could be a promising candidate for treatment of ischemic stroke.

Hyperbaric oxygen protects against PC12 and H9C2 cell damage caused by oxygen-glucose deprivation/reperfusion via the inhibition of cell apoptosis and autophagy

In this study,we investigated the protective effect of hyperbaric oxygen(HBO)on PC12 and H9C2 cell damage caused by oxygen-glucose deprivation/reperfusion and its possible mechanism.PC12 and H9C2 cell oxygen-glucose deprivation/reperfusion model were established.Cells were divided into a control group,model group,hyperbaric air(HBA)group and HBO group.The cell viability was detected by the CCK8 assay.Hoechst 33342 and PI staining assays and mitochondrial membrane potential(MMP)assays were used to detect cell apoptosis.The ultrastructure of cells,including autophagosomes,lysosomes,and apoptosis,were examined using a transmission electron microscope.The expression of autophagy-related proteins was detected by cellular immunofluorescence and immunocytochemistry.Our results showed that HBO can significantly improve the vitality of damaged PC12 and H9C2 cells caused by oxygen–glucose deprivation/reperfusion.HBO can significantly inhibit apoptosis of PC12 and H9C2 cells caused by oxygenglucose deprivation/reperfusion.Importantly,we found that the protective mechanism of PC12 and H9C2 cell damage caused by oxygen-glucose deprivation/reperfusion may be related to the inhibition of the autophagy pathway.In this study,the results of cellular immunofluorescence and immunocytochemistry experiments showed that the 4E-BP1,p-AKt and mTOR levels of PC12 and H9C2 cells in the model group decreased,while the levels of LC3B,Atg5 and p53 increased.However,after HBO treatment,these autophagy-related indexes were reversed.In addition,observation of the cell ultrastructure with transmission electron microscopy found that in the model group,a significant increase in the number of autophagic vesicles was observed.In the HBO group,a decrease in autophagic vesicles was observed.The study demonstrated that hyperbaric oxygen protects against PC12 and H9C2 cell damage caused by oxygen-glucose deprivation/reperfusion via the inhibition of cell apoptosis and autophagy.

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.

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.

Hyodeoxycholic acid protects the neurovascular unit against oxygen-glucose deprivation and reoxygenation-induced injury in vitro

Calculus bovis is commonly used for the treatment of stroke in traditional Chinese medicine. Hyodeoxycholic acid(HDCA) is a bioactive compound extracted from calculus bovis. When combined with cholic acid, baicalin and jas-minoidin, HDCA prevents hypoxia-reoxygenation-induced brain injury by suppressing endoplasmic reticulum stress-mediated apoptotic signaling. However, the effects of HDCA in ischemic stroke injury have not yet been studied. Neurovascular unit(NVU) dysfunction occurs in ischemic stroke. Therefore, in this study, we investigated the effects of HDCA on the NVU under ischemic conditions in vitro. We co-cultured primary brain microvascular endothelial cells, neurons and astrocytes using a transwell chamber co-culture system. The NVU was pre-treated with 10.16 or 2.54 μg/mL HDCA for 24 hours before exposure to oxygen-glucose deprivation for 1 hour. The cell counting kit-8 assay was used to detect cell activity. Flow cytometry and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling were used to assess apoptosis. Enzyme-linked immunosorbent assay was used to measure the expression levels of inflammatory cytokines, including interleukin-1β, interleukin-6 and tumor necrosis factor-α, and neurotrophic factors, including brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor. Oxidative stress-related factors, such as superoxide dismutase, nitric oxide, malondialdehyde and γ-glutamyltransferase, were measured using kits. Pretreatment with HDCA significantly decreased blood-brain barrier permeability and neuronal apoptosis, significantly increased transendothelial electrical resistance and γ-glutamyltransferase activity, attenuated oxidative stress damage and the release of inflammatory cytokines, and increased brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor expression. Our findings suggest that HDCA maintains NVU morphological integrity and function by modulating inflammation, oxidation stress, apoptosis, and the expression of neurotrophic factors. Therefore, HDCA may have therapeutic potential in the clinical management of ischemic stroke. This study was approved by the Ethics Committee of Experimental Animals of Beijing University of Chinese Medicine(approval No. BUCM-3-2016040201-2003) in April 2016.

Selenocysteine antagonizes oxygen glucose deprivation-induced damage to hippocampal neurons

Designing and/or searching for novel antioxidants against oxygen glucose effective strategy for the treatment of human isdlemic stroke. Selenium is deprivation （OGD）-induced oxidative damage represents an an essential trace dement, which is beneficial in the chemo- prevention and chemotherapy of cerebral ischemic stroke. The underlying mechanisms for its therapeutic effects, however, are not well documented. Selenocysteine （SeC） is a selenium-containing amino acid with neuroprotective potential. Studies have shown that SeC can reduce irradiation-induced DNA apoptosis by reducing DNA damage. In this study, the in vitro protective potential and mechanism of action of SeC against OGD-induced apoptosis and neurotoxicity were evaluated in HT22 mouse hippocampal neurons. We cultured HT22 cells in a glucose-free medium containing 2 mM Na2S402, which formed an OGD environment, for 90 minutes. Findings from MTT, flow cytometry and TUNEL staining showed obvious cytotoxicity and apoptosis in HT22 cells in the OGD condition. The activation of Caspa se-7 and Caspase-9 further revealed that OGD-induced apoptosis of HT22 cells was mainly achieved by triggering a mitochondrial-medi- ated pathway. Moreover, the OGD condition also induced serious DNA damage through the accumulation of reactive oxygen species and superoxide anions. However, SeC pre-treatment for 6 hours effectively inhibited OGD-induced cytotoxicity and apoptosis in HT22 cells by inhibiting reactive oxygen species-mediated oxidative damage. Our findings provide evidence that SeC has the potential to suppress OGD-induced oxidative damage and apoptosis in hippocampal neurons.

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.

Oxygen Glucose Deprivation Post-conditioning Protects Cortical Neurons against Oxygen-glucose Deprivation Injury: Role of HSP70 and Inhibition of Apoptosis

In the present study, we examined the effect of oxygen glucose deprivation（OGD） post-conditioning（PostC） on neural cell apoptosis in OGD-PostC model and the protective effect on primary cortical neurons against OGD injury in vitro. Four-h OGD was induced by OGD by using a specialized and humidified chamber. To initiate OGD, culture medium was replaced with de-oxygenated and glucose-free extracellular solution-Locke＇s medium. After OGD treatment for 4 h, cells were then allowed to recover for 6 h or 20 h. Then lactate dehydrogenase（LDH） release assay, Western blotting and flow cytometry were used to detect cell death, protein levels and apoptotic cells, respectively. For the PostC treatment, three cycles of 15-min OGD, followed by 15 min normal cultivation, were applied immediately after injurious 4-h OGD. Cells were then allowed to recover for 6 h or 20 h, and cell death was assessed by LDH release assay. Apoptotic cells were flow cytometrically evaluated after 4-h OGD, followed by re-oxygenation for 20 h（O4/R20）. In addition, Western blotting was used to examine the expression of heat-shock protein 70（HSP70）, Bcl-2 and Bax. The ratio of Bcl-2 expression was（0.44±0.08）% and（0.76±0.10）%, and that of Bax expression was（0.51±0.05）% and（0.39±0.04）%, and that of HSP70 was（0.42±0.031）% and（0.72±0.045）% respectively in OGD group and PostC group. After O4/R6, the rate of neuron death in PostC group and OGD groups was（28.96±3.03）% and（37.02±4.47）%, respectively. Therefore, the PostC treatment could up-regulate the expression of HSP70 and Bcl-2, but down-regulate Bax expression. As compared with OGD group, OGD-induced neuron death and apoptosis were significantly decreased in PostC group（P0.05）. These findings suggest that PostC inhibited OGD-induced neuron death. This neuro-protective effect is likely achieved by anti-apoptotic mechanisms and is associated with over-expression of HSP70.

Astragalus injection inhibits c-Jun N terminal kinase mRNA expression following oxygen-glucose deprivation and reintroduction in rat hippocampal neurons

BACKGROUND： In studies concerning cell injury induced by cerebral ischemia-reperfusion, current experiments have primarily focused on altered protein levels. In addition, the apoptotic proteins Bax and Bcl-2 have been thoroughly studied with regard to initiating neuronal apoptosis. OBJECTIVE： To establish an in vitro model of oxygen-glucose deprivation and reintroduction in the rat hippocampus to simulate cerebral ischemia-reperfusion injury; to observe c-Jun N-terminal kinase 3 （JNK3） mRNA expression in hippocampal neurons following Astragalus injection; and thus to determine changes in the signaling and downstream pathways of neuronal apoptosis at the cellular and molecular level. DESIGN, TIME AND SETTING： A randomized, controlled, cellular and molecular experiment was performed at the Department of Central Laboratory, Chengde Medical College from February to June 2008. MATERIALS： Astragalus injection, the main ingredient of astragaloside, was purchased from Chengdu Di＇ao Jiuhong Pharmaceutical Manufactory, China. JNK3 mRNA probe and in situ hybridization kit were purchased from Tianjin Haoyang Biological Technology, China, and JNK3 RT-PCR primers were designed by Shanghai Bio-engineering, China. METHODS： Primary cultures of hippocampal neurons derived from Sprague Dawley rats, aged 1 2 days, were established. After 8 days, the hippocampal neurons were assigned to the following interventions： model group, Astragalus group, and vehicle control group, cells were subjected to oxygen-glucose reintroduction after oxygen-glucose deprivation for 30 minutes in sugar-free Earle＇s solution and a hypoxia device, which contained high-purity nitrogen. The normal control group was subjected to primary culture techniques and was not treated using above-mentioned interventions. In addition, the Astragalus and vehicle control groups were treated with Astragalus injection （0.5 g/L raw drug） or sterile, deionized water at 2 hours prior to oxygen-glucose deprivation, respectively. MAIN OUTCOME MEASURES： JNK3 mRNA expression was measured by in situ hybridization and RT-PCR at 0, 0.5, 2, 6, 24, 72, and 120 hours after oxygen-glucose reintroduction. RESULTS： Hippocampal neuronal morphology was normal in the normal control group. Hippocampal neurons exhibited apparent apoptosis-like pathological changes in the model, as well as the vehicle control, groups. The apoptosis-like pathological changes in the hippocampal neurons were less in the Astragalus group. Results from in situ hybridization and RT-PCR showed that JNK3 mRNA expression significantly increased in hippocampal neurons from model group, as well as the vehicle control group, compared with the normal control group （P 〈 0.05）. In addition, JNK3 mRNA expression significantly decreased in hippocampal neurons of the Astragalus group, compared with the model group and vehicle control group （P 〈 0.05）. CONCLUSION： Astragalus injection inhibited apoptosis-related JNK3 mRNA expression following oxygen-glucose deprivation and reintroduction, and accordingly played a role in inhibiting hippocampal neuronal apoptosis.

Proliferation and Differentiation of Neural Stem Cells Co-Cultured with Cerebral Microvascular Endothelial Cells after Oxygen-glucose Deprivation

Various stem cells, including neural stem cells （NSCs）, have been extensively studied in stroke models, but how to increase neuronal differentiation rate of NSCs remains unresolved, particu- larly in a damaged environment. The purpose of this study was to investigate the effects of cerebral mi- crovascular endothelial cells （CMECs） on the neurogenesis of NSCs with or without oxygen-glucose deprivation （OGD）. The NSCs acquired from primary culture were immunostained to prove cell purity. Survival and proliferation of NSCs were determined after the co-culture with CMECs for 7 days. After removing the CMECs, NSCs were randomly divided into two groups as follows： OGD and non-OGD groups. Both groups were maintained in differentiation culture for 4 days to evaluate the differentiation rate. Mouse embryo fibroblast （MEF） cells co-cultured with NSCs served as control group. NSCs co-cultured with CMECs had an increase in size （on the 7th day： 89.80±26.12 μm vs. 73.08±15.01μm, P〈0.001） （n=12） and number [on the 7th day： 6.33±5.61/high power objective （HP） vs. 2.23±1.61/HP, P〈0.001] （n=12） as compared with those co-cultured with MEF cells. After further differentiation cul- ture for 4 days, NSCs co-cultured with CMECs had an increase in neuronal differentiation rate in OGD and non-OGD groups, but not in the control group （15.16% and 16.07% vs. 8.81%; both P〈0.001） （n=6） This study provided evidence that OGD could not alter the effects of CMECs in promoting the neuronal differentiation potential of NSCs. These findings may have important implications for the development of new cell therapies for cerebral vascular diseases.

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.

Proprotein convertase 1/3-mediated down-regulation of brain-derived neurotrophic factor in cortical neurons induced by oxygen-glucose deprivation

Brain-derived neurotrophic factor(BDNF)has robust effects on synaptogenesis,neuronal differentiation and synaptic transmission and plasticity.The maturation of BDNF is a complex process.Proprotein convertase 1/3(PC1/3)has a key role in the cleavage of protein precursors that are directed to regulated secretory pathways;however,it is not clear whether PC1/3 mediates the change in BDNF levels caused by ischemia.To clarify the role of PC1/3 in BDNF maturation in ischemic cortical neurons,primary cortical neurons from fetal rats were cultured in a humidified environment of 95%N_2 and 5%CO_2 in a glucose-free Dulbecco's modified Eagle's medium at 37℃for3 hours.Enzyme-linked immunosorbent assays and western blotting showed that after oxygen-glucose deprivation,the secreted and intracellular levels of BDNF were significantly reduced and the intracellular level of PC1/3 was decreased.Transient transfection of cortical neurons with a PC1/3 overexpression plasmid followed by oxygen-glucose deprivation resulted in increased PC1/3 levels and increased BDNF levels.When levels of the BDNF precursor protein were reduced,the concentration of BDNF in the culture medium was increased.These results indicate that PC 1/3 cleavage of BDNF is critical for the conversion of pro-BDNF in rat cortical neurons during ischemia.The study was approved by the Animal Ethics Committee of Wuhan University School of Basic Medical Sciences.

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.

Protective effect of luteolin-7-O-β-D-glucuronide against oxygenglucose deprivation-induced H9C2 cardiomyocytes injury

OBJECTIVE To investigate the protective effect and mechanisms of luteolin-7-O-β-dglucuronide(LGU) on oxygen glucose deprivation(OGD)-induced H9C2 cardiomyocytes injury.METH.ODS The protective effect of LGU on OGD-induced H9C2 cardiomyocytes death were investigated by MTT assay.The microfilament change of H9C2 cardiomyocytes was detected by phalloidin staining and the lactate dehydrogenase(LDH) leakage rate was also detected by LDH kit.In order to explore the possible mechanisms of LGU,ATP content,intracellular Ca^(2+) fluorescent intensity and concentra.tion,mitochondrial membrane potential(MMP)and the expressions of apoptosis-related proteins were detected by ATP kit,CLSM(Fluo-3/AM probe),Ca^(2+) kit,CLSM(JC-1 probe) and western blotting meth.od,respectively.RESULTS The inhibition of H9C2 cardiomyocyte survival rate inducedby OGD was improvedby pretreated with LGU in a concentrationdependent manner.The microfilaments injury as well as the increase of LDH leakage rate were also improvedby pretreated with LGU.The ATP content was significantly decreased,intracellular Ca^(2+) fluorescent intensity and concentration were significantly increased and the MMP was significantly decreased 4 hafter OGD.LGU significantly reversed the de.crease of intracellular ATP content,the increase of Ca^(2+) fluorescent intensity and concentration and the decrease of MMP.The release of cytochrome C,the expressionsof caspase-9 and caspase-3 in H9C2 cardiomyocytes were increased 16 h after OGD.LGUsignificantly inhibited the changes of these apop.tosis-related proteins.CONCLUSION LGU has a significant protective effect against OGD-induced H9C2 cardiomyocytes injury through inhibiting calcium overload,increasing ATP content,improving mi.tochondrial function and inhibiting apoptosis.

Oxygen-glucose deprivation regulates BACE1 expression through induction of autophagy in Neuro-2a/APP695 cells

Our previous findings have demonstrated that autophagy regulation can alleviate the decline of learning and memory by eliminating deposition of extracellular beta-amyloid peptide （Aβ） in the brain after stroke, but the exact mechanism is unclear. It is presumed that the regulation of beta-site APP-deaving enzyme 1 （BACE1）, the rate-limiting enzyme in metabolism of Aβ, would be a key site. Neuro-2a/amyloid precursor protein 695 （APP695） cell models of cerebral isch- emia were established by oxygen-glucose deprivation to investigate the effects of Rapamycin （an autophagy inducer） or 3-methyladenine （an autophagy inhibitor） on the expression of BACE1. Either oxygen-glucose deprivation or Rapamycin down-regulated the expression of BACE1 while 3-methyladenine up-regulated BACE1 expression. These results confirm that oxygen-glucose deprivation down-regulates BACE1 expression in Neuro-2a/APP695 cells through the introduction of autophagy.