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 an...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.展开更多
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 OG...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.展开更多
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 bee...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.展开更多
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 neur...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.展开更多
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 envir...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.展开更多
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, includin...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).展开更多
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 negat...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.展开更多
Recent research shows that the JNK1/2 signaling pathway plays a neuroprotective role against ischemia-reperfusion injury by cross-talk with other pathways.The present study investigated the effects of isoflurane and s...Recent research shows that the JNK1/2 signaling pathway plays a neuroprotective role against ischemia-reperfusion injury by cross-talk with other pathways.The present study investigated the effects of isoflurane and sevoflurane postconditioning on JNK1/2 pathway activity and neuronal cell viability after oxygen and glucose deprivation injury in hippocampal slices in vitro.Techniques used included population spike analysis,propidium iodide fluorescent staining,western blot assay,and the use of JNK1/2-specific pharmacological tools such as anisomycin (agonist) and SP600125 (inhibitor).We found that both isoflurane and sevoflurane inhibited JNK pathway activity and had neuroprotective effects against oxygen and glucose deprivation injury in slices of rat hippocampus in vitro.Postconditioning with volatile anesthetics exerted neuroprotective effects on nerve cells and preserved the function of the CA1 region by inhibiting JNK1/2 phosphorylation.This suppression of JNK1/2 activity could underlie the observed synergistic neuroprotective effect produced by volatile anesthetic postconditioning.展开更多
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 m...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.展开更多
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 ce...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.展开更多
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 g...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.展开更多
Bone marrow mesenchymal stem cell(MSC)-based therapy is a novel candidate for heart repair.But ischemiareperfusion injury leads to low viability of MSC.Dexmedetomidine(Dex)has been found to protect neurons against isc...Bone marrow mesenchymal stem cell(MSC)-based therapy is a novel candidate for heart repair.But ischemiareperfusion injury leads to low viability of MSC.Dexmedetomidine(Dex)has been found to protect neurons against ischemia-reperfusion injury.It remains unknown if Dex could increase the viability of MSCs under ischemia.The present study is to observe the potential protective effect of Dex on MSCs under ischemia and its underlying mechanisms.Specific mRNAs related to myocardial ischemia in the GEO database were selected from the mRNA profiles assessed in a previous study using microarray.The most dysregulated mRNAs of the specific ones from the above study were subject to bioinformatics analysis at our laboratory.These dysregulated mRNAs possibly regulated apoptosis of cardiomyocytes and were validated in vitro for their protective effect on MSCs under ischemia.MSCs were pre-treated with Dex at 10μM concentration for 24 h under oxygen-glucose deprivation(OGD).Flow cytometry and TUNEL assay were carried out to detect apoptosis in Dex-pretreated MSCs under OGD.The relative expressions of mitogen-activated protein kinase phosphatase 1(MKP-1)and related genes were detected by quantitative polymerase chain reaction and western blotting.Microarray data analysis revealed that Dex regulates MAPK phosphatase activity.Dex significantly reduced in vitro apoptosis of MSCs under OGD,which suppressed the synthesis level of Beclin1 and light chain 3 proteins.Dex down-regulated MKP-1 expression and attenuated an OGDinduced change in the mitogen activated protein kinase 3(MAPK3)signaling pathway.Dex increases the viability of MSC and improves its tolerance to OGD in association with the MKP-1 signaling pathway,thus suggesting the potential of Dex as a novel strategy for promoting MSCs efficacy under ischemia.展开更多
OBJECTIVE To explore the effect of icariside Ⅱ(ICS Ⅱ) on oxygen-glucose deprivation and reoxygenation(OGD/R)-induced injury in cerebral cortical neuronal cels.METHODS Primary cerebral cortical neuronal cells were de...OBJECTIVE To explore the effect of icariside Ⅱ(ICS Ⅱ) on oxygen-glucose deprivation and reoxygenation(OGD/R)-induced injury in cerebral cortical neuronal cels.METHODS Primary cerebral cortical neuronal cells were deprived of oxygen and glucose for 2 h to simulate ischemic stroke injury in vitro.The experiment was divided into 8 groups,which were control,control+ICSⅡ 25 μmol·L^(-1),OGD/R,OGD/R+ICSⅡ(6.25,12.5,25 μmol·L^(-1)),OGD/R+3-methyladenine(3-MA) and OGD/R+Rapamycin(Rap).The protective effect of ICS Ⅱ were detected by MTT assay and lactate dehydrogenase(LDH),respectively.Autophagic flux and autophagy related proteins expressions were detected by using adenovirus harboring tf-LC3 and Western blotting,respectively.RESULTS Compared with OGD/R group,the cell viability treated with ICSⅡwas elevated in a concentration-dependent manner,and the leakage rate of LDH was lowed.Moreover,ICSⅡ not only suppressed OGD/R-induced autophagic flux,but also inhibited the increase of LC3-Ⅱ/LC3-Ⅰ ratio and Beclin 1 after OGD/R insulted.CONCLUSION ICS Ⅱ exerts protective effects on OGD/R-induced cerebral cortical neuronal cells through inhibiting excessive autophagy.展开更多
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 relation...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.展开更多
Alcohol, a widely abused drug, has deleterious effects on the immature nervous system. This study investigates the effect of chronic in vitro ethanol exposure on the metabolism of immature rat cerebellar granular cell...Alcohol, a widely abused drug, has deleterious effects on the immature nervous system. This study investigates the effect of chronic in vitro ethanol exposure on the metabolism of immature rat cerebellar granular cells(CGCs) and on their response to oxygen-glucose deprivation(OGD). Primary CGC cultures were exposed to ethanol(100 mM in culture medium) or to control ethanol-free medium starting day one in vitro(DIV1). At DIV8, the expression of ATP synthase gene ATP5 g3 was quantified using real-time PCR, then cultures were exposed to 3 hours of OGD or normoxic conditions. Subsequently, cellular metabolism was assessed by a resazurin assay and by ATP level measurement. ATP5 g3 expression was reduced by 12-fold(P = 0.03) and resazurin metabolism and ATP level were decreased to 74.4 ± 4.6% and 55.5 ± 6.9%, respectively after chronic ethanol treatment compared to control values(P < 0.01). Additionally, after OGD exposure of ethanol-treated cultures, resazurin metabolism and ATP level were decreased to 12.7 ± 1.0% and 9.0 ± 2.0% from control values(P < 0.01). These results suggest that chronic ethanol exposure reduces the cellular ATP level, possibly through a gene expression down-regulation mechanism, and increases the vulnerability to oxygen-glucose deprivation. Thus, interventions which improve metabolic function and sustain ATP-levels could attenuate ethanol-induced neuronal dysfunction and should be addressed in future studies.展开更多
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 cul...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.展开更多
Scutellarin, a bioactive flavone isolated from Scutellaria baicalensis, has anti-inflammatory, anti-neurotoxic, anti-apoptotic and anti-oxida- tive effects and has been used to treat cardiovascular and cerebrovascular...Scutellarin, a bioactive flavone isolated from Scutellaria baicalensis, has anti-inflammatory, anti-neurotoxic, anti-apoptotic and anti-oxida- tive effects and has been used to treat cardiovascular and cerebrovascular diseases in China. However, the mechanisms by which scutellarin mediates neuroprotection in cerebral ischemia remain unclear. The interaction between scutellarin and nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) was assessed by molecular docking study, which showed that scutellarin selectively binds to NOX2 with high affinity. Cultures of primary astrocytes isolated from the cerebral cortex of neonatal Sprague-Dawley rats were pretreated with 2, 10 or 50 μM scutellarin for 30 minutes. The astrocytes were then subjected to oxygen/glucose deprivation by incubation for 2 hours in glucose-free Dulbecco's modified Eagle's medium in a 95% N2/5% CO2 incubator, followed by simulated reperfusion for 22 hours. Cell viability was assessed by cell counting kit-8 assay. Expression levels of NOX2, connexin 43 and caspase-3 were assessed by western blot assay. Reactive oxygen species were measured spectrophotometrically. Pretreatment with 10 or 50 μM scutellarin substantially increased viability, reduced the expression of NOX2 and caspase-3, increased the expression of connexin 43, and diminished the levels of reactive oxygen, species in astrocytes subjected to ischemia-'reperfusion. We also assessed the effects of scutellarin in vivo in the rat transient middle cerebral artery occlusion model of cerebral ischemia-reperfusion injury. Rats were given intraperitoneal injection of 100 mg/kg scutellarin 2 hours before surgery. The Bederson scale was used to assess neurological deficit, and 2,3,5-triphenyltetrazolium chloride staining was used to measure infarct size. Western blot assay was used to assess expression of NOX2 and connexin 43 in brain tissue. Enzyme-linked immunosorbent assay was used to detect 8-hydroxydeoxyguanosine (8-OHdG), 4-hydroxy-2-nonenal (4-HNE) and 3-nitrotyrosin (3-NT) in brain tissue. Immunofluorescence double staining was used to determine the co-expression of caspase-3 and NeuN. Pretreatment with scutellarin im- proved the neurological function of rats with focal cerebral ischemia, reduced infarct size, diminished the expression of NOX2, reduced levels of 8-OHdG, 4-HNE and 3-NT, and reduced the number of cells co-expressing caspase-3 and NeuN in the injured brain tissue. Furthermore, we examined the effect of the NOX2 inhibitor apocynin. Apocynin substantially increased connexin 43 expression in vivo and in vitro. Collectively, our findings suggest that scutellarin protects against ischemic injury in vitro and in vivo by downregulating NOX2, upregulating connexin 43, decreasing oxidative damage, and reducing apoptotic cell death.展开更多
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 essenti...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.展开更多
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 neuroprotect...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.展开更多
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 exogenou...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.展开更多
基金supported by the National Natural Science Foundation of China,No.81430102(to QGW)
文摘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.
基金supported by a grant from the Health Bureau of Henan Province,China(No.201003111)
文摘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.
基金the Natural Science Foundation of Hebei Province,No.C2006000865
文摘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.
基金supported by grants from the National Natural Science Foundation of China,No.81171090Natural Science Foundation of Chongqing Education Committee of China,No.KJ110313+1 种基金Foundation of Key State Laboratory of Neurobiology of Fudan University in China,No.10-08Foundation of Key Laboratory of Ministry of Education of the Third Medical Military University in China
文摘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.
文摘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.
基金supported by the National Natural Science Foundation of China,No.81771642(to WMX)the New Bud Research Foundation of West China Second University Hospital of China(to GQH)
文摘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).
基金supported by the National Natural Science Foundation of China,No.81771422(to ZY)
文摘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.
基金the Natural Science Foundation of Shihezi University,No.RCZX200688
文摘Recent research shows that the JNK1/2 signaling pathway plays a neuroprotective role against ischemia-reperfusion injury by cross-talk with other pathways.The present study investigated the effects of isoflurane and sevoflurane postconditioning on JNK1/2 pathway activity and neuronal cell viability after oxygen and glucose deprivation injury in hippocampal slices in vitro.Techniques used included population spike analysis,propidium iodide fluorescent staining,western blot assay,and the use of JNK1/2-specific pharmacological tools such as anisomycin (agonist) and SP600125 (inhibitor).We found that both isoflurane and sevoflurane inhibited JNK pathway activity and had neuroprotective effects against oxygen and glucose deprivation injury in slices of rat hippocampus in vitro.Postconditioning with volatile anesthetics exerted neuroprotective effects on nerve cells and preserved the function of the CA1 region by inhibiting JNK1/2 phosphorylation.This suppression of JNK1/2 activity could underlie the observed synergistic neuroprotective effect produced by volatile anesthetic postconditioning.
基金National Natural Science Foundation of China(8166070081260679)Ningxia Col ege First-Class Discipline Construction Project(Chinese Medicine)Funded Project(NXYLXK2017A06)
文摘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.
基金supported by the Zhejiang Traditional Chinese Medicine Science and Technology Plan Project(2021ZB027,2023ZL267)Zhejiang Medical and Health Platform Project of China(2019KY002,2019RC092).
文摘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.
基金supported by the National Natural Science Foundation of China,No. 81241022the Natural Science Foundation of Beijing,No. 7072023,7122045
文摘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.
基金This work was supported by grants from the 3×3 Clinical Scientist Fund of Sun Yat-sen Memorial Hospital(1320900026)the National Natural Science Foundation for Young Scientists of China(81600245)from the Guangdong Science and Technology Department(2020B1212060018).
文摘Bone marrow mesenchymal stem cell(MSC)-based therapy is a novel candidate for heart repair.But ischemiareperfusion injury leads to low viability of MSC.Dexmedetomidine(Dex)has been found to protect neurons against ischemia-reperfusion injury.It remains unknown if Dex could increase the viability of MSCs under ischemia.The present study is to observe the potential protective effect of Dex on MSCs under ischemia and its underlying mechanisms.Specific mRNAs related to myocardial ischemia in the GEO database were selected from the mRNA profiles assessed in a previous study using microarray.The most dysregulated mRNAs of the specific ones from the above study were subject to bioinformatics analysis at our laboratory.These dysregulated mRNAs possibly regulated apoptosis of cardiomyocytes and were validated in vitro for their protective effect on MSCs under ischemia.MSCs were pre-treated with Dex at 10μM concentration for 24 h under oxygen-glucose deprivation(OGD).Flow cytometry and TUNEL assay were carried out to detect apoptosis in Dex-pretreated MSCs under OGD.The relative expressions of mitogen-activated protein kinase phosphatase 1(MKP-1)and related genes were detected by quantitative polymerase chain reaction and western blotting.Microarray data analysis revealed that Dex regulates MAPK phosphatase activity.Dex significantly reduced in vitro apoptosis of MSCs under OGD,which suppressed the synthesis level of Beclin1 and light chain 3 proteins.Dex down-regulated MKP-1 expression and attenuated an OGDinduced change in the mitogen activated protein kinase 3(MAPK3)signaling pathway.Dex increases the viability of MSC and improves its tolerance to OGD in association with the MKP-1 signaling pathway,thus suggesting the potential of Dex as a novel strategy for promoting MSCs efficacy under ischemia.
基金National Natural Science Foundation of China(81560666)Program for Changjiang Scholarsand Innovative Research Team in University, China(IRT_17R113).
文摘OBJECTIVE To explore the effect of icariside Ⅱ(ICS Ⅱ) on oxygen-glucose deprivation and reoxygenation(OGD/R)-induced injury in cerebral cortical neuronal cels.METHODS Primary cerebral cortical neuronal cells were deprived of oxygen and glucose for 2 h to simulate ischemic stroke injury in vitro.The experiment was divided into 8 groups,which were control,control+ICSⅡ 25 μmol·L^(-1),OGD/R,OGD/R+ICSⅡ(6.25,12.5,25 μmol·L^(-1)),OGD/R+3-methyladenine(3-MA) and OGD/R+Rapamycin(Rap).The protective effect of ICS Ⅱ were detected by MTT assay and lactate dehydrogenase(LDH),respectively.Autophagic flux and autophagy related proteins expressions were detected by using adenovirus harboring tf-LC3 and Western blotting,respectively.RESULTS Compared with OGD/R group,the cell viability treated with ICSⅡwas elevated in a concentration-dependent manner,and the leakage rate of LDH was lowed.Moreover,ICSⅡ not only suppressed OGD/R-induced autophagic flux,but also inhibited the increase of LC3-Ⅱ/LC3-Ⅰ ratio and Beclin 1 after OGD/R insulted.CONCLUSION ICS Ⅱ exerts protective effects on OGD/R-induced cerebral cortical neuronal cells through inhibiting excessive autophagy.
基金supported by the National Natural Science Foundation of China(No.30971024)
文摘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.
基金supported by a grant of the Romanian National Authority for Scientific Research,project PN-II-PT-PCCA-2011-3,No 80/2012
文摘Alcohol, a widely abused drug, has deleterious effects on the immature nervous system. This study investigates the effect of chronic in vitro ethanol exposure on the metabolism of immature rat cerebellar granular cells(CGCs) and on their response to oxygen-glucose deprivation(OGD). Primary CGC cultures were exposed to ethanol(100 mM in culture medium) or to control ethanol-free medium starting day one in vitro(DIV1). At DIV8, the expression of ATP synthase gene ATP5 g3 was quantified using real-time PCR, then cultures were exposed to 3 hours of OGD or normoxic conditions. Subsequently, cellular metabolism was assessed by a resazurin assay and by ATP level measurement. ATP5 g3 expression was reduced by 12-fold(P = 0.03) and resazurin metabolism and ATP level were decreased to 74.4 ± 4.6% and 55.5 ± 6.9%, respectively after chronic ethanol treatment compared to control values(P < 0.01). Additionally, after OGD exposure of ethanol-treated cultures, resazurin metabolism and ATP level were decreased to 12.7 ± 1.0% and 9.0 ± 2.0% from control values(P < 0.01). These results suggest that chronic ethanol exposure reduces the cellular ATP level, possibly through a gene expression down-regulation mechanism, and increases the vulnerability to oxygen-glucose deprivation. Thus, interventions which improve metabolic function and sustain ATP-levels could attenuate ethanol-induced neuronal dysfunction and should be addressed in future studies.
基金supported by the National Natural Science Foundation of China,No.81070999the foundation of Xi’an Jiaotong University,No.95,2009+2 种基金Foundation of the Second Affiliated Hospital of Xi’an Jiaotong University,No.RC(GG)201109the US National Institutes of Health,No.NS046560the American Heart Association,No.0450142Z
文摘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.
基金financially supported by the National Natural Science Foundation of China,No.81303115,81774042,81771353the Natural Science Foundation of Guangdong Province of China,No.S2013040016915+3 种基金the Science and Technology Program of Guangzhou City of China,No.201508020050,201604020003the Pearl River S&T Nova Program of Guangzhou,the Postdoctoral Foundation of China,No.BBK42913K09,201003345,BBH429151701a grant from the Hong Kong Scholar Program,Guangzhou University of TCM 2017 High Level University Construction Program,No.A1-AFD018171Z11096a grant from the Specialty Program of Guangdong Province Hospital of Traditional Chinese Medicine of China,No.YN2016MJ07,YN2015QN16,YN2015B2025
文摘Scutellarin, a bioactive flavone isolated from Scutellaria baicalensis, has anti-inflammatory, anti-neurotoxic, anti-apoptotic and anti-oxida- tive effects and has been used to treat cardiovascular and cerebrovascular diseases in China. However, the mechanisms by which scutellarin mediates neuroprotection in cerebral ischemia remain unclear. The interaction between scutellarin and nicotinamide adenine dinucleotide phosphate oxidase 2 (NOX2) was assessed by molecular docking study, which showed that scutellarin selectively binds to NOX2 with high affinity. Cultures of primary astrocytes isolated from the cerebral cortex of neonatal Sprague-Dawley rats were pretreated with 2, 10 or 50 μM scutellarin for 30 minutes. The astrocytes were then subjected to oxygen/glucose deprivation by incubation for 2 hours in glucose-free Dulbecco's modified Eagle's medium in a 95% N2/5% CO2 incubator, followed by simulated reperfusion for 22 hours. Cell viability was assessed by cell counting kit-8 assay. Expression levels of NOX2, connexin 43 and caspase-3 were assessed by western blot assay. Reactive oxygen species were measured spectrophotometrically. Pretreatment with 10 or 50 μM scutellarin substantially increased viability, reduced the expression of NOX2 and caspase-3, increased the expression of connexin 43, and diminished the levels of reactive oxygen, species in astrocytes subjected to ischemia-'reperfusion. We also assessed the effects of scutellarin in vivo in the rat transient middle cerebral artery occlusion model of cerebral ischemia-reperfusion injury. Rats were given intraperitoneal injection of 100 mg/kg scutellarin 2 hours before surgery. The Bederson scale was used to assess neurological deficit, and 2,3,5-triphenyltetrazolium chloride staining was used to measure infarct size. Western blot assay was used to assess expression of NOX2 and connexin 43 in brain tissue. Enzyme-linked immunosorbent assay was used to detect 8-hydroxydeoxyguanosine (8-OHdG), 4-hydroxy-2-nonenal (4-HNE) and 3-nitrotyrosin (3-NT) in brain tissue. Immunofluorescence double staining was used to determine the co-expression of caspase-3 and NeuN. Pretreatment with scutellarin im- proved the neurological function of rats with focal cerebral ischemia, reduced infarct size, diminished the expression of NOX2, reduced levels of 8-OHdG, 4-HNE and 3-NT, and reduced the number of cells co-expressing caspase-3 and NeuN in the injured brain tissue. Furthermore, we examined the effect of the NOX2 inhibitor apocynin. Apocynin substantially increased connexin 43 expression in vivo and in vitro. Collectively, our findings suggest that scutellarin protects against ischemic injury in vitro and in vivo by downregulating NOX2, upregulating connexin 43, decreasing oxidative damage, and reducing apoptotic cell death.
基金supported by the Sci-Tech Development Project of Taian in Shandong,No.2016NS1058&2015NS2081the Sci-Tech Development Project of Linyi in Shandong,No.201515006
文摘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.
基金supported in part by the National Natural Science Foundation of China,No.81573644(to LMH),81573733(to SWX)the Tianjin 131 Innovative Team Project,China(to HW)+5 种基金the National Major Science and Technology Project of China,No.2012ZX09101201-004(to SWX)the Science and Technology Plan Project of Tianjin of China,No.16PTSYJC00120(to LMH)the Applied Foundation and Frontier Technology Research Program of Tianjin of China(General Project),No.14JCYBJC28900(to SXW)the National International Science and Technology Cooperation Project of China,No.2015DFA30430(to HW)the Key Program of the Natural Science Foundation of Tianjin of China,No.16ICZDJC36300(to HW)the Scientific Research and Technology Development Plan Project of Guangxi Zhuang Autonomous Region of China,No.14125008-2-5(to SXW)
文摘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.
基金supported by the Natural Science Foundation of Jilin Province, China, No. 201015181Jilin Province Science and Technology Development Projects, No.20120723
文摘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.