| In all neurological diseases, stroke is a leading cause of mortality and mobidity all over the world. Stroke can be calssified as ischemic and hemorrhagic. Ischemic stroke accounts for no less than 87% of all stroke cases and jeopardizes the quality of life because of its high morbidity, high recurrence rate and high disability rate. Ischemic stroke is the result of a transient or permanent reduction in cerebral arterial blood folw caused by residual tissue infarction via an embolus or local thrombosis which ultimately leads to cell death. Injuried brain tissues are divided into two parts, an ischemic core and an ischemic penumbra. The injury in ischemic penumbra is mostly induced by glucose/energy metabolic disorders and the pathological changes after reperfusion. If some effective measures could be made to restore the blood flow within a very short time window, the endangered cells in ischemic penumbra are possible to be salvaged because of the existence of collateral circulation in this area. During the past few decades, researchers are devoted to explore the effective treatment of cerebral ischemic stroke. Many new treatments emerged, including thrombolysis, anticoagulation, antiplatelet focus and transplantation of neural stem cell. What’s cost large amout of resources are the studies on neuroprotective drugs. Although more than 1000 therapeutics have been attempted in treanting cerebral ischemic stroke, but all these efforts were in vain. A rational explanation for this odyssey fate lies in that the ischemia triggered complex pathophysiological changes in brain tissues may have neurtralized the expected actions and made the treatment effects neligible. The major pathobiological mechanisms of cerebral ischemic injury include intracellular excitotoxicity, oxidative stress, apoptosis, and inlammation. For now, intravenous recombinant human tissue-type plasminogen activator (rtPA) remained as the only treatment for ischemic stroke which was approved by the US Food and Drug Administration (FDA). The mechanism of thrombolysis treatment is to lyse the embolus and restore the blood flow. Due to the very nearow time window, the application of IV rtPA thromolysis is still limited in real practice.Neurotrophins play a fundamental role in neurogenesis and neural plasticity, which including nerve growth factor (NGF), brain derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4/5), neurotropin-6 (NT-6), neurotropin-7 (NT-7). The protective functions of these soluble proteins are related to neurogenesis, neural plasticity, neuronal apoptosis and the synthesis of enzymes related to neuron. As one of them, BDNF is an effective growth factor in all kinds of nervous system and mainly distributed in the brain. BDNF is one of the most important neurotrophic factors in the brain. BDNF involved in maintaining, regulating and promoting the growth of neuron. BDNF can reduce the infarct volume, promote the recovery of neural function and improve the neurological deficit after local cerebral ischemia in animal experiments. BDNF improves the neural repair by promoting neuronal differentiation, neurogenesis, neural plasticity and survival during early development, thus influences the outcome of ischemic stroke. But there is no specific positive effect in clinical trial for BDNF despite of its certain neuroprotection demonstrated by numbers of researchers.Early growth response 1 (EGR1) also known as nerve growth factor inducible factor (NGFI-A), Zif268, Krox-24, Tis8. Structurally, EGR1 contains highly homologous DNA-binding domains with three zinc finger, which can recognize and bind GC-rich regions (5’-G(C/A)GGGGG(C/A)GGGG-3’) in the promoters of target genes. Immediate early genes (IEGs) can be activated quickly under a series of external stimuli and EGR1 is one of them. As a transcription factor, EGR1 could bind to the specific sequence in the promoter region of some factors and influence the reduction and expression of these factors with certain or some stimulation. The expression level of EGR1 in normal brain is relatively low, but a sharp increase can be induced by a variety of stresses including ischemia. Human brain could induce the transcription of EGR1 via one or multiple signal transduction pathways and activated EGR1 then bind with the specific binding sites in the promoter of targeted gene to generate varieties of biological functions. Brain tissues would induce massive EGR1 after ischemia or hypoxia. EGR1 plays an important role in regulating the growth, differentiation, development and proliferation of cell, especially in initiating and promoting inflammation. Over-expression of EGR1 regulates the key cytokines in the transcriptional level and results in secondary brain damage after cerebral infarction.PurposeAlthough increase BDNF expression may induce protective effects after ischemic stroke, the quantity may be insufficient. There are no evidences that BDNF could achieve the expected treatment effects in clinical trial. As a member of IEGs, EGR1 may infulence the outcome of ischemic stroke in a bad way. Reduction of the EGR1 expression level in vascular endothelial cell using siRNA technique shows neuroprotection after ishcemia. Considering the fact that a GC-rich region locates in the promoter of BDNF gene (UCSC Genome Bioinformatics website, access in Dec 29,2014), it is rational to presume that this GC-rich region may be recognized and bound by EGR1, and then regulate expression of BDNF. Therefore, BDNF and EGR1, one "good" factor and one "bad" factor, may interact with each other in brain tissues after ischemia. To test this hypothesis, we designed this study to determine whether EGR1 influenced the neurological functions after focal ischemia by interfering BDNF expression.MethodsWe performed this study in vivo and in vitro. In vivo, adult male C57BL/6 mice weighed 20 to 23g, provided by Model Animal Research Institute of Nanjing University (Nanjing, China), were used as subjects. To mimic ischemia, mice were subjected to middle cerebral artery occlusion (MCAO). To evaluate the effects of EGR1 on the expression of BDNF and the outcome of ischemic stroke, we constructed two types of lentiviruses, LV-EGR1 and LV-shEGRl, which can over-and under-express EGR1 respectively. Lentiviruses can be used for the following experiments after verification. Mice were randomized as:Sham (Sham+DMEM) group, Control (MCAO+DMEM) group, LV-control (MCAO+LV-control) group, LV-EGR1 (MCAO+LV-EGR1) group, LV-shEGR1 (MCAO+LV-shEGR1) group. Besides of Sham group, all other four groups were subjected to MCAO and were injected DMEM, LV-control, LV-EGR1, LV-shEGR1 respectively 6d before surgery. Sham operated mice underwent the same procedures without the insertion of filament.In vitro, we cultured primary neuron from fetal mice (16-17d) and performed oxygen-glucose deprivation (OGD) to mimic ischemia. Neurons in same batch were divided into five groups, Sham (Sham+DMEM) group, Control (OGD+DMEM) group, LV-control (OGD+LV-control) group, LV-EGR1 (OGD+LV-EGR1) group, LV-shEGRl (OGD+LV-shEGRl) group. All five groups were applied with DMEM, DMEM, LV-control, LV-EGR1, LV-shEGR1 respectively. Neurons were chanllenged with 2h OGD and harvested 24h later, which were proceed 48h after lentiviral addministration. Western blot analysis was performed to detect the expression of EGR1 in 293T cell, neuron and brain tissues after lentiviral infection at 72h,72h and 7d respectively.To clarify the effect of EGR1 on the outcomes of ischemic stroke, we conducted 2,3,5-triphenyltetrazolium chloride (TTC) staining to evaluate the infarct volume, Fluoro-Jade C staining to assess neuronal degeneration and terminal-deoxynucleoitidyl transferase mediated nick end labeling (TUNEL) to detect apoptotic cell. To explore how EGR1 could influence the outcome of ischemic stroke, we used western blot to measure the levels of EGR1 and BDNF in vivo and in vitro with or without the interference of lentiviruses. Besides, immunofluorescence staining was performed to locate EGR1 and BDNF in brain area and neuron in vitro. At last, we carried out electrohoretic mobility shift assay (EMSA) and chtromatin immunoprecipitation (ChIP) to determine that EGR1 was able to bind with BDNF promoter.Results Both EGR1 and BDNF expression were increased after focal cerebral ischemia in mice or oxygen-glucose deprivation in neurons without the interference of lentiviruses. The expression of EGR1 began to increase at 4h after reperfusion and reached a peak at 24h. Increased expression of BDNF was observed at 24h and sustained to 48h after reperfusion. EGR1 expression increased significantly after administration of LV-EGR1 and reduced remarkably with infection of LV-shEGR1 in 293T cell, in neuron cell and in brain tissues. In addtion, LV-EGR1 reduced the BDNF expression, while LV-shEGRl increased BDNF expression in neuron cell and brain tissues. Injection of LV-EGR1 significantly increased the infarct volumes after MCAO compared with DMEM controls (MCAO+LV-EGR1 vs. MCAO+DMEM:26.93±3.98% vs.20.61±2.38%, P<0.05), and with LV controls (MCAO+LV-EGR1 vs. MCAO+LV-control:26.93±3.98% vs. 21.13±1.39%, P<0.05). When the EGR1 expression was inhibited by LV-shEGR1, the infarct volumes were reversed as compared with DMEM controls (13.54±3.43% vs.20.61±2.38%, P<0.05) and LV controls (13.54±3.43% vs.21.13±1.39%, P<0.05). Injection of over-express EGR1 lentiviruses remarkable increased the numbers of FJC positive cells and TUNEL cells in the peri-infarct areas compared with DMEM controls and LV controls. However, injection of LV-shEGR1 reversed this trend. The elevated EGR1 and BDNF induced by ischemia or hypoxia were identified and a near inverse relationship between EGR1 and BDNF expressions was observed in brain tissues with lentiviral interference via western blot. As detected by immunofluorescence staining, the over-expressed EGR1 was co-localized with under-expressed BDNF in the primary neurons. While the EGR1 expression was suppressed by LV-shEGR1 infection in some primary neurons, the BDNF expression was increased in same cells. When the viruses were injected into the brains, similar trends were observed. Results of EMSA and ChIP assay confirmed that EGR1 could bind to the promoter of BDNF.ConclusionBoth EGRland BDNF expressions were elevated after ischemia or hypoxia. Ischemia induced EGR1 expression may exaggerate brain injury by reducing BDNF expression. Inhibiting EGR1 may become a potential treatment for improving outcomes of ischemic stroke. |
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