The Mechanism Of NF-κB Decoy ODNs Resisting The Inflammatory Injury In Primary Cortical Neurons After Oxygen Glucose Deprivation/Reoxygenation

BackgroudCerebral ischemia is the most life-threatening neurological disease,and is the third leading cause of death after heart disease and cancer,and in the elderly it is a leading cause of long-term disability.Many studies have been done for the disease both at home and board,which gradually deepens the comprehension of the ischemic injury.Howere,an ideal curative effect has not been achieved as yet.So it is necessary to research for a new and a stronger pointed therapy strategy.Now,the thrombolytic compound tissue plasminogen activator(t-PA) is the only Food and Drug Administration-approved agent for cerebral ischemia therapeutic window and is not suitable for many cerebral ischemia, in the other hand,many studies had showed that the inflammation response during reperfusion speeds the secondary brain damages after thromobolysis and play a key role in role in I/R.Inflammation acts as a double-edged sword in I/R.The timming,type of inflammatory mediator and the extent of its stimulation determines the balance between the bad versus good effects of inflammation.Uncontrolled numbers of the infiltrated macrophages and neutrophils increases free radical generation which is detrimental to vascular integrity leading to neuron death.Howere,microglial responses after ischemia is necessary for scavenging the necrotic debris,and the complement system that gets activated in post-ischemic brain initiates local inflammatory responses that ultimately contribute to neuronal survival and tissue remodeling.Howere, complement activation to an inappropriate extent promotes inflammatory mediator release and tissue injury.Brain ischemia/reperfusion has relationship with many factor,such as free radicals,the excitotoxicity,calcium over-loading,the dysfunction of mitchondria,inflammation and apotosis,among them,inflammation is a known precipitator of neuronal death after cerebral ischemia.Neurons' injury and dysfunction is a key event early in the pathogenesis of cerebrovascular disease,so it is important to protect the function and structure of neurons from injury.Degradation ofⅠκB induces NF-κB activation leading to the coordinated induction of multiple genes is involved in many inflammatory and immune cascades in I/R.NF-κB play a dynamic role in the survival and death of neuron in I/R.Much research has established that the activation of NF-κB enhances neuronal survival by preventing apoptosis.In contrast to the anti-apoptosis action of NF-κB,controversial results have appeared regarding the causative role of NF-κB to excitotoxicity.NF-κB dimer species exhibit different binding affinities to the different DNA sequences,the different protein-DNA complexes act on different gene promoters with different NF-κB binding sites.All of these differing conclusions were drawn from different experiment models including cell lines and tissues under different stimuli.NF-κB decoy strategy has been a powerful tool for gene therapy and gene regulation in the study of transcriptional rehulation.NF-κB decoy ODNs in dumbbell shape with the nuclear sequence which was similar to NF-κB cis-elements were synthetized in our study.Oliogodeoxynucleotides with high affinity for transcription factors may be used to bind the transcription factors and block the activation of target gene.Objectives(1) To culture and observe the morphology of primary cortical neurons, the cells were affirmed.(2) To establish a simple,stable and reliable model of oxygen glucose deprivation/reoxygenation(OGD/R) in primary cortical neurons.(3) To investigate the protein expression and the activation of NF-κB P65,P50 and c-Rel following OGD/R of primary cortical neurons in vitro.(4) To study the therapeutic effects of two different decoys:IgG-κB decoy ODNs and Bcl-x decoy ODNs on NF-κB downstream genes TNF-α, Bcl-xL,IL-1αand cell protection in primary cultured cortical neurons in an oxygen glucose deprivation(OGD) modelMethods(1) Primary neuronal cultures of cerebral cortex were obtained from neonatal Wistar rat(＜24h),and neurons were affirmed by immunocytochemical method,morphologies under Light Microscope.(2) Primary neuronal cultures of cerebral cortex were exposed to oxygen glucose deprivation(OGD) for various time(control group,OGD2h group,OGD4h group,OGD6h group),The neuronal injury was detected by the lactate dehydrogenase(LDH) relase and the tetrazolium salt 3-(4,5-dimethyl-2-thiazolyl)-2,5 diphenyl-2H tetrazolium bromide(MTT), and morphologic changes were studied under Light Microscopeto evaluate model of oxygen/glucose deprivation.(3) The protein level of NF-κB subunit P65,P50 and c-Rel in primary neuron was detected by Western Blot and immunocytochemistry after OGD/R(4) We applied different NF-κB decoy ODNs(IgG-κB decoy ODNs, Bcl-x decoy and scramble decoy ODNs) into primary cotical neurons,and then Western Blotting was used to detect the expression of NF-κB P65,P50 and c-Rel protein in neurons.The protein level of TNF-α,IL-1αin primary neuron was detected by ELISA,and the expression of TNF-αBcl-xL,IL-1αmRNA was detected by RT-PCR.Results (1) The characterization of primary cortical neuron was comfirmed based on the morphology and positive by the detetion of NSE andβ-tubulin 3.(2) The MTT and the LDH showed primary corticai neurons activity decreased and changed morphology and the mount of LDH release from injured neurons was increased significantly in OGD 4h group than in control group(P＜0.05).(3) The result of immunocytochemical method and Western Blotting indicated that the expression of P65,P50 protein gradually increased,there was significant different between OGD4h and control group(P＜0.05),and it was significantly highest on the OGD4h/R6h(P＜0.01).The expression of c-Rel protein begin increased on OGD4h/R2h,and it was significantly highest on the OGD4h/R12h(P＜0.05),and then it gradually decreased,but was still higher than that of the control group(P＜0.05).(4) We applied different NF-κB decoy ODNs(IgG-κBdecoy ODNs, Bcl-x decoy and scramble decoy ODNs into primary cotical neurons.The result of immunocytochemical method and Western Blot showed that IgG-κB decoy ODNs inhibited the expression of NF-κB P65 and P50 protein (P＜0.05),but no effect on c-Rel(P＞0.05),while Bcl-x decoy reduced the expression of NF-κB c-Rel and P50 protein(P＜0.05) but no effect on P65, scrambled decoy ODNs and Reagent have no effect on NF-κB subunit (P＞0.05). (5) The result of RT-PCR and ELISA showed that the mRNA and protein levels of TNF-α,Bcl-xL,IL-1αwere markedly elevated after OGD (P＜0.05),IgG-κB decoy ODNs decreased TNF-α,IL-1αmRNA and protein expressions in neurons(P＜0.05),but no effect on Bcl-xL mRNA (P＞0.05),Bcl-xL decoy ODNs suppressed Bcl-xL,IL-1αmRNA and protein expressions(P＜0.05),but no effect on TNF-αmRNA and protein (P＞0.05),scrambled decoy ODNs and Reagent have no effect on IL-1a,TNF-a,Bcl-xL protein and mRNA(P＞0.05)Conclusions(1) We isolated and cultured primary cortical neuron in vitro successfully.The method provided a useful way to study the cerebrovascular diseases in viro.(2) In vitro cerebral ischemia/reperfusion model may be induced easily by OGD in cultured neurons may be used to further study the mechanism of ischemic neuronal cell injury and its protection.(3) There are a small quantity of NF-κB P65,P50 and c-Rel protein expressions in control group,western Blotting and immune cytochemistry has shown that the NF-κB P65,P50 and c-Rel protein expressions increased after oxygen glucose deprivation/reoxygenation.The NF-κB P65,P50 protein begins to increase at 4h after oxygen/glucose deprivatio and keeps increasing at 2-6h after reoxygenation,reaches the summit at 6h after reoxygenation,and returns to the normal level at 24h,while the NF-κB c-Rel protein begins to increase at 2h after reoxygenation,reaches the summit at 12h after reoxygenation and still has a high expression at 24h after reoxygenation.The experimental results reveal that the expression pattern of NF-κB P65,P50 and c-Rel protein varies with different time points,which indicates that NF-κB P65,P50 may have an effect on the neuron in the early and middle stage after the cerebral ischemia reperfusion and NF-κB c-Rel primarily plays an important role in the middle and late stage.(4) OGD induced TNF-α,Bcl-xL,IL-1αmRNA and protein expression in primary cortical neurons.IgG-κB decoy ODNs inhibited the expression of NF-κB P65 and P50 protein,decreased TNF-α,IL-1αand Bcl-xL mRNA and protein expressions in neurons,while Bcl-x decoy ODNs reduced the expression of NF-κB c-Rel and P50 protein,suppressed Bcl-xL,IL-1αmRNA and protein.