The Mechanism Study Of Butylphthalide Resisting The Inflammatory Injury Following Oxygen Glucose Deprivation/Reoxygenation In Cortical Neurons

BackgroundIschemic cerebrovascular disease is a kind of common and frequent disease that severely imperils patients'lives. Many studies have been done for the disease both at home and abroad, which gradually deepen the comprehension of the ischemic injury and improve the prognosis.However, an ideal curative effect has not been achieved as yet. So it is necessary to search for a new and a stronger pointed therapy strategy. Brain ischemia/reperfusion has relationship with many factors, such as free radicals, the excitotoxicity, calcium over-loading, the dysfunction of mitochondria, inflammation and apoptosis. A good body of studies suggest the inflammatory response during reperfusion speeds the secondary brain damages and play a key role in I/R. While 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. The activation of neurons is a critical step on the onset of inflammatory responses and is associated with the up-regulation of a variety of adhesion molecules and cytokines under the regulation of NF-κB. Many scholars hold that the NF-κB/ IκB system may play a key role in inflammatory responses.Butylphthalide (NBP) is a Traditional Chinese Medicine which comes from apium. And it has been known as a powerful tool for therapy and gene regulation in the study of cerebral ischemia. NF-κB is a transcription factor that can regulate the activation of target gene. It is doubtful that NBP can block the activation of NF-κB. We will examine whether NBP inhibit the inflammation involved in blocking the activation of NF-κB.Objective(1) To isolate and to observe the morphology of neurons from newborn Wistar rat.(2) To establish a simple, stable and reliable model of oxygen glucose deprivation/reoxygenation (OGD/R) of neurons in vitro.(3) To investigate the protein expression and the activation of NF-κB p65 following OGD/R of neuron in vitro.(4) To investigate whether NBP could affect the protein expression of intercellular adhesion molecule-1 (ICAM-1) and inducible nitric oxide synthase (iNOS) following OGD/R in neurons.Methods Neurons of Wistar newborn rats were prepared by filtering through a mesh, centrifugation and trypsogen digestion. The neuron was affirmed by immunocytochemical method, morphologies under Light Microscope.Cells'injury in the model of OGD was evaluate by MTT assay, LDH release. Morphologic changes were studied under Light Microscope(LM). We studied the activation, the nuclear translocation of NF-κB p65 and the protein expression of ICAM-1 and iNOS affected by NBP in each group neuron by immunocytochemical method and RT-PCR.Results(1) The characterization of neuron was confirmed based on the morphology and immuocytochemistry positive by the detection of NSE.(2) The level of oxygen concentration was very low during the OGD. There was significant change in morphology neuron before and after OGD 4h under LM (P<0.05). The pattern of neuron injury indicated time-dependent through MTT assay and LDH release. Treated groups had significant differences compared with normal control group (P<0.05).(3) The protein expression of NF-κB p65 increased during the OGD/R. Compared with normal control group, the expression of NF-κB p65 was significantly higher than that of control group at OGD4h (P<0.05). It reached its peak after treated OGD4h/R2h-8h (P<0.05). There was a decrease at R12h, but it still was significantly higher than that of control group (P<0.05). At R24h, the expression of p65 was approximately equal to the normal level (P>0.05). The number of positive cells with NF-κB nuclear translocation reached its peak at reoxygenation 8h, which had significant differences compared with each group (P<0.05). Before OGD/R,NF-κB p65 mainly existed in cytoplasm, and cells showed"vacuole"phenomenon. After OGD/R, NF-κB p65 mainly existed in karyon and cells showed"solid"phenomenon.(4) NBP is proved it can add cellular vigor and decrease LDH release through MTT assay and LDH releaing assay.(5) The protein expression of ICAM-1 and iNOS in neurons after OGD4h/R8h decreased when treated by NBP through immunocytochemical and RT-PCR method(P<0.05).(6) There is statistics difference among every density NBP that it adds cellular vigor,decreases LDH release and decreases expression of ICAM-1 and iNOS in neurons after OGD4h/R8h(P<0.05). The same as NBP(100μmol/l) and PDTC(100μmol/l) (P<0.05).Conclusions(1) We isolated and cultured neurons in vitro successfully. The method provided an useful way to study the cerebrovascular diseases in vitro.(2) We have established a simple, stable and reliable model of OGD/R of neurons in vitro successfully.(3) NF-κB of neuron was activated quickly when treated by OGD. The protein expression of NF-κB reached its peak from OGD4h to OGD4h/R8h. The number of positive cells with NF-κB nuclear translocation reached its peak at reoxygenation 8h.(4) NBP is proved it can protect neurons through MTT assay and LDH releaing assay.(5) NBP significantly blocked OGD4h/R8h induced ICAM-1 and iNOS upregulation in neurons. It demonstrates that NBP can modulate inflammatory mediator in neurons after OGD/R, then they can protect neurons from injury during OGD/R. The NBP may be of potential pharmacological use for cerebral ischemia/reperfusion injury.(6) The effect of NBP(100μmol/l) that restrains activation of NF-κB is better than that of PDTC(100μmol/l), meanwhile the effect of high density of NBP is better than that of low density of NBP.(7) NBP is proved that there is no toxic side effect to neurons through MTT assay and LDH releasing assay.