The Expression Of NF-κB Is Induced By Ischemia/reperfusion Injury In Cultured Mice Primary Neurons And The Effects Of Flunarizine On The Expressin

In eukaryotic cells, NF-кB-dependent gene expression plays an important role in a number of biological frictions, including growth, differentiation, apoptosis, carcinomatous change of cells and ontogenesis. Recently, some studies demonstrates that NF-кB also exists in cerebral vessels endothelial cells, neurons, glial cells, and is tightly associated with the inflammatory response after cerebral ischemia/reperfusion. Up to now, more and more studies have been done in order to find the regulatory mechanisms of NF-кB in this pathologic process.Under normal conditions, binding of NF-кB as a complex to I KB inhibitor in the cytosol is the main cellar mechanism preventing NF-кB-dependent transcription. When activated by a number of extracelluar stimuli, including TNF-a , IL-1 or ultraviolet light, this multisubunit transcription factor dissociates from I K B and translocates as a p50/p65 dimer to the nucleus, and then induces the expression of genes encoding cell adhesion molecules, nitricoxide synthase(NOS), and cytokines.Here we reperfused the cultured mice primary neurons after ischemia-liketreat in vitro according to Matani et al, and then analyzed the expression and distribution of NF- K B in these neurons by immunofluorescence and flow cytometry to demonstrate the translocation process of NF- K B after activated. We also discussed the effects of Flunarizine on the expression of NF- K B in neurons after ischemia/reperfusion injury by treatment with Flunarizine, so we can further explain the protection mechanism of Flunarizine in the cerebral ischemia/reperfusion injury.In the experiment 1, low level of constitutive NF-кB p65 activity is observed only in cytoplasma of cultured neurons when not activated, and this transcription factor mainly distributes in axon hillock. After ischemia/reperfusion treatment, the intense nuclear localization and decrease in cytoplasma of NF-кB demonstrates the translocation of NF-кB from cytoplasma to nucleus. Flow cytometry assays show that the expression rate of NF-кB is very low in untreated cultured primary neurons, but after transient ischemia-like treatment, an obvious elevation of expression of NF-кB is found (P<0. 01) when the cells are reperfused for 15 min and, when reperfused for 30 min, the expression reaches it's peak (P<0. 01) , approximately 40-fold higher than those in control cultures. Then the expression rate decreases to a relatively low level (P<0. 01) when the cells are reperfused for 360 min. This indicates that NF-кB is activated and translocates to the nucleus in the process to act as a signal transducer and a transcription factor.In the experiment 2, flow cytometry assays show that Flunarizine can inhibit the expression of NF-кB obviously (P<0. 01). Conclusions in the experiment were drawn as follow:1 .NF-кB P65exists in cultured mice primary neurons.2. NF-KB P65 is lower in constitutive expression,and distrubites in cytoplasma of cultured neurons.3.NF-KB P65 is higher in cytoplasma of cultured neurons and traslocates from cytoplasma to nucleus after ischemia /reperfusion treatment.4.After ischemia /reperfusion treatment the expression of NF- K B P65 is quick and higher .After 15 min NF- K B P65expressed in nucleus and after 30 min the expression is the highestand subsequently the expression is decreased.5.A11 of conclusions illustrate that NF-KB P65 is actived ,and translocate from cytoplasma to nucleus and the effect of gene transcription adjustment and signal conduction is realized after ischemia /reperfusion treatment.6.The action of NF-KB P65 is inhibited significantly after Flunarizine treatment...