Study Of The Regulatory Effect Of NF-κB "Decoy" On COX-2 In Epileptic Rat

The epileptic harm to victims is brain injury due to recurrent seizure, and epileptic brain injury is mainly resulted from the inflammatory reaction in brain. Concerning the mechanism of the inflammatory reaction in brain, previous studies indicated that it was closely related to the increased expression of cyclooxygenase-2 (COX-2) and the elevated level of prostaglandins(PGs). COX-2 is the rate-limiting enzymes of PGs'synthesis, being activated ,it could not only lead to oxidative stress reaction ,but convert arachidonic acid into harmful substance such as PGs. Althoug COX-2 can be induced by some inflammatory factors and inflammatory relevant cytokines, its mechnism of activation and regulation in central nervous system and epileptic rat's brain is not clear.Activated nuclear factor kappa B ( NF-κB) could efficiently induce the transcription of extensive target genes, adjust the expression of immune and inflammatory response factor such as cell adhesion molecules and cytokines , play a regulating role in the course of inflammation and immunoreaction. Ample results of studies could prove that NF-κB is significant in epileptic brain injury , however whether it is the activation of NF-κB that makes one of its downstream target genes ---- COX-2's high expression in seizure or not is also not clear .Objective: Our study uses a"decoy"stratetgy to inhibit the activity of NF-κB. We designed and synthesized a double-stranded oligodeoxynucleo- tides(ODNs) ----κB-decoy ---- which matches with the cis-element of NF-κB and a corresponding ODNs with irrelevant scrambled sequence ---- scrambled-decoy, UsingκB-decoy to inhibit the activity of NF-κB in seizure, we detected the expression of COX-2. To do that we hope to explore the relationship of pathological changes in brain injury with the activity of NF-κB and the expression level of COX-2 ,clearify the regulating effect of NF-κB on COX-2 in seizure, and further understand the mechanism of brain pathological changes.Methods:Choose adult male SD rats whose body weight is in the range from 250g to 300g, and divide them randomly into three groups: experimental group (κB-decoy group) ,control group (Scrambled- decoy group) and normal group (saline-control group) .Use the rat stereotaxic instrument to take intraventricular injection ofκB-decoy- ExGen500 or Scrambled-decoy-ExGen-500 compound . Use Lithium- Pilocarpine to make rat's seizure model (40mg/kg). After seizure choose different time point to irrigate and fix the rats , use immuno-histochemistry to detect the activity of NF-κB and the expression of COX-2 in epileptic sensitive brain regions, use RT-PCR to detect the expression of COX- 2mRNA.Results: Immunohistochemistry of NF-κBp65 showed that: compared with control group, the positive staining in cytoplasm in experimental group was obviously deeper which indicated that NF-κB in experimental group was not translocated from cytoplasm into nucleus after stimulation, its activity was inhibited byκB-decoy. Immunohistochemistry of COX-2 showed that: compared with control group, the positive staining in experimental group was obviously shallower which indicated that the COX-2 expression level in experimental group was lower than in control group. RT-PCR of COX-2mRNA showed that: the expression level of COX-2mRNA in experimental group was lower than in control group (for the time point 4h after seizure).Conclusions:1.κB-decoy could inhibit the activity of NF-κB in epileptic rat's brain.2.κB-decoy could inhibit COX-2's expression in epileptic rat's brain.3. In epileptic rat's brain , NF-κB could positively regulate one of its downstream target genes ---- COX-2 gene.