Study On The Expression Of IRAK-1/4 Under Hypoxia In Rat B35 Neurons

Background and objective: The center nervous system diseases are characteristic with high mortality and high disability, while secondary brain injury is one of the most important factors during the occurrence and development of the injur, such as cerebral ischemia, energy metabolism, calcium overload, oxygen free radicals gathering,the toxic effects of excitatory amino acids, inflammation ,et al. Of the factors mentioned above, inflammation may play an important role in the pathological process of secondary brain injury,and bring us more and more attention. As we know, Interleukin-1 receptor activated kinases (IRAKs) are key mediators in inflammatory regulation. By the influence of inflammatory factors, IRAKs are activated which result in the transcription and expression of inflammatory cytokines, such as IL-1β, IL-6, TNF-α, and trigger the inflammatory cascade. Microglial cells, the brain resident macrophages, can trigger and sustain local inflammation process.In the previous study ,we found that activated microglia play an important role in neuroinflamation under hypoxia.Despite secrete the inflammatory factors, Neurons participate the immune response in the brain. Whether the family of IRAKs and the signal transduction systems exist in neurons, participate apoptosis regulation have not been fully recognized. On the basis of previous results,we investigated the dynamical changes of interleukin-1 receptor-associated kinases-1/4(IRAK-1/4)and neurons apoptosis in rat B35 neurons under hypoxia,aimed to further discuss the relationship between them and explore the mechanism of inflammatory damage which is mediated by IRAKs in center nerve system with hypoxia.Methods: B35 cells exposed to hypoxia of 3%O2,5%CO2,92%N2 were cultured for 1h,3h,6h,12h,24h,48h,72h and 96h respectively, the mRNA and protein expressions of IRAK-1/4 were detected by RT-PCR method and Western blot analysis, the expression of IRAK-1/4 in the cells were observed by laser scanning confocal microscope(LSCM), the concentrations of TNF-αand IL-6 were measured by ELISA method, while flow cytometry(FCM) was used to measured the cell apoptosis. After using the specific inhibitor of IRAK-1/4, the concentrations of IL-6, TNF-αwere measured, also the cell apoptosis. Result: (1).IRAK-1/ 4 which plays an important role in the TLR/IL-1R signaling cascade was observed in the neurons of center nerve system.(2). The mRNA and protein expressions of IRAK-1/4 increased after hypoxia for 1 hour and decreased gradually after reaching the highest level at 6h, remained at a high level at 12h, and then decreased gradually, after 48h the they decreased lower than that in normoxia.(3).the measurements of laser scanning confocal microscope(LSCM)showed that IRAK-1/4 were mainly distributed in the cytoplasm and the fluorescence intensity increased in 6h and 12h under hypoxia compared with normoxia, then decreased after 24h.(4). The concentrations of TNF-αand IL-6 increased obsiversly after hypoxia for 3 hour and reached the highest level gradually at 6h, then gradually decreased which higher than normal level all the time. After IRAK-1/4 inhibitor used, the concentrations of IL-6 and TNF-αunder hypoxia significantly decreased.(5).The apoptosis of neurons increased significantly after hypoxia for 6h, got to the peak after hypoxia for 12h, and maintained for a long time. After IRAK-1/4 inhibiting,the apoptosis of neurons decreased significantly consistently with the concentrations of TNF-αand IL-6 decreasing.Conclusion:(1) As the key factors of TLR/IL-1R signaling cascade,IRAK-1/4 exist in rat B35 neurons. The mRNA and protein expressions of IRAK-1/4 under hypoxia increased within 24h, then decreased gradually.(2) The expression of TNF-αand IL-6 under hypoxia reached the peak at 6h, and decreased gradually. After IRAK1/4 inhibitor used, the concentrations of TNF-αand IL-6 decreased significantly. (3) The apoptosis of neurons got to the highest level after hypoxia for 12 hour, and maintained at a relatively high level for a long time. The peak of apoptosis in neurons occurs after the expression of TNF-αand IL-6 got the highest level. After IRAK1/4 inhibitor used, the apoptosis of neurons also declined.