An Initial Study Of The Effects Of Hypoxia On IRAK-1 And IRAK-4 Expression In Murine N9 Microglia Cells

Background and Objective: Secondary brain injury (SBI) is the key factor that affects the outcome of traumatic brain injury by increasing the rate of mobility and disability, while the mechanisms of SBI are still not well known. Previous studies mainly focused on the following categories: 1. free radical damage, 2. abnormal calcium metabolism of neurons, 3. neurotransmitter theory, 4. abnormal clotting mechanism, 5. abnormal energy metabolism, 6. inflammation damage in the brain, etc. Inflammatory response, as a self-defense mechanism, is the most common pathophysiological process that helps to remove harmful substances, pathogenic microorganisms. But an excessive inflammatory response can also be an important reason to damage the body. The cascade inflammatory response leading to brain damage was considered to be the primary reasons resulting in the cell degeneration and necrosis in the brain. Since the cascade inflammatory response is hard to be controlled, it is important to give the intervention at the early stage. Interleukin-1 receptor associated kinase (IRAK) is a newly found key factor in the early signal transmit of inflammation. Microglia is the major cell participating the inflammation responses in the brain. Activated microglia can release a variety of inflammatory cytokines, which are closely related to the central nervous system diseases including SBI. Previous study mainly paid attention to the inflammatory cytokines released by microglia such as IL-1β, TNF-αNF-κB, while the existance of IRAK family in the microglia and its possible effects were rarely mentioned. This study was designed to investigate the possible effects of IRAK family on the microglia-mediated inflammatory response in the CNS under a condition of hypoxia by observing the change of IRAK-1,IRAK-4 and TNF-αexpression in the murine microglia as well as analyzing the relationship between them.Methods:The murine N9 microglia cells were first cultured under normal oxygen condition to detect the mRNA and protein level of IRAK-1 and IRAK-4. Then, cells were exposed at a condition of 3%O₂ for 1 h, 3 h, 6 h, 12 h and 24 h. The protein and mRNA level of IRAK-1 and IRAK-4 at each point were detected by western blotting and RT-PCR method repectively. The TNF-αlevel was estimated by ELISA. The cellular change of IRAK-4 was observed in terms of the immunofluorescence staining with the use of laser scanning confocal microscope (LSCM).Results:(1) IRAK-1 and IRAK-4, the key factors in the TLR/IL-1R signaling cascade, were observed in the normal cultured microglia cells.(2) The expression of mRNA and protein of IRAK-1 and IRAK-4 were upregulated in a time-dependent manner in the first 6 hours after hypoxia. Compared with the control cells, the upregulation of IRAK-1 and IRAK-4 began at 1h, showed statistic significance at 3h (P<0.01), peaked at 6h (P<0.01), remained at a higher level at 12h (P<0.01), and decreased at 24h after hypoxia.(3) The fluorescence intensity of IRAK-4 increased with the extension of anoxic time.(4) The concentration of TNF-αin the supernatant also increased in a time-dependent manner in the first 6 hours after hypoxia. The level of TNF-αincreased significantly at 3h (P<0.01), peaked at 6h (P<0.01), retained in a higher level at 12h (P<0.01), and decreased at 24h.(5) There were significant positive correlations between the protein expression of IRAK-1, IRAK-4 and TNF-αin microglia cells under a condition of hypoxia ( r =0.867 and 0.863 respectively, P < 0.05).Conclusions:(1)Our studies proved the existance of IRAK-1 and IRAK-4 in the central nervous system.(2) The mRNA and protein expression of IRAK-1 and IRAK-4 in in vitro cultured murine microglia cells were increased at the early stage of hypoxia.(3) There were significant positive correlations between the protein expression of IRAK-1, IRAK-4 in the microglia and the concentration of TNF-αdetected in the supernatant during a certain period of hypoxia.