| Soman is a potent inhibitor of acetylcholinesterase (AChE).The enzyme loses its activity after being phosphonylated, hence the released acetylcholine (Ach) accumulates at the synaptic cleft.Intoxication of soman gives rise to a series of signs and symptoms such as twitch,convulsion and death. Rapid deploy of army force in the high altitude area may encounte some medical problems resulting from acute hypoxia.The combined injury hypoxia and soman poisoning can enhance its toxicity. Clonal rat pheochromocytoma (PC12) cells are widely used as a model system for sympathetic ganglionlike neurons. In the presence of nerve growth factor (NGF), PC12 cells undergo mitotic arrest and differentiate both morphologically and biochemically into neuronal cells. PC12 cells may serve as a useful model for studying the interaction between hypoxia and cell death. JAK/STAT is a newly discovered intracellular signal transduction pathway that is activated by many cytokines and growth factors. Bingding of ligands to receptors leads to the activation of the JAK tyrosine kinase family, and the activated receptor-kinase complexes recruit members of the STAT family and activate them by phosphorylation . As a result, the phosphorylated STAT proteins dimerize, translocate into the nucleus, and bind response elements in the promoters of target genes to stimulate transcription. JAK-STAT signal transduction pathway is proven to take part in pathogenesis of various kinds of diseases.Objective: But its expression and roles in PC12 cells in hypoxic combined with soman intoxication remain unclear. The aim of this study is to explore the changes of JAK-STAT signal pathway in PC12 cells in hypoxia and soman intoxication in order to search a new idea about the pathogenesis, prevention and treatment of brain injury induced by hypoxia combined with soman intoxication.Methods: PC12 cells were cultured in RPMI-1640 and were treated with NGF(50 ng/ml) for seven days. The differentiated PC12 cells were then exposed to hypoxia in an incubator containing 5%CO2(carbon dioxide), 95% N2 (nitrogen) or(and) incubated withsoman (20μmol/L) for 2,6,12,24h. The expression level of Interleukin-6 mRNA, GP130 mRNA was detected by RT-PCR in PC12, the activity of IL-1β, IL-4, IL-6, IL-8, and IL-13, was mensurated by RIA or ELISA and the activity of tyrosine protein kinase in cells were detected by RIA .The expression of JAKs and STATs in PC12 cells and brain tissues was detected with Western blot, Northern blot, reverse transcription- polymerase chain reaction, Sanger's double strand DNA sequencing, immunohistochemical methods.Results:1. Hypoxia or soman intoxication remarkably reduce the survival rate of PC12 cells, the less survival rate were observed in PC12 cells culture under hypoxia combined with soman intoxication than soman intoxication or hypoxia condition. 2. Hypoxic condition and soman treatment could separately activate the expressions of IL-1β, IL-4, IL-6, IL-10, and IL-13 in PC12 cells. The expression level of IL increased gradually at the first 2 h under hypoxic condition (or after soman treatment), reached the highest level at 6 h (12h), and decreased at 12 h (24h) after the treatment, but still higher than that of normal control. With the induction of hypoxia (soman), the expression of IL-6 mRNA,GP130 mRNA raised at 2 h, reach its peak at 6h (12h), and decreased at 12 h (24h), but still higher than that of its normal control. The expression of GP130 protein increased at 2 h, met its peak summit at 6h (12h). 3. The activity of TKR in the cytoplasm and membrane of PC12 began to increase at 2 h after soman treatment or under hypoxic condition, continued to the summit in 12 or 6 h and decreased at 24 h (12h). 4. The expressions of mRNAs and protein of JAK1, JAK2 and JAK3 in cultured PC12 cells were increased markedly by 2 hour hypoxia(or soman treatment) that was reached their peak at 6h(12h). A significant decrease of the over expressions, however, was elucidated in 12h(24h) PC12 cells under the same incubative conditi... |
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