| Objective1. To establish and consummate experimental methods and conditions for primary cultured microglia isolated from rat cortices in vitro.2. To explore the effect of primary cultured microglia activated by lipopolysaccharide (LPS) on neuron in vitro.3. According to the effect of dl-3-n-butylphthalide (NBP) on microglial activation, attempt to expolre the possible new tagets of NBP which served as I class of national durgs.Methods Microglia cells and neurons were isolated from rat cortices for primary culture. Primary cultured microglia cells were stimulated with LPS to construct the activated microglia cell model in vitro. Neuron-rich cultures were stimulated with supernatant of microglia activated by LPS, and then the survival rate of neurons was evaluated by MTT assay. Microglia cells were fed with NBP prior to the addition of LPS, and then the morphology of microglia was observed while changes in inflammatory cytokines, TNF-alpha-, IL-lbeta and IL-10, were measured by enzyme-linked immunosorbent assay (ELISA). We also measured the levels of nitric oxide (NO) by the nitrate reductase method and evaluated the survival rate of neurons stimulated with NBP pretreatment microglial surpernatant by MTT assay under the same condition. The effect of NBP on activated microglia was assessed though comprehensive consideration of all of the above factors.Results Quantity and high purity microglia cells were cultured from rat cortices in vitro. The survival rate of neurons was decreased after applying supernatant of LPS-stimulated microglia to neuron cultures. NBP pretreatment microglia cells still showed as activated morphological characteristics, the expression and release of NO was inhibited while there were no significant changes in TNF-alpha> IL-lbeta and IL-10, and the survival rate of neurons stimulated with NBP pretreatment microglial surpernatant was higher than the control goup. The effect of NBP on activated microglia was dose-dependent.Conclusion Activated microglia cells can induce neuron death. NBP can’t inhibit the activation process of microglia, but can reduce the toxicity of the activated microglia on neuronal cell by inhibiting the expression and release of NO from activated microglia. In addition, the effect of NBP on activated microglia shows dose-dependent characteristics. |
