Effect of directive differentiation of microglia by SN50 on hypoxia-caused neurons injury in mice

Abstract

To explore the effect and mechanism of directive differentiation of microglia by SN50 on hypoxia-caused neurons injury in mice. The microglia were isolated and purified from brain tissue of new-born BALB/c mice through differential velocity adherent and vibration technique. The quantity of the microglia was identified by immunofluorescence staining of inducible nitric oxide synthetase (iNOS) and ionized calcium binding adapter molecule 1 (Iba1) and real-time fluorescence quantitative PCR (qRT-PCR) for special expression genes [iNOS, CD32, and interlenkin 10 (IL-10)]. Then the microglia were cultured with SN50, and the expressions of nuclear factor κB (NF-κB), differentiation-related genes (iNOS, CD11b, IL-10, and CD206), and apoptosis were detected by Western blot, qRT-PCR, and flow cytometry, respectively. The hypoxia model of neuron was established, and the cell apoptosis was evaluated by MTT after 0, 2, 6, 12, 24, and 48 hours of anoxic treatment. The apoptosis related markers (Bcl-2 and Caspase-3) were measured by Western blot and flow cytometry. In addition, the neurons after anoxic treatment were co-cultured with SN50 treated microglia (experimental group) and normal microglia (control group) for 24 hours. And the cell viability and apoptosis related markers (Bcl-2 and Caspase-3) were also measured. Immunofluorescence staining and qRT-PCR analysis showed that the cells expressed the specific proteins and genes of microglia. Compared with the normal microglia, the relative expressions of NF-κB protein and iNOS and CD11b mRNAs in the microglia treated with SN50 significantly decreased ( P<0.05), the relative expressions of IL-10 and CD206 mRNAs significantly increased ( P<0.05), and the cell apoptosis rate had no significant change ( P>0.05). Compared with the normal neurons, the cell viability, the relative expressions of Bcl-2 and Caspase-3 proteins after anoxic treatment significantly decreased ( P<0.05), while the relative expressions of cleaved-Caspase-3 protein and cell apoptosis rate of neurons significantly increased ( P<0.05). In the co-culture system, the cell viability, the relative expressions of Bcl-2 and Caspase-3 proteins were significantly higher in experimental group than those in control group ( P<0.05), while the relative expressions of cleaved-Caspase-3 protein and cell apoptosis rate were significantly lower in experimental group than those in control group ( P<0.05). SN50 can induce the microglia differentiation into M2 type through NF-κB pathway. The SN50-induced microglia can protect neurons from hypoxic injury.