Protection Effects Of Gas 6/Tyro 3/Akt Signal On Hypoxia Hippocampal Neurons

PartⅠEffect of hypoxia on apoptosis and Tyro 3 / Akt signal of hippocampal neurons with primary cultureAim: The primary culture of hippocampal neurons was established to detect effects of hypoxia on apoptosis and Tyro 3 / Akt signal of hippocampal neurons with primary culture.Methods: The primary culture of hippocampal neurons was prepared. Hippocampal neuron purity was identified with MAP 2 immunofluorescence assay. Neurons were randomly assigned as the normal, hypoxia A(12 hours after hypoxia), hypoxia B(24 hours after hypoxia) groups. Morphology of hippocampal neurons was detected by inversion microscope. Proliferative activity of hippocampal neurons was examined with MTT methods. Hippocampal neural apoptosis was observed through TUNEL methods. Expression of Tyro 3 in hippocampal neurons was detected with immunofluorescence assays. Akt phosphorylation level of hippocampal neurons was examined by Western blot.Results:1.Hippocampal nerve cell purity in primary culture reached 89% with MAP 2 immunofluorescence assays, which generally consistents with experimental needs, and we successfully established primary culture of hippocampal neurons. 2. Hypoxia suppressed proliferation activity of hippocampal neurons(P<0.01), and promoted nerve cell apoptosis(P<0.01). 3. Hypoxia inhibited Tyro 3 expression(P<0.01), and decreased Akt phosphorylation level in hippocampal neurons(P<0.01).Conclusions: Hypoxia suppresses proliferation activity of hippocampal neurons in primary culture, and promotes neural apoptosis. Inhibition of the Tyro 3 /Akt signal is involved in hypoxia induced hippocampal cell damage.Part Ⅱ Protection effects of Gas 6 / Tyro 3 / Akt signal on hypoxia hippocampal neurons with primary cultureAim: The primary culture model of hypoxic hippocampal neurons was established to explore protection effects of Gas 6 / Tyro 3 / Akt signal on hypoxia hippocampal neurons with primary culture.Methods: The primary culture model of hypoxia hippocampal neurons was prepared, and hippocampal neurons were randomly assigned as the normal, hypoxia, Gas 6(a), Gas 6(b), Gas 6(c) groups. Inversion microscope was used to dectect morphology of hippocampal neurons. MTT methods were conducted to examine proliferative activity of hippocampal neurons. Hippocampal neural apoptosis was observed through TUNEL methods. Double-label immunofluorescence assays were used to detect Gas 6 and Tyro 3 expression in hippocampal neurons. Akt phosphorylation level of hippocampal neurons was examined by Western blot.Results:1.Gas 6 obviously improved proliferation activity of hypoxia hippocampal neurons with primary culture(P<0.01), and inhibited hypoxia hippocampal neural apoptosis(P<0.01). 2.Gas 6 improved expression of Tyro 3(P<0.01), and increased Akt phosphorylation level(P<0.01) in hypoxia hippocampal nerve cell with primary culture.Conclusions: Gas 6 improves proliferation activity, inhibits apoptosis, increases Tyro 3 expression, and promotes Akt phosphorylation level in hypoxia hippocampal neurons with primary culture.Part Ⅲ Protection effects of Gas 6 / Tyro 3 / Akt signal on hippocampal neurons of ischemic ratsAim: Establishment of the ischemic rat model to investigate protection effects of Gas 6 / Tyro3 / Akt signal on hippocampal neurons in ischemic rats.Methods: A ischemic rat model was established by bilateral occlusion of common carotid arteries. The rats were randomly divided into the normal, ischemic, Gas 6(a), Gas 6(b), Gas 6(c) and Gas 6(d) groups. Gas 6 recombinant protein was injected into cella lateralis. TUNEL assays were used to detect the hippocampal neural apoptosis. Double-labeled immunofluorescence assays were conducted for Gas 6 and Tyro 3 expression in hippocampus. Western blot was done to analysis the phosphorylation level of hippocampal Akt.Results : 1. Gas 6 suppressed neural apoptosis in hippocampus of the ischemic rats. 2. The fluorescence intensity of hippocampal Tyro 3 and Gas 6 was significantly lower in the ischemic rats than that in the normal group, and Gas 6 significantly increased the fluorescence intensity of Tyro 3 in the ischemic rats(P<0.01). 3. The phosphorylation level of Akt in hippocampus was lower in the ischemic group compared with the normal group(P<0.01), and Gas 6 increased Akt phosphorylation level(P<0.01).Conclusions: Cumulatively, these findings suggest that inhibition of the Gas 6 / Tyro 3 / Akt pathway plays an important role in the hippocampal neural apoptosis of the ischemic rats, and Gas 6 improves this pathway and plays a role in neural protection for the ischemic rats.