Protective Effect Of Ligustilide Against Oxygen-glucose Deprivation-iduced Apoptosis In Rat Hippocampal Meurons

OBJECTIVEEstablish primary hippocampal neurons oxygen and glucose deprivation(Oxygen and glucose deprivation, OGD) model. To observe the effect of OGD model on hippocampal neurons, and the possible mechanism of protection on the damage of hippocampal neurons of ligustilide(Z-ligustilide, LIG) in the model.METHODSIdentification of primary cultured hippocampal neurons1 The hippocampal neurons from the new-born SD rat hippocampus area was separated for the primary hippocampal neurons culturing.2 The primary hippocampal neurons were stained by Nissl Staining Solution.3 The rat micro-tubule associated protein(MAP-2) immuno-fluorescence was used to identified hippocampal neuronsProtection effect of ligustilide on OGD injured hippocampal neurons1 The three air incubator(94% N2 and 5% CO2,) and low sugar medium(Dulbecco’s modified Eagle ’s medium, DMEM) method to establish the OGD model to simulate the cerebral ischemia in the body;MTT and LDH method were used to detected the survive rate and LDH releasing of the effect of LIG on the OGD injured hippocampal neurons.2 Inverted microscope was used to detect the growth changes of cell morphology of OGD injured hippocampal neurons.3 Transmission electron microscope was used to observe the ultrastructure changes of OGD injured hippocampal neurons.4 Hochest 33258 dyeing was used to observe the apoptosis morphology.5 FITC Annexin V/propidium iodide(PI) double staining flow cytometry was used to detect apoptosis rateResearch of the protection effect mechanism of on OGD injured hippocampal neurons1 Fluo-3 /AM fluorescent probe staining flow cytometry was used to detect the effect of LIG on calcium influx in the OGD injured hippocampal neurons.2 DCFH-DA fluorescent probe staining flow cytometry was used to detect the effect of LIG on the changes of reactive oxygen species(ROS)in the OGD injured hippocampal neurons.3 JC-1 staining flow cytometry was used to detect the effect of LIG on the mitochondrial membrane potential(MMP)in the OGD injured hippocampal neurons.4 Western-Blotting was used to detect the effect of LIG on transfer of Cyt-C in the OGD injured hippocampal neurons.5 Western-Blotting was used to detect the effect of LIG on the apoptosis related proteins Bax and Bcl-2 in the OGD injured hippocampal neurons.6 Western-Blotting was used to detect the effect of LIG on the apoptosis perform protein Caspase-3 in the OGD injured hippocampal neurons.Screening the pathways of LIG on the OGD injured hippocampal neurons1 FITC Annexin V/PI double staining flow cytometry was used to detect the effect of PI3K/Akt signal pathway on apoptosis.2 Western-Blotting was used to detect the effect of PI3K/Akt signal pathway on the expression of the downstream protein Bad.3 Western-Blotting was used to detect the effect of PI3K/Akt signal pathway on the apoptosis perform protein Caspase-3.RESULTSThe identification of primary cultured hippocampal neurons1 The primary cultured hippocampal neurons was pale purple after the Nissl staining,the dyeing result is positive2 MAP-2 immuno-fluorescence showed that the hippocampal neurons was fluorescent green and the experimental results was positive which show that the method of primary cultured hippocampal neurons was successfully.Protection effect of ligustilide on OGD injured hippocampal neurons1 Cell survive rate was detected by MTT, the results show that, compared with the OGD model group, different concentrations of ligustilide treatment group significantly increased OGD damage of hippocampus neuron cell survival rate, and was dose dependent.2 Inverted microscope observation, the OGD injured group hippocampus neuron cell shrinkage, shedding, fluid loss concentration appeared vacuolated. Meanwhile,the cell shed little and keep synaptic integrity in the ligustilide treatment group.3 Transmission electron microscopy results showed that the ultra-structure of cell apoptosis in OGD model group has obvious features, namely the appearance of apoptosis bodies. Compared with the OGD model group, ligustilide administration group organelle damage morphology is improved significantly, microvilli on cell surface, large nuclei, prominent nucleoli and can’t observe in the presence of apoptosis bodies.4 LDH test results show that, compared with the OGD model group, ligustilide treatment group can significantly reduce the release amount of LDH injured by OGD in hippocampal neurons.5 Hochest33258 staining showed that the control group, hippocampal neuronal nuclei were stained faint blue fluorescence, meanwhile the OGD model group cell nuclei showed strong fluorescence blue and the apoptosis nucleus also can be observed in condensed, or was chunky condensed. Compared with the model group, the number of apoptosis cells was significantly reduced and cell nucleus showed a weak blue-fluorescence in the LIG treatment group.6 FITC-Annexin V/PI double staining flow cytometry cell results show that ligustilide administration group can significantly reduce OGD induced apoptosis rate(P <0.01).Research of the protection effect mechanism of on OGD injured hippocampal neurons1 Ligustilide inhibited OGD induced hippocampal neuronal calcium ions flow.2 Ligustilide inhibited OGD induced hippocampal neurons ROS generation.3 Ligustilide inhibited OGD induced hippocampal neuron mitochondrial membrane potential(Mitochondrial membrane potential, MMP) decreased.4 The amount of Cyt-C release from the mitochondria into the cytoplasm was inhibited by ligustilide in OGD injured hippocampus neuron.5 Ligustilide inhibited the ratio of apoptosis protein Bax and anti apoptosis protein Bcl-2.6 Ligustilide inhibited the over-expression of cleaved Caspase-3 protein in OGD injured hippocampal neuron.Screening the pathways of LIG on the OGD injured hippocampal neurons1 PI3K/Akt signal pathway is involved in the activation of the ligustilide against apoptosis of hippocampal neurons damage effect of OGD.2 Ligustilide activated PI3K/Akt signaling pathway, and resulting in inhibiting the expression of its downstream protein Bad to inhibit apoptosis.3 Ligustilide activation of the PI3K/Akt signaling pathway, and participate in the inhibition of the expression of Caspase-3 protein to inhibit OGD induced apoptosis of hippocampal neurons.CONCLUSIONThe experimental results show that, the oxygen and glucose deprivation could induce apoptosis in the hippocampal neurons; ligustilide by inhibiting apoptosis, protect the OGD induced injury of hippocampal neurons. The neuro-protective effect possibly by reducing calcium influx, the generation of ROS, decreasing the mitochondrial membrane potential, reduced the degree of oxidation damage, and inhibit the apoptosis of hippocampal neurons induced by OGD. The molecular mechanisms of ligustilide research indicated that, activation of the PI3K/Akt signaling pathway, involved in the inhibition the releasing of Cyt-C by mitochondrial, inhibition of its downstream Bad protein over expression and inhibit the over expression of apoptosis executive Caspase-3 protein so as to achieve the purpose of anti-apoptosis.