Molecular Mechanisms Of Valproate's Neuroprotective Effects On Neurons Damages By Hypoxia And Ischemia

Objective: ①To investigate the effects of valproate on in in vitro cultured rat cortical and hippocampus neurons damages induced by hypoxia.②To investigate the effect of VPA pro-treatment on cerebral neurons damages in permanent MCAO rats.③ To analyze the mechanism of VPA above.Methods: ①Cortical and hippocampus neurons were cultured from P1 rats with no-serum method and then treated with VPA at 0.lmM, 0.5mM, 1.0mM and 20mM concentration respectively, followed by hypoxia and re-oxygen culturing. The growing status of the cells were observed with optical microscope. Data were analyzed by t test. ②SD rats were divided into control-, VPA micro-dose, VPA moderate-dose, VPA macro-dose and VPA ultra-dose group(0mg/kg, 50mg/kg, 100mg/kg, 200mg/kg 和 400mg/kg). VPA was administered by stamoch perfusion in advance. MCAO rat modal was established according Longa's method at day 7. Neural functions wore assessed at awaking time, 24h, 48h and 72h. After the last assessing, rats were killed and brains were removed, cut into slices and stained with 2% TTC. Infarction volum of every brain was calculated with graph software Data were analyzed by t test or oneway anova analysis. Neuroprotective effect of VPA was evaluated. By immunohistochemistry, expressions of pERK1/2, caspase-3, HSP70 and BrdU were studied, and the mechanism of VPA investigated.Results:①In cortical neuron hypoxia injury group, survival of cells was significant better in macro-dose group than that in control group(P<0.001) , but it was worse in ultra-dose (P<0.000) . In hippocampus neuron hypoxia injury group, survival of cells was significant better in moderate-dose group and macro-dose group than that in control group(P<0.05; P<0.05), but it was worse in ultra-dose(P<0. 05). ②On hypoxia and reoxygen day 5, processes were significant longer in macro-dose group and moderate-dose group (P<0.050H 和 P<0.022) , and so they were inhippocampus neurons. ③Cells treated with VPA were more strongly stained by Bcl-2 antibody than were they in control. ④Neural function value at 48h point was significant better in micro-dose and moderate-dose group than in control ( P<0.05); They were better in micro-dose group than were in control at 72h point. (P<0.05) .⑤Infarction volum in moderate-dose and macro-dose group decreased more than did in control (P<0.05; P<0.05) .⑥Neurons in cortex of VPA treatment group survived better than did in control (P<0. 05 ) , ⑦Expression of pERK1/2 in ischemic cortical and striatum of VPA treatment group was more stronger than was that in control.⑧Compared to control, expression of caspase-3 was decreased by VPA (P<0. 001 ) ⑨Compared to control, expression of HSP70 in cortex and hippocampus was significantly increased by VPA (P<0.001) . (P<0.001) . ⑩Compared to control group, BrdU positive cells were much more in hippocampus CA1 area (P<0. 001) . Conclusions:①VPA has definite neuroprotective effect on in vivo and in vitro neurons injury induced by hypoxia and ischemia. The neuroprotective effect is associated with VPA's dose level.②The neuroprotective mecharism of VPA is associated with increasing expression of HSP70 and inhibiting expression of caspase-3, and associated with stimulating ERK signal pathway.③VPA can promote the regeneration of injured hippocampus neurons.④ VPA can promote growth of neuronal processes.