葛根素对HIE新生大鼠神经及PI3KAkt信号通路的作用机制研究

Study on the Mechanism of Puerarin on Neurons and PI3K/Akt Signaling Pathway in Neonatal Rats with Hypoxic-ischemic Encephalopathy

目的观察葛根素对缺氧缺血性脑病(hypoxic-ischemic encephalopathy,HIE)新生大鼠的神经保护作用,分析神经保护作用是否涉及PI3K/Akt信号通路的参与以及可能的机制。方法 90只新生7日龄Wistar大鼠随机分为假手术组(n=10)、HIE组(n=40)及葛根素疗组(n=40),后两组又进一步分为3 h、6 h、12 h、24 h 4个时间点,每个时间点10只。参照Rice等和吴婉芳法,建立新生大鼠HIE模型。HE染色观察各组大鼠神经元形态变化,免疫组织化学法检测磷酸化Akt的表达。依据免疫组化的结果采用Western blot方法进一步检测PI3K/Akt信号通路表达。结果 90只Wistar大鼠模型全部成功,全部纳入结果分析。HE染色示葛根素治疗组大鼠大脑皮层和海马区神经细胞坏死率较HIE组明显减少。免疫组化结果示:随着时间的延长,葛根素治疗组磷酸化Akt蛋白阳性率较HIE组显著上调(P<0.01)。HIE组磷酸化Akt在12 h时达到高峰,但葛根素治疗组在24 h时达到高峰;在每组每个时间点葛根素组磷酸化Akt蛋白的阳性表达均高于HIE组(P<0.01)。假手术组亦有极少量表达。Western blot结果示:在各个时间点内,各组总Akt的表达一致;但磷酸化Akt的表达(Ser473和Thr308)在葛根素治疗组明显强于HIE组;且随着时间变化,其表达于上述免疫组化的检测大致一致。葛根素显著提高磷酸化Akt的水平,既可作用于Ser473磷酸化位点,也可作用于Thr308磷酸化位点。结论葛根素对HIE大鼠具有神经保护作用。葛根素可激活PI3K/Akt通路来发挥其对HIE新生大鼠神经元的保护作用,其机制可能与上调磷酸化Akt(Ser473)、磷酸化Akt(Thr308)表达水平,增加Akt磷酸化水平的表达有关。

Objective To observe the neuroprotective effect of puerarin in hypoxic-ischemic encephalopathy（HIE）rats model,and to investigate whether the PI3K/Akt signal pathway was involved in the neuroprotective effect of puerarin on neonate rat in HIE model and its possible mechanisms.Methods Ninety 7-day neonatal Wistar rats were randomly divided into sham operation group（n =10）,HIE group（n =40）and puerarin treatment group（n =40）.The last two group were further divided into 3 h,6 h,12 h and 24 h four time points,each of them had 10 rats.Rats model of HIE were established according to Rice’s and Wu Wanfang’s method,then HE stain was used to observe the morphological change,immunohistochemical method and western blot were used to detect the changes of PI3K/Akt pathway,including the expression level of total Akt,phosphorylated Akt（Ser473 and Thr308）in brains of rats.Results The HIE rats model had been established successfully and 90 Wistar rats were take into the results analysis.Compared with HIE group,HE result showed that the necrotic neurons in cortical layer and hippocampus were significantly reduced in puerarin treatment group.The immunohistochemical result proved that the expression of phosphorylated Akt in puerarin treatment group was apparently increased with time going by（P 〈0.01）.Phosphorylated Akt reached its peak after 12 h in HIE group while after 24 h in puerarin treatment group,and the expression of phosphorylated Akt in puerarin treated group was higher than that in HIE group in every time point（P 〈0.01）.There was extremely small amount expression in sham operation group.Western blot showed that the expression level of total Akt was at equal pace between three groups in every time point.Compared with HIE group,the phosphorylated Akt（Ser473 and Thr308）level was increased significantly in puerarin treatment group,what’s more,with time going by,the change of expression roughly conformed to the immunohistochemical result above.Puerarin could increase the levels of phosphorylated Akt by playing role on Ser473 phosphorylation site as well as Thr308 phosphorylation site.Conclusion Puerarin has neuroprotective effect on HIE rats model by activating the PI3K/Akt signal pathway.Its mechanism may be related to the up-regulation of phosphorylated Akt（Ser473 and Thr308）expression,and increased the expression of Akt phosphorylation.