安寐丹调控ROS/PARP-1/AIF通路改善睡眠剥夺模型神经元损伤

Anmeidan Regulates ROS/PARP-1/AIF Pathway to Alleviate Neuronal Damage in Sleep Deprivation Model

目的 探讨安寐丹对睡眠剥夺模型海马神经元凋亡及氧化应激的影响。方法 60只大鼠随机分为空白组、模型组、安寐丹组和褪黑素组,每组15只。安寐丹组给药18.18 g/(kg·d),褪黑素组给药100 mg/(kg·d),空白组和模型组给予等体积生理盐水。Ethovision视频分析系统评估大鼠行为学改变,HE染色观察海马神经元形态,透射电镜观察海马神经元超微结构,Tunnel染色观察神经元凋亡情况,生化检测血清超氧化物歧化酶(SOD)、丙二醛(MDA)和谷胱甘肽(GSH-PX)含量,免疫荧光检测海马B淋巴细胞瘤-2基因(Bcl-2)、Bcl-2相关X蛋白(BAX)、Caspase 3、Caspase 9及活性氧(ROS)的表达,蛋白免疫印迹法检测多聚ADP-核糖聚合酶1(PARP-1)和凋亡诱导因子(AIF)表达。结果 与空白组比较,模型组大鼠中央区探索时间和中央区穿行次数均下降(P<0.001,P<0.01),海马神经元数量减少,排列疏松紊乱,神经元凋亡指数升高(P<0.001),血清SOD活力和GSH-PX降低,MDA含量升高(P<0.001),海马BAX、Caspase 3、Caspase 9、PARP-1和AIF蛋白以及ROS含量上调,Bcl-2下调(P<0.001,P<0.01)。与模型组比较,安寐丹组和褪黑素组中央区探索时间和中央区穿行次数均升高(P<0.01,P<0.05),神经元凋亡指数降低(P<0.01),血清SOD和GSH-PX含量升高,MDA含量降低(P<0.001,P<0.01,P<0.05),海马BAX、Caspase 3、Caspase 9、PARP-1和AIF蛋白以及ROS含量下调,Bcl-2上调(P<0.001,P<0.01)。结论 安寐丹可改善睡眠剥夺模型氧化应激损伤和细胞凋亡水平,其机制可能与调控ROS/PARP-1/AIF信号通路有关。

Objective To investigate the effects of Anmeidan on hippocampal neuron apoptosis and oxidative stress in a sleep deprivation model.Methods Totally 60 rats were randomly divided into the control group,model group,Anmeidan group,and melatonin group,with 15 rats in each group.The Anmeidan group received Anmeidan at the dosage of 18.18 g·kg^(-1)·d^(-1),while the melatonin group received melatonin at the dosage of 100 mg·kg^(-1)·d^(-1).The control group and model group were given an equal volume of normal saline.The behavioral changes of the rats were evaluated using the Ethovision video analysis system.HE staining was used to observe the hippocampal neurons,transmission electron microscopy was used to observe the ultrastructure of hippocampal neurons,Tunnel staining was used to observe the neuronal apoptosis,and biochemical assays were conducted to measure the levels of superoxide dismutase(SOD),malondialdehyde(MDA),and glutathione peroxidase(GSH-PX)in the serum.Immunofluorescence staining was used to detect the expression levels of hippocampal B-cell lymphoma-2(Bcl-2),Bcl-2 associated X(BAX),Caspase 3,Caspase 9,and reactive oxygen species(ROS).Western Blot was used to detect the expression of poly ADP-ribose polymerase 1(PARP-1)and apoptosis-inducing factor(AIF).Results Compared with the control group,the model group showed a decrease in time in central and number of entries into central(P<0.001,P<0.01).The number of hippocampal neurons decreased,with a disorganized and sparse arrangement,and an increased index of neuronal apoptosis(P<0.001).The activities of serum SOD and GSH-PX were reduced,while the MDA content was increased(P<0.001).The expression of BAX,Caspase 3,Caspase 9,PARP-1,and AIF,as well as the ROS content in the hippocampus,were up-regulated,while the Bcl-2 protein level was down-regulated(P<0.001,P<0.01).Compared with the model group,the Anmeidan group and melatonin group showed an increase in time in central and number of entries into central(P<0.01,P<0.05).The index of neuronal apoptosis was decreased(P<0.01),and the levels of serum SOD and GSH-PX were increased,while the MDAcontent was decreased(P<0.001,P<0.01,P<0.05).The expression of BAX,Caspase 3,Caspase 9,PARP-1,and AIF,as well as the ROS content in the hippocampus were down-regulated,while the Bcl-2 protein level was up-regulated(P<0.001,P<0.01).Conclusion Anmeidan can improve oxidative stress damage and apoptosis levels in the sleep deprivation model,and the mechanism may be related to regulating the ROS/PARP-1/AIF signaling pathway.