运动预处理联合电针对血管性痴呆大鼠学习记忆能力及海马神经元铁死亡的影响

Effects of exercise preconditioning combined with electroacupuncture on learning memory capacity and hippocampal neuronal ferroptosis in rats with vascular dementia

目的:探讨运动预处理(exercise preconditioning,EP)联合电针(electroacupuncture,EA)调控海马铁死亡,对血管性痴呆(vascular dementia,VD)大鼠学习记忆能力的改善作用。方法:将72只雄性SD大鼠随机均分为非EP组和EP组,每组各36只。EP结束后进行VD模型制备,造模成功后将非EP组二次随机分为假手术(sham)组、模型组(VD组)和电针组(VD-EA组),各12只;EP组二次随机分为EP-sham组、EP-VD组和EP-VD-EA组,各12只。EP组所有大鼠进行4周的游泳运动训练,每周运动5 d,每天30 min。4周EP结束后,VD组、EP-VD组、EP-VD-EA组和VD-EA组大鼠进行VD模型制备,sham组和EP-sham组大鼠进行模拟VD模型制备的假手术。造模成功后第7天,EP-VD-EA组和VD-EA组大鼠进行为期4周的EA治疗,每周6 d,每天30 min。干预结束后采用Morris水迷宫评价大鼠学习记忆能力;尼氏染色法观察大鼠海马CA1区神经元形态;比色法检测大鼠海马组织中亚铁离子(Fe2+)、丙二醛(malondialdehyde,MDA)和还原型谷胱甘肽(reduced glutathione,GSH)含量;Western blot法测定大鼠海马组织中的铁死亡相关蛋白核因子E2相关因子2(nuclear factor E2-related factor 2,Nrf2)和谷胱甘肽过氧化物酶4(glutathione peroxidase 4,GPX4)蛋白表达水平。结果:与sham组比较,VD组大鼠平均逃避潜伏期延长,穿越平台次数减少(P<0.01);海马CA1区神经元排列松散紊乱,细胞形态不规则;海马Fe2+和MDA含量升高,GSH含量降低(P<0.01);海马Nrf2和GPX4蛋白表达水平下降(P<0.01)。与VD组比较,EP-VD组、EP-VD-EA组和VDEA组大鼠平均逃避潜伏期缩短,穿越平台次数增加(P<0.05);海马CA1区神经元排列较为整齐,细胞形态较规整;EP-VD组大鼠海马Fe2+和MDA含量显著降低(P<0.01),GSH含量升高(P<0.05);EP-VD-EA组和VD-EA组大鼠海马Fe2+和MDA含量显著降低,GSH含量显著升高(P<0.01);EP-VD组、EP-VD-EA组和VD-EA组大鼠海马Nrf2和GPX4蛋白表达水平显著升高(P<0.01)。结论:EP联合EA可改善VD大鼠学习记忆能力,其机制可能与减轻海马神经元内铁超载,维持机体氧化还原稳态,抑制铁死亡有关。

AIM:To investigate the effects of exercise preconditioning(EP)combined with electroacupuncture(EA)on learning and memory ability of rats with vascular dementia(VD),and to explore role of hippocampal ferroptosis in this process.METHODS:Seventy-two male SD rats were randomly divided into non-EP group and EP group,with 36 rats in each group.The rats were subjected to EP,and subsequently to establish the VD model.The rats from non-EP group were randomly divided into sham group,model group(VD group)and VD-EA group,each with 12 rats,while those in EP group were randomly divided into EP-sham group,EP-VD group and EP-VD-EA group,each with 12 rats.All rats in EP group underwent 4 weeks of swimming exercise training,5 d per week,30 min per day.At the end of the 4th week,the rats in VD,EP-VD,EP-VD-EA and VD-EA groups were used to induce the VD model,and the rats in sham and EP-sham groups received a sham surgery to simulate the VD model.On the 7th day after successful modeling,the rats in EP-VD-EA and VD-EA groups were treated with EA for 4 weeks,6 d per week,30 min per day.At the end of the intervention,the learning and memory ability of the rats was evaluated using Morris water maze.Neuron morphology in the CA1 area of rat hippocampus was observed through Nissl staining.Ferrous ion(Fe2+),malondialdehyde(MDA)and reduced glutathione(GSH)contents in the rat hippocampal tissues were quantified using the colorimetric assay.The expression levels of ferroptosis-related proteins,nuclear factor E2-related factor 2(Nrf2)and glutathione peroxidase 4(GPX4),in the hippocampal tissues were quantified by Western blot method.RESULTS:Compared with sham group,the rats in VD group exhibited longer mean evasion latency and decreased number of traversals across the plateau(P<0.01).The neurons in the CA1 region of the hippocampus were loose and disorganized,exhibiting an irregular cellular morphology.The hippocampal Fe2+and MDA content was elevated,and the GSH content was reduced(P<0.01).The protein levels of hippocampal Nrf2 and GPX4 were decreased(P<0.01).Compared with VD group,the rats in EP-VD,EP-VD-EA and VD-EA groups showed a shorter average escape latency and an increased number of traversals across the plateau(P<0.05).Neurons in the hippocampal CA1 area were more neatly arranged,showing regular cellular morphology.The hippocampal Fe2+and MDA contents of the rats in EP-VD group were significantly reduced(P<0.01),while the GSH content was elevated(P<0.05).Hippocampal Fe2+and MDA contents were significantly reduced and GSH contents were significantly increased in EP-EA and EA groups(P<0.01).The protein levels of hippocampal Nrf2 and GPX4 in EP-VD,EP-VD-EA and VD-EA groups were significantly increased(P<0.01).CONCLUSION:Exercise preconditioning combined with EA improves learning and memory ability in VD rats by reducing hippocampal intra-neuronal iron overload,maintaining organismal redox homeostasis,and inhibiting ferroptosis.