黑逍遥散级联调控氧化应激和细胞凋亡对阿尔茨海默病大鼠的神经保护作用  

Hei Xiaoyaosan Modulates Oxidative Stress and Apoptosis to Exert Neuroprotective Effect in Alzheimer's Disease Rats

作　　者：陈怡琴 杨娇 裴文丽 韩玉梅 王虎平[1,2,3] CHEN Yiqin;YANG Jiao;PEI Wenli;HAN Yumei;WANG Huping(Gansu University of Chinese Medicine,Lanzhou 730000,China;Gansu Key Laboratory of Traditional Chinese Medicine(TCM)Excavation and Innovative Transformation,Lanzhou 730000,China;Gansu Engineering Laboratory for New Products of TCM,Lanzhou 730000,China)

机构地区：[1]甘肃中医药大学,兰州730000 [2]甘肃省中医方药挖掘与创新转化重点实验室,兰州730000 [3]甘肃省中药新产品创制工程实验室,兰州730000

出　　处：《中国实验方剂学杂志》2025年第9期99-107,共9页Chinese Journal of Experimental Traditional Medical Formulae

基　　金：国家自然科学基金项目(82160862,81960828);甘肃省自然科学基金项目(22JR11RA113);第五批全国中医临床优秀人才研修项目(国中医药人教函〔2022〕239号);首批陇原青年英才项目(中共甘肃省委人才工作领导小组〔2022〕5号);甘肃省高校教师创新基金项目(2024A-083)。

摘　　要：目的:探究黑逍遥散调控蛋白激酶B(Akt)/糖原合成酶激酶-3β(GSK-3β)/核因子E_(2)相关因子2(Nrf2)/血红素加氧酶-1(HO-1)信号通路级联调控氧化应激和细胞凋亡防治阿尔茨海默病(AD)的作用及机制。方法:将90只4月龄雄性SD大鼠,随机分为空白组10只,假手术组(双侧海马各注射生理盐水1μL)10只,其余70只双侧海马各注射β淀粉样蛋白1-42(Aβ_(1-42))1μL溶液复制AD模型。造模结束1周后,筛选出造模成功大鼠50只,随机分为模型组、盐酸多奈哌齐组(0.45 mg·kg^(-1))及黑逍遥散高、中、低剂量组(15.30、7.65、3.82 g·kg^(-1))。1次/d,连续6周。Morris水迷宫实验观察各组大鼠学习记忆能力,苏木素-伊红(HE)染色分析AD大鼠海马形态学变化,原位末端标记法(TUNEL)染色观察大鼠海马CA3区神经元细胞凋亡情况,免疫荧光法(IF)检测大鼠海马CA1区神经元核抗原(NeuN)的表达,酶联免疫吸附测定法(ELISA)检测AD大鼠海马谷胱甘肽过氧化物酶(GSH-Px)、谷胱甘肽-S-转移酶(GST)、过氧化氢酶(CAT),实时荧光定量聚合酶链式反应(Real-time PCR)检测Akt、GSK-3β、Nrf2、HO-1 mRNA表达,蛋白免疫印迹法(Western blot)检测磷酸化(p)-Akt/Akt、p-GSK-3β/GSK-3β、Nrf2、HO-1相关蛋白的表达。结果:与空白组比较,模型组大鼠第5天游泳总距离显著增加(P<0.01),目标象限滞留时间百分比显著缩短(P<0.01),神经元细胞数量减少,排列紊乱,胞核固缩,细胞结构不同程度的受损,海马神经细胞凋亡率显著升高(P<0.01),NeuN含量显著降低(P<0.01),GSH-Px、GST、CAT活性显著降低(P<0.01),海马Nrf2、HO-1 mRNA表达显著降低(P<0.01),p-Akt/Akt、p-GSK-3β/GSK-3β、Nrf2、HO-1蛋白表达显著降低(P<0.01);与模型组比较,盐酸多奈哌齐组及黑逍遥散各剂量组第5天游泳总距离明显减少(P<0.05,P<0.01),目标象限滞留时间百分比明显延长(P<0.05,P<0.01),神经元细胞排列较整齐,胞核固缩减少,胞体清晰,海马神经细胞Objective:To explore the role and mechanism of Hei Xiaoyaosan in regulating the protein kinase B(Akt)/glycogen synthase kinase-3β(GSK-3β)/nuclear factor E_(2)-related factor 2(Nrf2)/heme oxygenase-1(HO-1)signaling pathway in cascade modulation of oxidative stress and apoptosis for preventing and treating Alzheimer's disease(AD).Methods:Ninety male SD rats of 4 months old were randomly assigned into a control group(n=10),a sham group(with injection of 1μL normal saline into bilateral hippocampi,n=10),and a modeling group(with injection of 1μL beta-amyloid 1-42 solution into bilateral hippocampi to induce AD,n=70).One week after modeling,50 successfully modeled rats were selected and randomly allocated into model,donepezil hydrochloride(0.45 mg·kg^(-1)),and high-,medium-,and low-dose(15.30,7.65,3.82 g·kg^(-1),respectively)Hei Xiaoyaosan groups.The rats were administrated with corresponding drugs once daily for six consecutive weeks.The Morris water maze was used to assess the learning and memory abilities of rats.Hematoxylin-eosin(HE)staining was performed to reveal hippocampal morphological changes in AD rats.Apoptosis in the hippocampal CA3 region was detected by terminaldeoxynucleotidyl transferase-mediated Nick end labeling.Immunofluorescence was used to visualize the expression of neuronal nuclear antigen(NeuN)in the CA1 region.Additionally,enzyme-linked immunosorbent assay was performed to assess the activities of glutathione peroxidase(GSH-Px),glutathione-S-transferase(GST),and catalase(CAT)in the hippocampus.Realtime PCR was conducted to measure the mRNA levels of Akt,GSK-3β,Nrf2,and HO-1,while Western blot was employed to determine the protein levels of phosphorylated Akt(p-Akt)/Akt,phosphorylated GSK-3β(p-GSK-3β)/GSK-3β,Nrf2,and HO-1.Results:Compared with the control group,the model group on day 5 showed an increase in total swimming distance(P<0.01),a reduction in the percentage of time spent in the target quadrant(P<0.01),reduced and disarranged neurons,nuclear condensation,varying degrees of

关 键 词：黑逍遥散 阿尔茨海默病 蛋白激酶B(Akt)/糖原合成酶激酶-3β(GSK-3β)/核因子E_(2)相关因子2(Nrf2)/血红素加氧酶-1(HO-1)信号通路 细胞凋亡 氧化应激 

分 类 号：R259[医药卫生—中西医结合] R289[医药卫生—中医内科学] R742[医药卫生—中医学]