刺五加苷B对疲劳小鼠学习记忆能力的改善作用及其激活Keap1/Nrf2/ARE信号通路的机制

doi:10.13481/j.1671-587x.20200417

摘要/Abstract

摘要： 目的：探讨刺五加苷B（ELB）对疲劳小鼠学习记忆能力的影响，阐明其可能的作用机制。方法：60只ICR小鼠随机分为对照组（灌胃给予蒸馏水，静坐实验）、模型组（灌胃给予蒸馏水，游泳实验）、ELB（C）组（灌胃给予80 mg·kg^-1 ELB，静坐实验）及ELB（M）组（灌胃给予80 mg·kg^-1 ELB，游泳实验），每组15只。持续给药4周后采用转棒实验观察小鼠的抗疲劳能力，采用Morris水迷宫实验和避暗实验检测疲劳小鼠的学习记忆能力，采用比色法检测小鼠血清中乳酸（LD）水平，微量酶标法检测小鼠血清中乳酸脱氢酶（LDH）活性，脲酶法检测小鼠血清中尿素氮（BUN）水平，羟胺法检测小鼠脑组织中超氧化物歧化酶（SOD）活性，硫代巴比妥酸法（TBA）检测小鼠脑组织中丙二醛（MDA）水平，酶联免疫吸附测定法（ELISA）检测小鼠脑组织中活性氧簇（ROS）、内皮型一氧化氮合酶（eNOS）、γ氨基丁酸（GABA）和谷氨酸（GLU）水平，分光光度法检测小鼠脑组织中三磷酸腺苷酶（ATPase，包括Na⁺K⁺-ATPase、Mg²⁺-ATPase、Ca²⁺-ATPase和Ca²⁺Mg²⁺-ATPase）活性，Western blotting法检测小鼠脑组织中Kelch样环氧氯丙烷相关蛋白1（Keap1）、核因子E2相关因子2（Nrf2）和血红素加氧酶1（HO-1）蛋白表达水平，HE染色法观察小鼠脑组织海马区神经元结构。结果：与对照组比较，模型组小鼠转棒时间明显缩短（P<0.05），水迷宫潜伏期明显延长（P<0.05），避暗潜伏期缩短（P<0.05），错误次数增加（P<0.05），血清中LDH活性和LD、BUN水平升高（P<0.05），脑组织中eNOS水平和SOD、Na⁺K⁺-ATPase、Mg²⁺-ATPase、Ca²⁺-ATPase、Ca²⁺Mg²⁺-ATPase活性及GLU/GABA比值降低（P<0.05），MDA和ROS水平升高（P<0.05），脑组织中Keap1蛋白表达水平升高（P<0.05），Nrf2和HO-1蛋白表达水平降低（P<0.05）；ELB（C）组小鼠转棒时间延长（P<0.05），但其余各检测指标差异均无统计学意义（P>0.05）。与模型组比较，ELB（M）组小鼠转棒时间延长（P<0.05），水迷宫潜伏期缩短（P<0.05），避暗潜伏期延长（P<0.05），错误次数减少（P<0.05），血清中LDH活性和LD、BUN水平降低（P<0.05），脑组织中eNOS水平和SOD、Na⁺K⁺-ATPase、Mg²⁺-ATPase、Ca²⁺-ATPase、Ca²⁺Mg²⁺-ATPase活性及GLU/GABA比值升高（P<0.05），MDA和ROS水平降低（P<0.05），脑组织中Keap1蛋白表达水平降低（P<0.05），Nrf2和HO-1蛋白表达水平升高（P<0.05）。与对照组比较，模型组小鼠海马区神经元排列松散，层次不清，部分海马区神经元形态有明显的病理学改变；与模型组比较，ELB（C）和ELB（M）组小鼠脑组织海马区结构层次清晰，神经元增多，排列较为紧密有序，海马区神经元形态和体积趋于正常。结论：ELB能够改善疲劳小鼠的学习记忆能力，其机制可能与调节Keap1/Nrf2/ARE信号通路的相关因子表达有关。

关键词: 刺五加苷B, 疲劳, 学习记忆能力, Kelch样环氧氯丙烷相关蛋白1, 核因子E2相关因子2

Abstract: Objective: To investigate the effect of eleutheroside B (ELB) on the learning and memory abilities in the fatigue mice, and to clarify its possible mechanism. Methods: A total of 60 ICR mice were randomly divided into control group (received distilled water by gavage, meditation experiment), model group (received distilled water by gavage, swimming experiment), ELB (C) group (received 80 mg·kg^-1 ELB by gavage, meditation experiment) and ELB (M) group (received 80 mg·kg^-1 ELB by gavage, swimming experiment) (n=15). After 4 weeks of continuous administration, the anti-fatigue abilities of the mice were observed with rotating rod experiment, and the learning and memory abilities of the fatigue mice were detected by morris water maze experiment and dark avoidance experiment; the levels of lactic acid (LD) in serum of the mice were detected by colorimetry, the activities of lactate deoxygenase (LDH) in serum of the mice were detected by microenzyme labeling method, the levels of urea nitrogen (BUN) in serum of the mice were detected by urease method, the activities of superoxide dismutase (SOD) in brain tissue of the mice were detected by hydroxylamine method,and the levels of malondialdehyde (MDA) in brain tissue of the mice were detected by thiobarbituric acid method; the levels of reactive oxygen species (ROS), endothelial nitric oxide synthase (eNOS), γ-aminobutyric acid (GABA) and glutamate (GLU) in brain tissue of the mice were detected by enzyme linked immunosorbent assay; the activities of adenosine triphosphase (Na⁺K⁺-ATPase, Mg²⁺-ATPase, Ca²⁺-ATPase, Ca²⁺Mg²⁺-ATPase) in brain tissue of the mice were detected by spectrophotometry.Western blotting method was used to detect the expression leves of Kelch-like ECH-associated protein-1 (Keap1),nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) proteins in brain tissues of the mice. HE staining was used to observe the structures of hippocampal neurons in brain tissue of the mice. Results: Compared with control group, the rotating rod time of the mice in model group was shortened (P<0.05), the water maze latency was extended (P<0.05), the dark avoidance latency was shortened (P<0.05), and the number of errors was increased (P<0.05); the activity of serum LDH and the levels of serum LD and BUN were increased (P<0.05); the level of eNOS, the activities of SOD, Na⁺K⁺-ATPase, Mg²⁺-ATPase and Ca²⁺-ATPase, Ca²⁺Mg²⁺-ATPase, and the GLU/GABA ratio in brain tissue of the mice were decreased (P<0.05), and the MDA and ROS levels were increased (P<0.05); the Keap1 protein expression level was increased (P<0.05),and the Nrf2 and HO-1 protein expression levels were decreased (P<0.05); the rotating rod time of the mice in ELB (C) group was prolonged(P<0.05), but the differences in the other indicators had no statistical significance (P>0.05).Compared with model group, the rotating rod time of the mice in ELB (M) group was prolonged(P<0.05), the water maze latency was shortened (P<0.05), the dark avoidance latency was prolonged (P<0.05), and the number of errors was reduced (P<0.05); the activity of serum LDH and the levels of serum LD and BUN were decreased (P<0.05), the level of eNOS and the activities of SOD, Na⁺K⁺-ATPase, Mg²⁺-ATPase and Ca²⁺-ATPase, Ca²⁺Mg²⁺-ATPase,and the GLU/GABA ratio in brain tissue of the mice were increased (P<0.05), and the MDA and ROS levels were decreased (P<0.05);the Keap1 protein expression level was decreased (P<0.05),and the Nrf2 and HO-1 protein expression levels were increased (P<0.05).Compared with control group, the arrangement of neurons in the hippocampus of the mice in model group was loose and unclear, the morphology of some hippocampal cells was changed, and there were obvious pathological changes.Compared with model group, the structures of the hippocampus region of the mice in ELB(C) and ELB(M) groups were clear, the number of neurons was increased,the arrangement was relatively tight and orderly, and the morphology and volume of hippocampal neurons tended to be normal. Conclusion: ELB can improve the learning and memory abilities of the fatigue mice, and its mechanism may be related to regulating the expressions of Keap1/Nrf2/ARE signaling pathway-related factors.

Key words: eleutheroside B, fatigue, learning and memory abilities, Kelch-like ECH-associated protein-1, nuclear factor E2-related factor 2

中图分类号:

R285.5

张倩, 冯晴霞, 周正乙, 李睿祺, 方雷, 杨秋楠, 盛瑜, 卢学春, 杜培革, 安丽萍. 刺五加苷B对疲劳小鼠学习记忆能力的改善作用及其激活Keap1/Nrf2/ARE信号通路的机制[J]. 吉林大学学报(医学版), 2020, 46(04): 771-778.

ZHANG Qian, FENG Qingxia, ZHOU Zhengyi, LI Ruiqi, FANG Lei, YANG Qiunan, SHENG Yu, LU Xuechun, DU Peige, AN Liping. Improvement effect of eleutheroside B on learning and memory abilities of fatigue mice and its mechanism of activating Keap1/Nrf2/ARE signaling pathway[J]. Journal of Jilin University(Medicine Edition), 2020, 46(04): 771-778.

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