醒脑益智针刺法对阿尔茨海默病大鼠认知功能及Nrf2/HO-1信号通路的影响

Effect of the brain-refreshing and intelligence-promoting acupuncture method on cognitive function and Nrf2/HO-1 signaling pathway in Alzheimer’s disease rats

关键词:  醒脑益智针刺法  阿尔茨海默病  认知功能  Nrf2/HO-1信号通路

Key words:  brain-refreshing and intelligence-promoting acupuncture method  Alzheimer’s disease  cognitive function  Nrf2/HO-1 signaling pathway

摘要:

[目的] 探讨醒脑益智针刺法对阿尔茨海默病（AD）大鼠认知功能的作用机制。[方法] 12只SPF级雄性SD大鼠随机分为对照组、假手术组、模型组和针刺组，每组3只。除对照组外，模型组和针刺组大鼠采用侧脑室注射链脲佐菌素（STZ）方法构建AD模型，假手术组大鼠则注射等剂量柠檬酸/柠檬酸钠缓冲液。造模成功后进行醒脑益智针法治疗，治疗4周，每周6次。针刺结束后进行Morris水迷宫评估认知功能，酶联免疫吸附（ELISA）法检测血清肿瘤坏死因子-α（TNF-α）和γ干扰素（IFN-γ）水平以及海马组织β淀粉样蛋白1-42（Aβ_1-42）含量，比色法检测额叶和海马组织氧化应激指标丙二醛（MDA）和活性氧（ROS）水平及海马组织总抗氧化能力（T-AOC）水平，实时荧光定量逆转录聚合酶链反应（qRT-PCR）和蛋白免疫印迹（Western blot）法检测额叶和海马组织核因子E2相关因子2（Nrf2）和血红素氧合酶-1（HO-1）及海马组织Kelch 样环氧氯丙烷相关蛋白1（Keap1）的mRNA和蛋白表达。[结果] 与对照组比较，假手术组第2~4天逃避潜伏期、跨越平台次数、目标象限停留时间，TNF-α、IFN-γ、Aβ_1-42、T-AOC、MDA和ROS水平以及Keap1、Nrf2和HO-1的mRNA和蛋白表达水平无显著性差异（P>0.05）；与对照组和假手术组比较，模型组第2~4天逃避潜伏期延长（P<0.05或P<0.01），跨越平台次数和目标象限停留时间减少（P<0.05），TNF-α、IFN-γ、Aβ_1-42、MDA和ROS水平升高（P<0.05或P<0.01），T-AOC水平降低（P<0.01），Keap1的mRNA和蛋白表达水平升高（P<0.01），而Nrf2和HO-1的mRNA和蛋白表达水平降低（P<0.05或P<0.01）；与模型组比较，针刺组第1~4天逃避潜伏期缩短（P<0.05或P<0.01），跨越平台次数和目标象限停留时间增加（P<0.05），TNF-α、IFN-γ、Aβ_1-42、MDA和ROS水平降低（P<0.05或P<0.01），T-AOC水平升高（P<0.01），Keap1的mRNA和蛋白表达水平降低（P<0.01），而Nrf2和HO-1的mRNA和蛋白表达水平升高（P<0.05或P<0.01）。[结论] 醒脑益智针刺法可改善AD大鼠认知功能障碍，其作用机制可能与激活Nrf2/HO-1信号通路有关。

Abstract:

[Objective] To explore the mechanism of the brain-refreshing and intelligence-promoting acupuncture method on cognitive function in rats with Alzheimer’s disease（AD）. [Methods] Twelve SPF male SD rats were randomly divided into control group，sham operation group，model group and acupuncture group，with 3 rats in each group. Except for the control group，the rats in the model group and the acupuncture group were injected with Streptozotocin（STZ） into the lateral ventricle to construct the AD model，and the rats in the sham operation group were injected with the same dose of citric acid/sodium citrate buffer. After successful modeling，the brain-refreshing and intelligence-promoting acupuncture method was used for treatment for 4 weeks，6 times a week. After acupuncture，the Morris water maze was used to evaluate cognitive function. The levels of serum tumor necrosis factor-α（TNF-α） and interferon-γ（IFN-γ） and the content of β-amyloid 1-42（Aβ_1-42） in hippocampus were detected by enzyme-linked immunosorbent assay（ELISA）. The levels of oxidative stress indexes malondialdehyde（MDA） and reactive oxygen species（ROS） in frontal lobe tissue and hippocampus and the total antioxidant capacity（T-AOC） in hippocampus were detected by colorimetry. The mRNA and protein expressions of nuclear factor E2-related factor 2（Nrf2） and heme oxygenase-1（HO-1） in frontal lobe tissue and hippocampus，and Kelch-like epichlorohydrin-related protein 1（Keap1） in hippocampus were detected by real-time quantitative reverse transcription polymerase chain reaction（qRT-PCR） and Western blot. [Results] Compared with the control group，there were no significant differences in the escape latency from days 2 to 4，the number of platform crossings，the time spent in the target quadrant，the levels of TNF-α，IFN-γ，Aβ_1-42，T-AOC，MDA and ROS，as well as the mRNA and protein expression levels of Keap1，Nrf2 and HO-1 compared with the control group（P>0.05）；compared with the control group and sham-operated group，the escape latency of the model group was prolonged from days 2 to 4（P<0.05，P<0.01）；the number of crossing the platform and the residence time in the target quadrant were decreased（P<0.05）；the levels of TNF-α，IFN-γ，Aβ_1-42，MDA and ROS were increased（P<0.05，P<0.01）；the level of T-AOC was decreased（P<0.01），and the mRNA and protein expression levels of Keap1 were increased（P<0.01），while the mRNA and protein expression levels of Nrf2 and HO-1 were decreased（P<0.05，P<0.01）；compared with the model group，the escape latency was shortened from days 1 to 4（P<0.05，P<0.01）；the number of crossing the platform and the residence time in the target quadrant were increased（P<0.05）；the levels of TNF-α，IFN-γ，Aβ_1-42，MDA and ROS were decreased（P<0.05，P<0.01）；the level of T-AOC was increased（P<0.01），the mRNA and protein expression levels of Keap1 were decreased（P<0.01），and the mRNA and protein expression levels of Nrf2 and HO-1 were increased in the acupuncture group（P<0.05，P<0.01）. [Conclusion] Brain-refreshing and intelligence-promoting acupuncture method improves cognitive dysfunction in AD rats，and its mechanism of action may be associated with the activation of Nrf2/HO-1 signaling pathway.