丰富环境对孕期慢性应激大鼠子代海马IGF-Ⅱ和NF-κB表达的干预研究

Intervention of enriched environment on expressions of IGF-Ⅱ and NF-κB in hippocampus of offspring of rats with chronic stress during pregnancy

  • 摘要:
    背景 生命早期生活环境对子代成长发挥着重要作用,孕期应激性事件会造成母体宫内环境改变,进而对子代生长发育产生影响。胰岛素样生长因子-Ⅱ(IGF-Ⅱ)和核因子-κB(NF-κB)蛋白主要分布在脑内与认知和情感记忆相关的区域,与单胺类神经递质去甲肾上腺素(NE)、多巴胺(DA)共同调节机体注意力和反应能力。丰富环境通过神经发生、突触发生、脑血管再生等神经重塑作用,在促进神经再生的同时可改善学习和记忆能力。
    目的 探讨丰富环境干预对孕期慢性应激大鼠子代海马突触相关蛋白IGF-Ⅱ和NF-κB表达的影响。
    方法 将20只Wistar雌鼠随机分为模型组和对照组,每组10只;15只Wistar雄鼠随机分为模型交配组10只和对照交配组5只。模型组选择慢性不可预知温和应激(CUMS)模型进行为期21 d的应激,在应激第3天按1:1雌雄比例合笼;对照组于正常环境饲养,同期按2:1雌雄比例合笼。雌鼠在应激前1d和应激第1、7、14天进行眼内眦静脉采血,利用放射免疫法测定血浆皮质醇质量浓度并计算皮质酮质量浓度。子鼠出生后21 d(PND21)断乳,将对照组的子鼠随机分为对照子鼠组和对照+丰富环境组,模型组的子鼠分为模型子鼠组和模型+丰富环境组,每组16只。进行丰富环境干预时,将子鼠每天暴露于丰富环境6 h(13:00-19:00),干预周期为30 d,未干预组于正常环境饲养。PND50时称重,并利用自制敞箱进行子鼠的行为学指标测定,指标包括水平运动得分和垂直运动得分。在PND55时处死各组子鼠,取海马组织。ELISA法检测子鼠海马组织单胺类神经递质NE和DA的水平,通过实时定量PCR和Western blotting法分别测定子鼠海马组织IGF-ⅡNF-κB mRNA和蛋白的相对表达水平。
    结果 模型组雌鼠在应激第7、14天血浆皮质酮质量浓度分别为(348.50±13.24)和(258.38±22.77)μg·L-1,均高于对照组雌鼠(224.00±39.84)和(218.78±16.68)μg·L-1P < 0.05),提示模型组大鼠在孕期处于应激状态。丰富环境干预后,PND50时模型+丰富环境组和对照+丰富环境组子鼠体重(191.70±18.31)和(208.80±21.40)g分别高于模型子鼠组和对照子鼠组(175.70±8.12)和(190.40±16.47)g(P < 0.05)。模型+丰富环境组子鼠水平及垂直运动得分分别为(46.10±9.26)和(23.50±7.99),高于模型子鼠组(24.40±8.30)和(16.30±4.42)(P < 0.05);对照+丰富环境组子鼠垂直运动得分为(27.20±9.94),较对照子鼠组(21.70±6.83)高(P < 0.05)。模型+丰富环境组子鼠NE和DA的水平(1.42±0.65)和(2.85±0.75)μg·L-1高于模型子鼠组(0.89±0.26)和(1.94±0.64)μg·L-1P < 0.05)。与模型子鼠组(0.50±0.19)和(0.96±0.30)相比,模型+丰富环境组(1.11±0.53)和(1.55±0.69)子鼠海马组织IGF-ⅡNF-κB mRNA的相对表达水平升高(P < 0.05)。模型+丰富环境组子鼠海马组织IGF-Ⅱ和NF-κB蛋白相对表达水平(0.83±0.10)和(0.67±0.27)高于模型子鼠组(0.40±0.07)和(0.30±0.16)(P < 0.05),对照+丰富环境组子鼠海马组织IGF-Ⅱ和NF-κB蛋白相对表达水平(1.41±0.11)和(0.75±0.30)高于对照子鼠组(0.76±0.07)和(0.59±0.07)(P < 0.05)。
    结论 丰富环境对孕期慢性应激大鼠子代的体重及运动能力有一定的促进作用,可能与其提高子鼠海马组织NE和DA水平以及突触相关蛋白NF-κB和IGF-Ⅱ的表达水平有关。

     

    Abstract:
    Background The early-life living environment plays an important role in the growth of offspring. Stressful events during pregnancy can induce changes in maternal intrauterine environment, which in turn affects the growth and development of offspring. Insulin like growth factor-Ⅱ (IGF-Ⅱ) and nuclear factor-κB (NF-κB) are mainly distributed in the areas related to cognition and emotional memory in the brain, they work together with monoamine neurotransmitters norepinephrine (NE) and dopamine (DA) to regulate the attention and response ability of the body. Enriched environment can improve learning and memory ability while promoting nerve regeneration through neurogenesis, synapsis, cerebrovascular regeneration and other neural remodeling effects.
    Objective  This experiment is designed to investigate the effects of enriched environmental intervention on the expressions of hippocampal synapse-associated proteins such as IGF-Ⅱand NF-κB in offspring of rats with chronic stress during pregnancy.
    Methods Twenty female Wistar rats were randomly divided into a model group and a control group of 10 rats each; 15 male Wistar rats were randomly divided into a model group of 10 rats and a control group of 5 rats. The female rats of the model group received chronic unpredictable mild stress (CUMS) for 21 d, and were caged at a 1:1 female-to-male ratio on the 3rd day of the CUMS protocol. The female rats of the control group were reared in normal environment and caged at a 2:1 ratio. Blood samples were collected from the iliac vein of the female rats 1 day before and 1, 7, and 14 days after the CUMS protocol, and measured for plasma cortisol content by radioimmunoassay to calculate corticosterone concentration. The offspring rats were weaned on postnatal day 21 (PND21). The control offspring rats were randomly divided into a control offspring group and a control + enriched environment offspring group. The model offspring rats were divided into a model offspring group and a model + enriched environment offspring group, with 16 pups in each group. The offspring rats of two enriched environment groups were exposed to enriched environment for 6 h (13:00-19:00) every day, and the intervention period was 30 d. The other offspring rats were raised in normal environment. The offspring body weight was weighed on PND50, and behavioral parameters were measured, including horizontal movement score and vertical movement score, using a self-designed open box. Then the offspring rats were sacrificed and hippocampal tissue samples were collected. The levels of monoamine neurotransmitters norepinephrine (NE) and dopamine (DA) in hippocampus of the offspring rats were detected by ELISA. The relative expression levels of IGF-Ⅱ and NF-κB mRNA and protein in hippocampus of the offspring rats were determined by real-time quantitative PCR and Western blotting, respectively.
    Results The plasma corticosterone concentrations of the female rats in the model group on the 7th and 14th days of stress(348.50±13.24) and (258.38±22.77) μg·L-1 were higher than those of the female rats in the control group(224.00±39.84) and (218.78±16.68) μg·L-1 (P < 0.05), suggesting that the female rats in the model group were stressed during pregnancy. After the enriched environment intervention, the body weights of the offspring rats in the model + enriched environment group and the control + enriched environment group on PND50(191.70±18.31) and (208.80±21.40) g were higher than those of the offspring rats in the model group and the control group(175.70±8.12) and (190.40±16.47) g (P < 0.05). The scores of horizontal movement and vertical movement of the offspring rats in the model + enriched environment group(46.10±9.26) and (23.50±7.99) were higher than the scores of the offspring rats in the model group(24.40±8.30) and (16.30±4.42) (P < 0.05). The vertical movement score of the offspring rats in the control + enriched environment group (27.20±9.94) was higher than that of the offspring rats in the control group (21.70±6.83) (P < 0.05). The levels of NE and DA of the model + enriched environment offspring group(1.42±0.65) and (2.85±0.75) μg·L-1 were higher than those of the model offspring group(0.89±0.26) and (1.94±0.64) μg·L-1 (P < 0.05). Compared with the model offspring rats(0.50±0.19) and (0.96±0.30), the relative expression levels of IGF-Ⅱ and NF-κB mRNA in hippocampus of the model + enriched environment offspring rats were increased(1.11±0.53) and (1.55±0.69) (P < 0.05). The relative expression levels of IGF-Ⅱ and NF-κB proteins in hippocampus of the model + enriched environment offspring rats(0.83±0.10) and (0.67±0.27) were higher than those of the model offspring rats(0.40±0.07) and (0.30±0.16) (P < 0.05); the relative expression levels of IGF-Ⅱand NF-κB proteins in hippocampus of the control + enriched environment offspring rats(1.41±0.11) and (0.75±0.30) were higher than those of the control offspring rats(0.76±0.07) and (0.59±0.07) (P < 0.05).
    Conclusion Enriched environment can promote the weight gain and exercise capacity of the offspring of chronically stressed rats during pregnancy, and can increase the expressions of monoamine neurons NE and DA and synaptic related proteins NF-κB and IGF-Ⅱ in offspring hippocampus.

     

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