Objectives: During pregnancy, pregnant women are prone to stress reactions due to external stimuli, affecting their own health and fetal development. At present, there is no good treatment for the stress reactions from pregnant women during pregnancy. This study aims to explore the effect of probiotics on abnormal behavior and hippocampal injury in pregnant stressed offspring.
Methods: SD pregnant rats were divided into a control group, a stress group, and a probiotics group, with 6 rats in each group. The control group was untreated; the stress group was given restraint stress on the 15th-20th day of pregnancy; the probiotics group was given both bifidobacterium trisporus capsules and restraint stress on the 15th-20th day of pregnancy, and the offspring continued to be fed with probiotics until 60 days after birth (P60). The offspring rats completed behavioral tests such as the open field test, the elevated plus maze test, the new object recognition test, and the barnes maze test at 60-70 d postnatally. Nissl's staining was used to reflect the injury of hippocampal neurons; immunohistochemical staining was used to detect the expression of microglia marker ionized calcium binding adapter molecule 1 (IBA-1) which can reflect microglia activation; ELISA was used to detect the content of plasma TNF-α and IL-1β; Western blotting was used to detect the expression of Bax, Bcl-2, and caspase-3.
Results: The retention time of offspring rats in the stress group in the central area of the open field was significantly less than that in the control group (P<0.01), and the retention time of offspring rats in the probiotic group in the central area of the open field was significantly more than that in the stress group (P<0.05). The offspring rats in the stress group stayed in the open arm for a shorter time than the control group (P<0.05) and entered the open arm less often than the control group (P<0.01); the offspring rats in the probiotic group stayed in the open arm for a longer time than the stress group and entered the open arm more often than the stress group (both P<0.05). The discrimination ratio for new to old objects in the offspring rats of the stress group was significantly lower than that of the control group (P<0.01), and the discrimination ratio for new to old objects in the offspring rats of the probiotic group was significantly higher than that of the stress group (P<0.05). The offspring rats in the stress group made significantly more mistakes than the control group (P<0.05), and the offspring rats in the probiotic group made significantly fewer mistakes than the stress group (P<0.05). Compared with the control group, the numbers of Nissl bodies in CA1, CA3, and DG area were significantly reduced in the offspring rats of the stress group (all P<0.001), the number of activated microglia in DG area of hippocampus was significantly increased (P<0.01), the contents of TNF-α and IL-1β in peripheral blood were significantly increased (P<0.05 or P<0.01), the protein expression level of Bcl-2 was significantly down-regulated, and the protein expression levels of Bax and caspase-3 were significantly up-regulated (all P<0.001). Compared with the stress group, the numbers of Nissl bodies in CA1, CA3, and DG area were significantly increased in the probiotic group offspring rats (P<0.001, P<0.01, P<0.05), the number of activated microglia in the DG area of hippocampus was significantly reduced (P<0.05), and the TNF-α and IL-1β levels in peripheral blood were significantly decreased (both P<0.05), the protein expression level of Bcl-2 was significantly up-regulated, and the protein expression levels of Bax and caspase-3 were significantly down-regulated (all P<0.001).
Conclusions: Probiotic intervention partially ameliorated anxiety and cognitive impairment in rats offspring of pregnancy stress, and the mechanism may be related to increasing the number of neurons, inhibiting the activation of hippocampal microglia, and reducing inflammation and apoptosis.
目的: 在怀孕期间孕妇极易对外界刺激产生应激反应，影响自身健康和胎儿发育。目前针对孕妇在孕期的应激反应没有很好的治疗方式，本研究旨在探索益生菌对孕期应激子代异常行为和海马损伤的作用。方法: 将SD孕鼠分为对照组、应激组、益生菌组，每组6只。对照组不做任何处理；应激组在母鼠怀孕第15~20天进行束缚应激；益生菌组在母鼠怀孕第15~20天进行束缚应激的同时给予双歧杆菌三联活菌胶囊，子代继续给予益生菌饲喂直至生后60 d(P60)。子代大鼠在出生后60~70 d完成旷场实验、高架十字迷宫实验、新物体识别实验和Barnes迷宫实验等行为学检测。采用尼氏染色反映海马神经元受损的情况；免疫组织化学染色检测小胶质细胞标志物离子钙接头蛋白1(ionized calcium binding adapter molecule 1，IBA-1)的表达，以反映小胶质细胞活化情况；ELISA检测血浆TNF-α、IL-1β的含量；蛋白质印迹法检测Bax、Bcl-2及caspase-3的表达。结果: 应激组子代大鼠在旷场中央区滞留时间明显少于对照组(P<0.01)，益生菌组在旷场中央区滞留时间明显长于应激组(P<0.05)。应激组子代大鼠在开放臂停留的时间短于对照组(P<0.05)，且进入开放臂的次数少于对照组(P<0.01)；益生菌组子代大鼠在开放臂停留的时间长于应激组，且进入开放臂的次数多于应激组(均P<0.05)。应激组子代大鼠的对新、旧物体的分辨比明显低于对照组(P<0.01)，益生菌组子代大鼠对新、旧物体的分辨比明显高于应激组(P<0.05)。应激组子代大鼠的犯错次数明显多于对照组(P<0.05)，益生菌组子代大鼠的犯错次数明显少于应激组(P<0.05)。与对照组比较，应激组子代大鼠CA1、CA3及DG区尼氏体数量均明显减少(均P<0.001)，海马DG区小胶质细胞活化的数量明显增加(P<0.01)，外周血TNF-α、IL-1β的含量显著增加(P<0.05或P<0.01)，Bcl-2的蛋白质表达水平明显下调，Bax及caspase-3的蛋白质表达水平明显上调(均P<0.001)；与应激组相比，益生菌组子代大鼠CA1、CA3及DG区尼氏体数量均明显增加(P<0.001，P<0.01， P<0.05)，海马DG区小胶质细胞活化的数量明显减少(P<0.05)，外周血TNF-α、IL-1β的含量显著减少(P<0.05)，Bcl-2的蛋白质表达水平明显上调，Bax及caspase-3的蛋白质表达水平明显下调(均P<0.001)。结论: 益生菌干预可以部分改善孕期应激子代大鼠的焦虑及认知功能损伤，其机制可能与增加神经元的数量、抑制海马小胶质细胞的活化、减轻炎症和凋亡有关。.
Keywords: apoptosis; cognitive function; inflammation; microglia; prenatal stress; probiotics.