针刺对宫内窘迫缺血缺氧脑损伤新生大鼠行为学及海马神经元自噬的影响

Acupuncture improves locomotor activity and learning-memory ability by improving hippocampal cellular autophagy in rats with fetal intrauterine distress

目的:观察针刺对宫内窘迫缺血缺氧脑损伤(HIBD)新生大鼠的行为学、海马神经元自噬水平及自噬相关蛋白Beclin-1、LC3表达的影响,探讨针刺调控海马神经元自噬改善新生儿HIBD的机制。方法:SD孕鼠6只,2只孕鼠以宫内窘迫延迟剖宫产法行HIBD造模,2只孕鼠作为HIBD模型新生大鼠代乳鼠,2只孕鼠自然分娩正常新生大鼠。自然分娩的9只新生大鼠为正常组,HIBD新生大鼠按体质量、性别因素分层随机分为模型组和针刺组,每组9只。针刺组大鼠于出生后第14天予"靳三针"针刺,每穴快速捻转10次,每日1次,持续14 d。旷场实验、Morris水迷宫实验观察各组自主活动能力、空间学习记忆能力;透射电镜法观察大鼠海马神经元超微结构、自噬小体形态和数量;流式细胞术检测海马组织自噬相关蛋白Beclin-1的阳性细胞百分比;Western blot法检测海马组织LC3的表达。结果:与正常组比较,模型组大鼠走出中央区域时间、逃避潜伏期、第1次穿越原平台时间均明显延长(P<0.01,P<0.05,P<0.001),中央区域路程和总路程、活动次数、目标象限停留时间、穿越原平台次数均明显减少(P<0.01,P<0.05),海马区神经元可见核膜锯齿样改变,核周间隙增宽,线粒体空泡化,内质网扩张,自噬小体增多,自噬相关蛋白Beclin-1、LC3Ⅱ/Ⅰ比值明显升高(P<0.01);与模型组比较,针刺组大鼠走出中央区域时间、逃避潜伏期、第1次穿越原平台时间明显缩短(P<0.05,P<0.01,P<0.001),中央区域路程和总路程、活动次数、目标象限停留时间、穿越原平台次数显著增加(P<0.01,P<0.05),海马区神经元可见完整连续核膜,细胞器形态较好,自噬小体明显增多,Beclin-1、LC3Ⅱ/Ⅰ比值明显升高(P<0.001)。结论:针刺可能通过上调Beclin-1和LC3Ⅱ/Ⅰ的表达,促进海马神经元自噬,改善HIBD新生大鼠的自主活动能力和空间学习记忆能力。

Objective To investigate the effect of acupuncture stimulation of head acupoints "Jin San Zhen"(JIN’s Three Acupuncture Needles Therapy) on behavior reactions, hippocampal neuronal autophagy and expression of autophagy associated proteins(Beclin-1 and light chain 3 Ⅱ/Ⅰ [LC 3 Ⅱ/Ⅰ]) in rats with hypoxic-ischemic brain damage(HIBD) due to fetal intrauterine distress, so as to reveal its underlying mechanisms in improving neonatal HIBD. Methods Pregnant SD rats were used in the present study. The HIBD model was established by delayed caesarean delivery and bilateral uterine arteries clipping for 10 minutes. The HIBD rats were randomly divided into model group and acupuncture groups(n=9 rats in each group). The other 9 rats delivered naturally were used as the normal control group. On day 14 after delivery, the neonatal rats in the acupuncture group received acupuncture stimulation of head acupoints("Nao San Zhen""Nie San Zhen" and "Zhi San Zhen") by twirling each needle leftward and rightward for 10 times, once a day for 14 d. The open field test and Morris water maze test were used to determine the locomotive activity and spatial learning-memory ability, respectively. The ultrastructure and autophagosomes in the hippocampal neurons were observed by transmission electron microscope. The contents and expression levels of Beclin-1 and LC3 Ⅱ/Ⅰ in the hippocampus tissues were detected by flow cytometry and Western blot, separately. Results Compared with the normal control group, the time to go out of the central region of open field test, and the escape latency and duration of first platform-quadrant-crossing of spatial exploration of Morris water maze tests were significantly increased(P<0.01,P<0.05,P<0.001), and the total distance and number of activities in the central region, and the target quadrant resistance time and number of platform-cros-sing remarkably decreased in the model group(P<0.01, P<0.05), suggesting a decline of both locomotor activity and learning-memory ability after modeling. The expression level(%) of Beclin-1 protein and ratio of LC3 Ⅱ/Ⅰ proteins were considerably increased in the model group(P<0.01). Following acupuncture interventions, the locomotor activity and spatial learning-memory ability were obviously increased(P<0.05,P<0.01,P<0.001), and the expression of Beclin-1 protein and ratio of LC3 Ⅱ/Ⅰ were further up-regulated relevant to the model group(P<0.001). Moreover, ultrastructural observation showed serrated change of nuclear membrane and widened perinuclear space, vacuolization in the mitochondria, dilation of endoplasmic reticulum and increase of autophagosomes in the hippocampal neurons in the model group. These situations were relatively milder in the acupuncture group. Conclusion Acupuncture of head acupoints of "JIN San Zhen" may increase locomotor activity and learning-memory abi-lity in rats with HIBD due to fetal intrauterine anoxia, which is closely with its effect in promoting hippocampal neuronal autophagy via up-regulating the expression of Beclin-1 and LC3 Ⅱ/Ⅰ.