二十二碳六烯酸對母體產後焦慮及雄性子代成鼠下視丘-腦下垂體-腎上腺軸對壓力反應之影響

Abstract

二十二碳六烯酸（Docosahexaenoic acid，簡稱DHA）是構成哺乳類動物神經細胞膜最主要的n - 3多元不飽和脂肪酸，為神經系統不可或缺之營養元素，並扮演許多重要的神經功能，如構成神經細胞膜，調節單胺類神經傳導物質，以及影響行為反應。在大腦發育時期，胎兒會經由胎盤及母乳中獲取DHA，大量的累積在神經細胞中。本研究旨要為探討「母體懷孕及泌乳期間，DHA對母親產後焦慮及子代下視丘-腦下垂體-腎上腺軸對壓力反應之影響」。 本研究首先欲探討「懷孕及泌乳期間胎兒是否會剝奪母體DHA，改變母體腦中DHA和其他脂肪酸組成，影響母體腦中血清胺代謝，而魚油補充是否可以減少母體腦中DHA流失及改善產後焦慮」。利用Sprague-Dawley懷孕母鼠，在懷孕及哺乳期間，餵食以葵花油為主的缺乏n-3不飽和脂肪酸飼料，並給予或不給予魚油補充，另外，將年齡相同的Sprague-Dawley母鼠餵食相同的飼料41天作為懷孕的對照組。結果顯示，在產後母鼠，其下視丘、海馬迴、額葉皮質、小腦、嗅球和視網膜DHA含量均顯著減少，腦幹、額葉皮質和海馬迴血清胺濃度下降，以及腦幹和額葉皮質血清胺代謝速率增加。在懷孕及哺乳期間給予魚油補充可以預防懷孕母鼠腦中的DHA流失，抑制腦幹單胺氧化酶MAO-A的活性，和減少十字迷宮類焦慮的行為表現。由以上的結果我們推論，懷孕生殖會消耗母體大腦DHA以及影響母體腦中血清胺代謝的調節，而魚油的補充可預防n-3脂肪酸缺乏飲食之母體腦中DHA的流失，以及改善產後焦慮行為。 第二部分的實驗則利用上述實驗母鼠的子代雄鼠，探討「大腦發育期間缺乏DHA對於雄性成鼠在遭遇壓力下所引發下視丘-腦下垂體-腎上腺軸(HPA axis)的活化機制與焦慮和憂鬱行為反應之影響」。利用Sprague-Dawley雄鼠，在大腦發育時期(胚胎至出生後第3週)或大腦發育趨於成熟後(出生後第三至第十週)餵食以葵花油為主的缺乏n-3不飽和脂肪酸飼料，控制組則給於魚油補充，作為含適量n-3不飽和脂肪酸組別，觀察十週大成鼠焦慮及憂鬱的行為反應，以及在遭遇壓力後血清中壓力荷爾蒙corticosterone和GABA合成酶glutamate decarboxylase(GAD 67)蛋白質的表現。另外，在動物麻醉的狀態下，直接注射GABA拮抗劑bicuculline methiodide至下視丘室旁核，觀察心跳、血壓和體溫等生理反應之變化。結果顯示，雄性子代成鼠在大腦發育期缺乏DHA顯著增加並延長約束壓力所引起的體溫變化及血清中皮質固醇的濃度上升，顯著增加GABAA受體拮抗劑所引起的心跳速率上升，以及增加十字迷宮測試中的類焦慮行為和強迫游泳行為測試的類憂慮行為反應。這些影響沒有在大腦發育成熟後餵食缺乏n-3不飽和脂肪酸飼料的成鼠中觀察到。由以上的結果我們認為，在大腦發育時期缺乏DHA會增加雄性子代成體後焦慮、憂鬱的行為表現，以及在遭受壓力下HPA axis的活性，期間可能經由影響GABA的合成或是調控GABAA受體，而影響GABA對HPA axis的抑制作用。 綜合以上研究結果顯示，DHA的缺乏是造成情感性疾病如焦慮、憂鬱的病因之一。在母體懷孕時缺乏n-3脂肪酸飲食下，魚油的補充能發揮其抗焦慮憂鬱的功效，有益於改善母體產後焦慮，及減少子代焦慮憂鬱之行為表現。期望此研究對於未來在情緒功能障礙的預防及治療能有所貢獻。

Docosahexaenoic acid (DHA, 22:6n-3), the major n-3 polyunsaturated fatty acids (PUFA), is specific enriched in the neuronal membrane. DHA plays an important role in neural function, such as the formation of neuronal membranes, modulation of monoamine neurotransmission, and even behavior changes. Most DHA accumulation rapidly in the brain occurs during brain development and is supplied via the placenta to the fetus and the breast-fed milk to the pup. The aims of this thesis were to examine the effects of DHA on maternal postpartum anxiety and adult offspring HPA axis responses to stress. In Part I, the study was designed to examine whether maternal brain DHA levels were depleted during pregnancy and lactation due to meeting the high demand of the developing nervous system in the offspring, and whether fish oil supplementation of maternal rats on an n-3 fatty acids deficient diet prevented depletion of maternal brain regional DHA levels and altered serotonin metabolism and had a postpartum anxiolytic effect. Pregnant rats were fed during pregnancy and lactation with a sunflower oil-based n-3 PUFA-deficient diet with or without fish oil supplementation, and the age-matched virgin rats were fed the same diets for 41 days. In both sets of postpartum rats, decreased DHA levels compared to those in virgin females were seen in the hypothalamus, hippocampus, frontal cortex, cerebellum, olfactory bulb and retina. Serotonin levels were decreased and turnover increased in the brainstem and frontal cortex in postpartum rats compared to virgin rats. Fish oil supplementation during pregnancy and lactation prevented the decrease in maternal brain regional DHA levels, inhibited monoamine oxidase-A activity in the brainstem and decreased anxiety-like behavior. We propose that the reproductive cycle depletes maternal brain DHA levels and modulates maternal brain serotonin metabolism to cause postpartum anxiety and suggest that fish oil supplementation may be beneficial for postpartum anxiety in females on an n-3 PUFA deficient diet. In part Ⅱ, the study was designed to evaluate whether brain development was the critical period in which DHA deficiency leads to dysregulation of the HPA axis in response to stress later in life. Rats were exposed to an n-3 fatty acid-deficient diet or the same diet supplemented with fish oil as an n-3 fatty acid-adequate diet either during the per-weaning period from the embryo to weaning at 3 wk-old or during the post-weaning period from 3- to 10-wk-old. We found that DHA deficiency during the pre-weaning period significantly increased and prolonged restraint stress-induced colonic temperature changes and serum corticosterone levels, caused a significant increase in GABAA antagonist-induced heart rate changes and enhanced depression-like behavior in the forced-swimming test and anxiety-like behavior in the plus-maze test in later life. These effects were not seen in male rats fed the n-3 fatty acid-deficient diet during the post-weaning period. These results suggest that brain development is the critical period in which DHA deficiency leads to excessive HPA responses to stress and elevated behavioral indices of depression and anxiety in adulthood. We propose that these effects of hypothalamic DHA deficiency during brain development may involve a GABAA receptor-mediated mechanism. The above-listed observations reveal that lack of DHA is of aetiological importance in mental disorders. These results suggest that fish oil supplementation show anxiolytic and antidepressant effects, and may be beneficial for the prevention of the maternal and child mental health disorders on an n-3 fatty acid-deficient diet.