探討大鼠腦部發育老化過程對突觸可塑性基因、參與DHA合成及嵌入細胞膜磷脂質酵素表現之影響

Abstract

二十二碳六烯酸（Docosahexaenoic acid, DHA；22:6n-3）主要富含在腦部，並嵌在細胞膜磷脂質以維持正常神經功能。DHA可經由前驅物α-次亞麻油酸（α-linolenic acid, ALA；18:3n-3）透過去飽和（desaturation）與加長碳鏈（elongation）合成DHA，並藉由Long-chain acyl-CoA synthetase（ACSLs）將DHA轉換為DHA-CoA，再靠Lyso-phosphatidate acyltransferase（LPAATs）將DHA-CoA嵌入磷脂質中。DHA在大鼠大腦發育期快速累積，隨著老化含量下降。給予停經後婦女雌激素替代療法，比起沒有給予雌激素替代療法的其血漿DHA含量較高，亦可延緩認知功能下降。因此，本論文的主旨主要要探討雌鼠隨著大腦發育、老化過程與去卵巢，攝食n-3脂肪酸缺乏的飲食、年齡與雌激素是否會影響參與DHA合成及嵌入細胞膜磷脂質的蛋白質與酵素和突觸可塑性相關基因表現。 本實驗第一部分利用母鼠在懷孕第一天開始餵食sunflower oil-based n-3脂肪酸缺乏或sunflower oil mixed with fish oil 之n-3脂肪酸充足的飼料，並在子代出生及離乳後，繼續讓子代食用n-3缺乏或充足的飼料直至犧牲。取子代出生第0.5、7、14、21、28、90、210、360、510天之腦部皮質。以氣相層析儀進行脂肪酸成分分析。以即時聚合酶鏈式反應分析mRNA。 本實驗發現腦部皮質DHA含量與突觸可塑性相關基因表現，包含synaptophysin、synapsin-1、PSD95、GluR1、NR1、NR2A、BDNF及CaMKII，會隨著大腦發育而逐漸增加至21到28天大，而後表現量達plateau至510天大。神經發育指標GAP-43與Drebrin在剛出生時表現量最高，隨著腦部發育表現量下降至14-21天大，而後成年期至510天大表現量皆很低。在攝食n-3脂肪酸缺乏飼料的情況下，腦部皮質DHA含量、Synapsin-1與PSD95基因表現量會低於攝食n-3脂肪酸充足飼料的組別。而參與DHA生成的酵素Fads2、Fads1、Elovl5、Elovl2、Aox、DBP 與TH，以及幫助DHA運輸至腦部的蛋白質FABP3、FABP5、FABP7、FATP4與FATP5，在剛出生時表現量最高，隨著腦部發育表現量下降至14-21天大，而後成年期至540天大表現量皆很低。相反地，腦部皮質參與將DHA嵌入磷脂質的酵素ACSL3、ACSL6、LPAAT4及LPEAT2，則會隨著大腦發育表現量逐漸增加至21-28天大，而後表現量達plateau至510天大。在大腦發育及老化過程，n-3脂肪酸飲食對這些酵素表現沒有影響。 本實驗第二部分探討雌激素影響，利用餵食Chow diet的雌鼠在六個月大時進行去卵巢手術（OVX）或假手術（Sham），然後再分成餵食sunflower oil-based n-3脂肪酸缺乏飼料或額外補充魚油的組別，飼養至12個月大時進行犧牲。本實驗補充魚油的組別，去卵巢的老鼠肝臟基因Fads2、Fads1與ACSL4的表現，顯著低於假手術組。此外，去卵巢老鼠的海馬迴中參與將DHA嵌入磷脂質的酵素ACSL1、ACSL3、ACSL6、LPAAT、LPEAT2、FABP3及FATP4，其表現量顯著低於假手術組。結果顯示，利用去卵巢剝奪雌激素會降低肝臟參與DHA合成及海馬迴參與將DHA嵌入細胞膜磷脂質的mRNA表現。 本實驗結果表示，腦部皮質DHA含量會在大腦發育期快速累積至成年，而後表現量達plateau至510天大。腦部參與DHA合成的酵素在剛出生時表現量最高，隨著腦部發育表現量下降，而後成年期至510天大表現量皆很低。相反地，腦部皮質參與將DHA嵌入磷脂質的酵素則會隨著大腦發育表現量逐漸增加至成年，而後表現量達plateau至510天大。因此我們推論，腦部參與DHA合成與參與將DHA嵌入細胞膜磷脂質的酵素，對於大腦發育期腦部DHA的堆積以及成年後的維持扮演重要的角色。

Docosahexaenoic acid (22:6n-3, DHA) is mainly specifically enriched in the brain and is primary anchored into neuronal membrane phospholipids for normal neurological function. DHA is biosynthesized from its precursor, α-linolenic acid (18:3n-3) via desaturases and elongases following by converted to DHA-Co-A by long-chain acyl-CoA synthetase (ACSLs) and then incorporated into phospholipids by Lyso-phosphatidate acyltransferase (LPAATs). DHA accumulates rapidly during brain development and is reduced in aged rats. Postmenopausal women with estrogen replacement therapy have higher plasma DHA levels and delay cognitive decline than those without hormone replacement therapy. The aim of this thesis was to examine the effect of n-3 fatty acid-deficient diet, estrogen and age on the enzyme involved in DHA biosynthesis and incorporation into membrane phospholipids as well as the synaptic plasticity-related gene expression during brain development, in aging and ovariectomized (OVX) female rats. Rats exposed to a sunflower oil-based n-3 fatty acid-deficient diet or sunflower oil mixed with fish oil as an n-3 fatty acid-adequate diet from in utero via maternal intake. After weaning, pups were maintained on the same diet as their dams till sacrificed at age of 0.5, 7, 14, 21, 28, 90, 210, 360 and 510 days old. The fatty acid composition and mRNA expression in cortex were analyzed by GC and RT-qPCR, respectively. The brain DHA levels and synaptic plasticity-related gene expression including synaptophysin, synapsin-1, PSD-95, GluR1, NR1, NR2A, BDNF and CaMKII were increased during brain development up to the age of 21-28 days old and then plateau up to the examined age at 540 days old. The neuronal development marker, GAP-43 and Drebrin mRNA expression in cortex were high at newborn following decreased during development down to 14-21 day old and then remain constant low at adult and in aging to the age of 540 days old. The brain DHA levels as well as gene expression of synapsin-1 and PSD-95 were significantly lower in rats fed an n-3 fatty acid-deficient diet compared the rats fed n-3 adequate diet. The expression of the enzyme involved in DHA biosynthesis, Fads2, Fads1, Elovl5, Elovl2, Aox, DBP and TH as well as the protein for the DHA uptake into brain, FABP3, FABP5, FABP7, FATP4 and FATP5 were high at newborn following decreased during development down to 14-21 day old and then remain constant low throughout to the age of 540 days old. In contrast, the expression of the enzyme involved DHA incorporation into phospholipids, Acsl3, Acsl6, Lpeat2 and Lpaat4 were increased during brain development up to the age of 21-28 days and then plateau up to the examined age at 540 days old. No main effect of n-3 diet on those enzymes expression during developing and in aging cortex. In study of the estrogen effect, Chow diet fed female rats were subjected to OVX or sham operation (sham) at 6 months old and then fed n-3 fatty acid-deficient diet without or with fish oil supplementation as n-3 fatty acid-adequate groups till sacrificed at 12-month-old. The Fads2, Fads1 and Acsl4 gene expression in liver were significantly reduced in rats fed n-3 adequate diet with OVX compared to the rats with sham operation. In addition, in hippocampus, the gene expression of the enzyme involved DHA incorporation into phospholipids, Acsl1, Acsl3, Acsl6, Lpeat2 and Lpaat4 as well as FATP4 and FABP3 were significantly decreased in OVX rats compared to the sham. It was concluded that estrogen deprivation by OVX did reduce mRNA expression of enzymes involved in DHA biosynthesis in liver and in DHA incorporation into membrane phospholipids in hippocampus. It was concluded that the brain DHA levels were accumulated rapidly during brain development and then plateau without change up to the examined age at 540 days old. The expressing of the enzyme involved in DHA biosynthesis in brain was high at new born, following decreasing during brain developing and then remain constant low in aging. In contrast, the expression of enzyme involved DHA incorporation into phospholipids in brain were increased during brain development and then plateau up to the examined age at 540 days old. We proposed that the enzyme involved in DHA biosynthesis in brain and the enzyme involved DHA incorporation into neuronal membrane may play important roles for the DHA accumulation during brain development and its maintenance in aging brain, respectively.