檳榔鹼對小鼠味蕾和斑馬魚胚胎體節的影響

Abstract

檳榔鹼 (arecoline)是檳榔 (betel quid) 中含量最多的生物鹼 (alkaloid)，嚼食檳榔會引起口腔之發炎、疾病及癌症，對於懷孕婦女更會造成早產及胎兒發育不良、體重過低等問題。檳榔鹼或嚼食檳榔對口腔之影響，已有甚多之研究，但口腔中的味蕾，是味覺的感受器官，若味蕾受到影響，可能會引起味覺異常，但鮮有研究指出檳榔鹼對味蕾的影響，故本論文之第一部分是研究檳榔鹼對味蕾的影響。另外，雖有甚多以不同動物針對檳榔鹼對懷孕的影響進行研究，但均做長時間的檳榔鹼接觸；因此本論文之第二部分選用斑馬魚為實驗動物，作短時間的檳榔鹼接觸，以模擬孕婦懷孕初期接觸檳榔鹼的影響。 實驗一：味蕾 (taste bud) 位在口腔舌頭表面乳突下的味覺接受器，若味蕾受到影響就有可能會影響味覺，但鮮有研究指出檳榔鹼對味蕾的影響。 實驗一係將雄性小黑鼠(C57BL/6)分成接受檳榔鹼1、2、3、4週的實驗組以及未接受檳榔鹼的控制組。將檳榔鹼2 mg/kg 一天兩次以腹腔注射方式給予各組之實驗小鼠；觀察 (1) 輪廓狀味蕾外形、大小、數量和內含味蕾細胞數；(2) 味蕾細胞超微結構分析；(3) 免疫螢光染色分析味覺接受器 (T1R1、T1R2、T1R3和T2R)以及味覺相關蛋白 (α-gustducin, PLCβ2 和 SNAP25) 的表現；(4) 在動物飲用含1%蔗糖之飲水後，統計孤立束核和味覺皮質區c-fos的表現量；(5) 紀錄動物的體重、攝食和飲水以及雙瓶喜好度行為。另外，要了解檳榔鹼對味蕾細胞更替率的影響，在各組實驗小鼠接受設定的檳榔鹼後，將BrdU 50mg/kg以腹腔注射方式給予，標定味蕾新生細胞，(6)統計在接受檳榔鹼後，味蕾內第1、3、7和14天被BrdU標定細胞數；(7)並且以RP-PCR測定舌頭上皮cyclin B2、E2F1、p53和bax味蕾更替相關基因的表現量。 結果發現，實驗小鼠接受檳榔鹼後，(1)小鼠味蕾的數量、大小及內含味蕾細胞數不會改變；(2) 味蕾第二型細胞內之粒線體、內質網明顯腫脹，低密度不規則顆粒變多，雙環小泡和密心顆粒結構腫脹且不規則，自噬小體數量明顯增加；(3) 免疫螢光染色之味蕾切片顯示甜味接受器 (T1R2和T1R3)表現量減少；(4) 含1%蔗糖之飲水實驗結果，顯示孤立束核和味覺皮質區c-fos的表現量降低；(5) 減少食物的攝取量導致體重沒有增加，並改變對甜味覺的喜好度。另外，(6)減少輪廓狀味蕾新生細胞數並縮短新生味蕾細胞的存活週期；(7) cyclin B2、E2F1表現量隨接受檳榔鹼時間的延長而減少，p53和bax則相反隨接受時間的延長而上升。 本研究顯示，長時間接受檳榔鹼會影響味蕾的超微結構，並改變味覺辨識能力及味蕾新生週期，這些可能會導致味覺異常或營養不足，進而影響發育生長。 實驗二：近年來，利用不同動物模式的研究，證實檳榔鹼能造成胚胎發育遲緩，現有已知的研究結果亦證實，長時間接觸檳榔鹼的斑馬魚胚胎，其發育過程也會受到影響。本實驗亦選用斑馬魚作實驗動物，但做短時間的檳榔鹼接觸，以模擬嚼食檳榔婦女在懷孕初期未能警覺懷孕而短暫接觸檳榔鹼對胎兒發育的影響。 將斑馬魚的受精卵在受精後4小時 ( 4hpf，hours post fertilization)內將其分組，各組受精卵分別以0.001%、0.01%、0.02%、0.04%的檳榔鹼處理20小時，並於24、48、72、96、120 hpf收集其胚胎，記錄 (1) 胚胎存活與孵化率；(2) 觀察胚胎發育外形的變化；(3) 紀錄胚胎魚泳游功能；(4) 並利用免疫螢光染色及穿透式電子顯微鏡觀察骨骼肌生長情形；(5) 利用RT-PCR及JC-1的方式釐清骨骼肌內的粒線體是否受到影響。 結果顯示，當斑馬魚胚胎接觸之檳榔鹼濃度提升時，各組斑馬魚之存活率、孵化率降低，外形明顯異常、體長縮短，且運動功能游泳距離縮短；免疫螢光染色可見體節(somite)內原本緻密排列的骨骼肌纖維，隨著接觸之檳榔鹼濃度的上升而顯得越不整齊；超微結構的觀察中，隨接觸之檳榔鹼濃度的上升，肌小纖維(myofibrils)間出現明顯的空泡狀、粒線體也受到嚴重破壞；透過RT-PCR觀察與粒線體相關的基因如sdhb、coxI、cox4i1、atp5a1、atp5f1在48及96hpf各基因的表現量，皆隨接觸之檳榔鹼濃度的上升而下降；此外，JC-1檢測結果顯示，隨接觸之檳榔鹼濃度的上升，粒線體膜電位受到影響的比例也會增加。 由以上之研究結果，吾人得知：檳榔鹼會造成胚胎的存活率下降、游泳活動能力受限、胚胎發育遲緩、骨骼肌發育受損、粒線體結構及功能受到破壞。由此觀之，雖短時間之接觸檳榔鹼，亦可能對受孕、早產及胎兒之發育造成嚴重之影響。

Arecoline, a major alkaloid in areca nuts, is involved in the pathogenesis of inflammatory oral diseases, cancers and in the developmentally toxic effect of lowering weight and retarding growth of the embryo. Experiment I：Mammalian taste buds are the basic structural unit for detecting taste stimuli in the oral cavity, however, the effects of arecoline on the taste bud are poorly understood. We injected arecoline intraperitoneally into C57BL/6 mice twice daily for 1 - 4 weeks and aimed to revealthe taste bud morphology, life span and gustatory functional activity by immunohistochemistry (IHC) and electron microscopy. At end of arecoline treatment, the animals were sacrificed through perfusion, and the vallate papillae were excised and processed for electron microscopy and immunohistochemistry (IHC) analysis of taste receptor proteins (T1R2, T1R3, T1R1 and T2R) and taste associated proteins (α-gustducin, PLCβ2 and SNAP25). Expression levels of c-fos were detected in the solitary nucleus and gustatory cortex. Body weight, food intake and water consumption were recorded during the treatment period every other day. After arecoline treatment, a two-bottle preference test between water and 1 % sucrose was performed for 4 weeks. For study of taste bud cells life-span, mice were also injected with BrdU (50 mg/kg i.p.) at end of arecoline treatment and then perfused at day 1 to day 14 following BrdU administration. The results demonstrated that (1) arecoline treatment did not change the number and size of the taste buds, and the number of taste bud cells was not affected, (2) electron microscopy revealed the swollen mitochondria, dilated endoplasmic reticulum cisternae, and numerous irregular autophagosomes accumulated in type II cells, (3) IHC demonstrated a decrease in the number of taste receptor T1R2- and T1R3-expressing cells, (4) the level of c-fos expression was decreased in the solitary nucleus and gustatory cortex, (5) the body weight and food intake amount were markedly reduced, (6) the sweet preference behaviour was-reduced, (7) the number of BrdU-labeled taste bud cells was significant reduced at 1, 3, 7 and 14 days, and (8) PCR array experiments showed that the expression of cyclin B2 and E2F1, was markedly downregulated, but the expression of p53 and bax was markedly upregulated by arecoline in the circumvallate. We conclude that the long-term arecoline injection alters the morphology of type II taste bud cells, retards the growth of mice from puberty to adulthood, affects gustatory discrimination competencies, also inhibits taste progenitor cells proliferation and shorten life span of renew taste bud cells. Experiment II：The effects of arecoline on birth defects have been explored in many species, including chicken, mice, and zebrafish. The effects of arecoline on embryos after long-term exposure are well established, however, the effects of short-term arecoline exposure to embryos are not fully understood. Using zebrafish as an animal model, we studied the effects of short-term exposure of arecoline on zebrafish embryos to mimic the areca nut-chewing woman during early pregnancy. Arecoline, at concentrations from 0.001 to 0.04%, was administered to zebrafish embryos from 4 to 24 hpf (hours post fertilization). The morphological changes, hatching and survival rates, body length, and somitic skeletal muscle fiber structure were investigated by immunohistochemistry (IHC), confocal microscopy, and conventional electron microscopy. With exposure of zebrafish embryos to the increasing concentrations of arecoline, we observed a significant decline in the hatching and survival rates, general growth retardation, lower locomotor activity and impairment of swimming ability. Immuno-fluorescence staining demonstrated a loose arrangement of myosin heavy chains, and ultrastructural observations revealed an altered arrangement of somitic myofibril and swelling of the mitochondria. In addition, the results from flow-cytometry, JC-1 staining to assay mitochondria activity, and RT-PCR analysis of mitochondrial functional gene expression, revealed mitochondrial dysfunctions after exposure to arecoline. We confirmed that short-term arecoline exposure resulted in retarded embryonic development and decreased locomotor activity due to defective somitic skeletal muscle development and mitochondrial dysfunction.