檳榔生物鹼調節3T3-L1前脂肪細胞的生長;Betel nut alkaloids regulate 3T3-L1 preadipocyte growth

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Title:	檳榔生物鹼調節3T3-L1前脂肪細胞的生長;Betel nut alkaloids regulate 3T3-L1 preadipocyte growth
Authors:	田子函;Tian,Zi-Han
Contributors:	生命科學系
Keywords:	檳榔生物鹼;檳榔鹼;3T3-L1前脂肪細胞;細胞週期;Betel nut alkaloids;arecoline;3T3-L1 preadipocyte;cell cycle
Date:	2014-10-22
Issue Date:	2014-11-24 15:19:17 (UTC+8)
Publisher:	國立中央大學
Abstract:	檳榔是亞洲最廣泛消費的物質之一，其檳榔生物鹼（Betal nut alkaloids），尤其是檳榔鹼(arecoline)，被發現存在於檳榔嚼食者的唾液。雖然檳榔鹼已被證實在3T3-L1脂肪細胞會調節葡萄糖攝取和脂肪生成，但檳榔鹼對前脂肪細胞生長的作用之相關訊息是很少的。利用3T3-L1前脂肪細胞，我們觀察到檳榔鹼，但不包括其他結構上相似的生物鹼，如arecaidine和guvacine，對3T3-L1前脂肪細胞會劑量依賴性和時間依賴性地降低細胞生存率。流式細胞儀分析細胞週期的變化後顯示，處理檳榔鹼但並非arecaidine或guvacine， 24和48小時3T3-L1前脂肪細胞的細胞群體分佈中，G1期會減少並且增加S期和G2 / M期。進一步的西方點墨法分析結果顯示，檳榔鹼在24小時使G1檢查點刺激蛋白質總量降低，如p21，p27和AMP活化蛋白激酶（AMP-activated protein kinase）的蛋白質總量，並減少了G2檢查點調控蛋白的總量如CDK1，增加細胞週期素(cyclin) B1和p53的含量，但沒有改變CDK2蛋白總量。有趣的是，arecaidine和guvacine改變p21和p53的蛋白總量但不改變p27，CDK1，CDK2和cyclin B1的蛋白總量。預處理穀胱甘肽活化劑例如N-乙酰半胱氨酸（NAC），抑制了檳榔鹼所誘導的細胞生存率之減少和細胞週期的G1期的分佈之降低。此外，NAC阻斷檳榔鹼所增加的S和G2/M期細胞分布以及活性氧（ROS）的產生。另外，預處理NAC阻止了 arecoline 所降低的p21, p27，CDK1和AMPK蛋白，和增加的p53和cyclin B1蛋白質總量。不過毒蕈鹼型乙酰膽鹼受體（muscarinic acetylcholine receptor）拮抗劑，如阿托品(atropine)，並沒有阻止檳榔鹼所降低細胞存活率，但進一步地強化檳榔鹼刺激ROS的產生。另外GABA及其B型受器抑制劑-saclofen皆無法抑制arecoline所降低的細胞存活率。總結，檳榔生物鹼成分中會選擇性的僅arecoline會透過ROS而非mAchR和GABA B型受器的路徑，改變p53、p21、p27、CDK1、cyclin B1及AMPK蛋白表現量，進而調控細胞週期的變化，使得3T3-L1前脂肪細胞的生長受到抑制。;Betel nut is one of the common substances consumed in Asia, and betel nut alkaloids (BNAs), especially arecoline, are found to be present in the saliva of areca nut chewers. Although arecoline has been shown to regulate glucose uptake and adipogenesis in 3T3-L1 adipocytes, little information is known about the action of arecoline on growth of preadipocytes. Using 3T3-L1 preadipocytes, we observed that arecoline, but not other structurally related alkaloids, such as arecaidine or guvacine, reduced cell viability of 3T3-L1 preadipocytes in dose- and time-dependent manners. Flow cytometric analysis of the cell cycle indicated that exposure to arecoline, but not arecaidine or guvacine, for 24 and 48 h decreased the cell population in G1 stage and increased the cell population in S stage and the G2/M stage of 3T3-L1 preadipocytes. Further Western blot analysis showed that arecoline at 24 h reduced levels of the G1 checkpoint-stimulating proteins, such as p21, p27 and AMP-activated protein kinase (AMPK), decreased levels of the G2 checkpoint-controlling protein, such as CDK1, increased cyclin B1 and p53 protein level, and unaltered CDK2 protein level. Interestingly, arecaidine and guvacine altered p21 and p53 protein levels and unaltered p27, CDK1, CDK2, and cyclin B1 protein levels. Pretreatment with N-acetyl-cysteine (NAC), a glutathione activator, suppressed the arecoline-induced decreases in levels of cell viability and G1 phase of the cell cycle. Moreover, NAC blocked the arecoline-increased percentages of S and G2/M stages of the cell cycle and levels of reactive oxygen species (ROS) production. In addition, NAC prevented the arecoline-decreased levels of p21, p27, CDK1 and AMPK proteins and the arecoline-increased levels of p53 and cyclin B1. However the muscarinic acetylcholine receptor (mAChR) antagonist, such as atropine, did not block the arecoline-reduced cell viability but enhanced further arecoline-stimulated ROS production. Neither GABA nor its type B receptor antagonist, such as saclofen, blocked arecoline-suppressed cell viability. In conclusion, arecoline is the selective ingredient from BNAs to inhibit 3T3-L1 preadipocyte growth through alterations of the cell cycle possibly in p53-, p21-, p27-, CDK1-, cyclin B1, AMPK- and ROS-dependent pathways and GABA B receptor- and mAChR-independent pathways.
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