【作者】 郝丽萍；

【导师】 孙秀发；

【作者基本信息】 华中科技大学 ， 营养与食品卫生学， 2005， 博士

【摘要】 全球酒精消耗量近几十年来一直上升，2004年全球酒精饮料消费接近2亿吨，中国居首。酒精对健康的影响具有“双刃剑”的作用：适度饮酒能够明显降低动脉粥样硬化和冠心病的发生率，降低由此导致的死亡率；但是饮酒对机体的危害更不容忽视，过量饮酒是当今世界范围内的一个重要的公共卫生问题。随着世界范围内糖尿病发病率的快速增加，饮酒与糖尿病之间存在的相关性已经受到广泛的关注。大量的流行病学研究显示，酒精与2型糖尿病的发生存在U型关系，长期规律性适量饮酒降低患糖尿病的危险性，而长期过量饮酒却增加患糖尿病尤其是患2型糖尿病的危险性。但有关酒精与糖尿病关系的实验研究目前还比较少，机理还远未阐明。尽管目前对胰岛素抵抗与胰岛素不足在2型糖尿病的发生、发展中所起的核心作用仍有争论，但是胰岛β细胞功能缺陷无疑在糖尿病的发生发展中起着决定性作用。因此，酒精对胰岛β细胞分泌胰岛素功能的影响及机制的探讨在饮酒与糖尿病关系的研究中是一个必不可少的环节。糖尿病的发病是由于胰岛β细胞功能障碍造成体内胰岛素绝对或相对缺乏导致的，以往的研究人们更多关注的是1型糖尿病中β细胞的破坏，由自身免疫T细胞介导的β细胞凋亡在1型糖尿病发病中的作用已得到普遍的认可。近年来，由于研究方法的进步和对疾病认识的深入，越来越多的证据表明，2型糖尿病的胰岛β细胞功能衰竭与β细胞凋亡有关，胰岛β细胞凋亡在2型糖尿病中的作用已越来越受到人们的重视，因此，酒精是否可以通过诱导胰岛β细胞凋亡途径导致糖尿病发生的危险性升高是本研究的另一个重要内容。本课题拟在体外观察酒精直接对胰岛β细胞胰岛素分泌功能和凋亡的影响并探讨相关机制，以期揭示酒精与糖尿病患病危险性之间关系的机理，为指导人们合理消费酒精饮料提供科学依据。本研究主要包括三部分，现将结果报告如下：第一部分大鼠胰岛的原代培养及小鼠胰岛瘤细胞(NIT-1细胞)培养方法的建立一．大鼠胰岛的原代培养及鉴定目的建立大鼠胰岛的原代培养方法，为本课题研究提供足够纯净、存活且具有良好功能的胰岛。方法从胆总管逆行灌注KRBB，外置胶原酶消化，随后使用单一的Histopaque-1077作为分离剂来制备大鼠胰岛，并用双硫腙染色法，AO／PI双染色法，胰岛素分泌功能测定，电镜等方法对分离效果进行评价。结果成年Wistar大鼠(10只)胰腺消化后平均能获得540±84胰岛／胰腺，纯化后获得005±81胰岛／胰腺；本方法分离的胰岛纯度可达90％，存活率可达99％，纯化后胰岛体外葡萄糖刺激实验发现在高糖刺激下胰岛素分泌量显著增加，电镜观察胰岛β细胞内充满核心分泌颗粒，说明胰岛β细胞的功能良好。结论通过对胰岛分离纯化方法的摸索和改进，我们认为本实验使用单一的Histopaque-1077进行纯化，是一种能够获得大量纯度较高且活性好的胰岛的高效简便的分离方法，为深入进行相关研究奠定了基础。二．小鼠胰岛瘤细胞(NIT-1细胞)培养及胰岛素分泌功能鉴定目的分泌胰岛素的细胞为胰岛β细胞，为了更深入研究酒精对胰岛β细胞功能和凋亡的影响，我们把一种胰岛β细胞系即小鼠胰岛瘤细胞(NIT-1细胞)亦作为研究对象。对NIT-1细胞分泌胰岛素功能进行鉴定并选择最适合的培养条件是本部分的研究目的。方法通过MTT法选择合适浓度葡萄糖的培养基；用免疫细胞化学法鉴定NIT-1细胞胰岛素分泌能力；用不同浓度葡萄糖刺激胰岛素分泌实验评价NIT-1细胞对糖刺激后胰岛素分泌的反应能力。结果当培养液中葡萄糖浓度在2.8～11.2mmol／L的时候，细胞生长处于最佳状态，葡萄糖浓度超过11.2mmol／L时，对细胞的生长呈现抑制作用。免疫细胞化学实验证实，几乎所有的细胞胞浆均被染成棕黄色，说明此细胞具有良好的胰岛素分泌功能。NIT-1细胞对不同浓度葡萄糖刺激有良好的反应能力，随着葡萄糖浓度升高，胰岛素分泌增加，但超过11.2mmol／L后，胰岛素分泌呈下降趋势。结论NIT-1最适宜的培养条件为含5.6mmol／L葡萄糖浓度的DMEM培养基。用于本实验研究的NIT-1细胞具有良好的胰岛素分泌功能和对不同浓度葡萄糖刺激的良好的反应能力。第二部分酒精对胰岛β细胞分泌胰岛素功能的影响及相关机制研究一．酒精对胰岛β细胞分泌胰岛素功能影响的研究目的观察不同剂量酒精作用不同时间后，葡萄糖刺激的胰岛素分泌(GSIS)情况从而揭示酒精对胰岛β细胞分泌胰岛素功能影响的规律。方法1．观察不同剂量酒精(0、50、100、200、400mmol／L)作用24小时后对NIT-1细胞形态的影响。2．酒精对NIT-1细胞胰岛素分泌的影响(放免法测定胰岛素)：(1) 100mmol／L酒精作用24小时后，NIT-1细胞对不同浓度葡萄糖(0、1.4、2.8、5.6、11.2、22.4mmol／L)刺激的胰岛素分泌情况。(2)不同剂量酒精(0、50、100、200、400mmol／L)作用不同时间(6、12、24小时)后，测定5.6、22.4mmol／L葡萄糖刺激的胰岛素分泌水平。3．酒精对原代培养大鼠胰岛(β细胞)胰岛素分泌的影响：(1)不同剂量酒精(0、10、50、100、200mmol／L)作用12小时后，先后用含5.6、22.4mmol／L葡萄糖的KRBB液孵育2小时，取上清测胰岛素。(2)酒精(0mmol／L、100mmol／L)作用6、12、24小时后，先后用含5.6、22.4mmol／L葡萄糖的KRBB液孵育2小时，取上清测胰岛素。结果1．低剂量酒精对NIT-1细胞形态影响较小，基本无改变；但高剂量酒精组特别是400mmol／L剂量组细胞形态变化较大，胞内颗粒状物质增多，细胞皱缩，变圆甚至浮起。2．酒精对NIT-1细胞胰岛素分泌的影响：(1)对照组和剂量组GSIS均与葡萄糖浓度有关，变化趋势相似，即随着葡萄糖浓度升高，胰岛素分泌先是逐渐增强，葡萄糖浓度5.6mmol／L时达最高峰，然后又逐渐下降。但是，葡萄糖浓度相同时，与对照组相比，酒精作用后抑制了胰岛素分泌，其中2.8、5.6、22.4mmol／L葡萄糖刺激的胰岛素分泌，酒精的抑制作用呈显著性。因此，在后续的实验中，我们选取5.6mmol／L(生理状况下葡萄糖浓度)及22.4mmol／L(高糖)作为GSIS的葡萄糖浓度。(2)酒精剂量和作用时间的影响：酒精作用6小时，低糖(5.6mmol／L)和高糖(22.4mmol／L)刺激NIT-1细胞的胰岛素分泌增加。酒精作用12小时和24小时后，各剂量组胰岛素分泌量下降(个别低剂量组例外)。酒精剂量、作用时间相同，高糖刺激后胰岛素分泌量低于低糖刺激后胰岛素分泌量。酒精剂量与NIT-1细胞胰岛素分泌量存在着相关性。相同剂量酒精作用时间延长降低NIT-1细胞胰岛素分泌量。3．酒精对原代培养大鼠胰岛(β细胞)胰岛素分泌的影响：(1)酒精剂量影响：低糖(5.6mmol／Lglucose)和高糖(22.4mmol／L glucose)刺激后胰岛素分泌与酒精剂量呈倒“U”型相关。与对照组相比，低剂量酒精组胰岛素分泌升高，差异有极显著性；高剂量酒精组胰岛素分泌下降，差异有显著性。说明酒精对原代培养大鼠胰岛β细胞胰岛素分泌的影响具有剂量依赖性。(2)酒精对原代培养大鼠胰岛β细胞胰岛素分泌的影响有时间依赖性。随酒精作用时间延长，酒精组低糖和高糖刺激的胰岛素分泌先升高(6h)，然后下降(24h)。高糖刺激的胰岛素分泌变化更为显著，与对照组比较，差异有显著性。结论酒精对胰岛β细胞胰岛素分泌的影响与酒精作用时间、剂量有关。二．酒精对NIT-1细胞胰岛素(INS)、细胞色素氧化酶Ⅱ(Cytochrome oxidaseⅡ，COXⅡ)以及大鼠原代培养胰岛INS mRNA表达的影响目的选取在葡萄糖刺激胰岛素分泌过程中的两个关键环节即胰岛素基因和细胞色素氧化酶Ⅱ基因mRNA表达的变化探讨酒精对胰岛β细胞分泌胰岛素功能影响的机理。方法1．NIT-1细胞0mmol／L、50mmol／L、100mmol／L、200mmol／L、400mmol／L酒精作用6h、12h、24h后，RT-PCR法检测INS、COXⅡmRNA表达水平变化。2．大鼠原代培养胰岛(β细胞)0、50、100、200mmol／L酒精作用12h后，RT-PCR法检测INS mRNA表达水平变化；0、100mmol／L酒精作用6h、12h、24h后，RT-PCR法检测INS mRNA表达水平变化。结果1．酒精对NIT-1细胞INS、COXⅡmRNA表达的影响与酒精作用时间和剂量有关。酒精作用6h，各剂量组(除400mmol／L组外)INS mRNA表达升高，酒精作用12h、24h各剂量组INS mRNA表达均降低。相同剂量酒精作用时间延长降低NIT-1细胞INS mRNA表达水平。酒精作用6h，COXⅡmRNA表达升高，100，200mmol／L剂量组表达呈显著性升高(p＜0.05)，酒精作用12h，表达开始降低，24h，400mmol／L剂量组表达呈显著性降低(p＜0.05)。2．酒精对大鼠原代培养胰岛(β细胞)INS mRNA表达水平的影响与酒精作用剂量和时间有关。低剂量(50mmol／L)表达升高，随着剂量增加，表达下降；短时间(6h)表达上升(P＜0.05)，随着时间延长，表达开始下降，孵育24h，Ins mRNA表达水平与对照组比较，差异有显著性(P＜0.05)。结论酒精对胰岛β细胞INS mRNA、COXⅡmRNA表达改变可能是其影响胰岛素分泌功能改变的有关机制。第三部分酒精对NIT-1细胞凋亡作用一．酒精对NIT-1细胞凋亡的影响目的观察酒精对NIT-1细胞凋亡的影响方法不同剂量的酒精(0mmol／L、50mmol／L、100mmol／L、200mmol／L、400mmol／L)作用不同时间(6h、12h、24h)后，乳酸脱氢酶(LDH)释放实验检测酒精对NIT-1细胞的细胞毒性作用；不同剂量的酒精(剂量同上)作用24h后，单细胞凝胶电泳实验(SCGE)，琼脂糖凝胶电泳法，AnnexinV／PI双染色法流式细胞仪检测NIT-1细胞凋亡情况。结果1．酒精对NIT-1细胞的毒性作用：酒精作用6h对NIT-1细胞的毒性作用并不明显。酒精作用12h、24h，高剂量酒精能够对细胞膜造成损伤，LDH活性升高。酒精作用6h、12h、24h，LDH活性均与酒精剂量正相关。相同剂量酒精作用时间延长增强LDH活性。2．SCGE实验结果：酒精对NIT-1细胞DNA损伤与酒精剂量有关。低剂量损伤不明显，高剂量(200、400mmol／L)组DNA迁移率，DNA损伤程度分级，DNA平均迁移长度与对照组比较差异有显著性。3．琼脂糖凝胶电泳结果：400mmol／L酒精剂量引起NIT-1细胞典型的梯状条带，200mmol／L酒精剂量引起少量的梯状条带，而50mmol／L、100mmol／L剂量组则未见明显梯状条带。4．AnnexinV／PI检测早期细胞凋亡：200、400mmol／L酒精剂量组NIT-1细胞凋亡率升高，与对照组相比差异有极显著性。结论：酒精可以诱导NIT-1细胞发生凋亡。二．酒精对NIT-1细胞凋亡机制的探讨目的从氧化应激的角度探讨酒精诱导NIT-1细胞凋亡机制方法NIT-1细胞不同剂量酒精(0mmol／L、50mmol／L、100mmol／L、200mmol／L、400mmol／L)作用6h，12h，24h后进行相关指标检测。1．脂质过氧化及抗氧化指标MDA，GSH，SOD，GSH-Px的检测。2．RT-PCR法检测与凋亡有关的基因Bcl-2，Bax mRNA基因表达。3．RT-PCR法检测Caspase-3 mRNA基因表达。4．Caspase-3酶活性的测定：Ac-DEVD-pNa(Caspase-3活性显色底物)法。结果1．酒精对小鼠NIT-1细胞MDA、SOD、GSH、GSH-Px的影响：剂量组MDA含量升高，与对照组比，200mmol／L、400mmol／L剂量组差异有极显著性。随着剂量升高，SOD和GSH-Px活性降低，GSH含量下降。与对照组比，200mmol／L、400mmol／L剂量组SOD活性差异有极显著性，400mmol／L剂量组GSH-Px活性及GSH含量差异有极显著性。MDA含量、SOD活性、GSH水平、GSH-Px活性与酒精剂量作相关分析后的相关系数(r)分别为：0.9034、-0.7258、-0.6201、-0.6222，p＜0.01。2．Bcl-2／Bax与酒精作用剂量和时间有关。6h，随着酒精剂量增加，Bcl-2／Bax先升高后降低。12h，400mmol／L剂量Bcl-2／Bax下降，与对照组比差异具有显著性。24h，各剂量组表达均呈现下降趋势，与对照组比，400mmol／L剂量组差异具有极显著性。3．Caspase-3 mRNA表达水平与酒精剂量和作用时间相关。随着酒精剂量的增大，作用时间的延长，Caspase-3 mRNA表达增加。400mmol／L剂量组作用24h，表达水平与对照组比，差异具有显著性。4．caspase-3活性的变化与酒精作用的时间和剂量有关。与对照组相比，酒精作用6h，caspase-3活性的变化不明显；作用12h，200mmol／L、400mmol／L剂量组caspase-3活性分别呈显著、极显著性升高；24h，100mmol／L剂量组caspase-3活性显著性升高，200mmol／L、400mmol／L剂量组caspase-3活性极显著性升高。结论：高剂量酒精导致NIT-1细胞凋亡可以通过引起小鼠NIT-1细胞氧化抗氧化失衡，处于氧化应激态，ROS升高并上调Bax的表达，Bcl-2／Bax比值降低，Caspase-3被激活，最终导致细胞内重要蛋白质降解失活及DNA断裂，进而引起细胞凋亡。因此，酒精通过诱导胰岛β细胞凋亡使胰岛β细胞功能障碍可能是酒精导致糖尿病发生的重要机制之一。创新点：1．通过对胰岛分离纯化方法的摸索和改进，使用单一的Histopaque-1077作为分离剂来制备大鼠胰岛是能够获得大量纯度较高且活性好的胰岛的一种高效并且简便的分离方法。2．饮酒与2型糖尿病相关性方面的研究报道多为动物实验、流行病学调查，体外实验研究酒精直接对胰岛β细胞功能影响的报道很少，有关机制的研究几乎未有涉及。本实验以原代培养的大鼠胰岛(β细胞)和NIT-1细胞为研究对象较系统地阐述了酒精对胰岛β细胞胰岛素分泌功能影响的时效关系，并通过测定胰岛素基因表达、细胞色素氧化酶Ⅱ基因表达探讨酒精影响胰岛β细胞分泌胰岛素功能的机理。3．研究酒精对胰岛β细胞凋亡影响的报道几乎未见。本研究发现酒精通过诱导胰岛β细胞凋亡使胰岛β细胞功能障碍可能是酒精导致糖尿病发生的重要机制之一，为我们进行酒精导致糖尿病发生机制的研究开辟了一个新领域。更多还原

【Abstract】 The amount of alcohol beverage consumed in the world has being increased in recent decades. The total amount of alcohol consumption of the globe was nearly 200million tons and the nation that had the largest consumption was China. Alcohol is a two-edged sword. Light to moderate alcohol intake can reduce the incidence of atherosclerosis and coronary heart disease, thus the mortality rates due to these diseases are low. But. at the same time, the harmful effect of heavy alcohol consumed should not be neglected and the alcohol abuse has being an important public health problem. Because of the rapid increase in the incidence of diabetes worldwide, the relationship between alcohol intake and the risk of diabetes has been noticed widely. Many epidemiological researches suggested there exists a U-shaped relationship between the amount of alcohol consumption and the risk of diabetes. Chronic regular mild to moderate alcohol consumption among healthy people may be associated with increased insulin sensitivity and a reduced risk of type 2 diabetes, while excessive consumption can impair glycacmic control and increase the risk of type 2 diabetes. But until now. the mechanism of the diabetogenic action of alcohol remains unexplored. Type 2 diabetes is characterized by insulin resistance and impaired insulin secretion. Although the relative contribution of insulin resistance versus insulin deficiency remains a matter of controversy, there is considerable evidence that deficient β-cell function plays a dominating role in the establishment of overt diabetes. So, it is necessary to study the effect of alcohol on β-cell function and the mechanism involving it. Diabetes is believed to occur due to absolute or relative deficiency of insulin when β-cell function is impaired. In the past, too much researches focused on β-cells destroyed in type 1 diabetes and it is generally accepted that β-cell destruction occurs mainly by apoptosis. Recently, more and more evidence demonstrated that the failure of β-cell function is also related to apoptosis, so the role of β-cell apoptosis during the development of type 2 diabetes spurred the interest of research in this area. Another aspect of this research is whether alcohol could increase the risk of diabetes due to inducing apoptosis of pancreatic islet β-cells.In the present study, we examined the direct effect of alcohol on insulin secretion and apoptosis of pancreatic islet β-cells in vitro. At the same time, we investigated the potential mechanism involving. From this study, we hope to elucidate some understanding about the mechanism between the alcohol and diabetes and provide scientific advice for healthy drinking. The experiments were designed and the main results were summarized as follows:Part I Establishment of culturing methods of primary rat pancreaticislet and mouse insulinoma cells (NIT-1 cells)Primary culture of rat pancreatic islet and identificationObjective To establish the method of primary rat pancreatic islet in order to provide enough purified, survival and functional rat islets for next experiments in this study.Methods Islets were isolated from male Wistar rats by ductal injection of pancreas with KRBB, then collagenase digestion and a one-step single-layer Histopaque-1077 purification. Evaluate the islet quality by dithizone stain, AO/PI stain, electron microscope and determination the function of glucose stimulated insulin secretion (GSIS).Results 540 ± 84 islets and 335 ± 81 islets were obtained in 1 rat pancreas after digestion and purification individually, with purity over 90% and living islets 99%. The results of GSIS demonstrate a functional profile of glucose sensitivity. Electron microscope of the isolated islet showed a healthy, well-preserved intracellular structure containing insulin granules in the β-cell cytoplasm.Conclusion This is a simple and effective method to gain enough and functional integrity rat islets. Therefore, the situation was capable of further research. Culture of mouse insulinoma cells (NIT-1 cells) and identification of insulinsecretion functionObjective Insulin is secreted by pancreatic islet β-cells. So, we use a kind of β-cell line (NIT-1 cells, established from mouse insulinoma) for further study the influence of alcohol on β-cells function and apoptosis. The aim of this part of study is to identify the function and establish the suitable culture condition of NIT-1 cells.Methods Choose the culture medium through MTT assay. Identify the insulin producing capability of NIT-1 cells by immunocytochemical method and Identify the responsiveness to secretagogues with glucose stimulated insulin secretion method.Results NIT-1 cells grew well when the glucose concentration in the medium was between 2.8 and 11.2mmol/L. But the growth of cells was inhibited when the glucose concentration exceeded 11.2mmol/L. Using immunocytochemical method, almost all of the cells showed brown staining pattern over the cytoplasm, thus implying the good capability of insulin biosynthesis in NIT-1 cells. Insulin secretion is responsive to glucose concentration well in the medium. The stimulation was increased with increasing glucose concentration and began to dwindle when the glucose concentration was higher than 11.2mmol/LConclusion The suitable concentration of glucose in DMEM culture medium was 5.6mmol/L for NIT-1 cells. NIT-1 cells used in this study showed satisfactory function of insulin secretion and responsiveness to GSIS.Part II Effect of alcohol on insulin secretion of islet β-cells and study of related mechanismEffect of alcohol on insulin secretion of islet β-cellsObjective To examine primary cultured rat pancreatic islet (β cells) and NIT-1 cells insulin secretion in response to a glucose challenge following exposure to different concentrations of alcohol. Therefore, we can conclude how the alcohol influence the β-cells function., Methods1. To observe alcohol-induced changes in cell morphology, NIT-1 cells were examined by phase-contrast microscopy after cells were treated with alcohol atconcentrations of 0, 50, 100, 200 and 400 mmol/L for 24 h.2. Effect of alcohol on insulin secretion in NIT-1 cells(1) NIT-1 cells were treated with 100mmol/L alcohol for 24h and insulin release wasmeasured by radioimmunoassay after 2h incubation in KRBB containing 0, 1.4, 2.8, 5.6, 11.2 and 22.4mmol/L glucose.(2) NIT-1 cells were exposed to various concentrations of alcohol (0, 50, 100, 200, 400mmol/L) for 6, 12 and 24 h and insulin release was measured by radioimmunoassay after 2h incubation in KRBB containing 5.6 and 22.4mmol/L glucose.3. Effect of alcohol on insulin secretion in primary cultured rat pancreatic islet (β-cells):(1) Islets (β-cells) were exposed to various concentrations of alcohol (0, 10, 50, 100, 200mmol/L) for 12 h and insulin release was measured by radioimmunoassay after 2h incubation in KRBB containing 5.6 and 22.4mmol/L glucose.(2) Islets (β-cells) were exposed to various concentrations of alcohol (0,100 mmol/L) for 6, 12 and 24h and insulin release was measured by radioimmunoassay after 2h incubation in KRBB containing 5.6 and 22.4mmol/L glucose.Results1. Morphological changes induced by low concentrations of alcohol were little. But NIT-1 cells of high concentrations (especially 400mmol/L) of alcohol treated groups presented with cell shrinkage, irregularity in shape and cellular detachment.2. Effect of alcohol on insulin secretion in NIT-1 cells(1) The tendency of GSIS in both untreated and treated group was similar and the insulin secretion was correlated with the concentration of glucose. With increasing glucose concentration, the insulin release was increased first and reached the peak at 5.6 mmol/L glucose, then began to dwindle. But at the same concentrations of glucose, alcohol inhibited insulin secretion compared to the untreated control group. During incubation with 2.8, 5.6 and 22.4mmol/L individually, the inhibitory effects were significant. So, in the next GSIS experiments, the concentrations of glucose were 5.6 and 22.4 mmol/L.(2) At 6h, alcohol increased the glucose-stimulated insulin secretion. At 12h and 24h, alcohol decreased the insulin secretion. When alcohol concentration and exposure time were same, insulin release response to 22.4 mmol/L glucose was less than those to 5.6mmol/L glucose. The insulin secretion was correlated with the alcohol concentration. The insulin secretion was decreased after alcohol exposure time prolonged even if the dosage of alcohol was identical.3. Effect of alcohol on insulin secretion in primary cultured rat pancreatic islet (β-cells)(1) There was a reverse U-shaped association between GSIS (stimulated with both 5.6 and 22.4 mmol/L glucose) and alcohol dosage. Compared to untreated group, insulin release was increased significantly after exposed to low dosage of alcohol, but decreased markedly after exposed to high dosage of alcohol.(2) The effect of alcohol on insulin secretion (incubated with 5.6 and 22.4 mmol/L glucose) in islet (β-cells) was time dependency. At 6h, alcohol increased the GSIS, but alcohol decreased the GSIS at 24h. The effect of alcohol on GSIS at 22.4 mmol/L glucose was more significant than those at 5.6 mmol/L glucose.Conclusion: Depending on the timing and concentrations, alcohol can exert both stimulatory and inhibitory effects on insulin secretion.Effect of alcohol on INS and Cytochrome oxidase II mRNA expression in NIT-1 cells and INS mRNA expression in primary cultured rat pancreatic islets (β-cells)Objective To investigated the mechanism of the effect of alcohol on β-cell function, INS mRNA expression and Cytochrome oxidase II mRNA expression were observed because they are the key points related to the procedure of insulin secretion.Methods1. After NIT-1 cells were exposed to various concentrations of alcohol (0, 50, 100, 200, 400mmol/L) for 6, 12 and 24h, the levels of insulin and COXII mRNA expression were analyzed by RT-PCR method. 2. After primary cultured rat pancreatic islets were treated with various concentrations of alcohol (0, 50, 100, 200 mmol/L) for 12h, the levels of insulin mRNA expression were analyzed by RT-PCR method. After islets were treated with alcohol (0, 100 mmol/L) for 6, 12 and 24h, the levels of insulin mRNA expression were analyzed by RT-PCR method.Results1. The effect of alcohol on INS and COX II mRNA expression in NIT-1 cells were depended on the time and concentration. INS and COX II mRNA expression were upgraded at 6h and began to decrease at 12h. At 24 h, high alcohol group decreased COX II mRNA expression significantly.2. The effect of alcohol on INS mRNA expression in islets (β-cells) were depended on the time and concentrations. INS mRNA expression was upgraded at low concentration (50mmol/L), but began to decrease with the concentrations increasing. INS mRNA expression was upgraded (P<0.05) at 6h and began to decrease at 12h. At 24 h, the expression decreased significantly compared to untreated group (p<0.05).Conclusion: Influence of alcohol on INS mRNA、 COX II mRNA expression in β-cells may be involved in the effect of alcohol on insulin secretion.Part III Influence of alcohol on apoptosis in NIT-1 cellsEffect of alcohol on apoptosis in NIT-1 cellsObjective To investigate whether alcohol induces apoptosis in NIT-1 cells.Methods After NIT-1 cells were treated with alcohol at concentrations of 0, 50, 100, 200 and 400 mmol/L for 6h, 12h and 24h, the LDH activity released in the media was measured in order to determine the cytotoxicity of alcohol. After NIT-1 cells were exposed to various concentrations of alcohol (same as above), single cell gel electrophoresis (SCGE), agarose gel electrophoresis and AnnexinV/PI methods were performed to detect the apoptosis induced by alcohol.Results1. At 6h, alcohol induced no significant cytotoxic effect in NIT-1 cells. At 12h, 24h, high concentration alcohol damaged the plasma membrane and the LDH activity increased. At 6h, 12h and 24h, the activity of LDH was positive correlation to the dosage of alcohol. At the same dosage of alcohol, the LDH activity was increased after the exposure time prolonged.2. SCGE results showed that low concentration alcohol induced no obviously DNA damage in NIT-1 cells, but the rate of DNA migration, degree of DNA damage and average length of DNA migration of 200 and 400 mmol/L alcohol-treated groups were significantly changed compared to those of untreated group .3. The results of DNA fragmentation assay showed that 400 mmol/L alcohol treatment induced a classic "DNA ladder" pattern. 200 mmol/L alcohol-treated group had a few "DNA ladder" pattern and the other groups appeared no "DNA ladder" pattern.4. The results of Annexin V/PI assay indicated that the apoptosis rates of NIT-1 cellsin 200 and 400mmol/L alcohol-treated groups were higher than that of untreated group. Conclusion Alcohol induced apoptosis in NIT-1 cellsMechanism of the effect of alcohol on apoptosis in NIT-1 cells Objective To study the mechanism of alcohol induced apoptosis via oxidative stress Methods After NIT-1 cells were exposed to various concentrations of alcohol (0,50, 100, 200,400mmol/L) for 6, 12 and 24h, the experiments were performed as follows: 1 .Malondialdehyde (MDA) content, Glutathione (GSH) level, Superoxide dismutase(SOD) and Glutathione peroxidase (GSH-Px) were measured in NIT-1 cells to evaluatethe oxidative damage degree.2. The levels of Bcl-2, Bax mRNA expression were determined by RT-PCR method.3. The level of Caspase-3 mRNA expression was measured by RT-PCR.4. Caspase-3 enzyme activity was measured using Ac-DEVD-pNa which is the substrate of Caspase-3 enzyme.Results1. The MDA content was higher, the SOD activity, GSH-Px activity and GSH content were lower in alcohol group than those of control group. The MDA content, SOD activity, GSH-Px activity and GSH content were correlated with the concentrations of alcohol and the correlation coefficients were 0.9034, -0.7258, -0.6201, -0.6222 (p<0.01) respectively.2. At 6h, the ration of Bcl-2/Bax expression was increased first, then decreased with the increasing of alcohol dosage. At 12h and 24h, the ration was decreased. By treatment at 400mmol/L, the decrease was dramatically compared to control group.3. Alcohol increased the caspase-3 mRNA expression at high concentrations of alcohol and long exposure time. At 24h, the caspase-3 mRNA expression was increased significantly by treatment at 400mmol/L.4. Alcohol increased the caspase-3 enzyme activity by a dose and time dependent manner. After 6h exposure to alcohol, the enzyme activity changed little. After 12h, the enzyme activity was seen to be increased dramatically by treatment with alcohol at 200 and 400 mmol/L respectively. After 24h, the enzyme activity was increased markedly by treatment with alcohol at 100, 200 and 400 mmol/L respectively.Conclusion Higher concentrations of alcohol induced imbalance of oxidative and antioxidative ability in NIT-1 cells, then oxidative stress further leads to apoptosis. Based on the results, alcohol appears to activate specific intracellular death-related pathways leading to bax-dependant caspase-3 activation and the induction of apoptosis in NIT-1 cells. From the results of the present study, it could be suggested that alcohol-induced apoptosis in NIT-1 cells is a possible pathologic mechanism of alcohol-related diabetes.更多还原