【作者】 王春旭；

【导师】 李和； 邹思革；

【作者基本信息】 华中科技大学 ， 人体解剖学与组织胚胎学， 2012， 博士

【摘要】 1.研究背景目前全人类都面临一个人口老龄化的问题。由于人口老龄化导致的生产力下降,严重制约了社会经济的发展。衰老是机体生理状态下降的一种过程,衰老增加疾病易感性从而导致死亡。与衰老相关的寿命长短和健康指标的下降受基因和环境的调节。研究表明,通过调节饮食成分和基因表达可延缓衰老,延长寿命。流行病学研究显示,人类寿命的延长和老年相关疾病发病率的降低除了通过体育锻炼以外,应用最广泛和重复性最高的方法是在多种动物模型上限制卡路里,即在保证足够营养的条件下适当的减少能量。卡路里限制可以在酵母菌、线虫、果蝇、小鼠、大鼠和非人类的灵长类——恒河猴等动物中有效延长寿命和减轻衰老相关的慢性疾病。卡路里限制虽然能有效的延长寿命,同时还能产生降低血脂、胆固醇含量等有益效果。但是也会产生一些明显的副作用,例如影响机体的新陈代谢,最明显的作用就是会使雌性动物产卵量下降和导致不孕不育等危害。那么有没有既能有效延长寿命又能减少严重副作用的方法,人们试图发现替代卡路里限制的方法。而最新的营养基因学研究显示糖和蛋白质含量的变化在几种生物包括：果蝇、金鱼和墨西哥果蝇可以显著的影响寿命。在果蝇模型上,糖与蛋白质的比例变化显著调节寿命。标准饮食中含10%的糖和10%酵母(酵母是标准饮食中唯一的蛋白质来源)；高糖低蛋白饮食含18%的糖和2%的酵母(糖和酵母的比例为9：1)；而低糖高蛋白饮食含2%的糖和18%的酵母(糖和酵母的比例为1：9)；在标准饮食的基础上稀释4倍,即含2.50%的糖和2.5%酵母(0.25倍的饮食)即通常所说的卡路里限制饮食。以上饮食都添加了1.5%的琼脂。本人通过前期的研究已证实,糖和酵母的比例变化对野生型果蝇寿命有显著的影响：在总卡路里相同的条件下,与标准饮食相比较,无论雄性还是雌性果蝇,高糖低蛋白饮食延长果蝇的平均寿命,而低糖高蛋白饮食缩短果蝇的平均寿命。卡路里限制饮食(总卡路里减少)对果蝇寿命的延长位于高糖低蛋白饮食组和标准饮食组之间。这些结果说明饮食成分在寿命的调节中起重要作用。此外寿命与动物种系的基因类型也有密切关系。在不同饮食对小鼠寿命影响的研究中,40个重组培育的品系在不同的饮食成分下,饮食成分限制仅可延长部分亚类小鼠品系的寿命,而对某些品系甚至使寿命缩短。由此提示,饮食成分限制对寿命的延长作用还依赖于生物体的基因背景。因此评价各种环境因素和基因的相互作用对寿命的调节具有重要意义。人们在自然界,特别是水果和蔬菜当中,发现一些抗氧化物质,这些物质可以产生类似卡路里限制的作用,被称为卡路里模拟性物质。这些物质可以明显的延缓衰老,降低机体的新陈代谢。有报道认为巴西莓和白藜芦醇是卡路里模拟性物质。巴西莓是一种棕榈树结的果实,最初生长于亚马逊河流域,常用来酿制啤酒和作为食品添加剂。巴西莓是一种强抗氧化剂,可以对抗氧化损伤。巴西莓含有多种植物化学物质,特别是花色苷、原花青素和其他的黄酮类,具有很强的抗氧化活性,特别是对抗超氧化物和过氧化自由基。白藜芦醇是一种存在于葡萄皮当中的植物抗毒素,是红酒中的有效成分,可以降低高脂饮食人群的心血管疾病的发病率。白藜芦醇可以减缓许多疾病的进程,如癌症、心血管疾病和神经系统疾病。添加白藜芦醇后可以推迟与衰老相关的认知功能下降,改善胰腺β细胞的功能,减少大鼠的心肌肥厚。白藜芦醇对果蝇寿命是否能够延长存在争议。而且白藜芦醇如果能够延长果蝇的寿命,那么白藜芦醇延长寿命是否和食物的成分有关。以及白藜芦醇和食物的相互作用调节寿命的机制还不太清楚。本研究试图明确：①巴西莓和白藜芦醇是否能够延长果蝇的寿命；②巴西莓和白藜芦醇与不同食物的相互作用对果蝇寿命的影响；③巴西莓和白藜芦醇延长果蝇寿命的分子机制。2.研究方法(1)通过调节食物的蔗糖、酵母和多脂酸含量制备各种成分的果蝇饮食。添加巴西莓和白藜芦醇制备特定饮食。(2)记录群体果蝇的生存时间,描绘果蝇的寿命曲线和计算果蝇群体的平均寿命和最大寿命。(3)应用定量PCR观察各种转录因子的活性。(4)应用RNA干扰技术构建超氧化物歧化酶1功能减弱的果蝇。(5)用毛细管喂养法测定不同处理组之间的食物摄入量。3.结果(1)各种浓度的巴西莓和白藜芦醇对标准饮食的野生型果蝇及高糖低蛋白饮食的野生型果蝇的寿命均没有影响。(2)高蛋白低糖饮食条件下,白藜芦醇的浓度达到200μmol/L可以延长野生型雌性果蝇。(3)高脂饮食条件下,添加2%的巴西莓可以显著延长雌性果蝇的寿命；白藜芦醇的浓度达到400μmol/L可以延长雌性果蝇的寿命。(4)对于超氧化物歧化酶1干扰的果蝇,白藜芦醇的浓度达到200μmol/L可以延长标准饮食的雌性果蝇的寿命；白藜芦醇的浓度达到400μmol/L可以延长高脂饮食的雌性果蝇的寿命。而添加0.5%到2%的巴西莓果浆可以延长标准饮食和高脂饮食雌性果蝇的寿命。以上的各种情况对雄性果蝇的作用影响轻微,甚至有轻微缩短雄性果蝇的寿命。说明饮食营养组成对白藜芦醇和巴西莓在寿命效果的影响具有性别特异性。(5)用毛细管喂养法测量,添加巴西莓和白藜芦醇的无琼脂高脂饮食的雌性果蝇连续3天每日食物摄取量。巴西莓和白藜芦醇没有改变这些果蝇每日的食物摄取量,表明巴西莓和白藜芦醇不影响食物摄入量。(6)白藜芦醇对寿命的延长与下调衰老相关途径的基因有关。在低糖高蛋白饮食雌性果蝇,白藜芦醇下调6种基因：胰岛素样蛋白3和5(dllp3和dIlp5),GstDl和热休克蛋白68(Hsp68),过氧化物氧化酶基因Prx2540-1和Prx6005。由此提示,添加白藜芦醇能通过减少氧应激反应促进果蝇的存活。在高脂饮食雌性果蝇,白藜芦醇下调低糖高蛋白饮食的6种基因其中的5种：dIlp3,dllp5,gstD1,hsp68和Prx2540-1,同时还减少了Prx2540-2的转录水平。由此进一步支持添加白藜芦醇可以减少果蝇的氧应激损伤。巴西莓上调了1(2)efl基因,抑制了Pepck基因的转录。4.结论(1)巴西莓增加高脂饮食雌性果蝇的寿命。白藜芦醇增加高蛋白饮食和高脂饮食雌性果蝇的寿命,寿命的延长具有性别特异性,而这种性别特异性的寿命延长又受到饮食成分的调节。(2)巴西莓和白藜芦醇可延长标准饮食下超氧化物歧化酶1RNA干扰果蝇的寿命。对高脂饮食下超氧化物歧化酶1RNA干扰果蝇寿命的延长更加进一步支持巴西莓和白藜芦醇可以减少果蝇的氧应激损伤。(3)白藜芦醇下调氧化应激相关基因的转录水平,说明白藜芦醇通过减少氧应激反应促进果蝇的存活。(4)白藜芦醇成分依赖性延长寿命说明营养成分在干预衰老的过程中起重要作用。(5)巴西莓和白藜芦醇不改变果蝇的食物摄取量,说明巴西莓和白藜芦醇的有益作用不是通过卡路里限制起作用。更多还原

【Abstract】 1. BackgroundAging is a problem for the entire human. Due to this, the ecnomics is obviously inhabited. Aging is a process that leads to physiologic decline and an increased vulnerability to disease and death. While the process of aging is complex, advances in aging research have shown that age-related declines in lifespan and healthspan can often be delayed through genetic and environmental manipulation, e.g. diet or pharmacological agents.Epidemiological studies propose that extension of the human lifespan or the reduction of age associated diseases may be achieved by physical exercise and caloric restriction. Calorie restriction (CR) is the most widely used experimental paradigms that extend lifespan in model organisms, which is a condition that calories nutrients are modestly reduced while maintaining adequate nutrition. CR has been reported to extend lifespan and attenuate age-related diseases in a wide variety of organisms including yeast, worms, flies, mice, rats and probably non-human primates, rhesus monkeys.Alrough CR can effectively extend the lifespan, and at the same time reduce dyslipidemia and cholesterol, but it also can produce some obviously side effects, such as influence the body metabolism and make the female animal less eggs and infertility. So people want to find some ways replace CR. Recent studies suggest that dietary composition or the ratio of carbohydrate relative to protein is more important than CR in modulating lifespan.In flies, the ratio of carbohydrate and protein can obviously modulating lifespan. The standard base diet contained 10% sugar and 10% yeast extract in weight/volume; the CR diet had 2.5% sugar and 2.5% yeast extract; the high sugar-low protein diet consisted of 18% sugar and 2% yeast extract; the low sugar-high protein diet contained 2% sugar and 18% yeast extract; and the high-fat diet had 10% sugar,10% yeast extract and 2% palmitic acid. Compare with the standard food, high sugar diet extend the lifespn in both male and female flies, while high protein diet reduce the lifespan. CR diet extend the lifespan between the standard diet and high sugar diet. So the composition of diet play an important role in modulating lifespan.In mice, a study of lifespan in approximately 40 recombinant inbred lines under dietary restriction condition indicates that dietary restriction only extends lifespan in a subset of these mouse lines and even shortens lifespan in some. This suggests that the prolongevity effect of dietary restriction depends on genetic background of the organism.Diets rich in botanicals are known to have numerous health benefits to humans. Clinical trials, animal model and cell-based studies, and biochemical analyses demonstrate that plants contain a wide range of nutrients and biologically active phytochemicals that provide numerous health benefits. Fruits and vegetables are rich in phytochemicals, nutrients and anti-oxidants and their consumption may retard aging. Some of them were called the CR-mimic substances. Acai and resveratrol were regarded as the CR-mimic substances.Acai is a fruit from the palm tree, Euterpe oleracea Mart, indigenous to the Amazon River area in South America. It is commonly used to make beverages, and served as a food additive, and is even used in folk medicine. Acai contains numerous kinds of phytochemicals, particularly, anthocyanins, proanthocyanidins and other flavonoids. Acai possesses unusually high anti-oxidant activity compared to other plant foods based on various anti-oxidant assays, particularly against the superoxide and peroxyl radicals.Resveratrol is identified as a natural compound from grapeskins. It demonstrates that there is an inverse correlation between red wine consumption and the incidence of cardiovascular disease under the fat-rich diets. Resveratrol maybe play an important role in that. After that, there were suceessive reports resveratrol can prevent or slow the progression of some diseases, such as cancer, cardiovascular disease and neurological disorders. However, the mechanisms underlying the interaction of resveratrol and dietary nutrients in modulating lifespan remain elusive. So here we first investigated whether the resveratrol can extend the Drosophila melanogaster’s lifespan or not. Second, we investigated the effect of resveratrol on lifespan of Drosophila melanogaster fed diets differing in the concentrations of dietary composition. Finally we want to investigated the underline mechanicism.2. MethodsWe record the life curve of Drosophila melanogaster and calculate the mean lifespan and maximam lifespan. Then we compare the different concentration of acai and resveratrol and then vary the dietary composition influence on Drosophila melanogaster’s lifespan. Four types of sugar and yeast extract based diets as control diets were prepared. Then we applied the qRT-PCR to detect the transcription of genes in aging-related pathways, including antioxidant peroxiredoxins, insulin-like peptides involved in insulin-like signaling and several downstream genes in Jun-kinase signaling involved in oxidative stress response. After that, we constructed the SOD1 mutant Drosophila melanogaster useing the RNA interference to find the result of acai and resveratrol on SOD1RNAi Drosophila melanogaster. Finally, we test the food intake between the different treated groups. Food intake was measured using the capillary feeder method (CAFE).3. ResultsAcai can extend the flies lifespan fed on high fat diets. Resveratrol at up to 200μmol/L in diets did not affect lifespan of wild-type female flies fed a standard, restricted or high sugar-low protein diet, but extended lifespan of females fed a low sugar-high protein diet. resveratrol at 400μmol/L extended lifespan of females fed a high-fat diet. Lifespan extension by resveratrol was associated with down-regulation of genes in aging-related pathways, including antioxidant peroxiredoxins, insulin-like peptides involved in insulin-like signaling and several downstream genes in Jun-kinase signaling involved in oxidative stress response. Furthermore, acai and resveratrol increased lifespan of superoxide dismutase 1 (sod1) RNAi females fed a standard or high-fat diet. No lifespan extension by resveratrol was observed in wild-type and sod1 RNAi males under the culture conditions in this study. We also measured daily food intake of female flies fed with the agar-free high-fat diet supplemented with or without 400μmol/L resveratrol for three consecutive days using the CAFE method. Resveratrol did not change the daily food intake in these flies, indicating that lifespan extension by resveratrol in female flies fed the high-fat diet is not due to the difference in food intake.Six genes were found down-regulated by resveratrol. Among them are Drosophila insulin-like peptide 3 and 5 (dIlp3 and dIlp5) involved in insulin-like signaling. Supplementation of resveratrol also down-regulated glutathione S transferase D1(GstDl) and heat shock protein 68 (Hsp68), two downstream targets of Jun kinase (JNK) signaling pathway, which is a major oxidative stress response pathway and is known to modulate lifespan in several model organisms. In addition, two peroxiredoxin (Prx) genes, Prx2540-1 and Prx6005, were down-regulated by supplementation of resveratrol. Taken together, these findings suggest that supplementation of resveratrol promotes the survival of flies by reducing oxidative damage.Five of the six genes, dIlp3, dIlp5, gstD1, hsp68 and Prx2540-1, down-regulated by resveratrol in flies fed the low sugar-high protein diet, were also down-regulated by resveratrol in flies fed the high fat diet. Supplementation of resveratrol also reduced the transcript level of Prx2540-2 instead of Prx6005. These findings are consistent with what we observed in the case of the low sugar-high protein diet and further support the notion that supplementation of resveratrol can reduce oxidative damage in flies. Acai can upregulated the 1(2)efl genes and downregulated the Pepck genes.4. ConclusionThe prolongevity effect of acai and resveratrol are influenced by dietary composition, only appear on the high fat or high protein diet. And this effect are gender-specific. They can influence the female, not male. The prolongevity effect on sodl RNAi are further support this. Acai and resveratrol promotes the survival of flies by modulating genetic pathways that can reduce cellular damage. This study reveals the context-dependent effect of resveratrol on lifespan and suggests the importance of dietary nutrients in implementation of effective aging interventions using dietary supplements.更多还原