【作者】 林宁；

【导师】 蔡东联；

【作者基本信息】 第二军医大学 ， 军事预防医学， 2010， 博士

【摘要】 研究目的肥胖指体内脂肪积累过多,是由于能量摄入大于消耗所致。肥胖是一种慢性疾病,同时也是心脑血管疾病、2型糖尿病、骨关节病、某些肿瘤等慢性病和社会心理障碍的重要危险因素,也是导致早死、致残、影响生命质量和增加各国财政负担的重要公共卫生问题。近几十年来,随着经济发展和生活方式变化,全球肥胖率呈现持续上升趋势,预防肥胖的流行已成为全球面临的最重大的公共卫生挑战。目前的肥胖综合干预治疗安全可靠,但是受到饮食、运动、静息式生活方式等多种综合因素影响,效果较难维持；而药物减肥对于身体的副作用较大或者尚不明确,且药物减肥与手术治疗都同样存在反弹的问题。寻找安全有效的减肥保健品成为一条可能的途径。与此同时,在饮食诱导肥胖模型的研究中,人们发现某一特殊食物使一部分个体发展为肥胖。而另一部分吃相同食物的个体没有发生肥胖。因此,有人提出食物诱导肥胖抵抗假说：即致肥胖饮食在体内产生一种代偿反应,使机体能够预防肥胖的发生。据文献报道同一品系、同一批小鼠饲用相同的高脂饲料,其中部分发生肥胖,部分不发生肥胖,前者被称为肥胖倾向(obesity prone, OP),而后者被称为肥胖抵抗(obesity resistance, OR),具体机制尚不十分清楚。本文旨在通过探讨植物提取物单体对于肥胖小鼠的预防肥胖的作用及机制的同时,排除肥胖抵抗对于肥胖研究的影响.比较单体与饮食改变的作用效果强弱,为进一步开发预防肥胖的保健品提供依据。人参(panax ginseng C. A. Meyer)是我国传统的名贵中药,具有大补元气、补气益肺、生津止渴、安神益智等功效,属五加科草本植物。中国古老的药学典籍《神农本草经》把人参列为上品,言其具有“主补五脏、安精神、定魂魄、止惊悸、明目、开心益智,久服有轻身延年之功效”。人参素有“百草药王之美称,应用于中医临床已有二千多年的历史,由于其具有广泛的药理作用和医疗用途,因而受到国内外众多学者的重视,人参许多新的生物活性也相继被发现。近年来,一些临床研究和动物实验表明人参和西洋参及其主要活性成分人参皂甙具有降低血糖,增加胰岛素敏感性,减轻体重,调节糖脂代谢的作用,但其作用机制并不十分清楚。有研究发现人参提取物能促进糖尿病小鼠脂肪组织PPARγ的mRNA和蛋白水平,其中人参皂甙Rb1经体外细胞实验证实作为PPARγ配体,通过上调PPARγ2, C/EBPα的表达促进脂肪细胞的脂肪形成,增加基础和胰岛素刺激的葡萄糖利用,同时抑制基础脂解。表明人参和人参皂甙抗糖尿病的作用可能与促进脂肪细胞分化,胰岛素敏感性和抑制基础脂解有关。此外,动物实验发现Rb1通过中枢NPY和外周CCK作用对高脂饮食诱导的肥胖小鼠有减轻体重和脂肪含量的作用。本课题基于现今关于肥胖和肥胖抵抗的最新研究进展,用高脂、高能饲料制备符合临床特点的肥胖与肥胖抵抗型C57/BL6小鼠模型,研究Rb1对肥胖预防的效果,比较Rb1对于肥胖的作用与单纯改变饮食对于代谢和食欲的调节差异。进行为期3w的Rb1干预实验,观察小鼠进食量,体重变化以及血脂,血糖变化,并采用ELISA和实时荧光定量PCR技术,检测肥胖与肥胖抵抗型C57/BL6小鼠在Rb1干预后中枢和外周血的NPY以及PYY的水平,为进一步探讨人参皂甙Rb1的对于肥胖防治机制及功能性保健食品的开发提供实验依据。研究方法清洁级雄性健康野生型C57/BL6小鼠70只,体重(21.6g±1.3g),按体重排序后读取电脑随机数字表抽取8只作为空白对照组,剩余62只用于制备肥胖与肥胖抵抗型小鼠模型。空白对照组(N)：每鼠每天自由饮水及进食普通饲料,造模组进食高脂饲料连续12w后称量体重时,参考体重大于N组的平均体重+1.96倍标准差和大于正常体重的20%的标准,将体重最大的20只作为肥胖组(DIO)；肥胖抵抗组(DR)：参考体重小于C组的平均体重+1.0倍标准差,将体重最小的10只作为DR组,余弃除。小鼠自由进食,每天观察小鼠的一般状况、饮食变化、行为(自主活动、精神状态)变化、毛发变化,每天称量饲料,每周称量小鼠体重。造模完成后记录48小时各组小鼠的进食量和能量摄入量作为基础参考值。造模12w后根据体重分为肥胖型小鼠、肥胖抵抗型小鼠和正常对照组,肥胖组抽取5只为继续高脂喂养组(HF),5只高脂饮食变为低脂普通饮食的饮食改变组(DC),余10只为Rb1干预组。于第13w开始时给予DIO和DR组小鼠Rb1腹腔注射,同时给予普通对照组10%的乙醇稀释液进行注射3w。在注射3w结束时,小鼠禁食12h后,摘眼球取血,用于测定TC、TG、HDL、LDL、glucose, NPY和PYY水平；血取净后,立即进行解剖,留取左叶肝脏于10%福尔马林溶液中固定,用于病理检测；同时迅速分离下丘脑,取完整小肠和小肠生理盐水漂净后置于液氮中保存,用于NPY和NPY Y2以及PYY测定。3w内每日记录各组小鼠进食量,称取每只小鼠体重,观察小鼠变化。免疫荧光酶联免疫吸附法测定血清中NPY, PYY的表达水平。两步法实时荧光定量逆转录聚合酶链反应检测下丘脑组织和肠道组织的NPY,NPY Y2以及PYYmRNA表达。选择SPSS 13.0软件行统计学分析,P<0.05被认为有统计学差异。结果l、高脂、高能饲料制备肥胖与肥胖抵抗小鼠模型建立DR与DIO组体重有显著性差异,DR与N组体重无显著性差异,DIO与N组体重有显著性差异。2、人参皂甙Rb1对肥胖与肥胖抵抗型C57/BL6小鼠体重和代谢的影响。病理结果显示,HF组小鼠肝脏出现严重的脂肪变性,而其余各组的肝脏病理正常。DIO小鼠的能量摄入量高于其他各组,尽管DR组小鼠的进食量低于N组小鼠,但是其能量摄入量基本相当。Rb1进行干预治疗以后,DIO-Rb1组小鼠的体重在第1w内急剧下降,后2w下降速率降低趋于平稳,基本维持在跟饮食组小鼠减重后的水平。DC组小鼠的体重3w内也下降,第1w内的降低速率最大,后平稳的下降。空腹血糖结果显示：与普通对照组相比,HF组与DC组的血糖与之有差异,其余各组均无显著性差异；与高脂组相比,各组血糖均有显著性差异；血脂结果显示：与N组相比,DIO-Rb1组的TCH和HDL与之有统计学差异,DR-Rb1组的HDL和LDL与之有统计学差异,HF组的TCH和HDL与之有统计学差异；与DIO-Rb1组相比,N组和DR-Rb1组的TCH和HDL与之有统计学差异,DR-Rb1组的TCH和LDL与有统计学差异；与HF组相比,各组的HDL与之均有差异,N, DIO-Rb1, DC组的TCH与之有统计学差异,DR-Rb1的LDL与之又统计学差异(P<0.05)。3、血清NPY, PYY在人参皂甙Rb1治疗饮食诱导肥胖与肥胖抵抗型小鼠中的表达。DIO-Rb1组的NPY值与DC和HF组相比有统计学差异,使用Rb1注射的两组与N组之间无统计学差异,其余各组之间无统计学差异；除DIO-Rb1以外,N组PYY值与其余各组都有统计学差异,N组与DIO-Rb1组跟HF之间有统计学差异,与DIO-Rb1组相比,DR-Rb1, DC, HF组的PYY值都与之有统计学差异(P<0.05)。4、下丘脑NPY, NPY Y2的mRNA,小肠PYY的mRNA在饮食诱导肥胖与肥胖抵抗型小鼠中的表达。N, DIO-Rb1, DR-Rb1, DC组下丘脑的NPY mRNA水平明显低于HF组,这四组间没有显著性差异(P<0.05)；N, DIO-Rb1, DR-Rb1, DC组下丘脑的NPY-Y2 mRNA水平与HF相比有显著性差异,但是只有DIO-Rb1组的水平低于HF组,其他各组均高于HF组。在小肠内PYY mRNA的水平来看,N, DIO-Rb1, DR-Rb1, DC各组显著低于FHF组(P<0.05),其余各组间没有统计学差异。结论1.用高脂高能饲料成功制备肥胖与肥胖抵抗型小鼠模型。2. Rb1腹腔注射可以对肥胖小鼠起到降低体重,改善血糖的作用,对血脂的改变主要体现在Tch和HDL水平上。Rb1的作用体现在第1w内的迅速将体重作用上,小鼠的进食量和能量摄取均再第1w内显著下降3.Rb1通过提高血清的PYY水平,抑制了下丘脑NPY Y2的mRNA表达,从而降低了血清NPY水平,降低了下丘脑NPY mRNA表达,进而抑制了小鼠的食欲,起到了降低体重的作用。4.Rb1腹腔给药的效果在血脂改变上与饮食改变相比无显著性差异,但是在血糖以及肠道—中枢食欲调节上有显著性差异。更多还原

【Abstract】 AimsObesity is a major predisposing factor for cancer, type II diabetes, hypertension and coronary heart diseases with their enormous costs both socially and financially. Therefore, there is a pressing demand to research the mechanism of obesity so that effective treatment regimes to deal with its rising prevalence may be devised. It is well believed that energy accumulation can lead to obesity, and the high-fat and high-energy food can especially easily complete the task. However, in both rodents and humans, some become obese while others remain lean when raised the same high-fat and high-energy diet. The previous work has shown that when C57/BL6 mice are fed a high fat diet some mice develop obesity whist others remain lean. This has also been shown in early studies in rats fed a palatable high fat/high sugar diet. The study on this point has showed interesting results on their difference on appetite control system. In the central neural regulatory systems, neuropeptide Y plays an important role in appetite and energy balance. NPY consists of 36 ami no acids, and is a powerful neurochemical stimulator in feeding. PYY is a member of the pancreatic polypeptide family whose structure and function is related to NPY. PYY is mainly secreted from the endocrine L cells of the gastrointestinal tract and is related into the blood stream in response to the ingestion of food, especially fat. PYY has a high affinity for Y2 receptors, and upon binding can inhibit food intake via the hypothalamic arcuate nucleus.Ginsenosides, the main component of Panax ginseng root, has been reported to show several pharmacological actions on the peripheral Metabolism of glucose and lipid and on endocrine secretion. Recent studies showed that administration of Panaxoside Rbl in high fat diet group reduced the body weight, total food intake, fat content, serum ieptin and NO to a level equal or below the normal diet group in SD rats. In vitro study revealed that Rbl promoted adipogenesis in 3T3-L1 cells by enhancing PPARγ2 and C/EBPαgene expression. Little is known about the specific effect and feature of Rbl on anti-obesity in DIO and DR mice, especially compared with diet pattern change, as well as its effect with time change. Besides, the literature revealed that Rbl was a suppressor in central modulation of feeding in the rat through NPY pathways. Our study aimed to find the change of peripheral NPY and PYY between groups.Ginsenoside Rbl, the main component of ginsenosides, have been reported to show a variety of efficacies such as anticancer, antihypertension, antidiabetes, and antinociception. In the present study, the anti-obesity effect of Ginseng Panaxosides Rb1 on diet-induced obese mice were evaluated.MethodsSeventy C57/BL6 male mice were randomly divided into chow diet (N, n=8) group and high fat diet group (n=62). After fed with high fat and energy diet for 12 weeks, the mice were divided into two groups, including an obesity group (DIO, n=20) and obesity-resistant group (DR, n=10) according to the highest and lowest weight gain, with other mice discarded. Obesity group was then subgrouped into DIO-Rb1 injection group (n=10), diet change to normal group (DC,n=5) and no intervention group (HF,n=5). Intraperitoneal injection of Rb-1 was received daily by mice in DIO-Rb1 and DR group for 3 weeks. Body weight and food intake were observed, and fasting blood glucose, lipids was examined by biochemical analyzer. In addition, we also determined the secretion and expression of neuropeptide Y (NPY), Y2 receptor, and Peptide YY (PYY) by enzyme linked immunosorbent assay (ELISA) and real-time RT-PCR.ResultsEffects of Rbl on the body weight and food intakeBody weight is a gold standard of obese. So we firstly investigated the effect of Rbl on body weight of obese mice. After fed with high-fat diet for 12 weeks, the body weight of diet-induced obese (DIO) mice was significant increase. However, after treatment with Rb1 for 3 weeks, the body weight of DIO-Rb1 group was obvious decrease when compared with that in DIO group. In addition, we also detected the food intake after treatment with Rb1. We found that food intake was also decrease in DIO-Rbl group as compared with that in DIO group.Effects of Rbl on the blood glucose and lipidsThe blood glucose and lipids are also important indices of obese. Thus, we also detected the effects of Rb1 on fasting blood glucose and lipids. After fed with high-fat diet for 12 weeks, the fasting blood glucose and total cholesterol (TC) of DIO mice were significant increase. Whereas, after treatment with Rb1 for 3 weeks, the fasting blood glucose and total cholesterol of DIO-Rb1 group were obvious decrease when compared with those in DIO group. Additionally, there was no significant difference in triglyceride(TG) among three groups.Effects of Rb1 on the serum levels of PYY and NPYIt is well known that PYY and NPY are involved in the obese, so we next measured the serum levels of PYY and NPY to explore the possible action mechanisms of Rb1. As shown in table 4, after fed with high-fat diet for 12 weeks, the serum PYY of DIO mice was significant decrease. Rb1 could inhibit the decreasing of serum PYY of DIO mice. On the contrary, after fed with high-fat diet for 12 weeks, the serum NPY of DIO mice was significant increase. Rb1 could reverse the increasing of serum NPY of DIO mice.Effects of Rb1 on the mRNA levels of NPY, NPY-2R and PYYThe mRNA levels of NPY in hypothalamus were significantly lower in N and DIO-Rb1 groups than that in DIO group (P<0.05). Similar pattern was observed in NPY-2R level. Rb1 could reverse the increasing of NPY-2R expression to normal level. The mRNA level of PYY in intestine tract in DIO group was lower than that in any other groupConclusionsTaken together, our results indicate that the ginsenoside Rb1 may be useful in the treatment of obesity via modifying the serum content and mRNA expression of NPY, Y2 receptor and PYY.更多还原