【作者】 向雪松；

【导师】 杨月欣； 王竹；

【作者基本信息】 中国疾病预防控制中心 ， 营养与食品卫生学， 2010， 博士

【摘要】 研究背景随着社会经济的发展和人们膳食结构和生活方式的改变,糖尿病(diabetes,DM)已经成为严重危害人类健康的慢性疾病。饮食和营养治疗是糖尿病治疗的重要组成部分。通过膳食和保健食品预防和控制糖尿病成为一种趋势。辅助降血糖保健食品主要是天然植物性食品或提取物,其来源广泛,降糖效果和作用机理也不尽相同,这些因素给功能性食品评价带来挑战。目前的保健食品评价标准仅给出一种类似1型DM的化学诱导动物模型,显然不能满足当前保健食品多样化发展的要求。为了适应功能食品评价的发展要求,科技部提出了功能性食品评价技术的研究的十一五课题。近年有人考虑膳食在糖尿病发病进程中的因素,提出膳食诱导联合小剂量链脲佐菌素(streptozotocin, STZ)的造模方法。但是这种模型的造模条件,高脂饲料配方、高脂喂养时间和STZ注射剂量等缺少统一的操作,此外该模型的代谢特征,以及在评价不同来源、不同作用机理的辅助降糖功能食品中的应用性还未见较系统的研究。因此,针对这些问题我们在以往研究中主要对造模方法进行了初步探讨,本研究主要从模型血生化代谢特点、组织形态学和分子生物学特征,从整体到组织再到细胞水平全方位深入研究该模型是否具有2型糖尿病特点；并通过促胰岛素分泌剂和胰岛素增敏剂药物进行验证和评估该模型的胰岛素抵抗水平和β细胞功能；最后在具体的功能食品评价中对该模型的应用性进行探讨。研究目的1.建立高脂高糖饲料联合小剂量STZ制造2型DM实验动物模型,并探讨其模型特征；2.通过重复性研究和药物评价验证模型的可复制性和适用性3.模型在表没食子儿茶素没食子酸酯(epigallocatechin-3-gallate, EGCG)和葛根黄酮,不同来源和不同机制的功能性食品降血糖评价中的适用性方法：1、2型糖尿病大鼠最佳造模条件确立1.1饲料中猪油、蔗糖配比对造模的影响55只大鼠根据体重随机分为普通饲料组(N)和高脂高糖饲料组(HFS)。HFS饲料依据猪油(F)、蔗糖(S)水平设计为1OF10S(含10%猪油10%蔗糖,其余类推)、10F20S、20F10S组。观察每组大鼠8周内体重、空腹血糖(FBG)和葡萄糖耐量实验后2h血糖(PBG)、血脂水平变化。然后,HFS喂养大鼠腹腔注射25 mg/kg STZ,对照组同法注射缓冲液。观察大鼠饮食、饮水、排尿量、毛色、体重变化及死亡情况；注射后连续2周观察FBG、PBG的稳定性,确立适宜饲料配方。1.2 STZ注射剂量对造模的影响75只大鼠根据体重、血糖水平随机分为6组：正常对照组(10只)、普通饲料STZ组(15只)、高脂对照组(10只)、低剂量STZ组(15只)、中剂量STZ组(15只)、高剂量STZ组(15只)。正常对照组和普通饲料STZ组给予普通饲料,其余组给予10F20S饲料。喂养8周后,分别一次性腹腔注射0、25、0、20、25、30mg/kg,bw STZ。继续高脂饲料喂养,观察大鼠饮食、饮水、排尿量、毛色变化,记录每周体重变化、死亡情况；连续监测7wFBG、PBG变化,确定血糖稳定情况和成模率,根据血糖水平以及胰岛组织形态学检测确立最适造模条件。2、2型糖尿病大鼠模型可复制性及特征研究选择75只健康雄性Wistar大鼠按照以上确立的条件(10F20S喂养+25mg/kg,bw STZ)造模。另选正常对照组(N组)10只和高脂饲料对照组(HF组)10只。HFS喂饲8周后测定FBG、PBG、空腹INS、TG、TC、ISI,确立胰岛素抵抗发生。然后STZ造模,2周后筛选糖尿病模型大鼠；继续喂养5周后处死。观测血生化指标：ALT、AST、CR、LDL、HDL、TG、TC、GLU、INS;组织形态学指标：胰腺、肝、肾、心组织的HE染色,胰岛的p细胞的胰岛素免疫组织化学；胰岛素信号传导指标：各组大鼠肝脏和肌肉中胰岛素信号传导相关蛋白InsR、IRS、PPARγ的表达情况,确立模型特征。通过统计学分析确立模型的可复制性。3、2型糖尿病大鼠模型的验证分别以胰岛素促分泌剂瑞格列奈和胰岛素增敏剂罗格列酮为阳性对照物对上述模型大鼠的胰岛素抵抗水平和p细胞功能进行评估和验证。挑选正常对照组(N组)大鼠10只；以及2型糖尿病造模成功的大鼠27只,根据血糖随机分为3组,每组9只,各组每天分别给予空白对照(蒸馏水)、胰岛素促进剂瑞格列奈(1mg/kg)、胰岛素增敏剂罗格列酮(0.5mg/kg)灌胃(分别为DM组、Rep组、Ros组)。连续喂养5周,再次进行OGTT试验后,取血并处死大鼠。从血生化指标、组织形态学、胰岛素信号传导三个方面评价瑞格列奈和罗格列酮的作用特征,具体指标同上。4、2型糖尿病大鼠模型在辅助降糖功能性食品评价中的应用4.1葛根素对2型糖尿病大鼠的影响及其作用机制研究挑选正常对照组(N组)大鼠10只；以及2型糖尿病造模成功的大鼠36只,根据PBG和FBG随机分为4组,模型对照组(DM组,蒸馏水灌胃)、葛根素低剂量组(P1组,葛根素50mg/kg灌胃)、中剂量组(P2组,100mg/kg)、高剂量(P3组,200mg/kg)。5周后进行OGTT试验,取血并处死大鼠。检测血清ALT、AST、CR、LDL、HDL、TG、TC、GLU、INS;总超氧化物歧化酶(Total superoxide dismutase, T-SOD)、丙二醛(Malondialdehyde, MDA)、谷胱甘肽过氧化物酶(Glutathione peroxidase, GSH-PX)、8-羟基脱氧鸟苷(8-OHdG)水平；胰腺、肝、肾、心等组织的HE染色,胰岛的p细胞的胰岛素免疫组织化学；各组大鼠肝脏和肌肉中InsR、IRS、PPARγ、NF-κB蛋白的表达情况。通过抗氧化水平、炎症信号传导和胰岛素信号传导等方面研究葛根素的降血糖作用,并探讨其可能的作用机制。4.2表没食子儿茶素没食子酸酯(EGCG)对2型糖尿病大鼠的预防作用挑选正常对照组(N组)大鼠10只；以及胰岛素抵抗模型大鼠40只,根据血糖随机分为4组,分别为HFS对照组(H组)、EGCG低剂量(E1)、中(E2)、高剂量组(E3)。EGCG一次性降血糖作用：N组、H组、E1组、E2组和E3组分别给予蒸馏水、蒸馏水、200mg/kgEGCG、700mg/kgEGCG、1000mg/kgEGCG一次性灌胃。20 min后进行OGTT实验,计算OGTT曲线下面积(area under the curve, AUC),观察EGCG对血糖应答的影响。EGCG干预10天后降血糖作用：N组、H组、E1组、E2组和E3组大鼠分别给予蒸馏水、蒸馏水、EGCG低剂量(20mg/kg)、EGCG中剂量(50mg/kg)、EGCG高剂量(200mg/kg)灌胃。EGCG干预10天后,进行OGTT试验,计算OGTT-AUC。EGCG干预5周后降血糖作用：按照短期实验使用剂量,用EGCG干预5周测定OGTT,计算OGTT-AUC.然后各组大鼠乙醚麻醉,腹主动脉取血测定ALT、AST、CR、LDL、HDL、TG、TC、GLU、INS;进行胰腺、肝、肾、心组织的HE染色,观察EGCG对大鼠发生胰岛素抵抗的影响。结果：1、2型糖尿病大鼠模型的建立1.1饲料中猪油、蔗糖配比对造模的影响高脂喂养至第8周,各HFS组出现胰岛素抵抗现象。体重已经超出N组10%,但HFS组间未见统计学差异(P<0.05)。和对照组相比,HFS各组的PBG均有所升高(P<0.05),胰岛素敏感指数(ISI)下降(P<0.05),其中10F20S的ISI值最低,说明10F20S易于诱发胰岛素抵抗。进一步采用小剂量STZ造模,大鼠出现明显的体重下降、血糖升高(和对照组相比,P<0.05)等糖尿病体征,造模2周后血糖趋向稳定。和其他组相比,20F10S组血糖升高最明显(P<0.05),其中PBG超过30mmol/L的有4只,同时死亡数较高；10F20S组血糖升高相对温和,PBG介于11.1-20mmol/L之间。造模成功率10F20S和20F10S均为73.3%,10F10S为66.6%。说明10F20S组造模成功率较高且血糖中度升高。1.2 STZ注射剂量对造模的影响STZ注射剂量与造模后血糖水平明显相关。高剂量STZ组血糖显著升高、胰岛素严重缺乏,接近1型糖尿病特征；而普通饲料和低剂量STZ组则血糖升高不明显(P<0.05)。中剂量STZ组血糖中度升高,PBG介于11.1mmol/L-20.0mmol/L之间,具有2型糖尿病血糖特征。连续7周的OGTT血糖监测显示：血糖初期波动较大和注射剂量有关,高剂量组血糖上升最多,波动也最大；中剂量组血糖在2周后基本达到稳态,在之后的7周内血糖较稳定。胰岛HE染色和免疫组化染色观察显示中剂量STZ组胰岛结构完整,p细胞数量和INS分泌虽有所减少,但接近正常大鼠。高剂量组胰岛则损伤太大,而低剂量组能代偿损伤不引起高血糖,均不适合造模。以上实验确立饲料配方选择10%猪油、20%蔗糖,STZ剂量选择25mg/kg.bw,造模条件最佳。2、2型糖尿病大鼠模型的可复制性及其特征研究8周高脂喂养后大鼠已经出现胰岛素抵抗现象：大鼠明显肥胖,体重显著增加,空腹血糖和餐后血糖升高,ISI明显低于正常对照组(P<0.05)。在随后15周时的组织形态学检测中可见,胰岛增生肥大,p细胞数目增多但分泌功能障碍,肝脏出现明显的小泡样脂肪变性。胰岛素信号传导途径中IRS-2和PPARγ蛋白表达水平显著下降(P<0.05)。在胰岛素抵抗大鼠基础上小剂量腹腔STZ注射,大鼠出现以下变化：1)FBG(8.14±5.87mmol/L)和PBG(17.23±8.98mmol/L)均温和升高,胰岛素水平与N组无差异,ISI低于N组(P<0.05),肝糖原增加为N组的5倍。此外成模率为71.6%,与我们前两次的73.3%结果基本一致。2)组织形态学检测：小剂量STZ仅仅特异性损伤了大鼠的胰腺,对其他脏器损伤均较小。肝脏中有小泡样脂肪变性。胰岛数目虽然相对下降,但结构正常,仍有足够多的正常β细胞来维持胰岛素的分泌。与人类2型糖尿病前期和早期形态学改变相似。3)糖尿病大鼠胰岛素传导途径障碍：InsR蛋白水平均接近正常组大鼠；外周组织中IRS-2和PPARγ表达水平显著下降。证明通过8周高脂喂养和小剂量STZ注射,可成功诱导出与人类2型糖尿病相似的大鼠模型。统计结果显示,2次独立实验表现了类型的造模情况,说明本方法具有可复制性。3、2型糖尿病大鼠模型的验证经过胰岛素促进剂(瑞格列奈)和胰岛素增敏剂(罗格列酮)5周灌胃治疗后,能够显著改善大鼠的FBG和PBG水平(P<0.05)；胰岛素免疫组化染色显示罗格列酮和瑞格列奈能提高p细胞数量和细胞质内棕色颗粒水平,瑞格列奈对刺激β细胞胰岛素分泌作用稍强于罗格列酮；罗格列酮和瑞格列奈降血糖作用可能与提高外周组织中IRS-2和PPARγ表达水平相关,罗格列酮在大鼠肝脏中提高PPARγ水平明显,是瑞格列奈的1.4倍。瑞格列奈通过促进β细胞胰岛素分泌,罗格列酮通过改善胰岛素抵抗均能降低模型大鼠血糖,从另一个方面证实了该模型仍有足够的β细胞数量和功能维持正常血糖水平,同时胰岛素抵抗是导致模型大鼠高血糖的原因之一。4、2型糖尿病大鼠模型在辅助降糖功能性食品评价中的应用研究4.1葛根素对2型糖尿病大鼠的影响葛根素通过5周干预后,能显著改善DM大鼠的各项指标。1)血生化指标：与糖尿病对照组比较,葛根素能降低糖尿病大鼠的FBG和PBG水平,改善ISI,降低血脂水平,改善肝肾功能,且存在剂量反应关系(P<0.05)。2)组织形态学检测：葛根素干预后,随着剂量的增加,胰腺中胰岛数逐渐增多,边界逐渐变清楚,空泡变性减少,p细胞数量和胰岛素含量增加,但仍未达到正常组水平。3)氧化应激：葛根素能够提高糖尿病大鼠的抗氧化能力,提高SOD水平,同时反应机体氧化产物的8-OHdG和MDA水平则降低(P<0.05)。4)胰岛素信号传导途径：葛根素抑制外周组织(肝脏、肌肉)中NF-KB水平的表达,减轻外周组织中炎症过程,改善了胰岛素信号转导中的IRS-2和PPARγ的表达(P<0.05)。4.2EGCG对2型糖尿病大鼠的预防作用EGCG降血糖效果与其在体内作用时间长短有关。在本研究中,通过分析EGCG三个不同作用时间对血糖的影响：发现一次口服EGCG对胰岛素抵抗大鼠的血糖影响不明显。连续10天的干预后,EGCG的中、高剂量组有改善大鼠糖耐量的趋势,降低餐后血的作用(P<0.05)。随着干预期的延长,EGCG作用越明显。连续5周EGCG干预后,EGCG降低血糖的同时(P<0.05),改善肝脏脂肪变性,促进胰岛增生以及改善β细胞功能。结论：1、确定10%猪油20%蔗糖的高脂膳食喂养8周能建立胰岛素抵抗大鼠模型,在此基础上一次性腹腔注射25 mg/kg,bw STZ能建立2型糖尿病大鼠模型。2、通过在不同动物饲养中心,不同批次大鼠的造模中证实该模型有较好的稳定性和复制性。3、通过对模型大鼠血生化指标、组织形态学检测和胰岛素信号传导途径等特征的研究,发现该模型具有胰岛素抵抗和p细胞功能障碍等2型DM典型特征。4、选择特异性强的阳性药物——胰岛素增敏剂和促胰岛素分泌剂进行干预,验证该模型确实存在胰岛素抵抗和β细胞功能下降,并证实该模型在这两类作用机制的功能物质中有较好的适用性。5、该模型在黄酮类葛根素和多酚类EGCG降血糖功能评价应用中,证实模型能够应用于改善胰岛素信号传导、抗氧化、抗炎和抑制α-糖苷酶活性等机制的降血糖功能评价。研究同时表明,葛根素降血糖的作用可能与其改善胰岛素信号传导,提高机体抗氧化水平,抑制炎症信号途径NF-KB蛋白表达水平有关。长期给于EGCG可具有降血糖效果,其作用机制可能与提高体内的抗氧化能力和抑制α-糖苷酶活性有关。本研究的创新之处：1.全面探讨了大鼠膳食配比和STZ注射剂量对造模的影响,为解释模型和标化模型提供了依据。2.从模型血生化代谢特点、组织形态学和分子生物学特征,从整体到组织再到细胞水平全方位深入研究该模型的特征。3.首次探讨了模型的稳定性、复现性,对关键技术条件进行了摸索,为提出功能食品评价动物模型的建议提供了参考。4.首次初步探讨了模型在不同作用机制的功能食品评价中的应用性。更多还原

【Abstract】 BackgroudsWith the socio-economic development and people’s diet and lifestyle changes, diabetes (DM) has become a serious hazard to human health. Diet and nutrition therapy is an important part of diabetes treatment. Preventing and controlling diabetes by diet and functional foods become a trend. Hypoglycemic functional foods mainly originate from natural plant foods or their extracts, their wide variety of sources and different mechanisms of hypoglycemic make a great challenge to the evaluation of functional foods.Current standards of health food evaluation only given a similar type 1 DM chemically induced animal model, clearly can not meet the requirements of the multiform development of health food. Therefor we initiated this study with the objective of developing a suitable type 2 diabetic rat model that would on the one hand closely mimic the natural history of the disease events (from insulin resistance to beta cell dysfunction) as well as metabolic features of human type 2 diabetes and useful for the investigation as well as preclinical testing of various functional foods viz. insulin sensitizers and insulinotropics for the treatment of type 2 diabetes.Objective1. To develop a rat model that replicates the natural history and metabolic characteristics of human type 2 diabetes.2. To indentify the reproducibility and applicability of the animal models3. To verify the applicability of the established animal models by drug and fuctional foods assessment.4. To study the preventive effects and possible mechanisms of Pueraria and EGCG on the regulating blood glucose.Materials and methods1. The establishment of type 2 diabetic rat model1.1 Effects of high-fat diet ratio on the modeling 55 healthy Wistar rats were randomly assigned to normal group and high-fat-sugar feeding group (HFS) based on initial body weight. According to the ratio of lard (F) and sucrose (S) in feeds, HFS feeding group diets were repartition to 10F10S (representing containing 10% lard and 10% sucrose in feeds, same below),10F20S, 20F10S. Body weight was weekly recorded. At the 8th week, fasting blood glucose and 2h postload glucose after oral glucose torlerence test (OGTT) were tested. Blood lipid level were detected. The influence of fat and sugar in diets on body weight gain, glucose response and lipid was analyzed.Then, each dietary group was injected intraperitoneally (i.p.) with low dose of STZ(25 mg/kg). Fast plasma glucose and OGTT were carried out every week. The influence of dietary regimens on the animal model was envaluated based on integration of injury intensity, blood glucose levels and mortality.1.2 Effects of STZ injection dose on the modelingThe rats were allocated into two dietary regimens consisting of 20 and 55 rats by feeding either common or hight-fat diet. Base on body weight, blood glucose and lipid level,55 HFS-fed rats were randomly divided into 4 groups:STZO, STZ20, STZ25 and STZ30. After fasted for overnight, each groups were respectively intraperitoneally injected with 0,20,25,30 mg/kg.bw of streptozotocin (STZ). The basis of observation on the relationship between dose depended blood glucose response and death, the appropriate dose of STZ used in model made was indentified. Then,5 weeks of blood glucose monitor was preceeded in each groups. The influence of STZ injection dose on the modeling was evaluated based on integration of injury intensity, blood glucose levels, mortality and islet morphology.2. The replication and characteristics of type 2 diabetes rat modelThe rats were allocated into two dietary regimens consisting of 10 and 85 rats by feeding either common or high-fat diet. Eight weeks later, blood lipid was measured and OGTT was carried out in each group and insulin sensitivity index (ISI) was calculated and compared.Then 75 high-fat diet rats were injected intraperitoneally (i.p.) with low dose of STZ(25 mg/kg). Fast plasma glucose and OGTT were carried out 2 weeks later to make sure that type 2 diabetes was induced in rats.10 rats with type 2 diabetes and 10 common or high-fat diet rats were chosen and continued on their original diets for 5 weeks. The body weight and ALT, AST, CR, LDL, HDL, TG, TC, GLU and INS were carried out. The pancreatic, kidney, liver, heart tissues were stained with hemotoxylin and erosin. The expressions of InsR, IRS, PPARy were examined using Western blot method.3. The validation of Type 2 diabetic rat model10 normal control rats (N) and 27 rats with type 2 diabetes were selected and then assign to insulin secretion agent repaglinide (1mg/kg), insulin sensitizer rosiglitazone (0.5mg/kg) or distilled water.Each group continued on their original diets for 5 weeks. Five weeks later, the body weight and ALT, AST, CR, LDL, HDL, TG, TC, GLU and INS were carried out. The pancreatic, kidney, liver and heart tissues were stained with hemotoxylin and erosin. The expressions of InsR, IRS and PPARy were examined using Western blot method.4. The applicability of Type 2 diabetic rat model for the assessment of fuctional food4.1 Effects of Puerarin on rats with type 2 diabetes10 normal control rats (N) and 36 rats with type 2 diabetes were selected and then assigned to low dose of puerarin (50mg/kg), middle dose of puerarin (100mg/kg) high dose of puerarin (200mg/kg), or distilled water.Each group continued on their original diets for 5 weeks. Five weeks later, the body weight and ALT, AST, CR, LDL, HDL, TG, TC, GLU and INS were carried out. The pancreatic, kidney, liver and heart tissues were stained with hemotoxylin and erosin. The expressions of InsR, IRS, PPARy, and NF-κB were examined using Western blot method.4.2 Effects of EGCG on the prevention of type 2 diabetesSelected 10 normal control rats (N); 40 rats with insulin resistant were randomly divided into 4 groups:H, E1, E2 and E3 group.One time hypoglycemic effect of EGCG:single intragastric administration were given to N, H, E1, E2 and E3 group with distilled water group, distilled water, EGCG low dose (200mg/kg), EGCG middle dose (700mg/kg) or EGCG high dose (1000mg/kg). OGTT were carried out.Short-term hypoglycemic effect of EGCG:after one time experiment, group N, H, E1, E2 and E3 group were given distilled water, distilled water, EGCG low dose (20mg/kg), EGCG middle dose (50mg/kg), EGCG high dose (200mg/kg) orally. After EGCG intervention 10 days, OGTT were carried out.Long-term hypoglycemic effect of EGCG:After EGCG interference 5 weeks. The body weight, ALT, AST, CR, LDL, HDL, TG, TC, GLU and INS were carried out. The pancreatic, kidney, liver and heart tissues were stained with hemotoxylin and erosin.Results1. The establishment of type 2 diabetic rat model1.1 Effects of high-fat diet ratio on the modelingInsulin resistance was induced in high fat diet feeding rats 8 weeks later. The weight of HFS groups has exceeded 10% of N group, but no significant difference amog HFS groups (P<0.05). The PBG of HFS groups increased also (P<0.05), insulin sensitivity index (ISI) was significantly lower than the control group (P<0.05). After STZ injection, body weight decreased rapidly in the first week.2 weeks later, body weight begun to stabilize. The level of blood glucose in 20F10S increased significantly (p<0.05). The level of PBG in HFS groups significantly increased compared with the pre-modeling (p<0.05).20F10S had the highest level of blood glucose, there were 4 rats’PBG over 30mmol/L. The level of FBG in 10F20S group was not obvious increased, but the PBG increased significantly (p<0.05), range of 11.1-20mmol/L. The achievement ratio in 10F20S and 20F10S were 73.3%,10F10S was 66.6%.1.2 Effects of STZ injection dose on the modelingThe 20mg/kg of STZ injection group had low successful rate(30%); The 25mg/kg of STZ injection group had the highest rate (73.3%) of establishing model successfully, and stable moderate hyperglycemia. Islet structural integrity, but the number ofβ-cell and cytoplasmic brown granules decreased; The 30mg/kg of STZ injection group had the highest mortality and highest blood glucose, the number of islets decreased obviously, small islets more common, islet structure is not complete.2. The replication and characteristics of type 2 diabetes rat modelInsulin resistance was induced in high fat diet feeding rats 8 weeks later. The insulin resistant rats had higher body weight and levels of FBG and PBG compared to control group(P<0.05). Islets mass enlarged in HFS groups,but the expression of insulin was lower than N group. The expressions of IRS-2 and PPARy decreased significantly.After low-dose intraperitoneal injection of STZ, rats had the following changes:1) FBG (8.14±5.87mmol/L) and PBG (17.23±8.98mmol/L) were moderately elevated. ISI was less than N group (p<0.05), liver glycogen increased 5 times above the N group. The achievement ratio was 71.6%.2) histological detection:Islet structural integrity, but the number ofβ-cell and cytoplasmic brown granules decreased; the liver were small bubble-like steatosis 3) The expressions of IRS-2 and PPARγdecreased significantly in the Peripheral tissue.3. The validation of Type 2 diabetic rat modelAfter 5 weeks of insulin promoter (repaglinide) and insulin sensitizer (rosiglitazone) treatment, FBG and PBG levels in rats were significantly improved (p<0.05); Insulin immunohistochemistry:rosiglitazone and repaglinide could improveβ-cell volume and cytoplasmic brown granular level, repaglinide stimulateβ-cell insulin secretion was slightly stronger than rosiglitazone; rosiglitazone and repaglinide could increase the expressions of IRS-2 and PPARγ.4. The applicability of Type 2 diabetic rat model for fuctional foods assessment.4.1 Effects of Puerarin on rats with type 2 diabetesPuerarin could significantly improve the indicator level of DM rats by 5 weeks intervention.1) Blood biochemical indicators:compared with the diabetic control group, puerarin could reduce the levels of FBG and PBG in diabetic rats, improve the ISI, improve liver and kidney function, and had dose-response relationship (p<0.05).2) Histological detection:with the dosage increased, the number of pancreas islets gradually increased, vacuolar degeneration decreased, the number ofβcells and insulin content was increased, but not yet reached the level of the normal group.3) Oxidative stress:puerarin could increase the antioxidant capacity of diabetic rats, increase the level of SOD and reduce the level of 8-OHdG and MDA (p<0.05).4) Insulin signaling pathway:puerarin inhibited the expression of NF-κB in peripheral tissue (liver, muscle) and increased the expressions of IRS-2 and PPARy.4.2 Effects of EGCG on the prevention of type 2 diabetesThe hypoglycemic effect of EGCG was concerned with the length of action time. In this study, we analyzed the effects of EGCG on blood sugar in three differern stages. There was no obvious effect in one time intervention. After 10 consecutive days of intervention, EGCG’s middle and high dose group had a trend to improve glucose tolerance and reduce the level of postprandial blood (P<0.05). After 5 weeks intervention, EGCG lowered blood glucose (P<0.05), improved liver steatosis and isletβcell function.Conclusions1. Through study the effects of high-fat diet ratio and STZ injection dose on the modeling, we established the type 2 diabetic rat model with 10% lard and 10% sucrose high-fat diet and low dose of STZ(25 mg/kg) injection.2. The hyperglycemia models have strong consistency in duplication checks, and are suitable for long-term observation in prevention and intervention studies.3. These fat-fed/STZ-treated rats simulate natural disease progression and metabolic characteristics typical of individuals at increased risk of developing type 2 diabetes because of insulin resistance.4. Insulin secretion agent repaglinide and insulin sensitizer rosiglitazone, which could reduce blood glucose, confirmed from another aspect that the model still had adequate number ofβ-cell with function of contolling blood glucose level, and insulin resistance was one of the reasons leading to high blood sugar.5. The model had a good application in different kinds of functional foods and could reflect the different mechanisms (antioxidant, anti-inflammatory, insulin resistance).6. Puerarin could improve insulin signal transduction, its hypoglycemic effect might be related to raising the level of antioxidant, as well as inhibition the expression of NF-κB in inflammatory signaling pathway.7. The hypoglycemic effect of EGCG was concerned with the length of action time.The mechanism might be related to increasing the body’s antioxidant capacity and its a-glucosidase inhibitor effects.更多还原