【作者】 毛雪琴；

【导师】 夏作理； 陈连璧；

【作者基本信息】 山东大学 ， 生理学， 2008， 博士

【摘要】 糖尿病(diabetes mellitus,DM)是由胰岛素绝对或相对不足引起的以糖代谢紊乱为主要表现的临床综合征,长期糖尿病可引起多个系统器官的慢性并发症,是患者致残与致死的主要原因,对病人的生活质量产生严重的影响。目前的治疗措施还远达不到预期的治疗目标:在严格控制血糖正常水平,避免代谢紊乱和慢性并发症的发生,维持β-细胞功能,防止病程进展。因而研究改进糖尿病治疗新方法,开发安全有效、使用方便的抗糖尿病新药具有非常重要的现实意义。实验证明,微量元素钒的抗糖尿病作用一直受到临床重视,但该作用需要较高的剂量,会引起多系统毒副反应;而有机钒化合物的降血糖作用优于无机钒化合物,且无明显副作用,如BMOV在国外已经获得注册,并应用于临床。鹰嘴豆(chickpea,学名Cicer arietnum L.)营养丰富,并具有极高的药用价值,被我国药典收录。研究发现,鹰嘴豆种子的芽胚中含有丰富的异黄酮、鹰嘴豆芽素A、C和鹰嘴豆谷芽素B等抗氧化成分,对试验动物及人体均具有肯定的降低血糖、血压和血脂的作用。糖尿病病理过程会导致学习记忆功能的障碍,是糖尿病常见而严重的并发症之一。已有研究发现,长期糖尿病会加速阿尔茨海默病(AD)的早期发病。大脑海马区是学习、记忆的重要解剖基础和神经中枢。本论文以Wistar雄性大鼠为实验动物,在链脲佐菌素(Streptozotocin,STZ)诱发的糖尿病动物模型上,以糖脂代谢、症状体征和海马区神经细胞形态学、功能学和分子生物学等为观察指标,研究了富钒鹰嘴豆芽(Vanadium-enrichedchickpea sprouts,VCS)对糖尿病的治疗作用及其作用机理。目的:1.培育鹰嘴豆芽(Chickpea sprout,CS)及VCS。2.筛选对糖尿病大鼠安全、有效、易吸收的最佳浓度富钒鹰嘴豆芽作为糖尿病模型大鼠的干预饲料。3.研究VCS对糖尿病的治疗作用及其作用机理。方法:1.采用Burguillo PD的方法培育CS和分别由8个浓度(50μg/mL、100μg/mL、200μg/mL、400μg/mL、600μg/mL、800μg/mL、1600μg/mL、2000μg/mL)原钒酸钠水溶液培育的的VCS;2(1).采用生物样品分析分解方法,利用全谱直读等离子体发射光谱仪测定不同浓度VCS中的总钒含量;同法测定正式试验后各实验组大鼠每日钒摄入量及大鼠血浆、重要脏器中钒含量。2(2).采用对Wistar雄性大鼠(购自华中科技大学同济医学院,SCXK2004-0007)一次性尾静脉注射链脲佐菌素(Streptozotocin,STZ)45mg/kg的方法建立糖尿病模型,随机分组,分别给予5%的CS和各种浓度VCS饲料进行干预。2(3).根据4w的预实验,观察实验动物的血糖变化、“三多一少”症状、精神状态和行为学变化,选取安全、有效的VCS作为实验用干预饲料。3(1).将链脲佐菌素(Streptozotocin STZ)诱发的糖尿病模型大鼠分成5组,分别为:糖尿病对照(D)组、0.5mg/mL原钒酸钠治疗(V)组、鹰嘴豆芽治疗(VC)组、0.2mg/mL原钒酸钠联合鹰嘴豆芽治疗(V+CS)组、富钒鹰嘴豆芽治疗(VCS)组,另设正常对照(C)组。干预8w后,观察各组实验大鼠血糖和血脂等指标的变化情况;以Morris水迷宫检测各组实验大鼠的空间学习记忆能力。3(2).大鼠海马组织石蜡切片HE染色光镜观察,透射电子显微镜观察,免疫组织化学分析、蛋白质免疫印记(Western-blotting)以及实时定量PCR(RealTime-PCR)方法,分别对海马神经细胞的形态变化、细胞凋亡情况、凋亡相关因子Caspase3、Bax和Bcl-2的表达,以及App,IGF-1和IGF-1R的表达进行定位和定量研究,以探讨VCS对学习记忆功能影响的可能机制。4.统计学处理将实验数据输入SPSS11.5统计软件数据库中,以均数±标准差(m±SD)表示,两组之间比较采用t检验,多组间比较采用ANOVA方差分析,相关性分析采用直线相关法。以P＜0.05表示有统计学意义。结果:1.CS和VCS均生长良好;原钒酸钠溶液对豆芽的生长存在一定程度的抑制作用,但不同浓度之间差异不明显;2(1).8种不同浓度富钒鹰嘴豆芽及饲料中的钒含量随培育液中原钒酸钠浓度的变化而变化,存在剂量依赖关系,但不是倍数关系。2(2).大鼠一次性尾静脉注射Streptozotocin(STZ)45mg/kg9d后,出现典型的多饮、多尿、血糖升高等糖尿病表现。成功率92%,死亡率4%。2(3).根据4w的预实验观察记录发现,100μg/mL原钒酸钠培育的VCS干预组实验动物的血糖控制稳定、“三多一少”症状明显改善、精神状态和行为学变化与正常对照组无显著性差异,且未发现钒明显的毒副反应,故选取100μg/mL原钒酸钠培育的VCS作为实验用干预饲料,即最佳浓度的VCS。3(1).实验8w后,与正常对照组(C)比较,糖尿病组(D)大鼠出现明显“三多一少”症状;血糖升高4倍;血浆胰岛素(Ins)降低57.63%,血浆瘦素水平明显降低;血浆脂质水平明显升高,HDL-c明显下降;糖化血红蛋白(GHbA1)升高42.28%,肝肾功能明显受损;口服葡萄糖耐量(OGTT)、血浆胰岛素(OGIRT)释放试验明显受损,各自曲线下面积(AUC)明显异常。而各干预组的上述指标均有不同程度的改善,其中以VCS组改善最明显,“三多一少”症状消逝,血糖水平降低58.60%(P＜0.05);血浆胰岛素水平明显增高(P＜0.05)且接近正常;血浆瘦素水平恢复;血浆低密度脂蛋白(LDL-c)、甘油三酯(TG)和总胆固醇(TC)明显下降(P＜0.05),HDL-c明显上升;GHbA1降低32.81%,BUN和Cr恢复,谷丙转氨酶(ALT)降低39.98%。而OGTT和OGIRT得到显著改善,曲线下面积(AUC)得到恢复。其他各组效果依次为:V+CS＞CS～V。其中V和V+CS组对脂质代谢障碍、血浆LEP水平、血浆糖化血红蛋白(GHbA1)、受损的肾脏功能、异常的OGTT和OGIRT、明显减少的OGIRT-AUC无显著改善作用,尤其是V治疗组,甚至有加重AUC减少的作用。糖尿病8w后,大鼠游泳能力达3小时以上,证实糖尿病大鼠肢体活动能力未见改变,以Morris水迷宫实验观察糖尿病大鼠的学习记忆能力。结果糖尿大鼠在训练后第5天测试潜伏期延长、搜寻平台策略成绩较正常对照组降低。各治疗组大鼠潜伏期明显缩短,搜寻平台策略成绩也明显升高;其中以VCS组改善作用最明显。3(2).大鼠海马形态学和分子生物学方面8w后,HE染色组织病理学检查结果显示:C组大鼠海马轮廓清楚,细胞形态完整,细胞紧密有序,排列整齐,结构清晰,胞浆染色均匀,核仁清晰可见;而D组大鼠海马轮廓欠清,CA1区可见细胞形态不完整,排列紊乱疏松,数目减少,胞体肿胀,结构不清,存在空泡样变性;表明糖尿病大鼠海马神经元细胞受损。各治疗组大鼠海马细胞形态变化各不相同,VCS组大鼠海马细胞数目明显增多,形态较完整,排列较紧密、有序,胞体轻度肿胀,核仁较清晰,其余组效果依次为:V+CS＞CS＞V。。透射电镜超微结构观察结果发现,C组大鼠海马神经元细胞核形态规则,核大而圆,染色质均匀分布,核仁明显,核膜光滑连续,双层结构清楚;核周边细胞器丰富,结构完整,粗面内质网网腔均匀一致,表面有丰富的核糖核蛋白颗粒;线粒体发达,排列密集;高尔基器扁平,极性明显;神经髓鞘排列规整,纤维清楚。与C组比较,D组大鼠海马神经元出现明显的凋亡改变:细胞体积变小,胞质浓缩;细胞核固缩、碎裂,核内染色质浓缩、颗粒化、边集现象;核膜可见不规则凹陷、裂解、不连续;细胞器明显减少,结构不清,线粒体肿胀、破坏、溶解;粗面内质网扩张、断裂、脱颗粒;高尔基器腔扩张。神经髓鞘破坏严重,溶解状态,鞘膜破坏,神经纤维不清楚,突触间隙增宽。各治疗组大鼠海马细胞的超微结构均得到不同程度改善,神经细胞密度增高。突触间隙缩小,其中以VCS组改善最明显,其余各组效果为:V+CS＞CS＞V。免疫组化分析结果发现,调亡促进蛋白Bax在C组表达不明显,但在D组的表达为强阳性(胞浆内充满明显的棕褐色颗粒);各治疗组结果比较,以VCS组效果最好,阳性表达最弱,其余三组的阳性表达强度依次为:V＞CS＞V+CS。作为调亡抑制因子,Bcl-2在各组的表达差异并不明显。Active caspase3在C组的表达并不明显,但在D组却呈强阳性表达;而各治疗组的表达情况以VCS组最弱,其余三组均有阳性表达,强度依次V＞CS＞V+CS。IGF-1在C组的表达呈现强阳性,而D组大鼠的蛋白表达明显降低;各治疗组的表达以VCS组表达最强,其余依次为V+CS＞CS＞V。相应的胰岛素样生长因子1受体(IGF-1R)蛋白表达在C组呈现弱阳性,D组出现了代偿性上调,表达强阳性,各治疗组中,VCS组的表达最弱,其次是V+CS组,而CS组和V组仍然呈现阳性表达。Western blotting结果,与C组比较,D组大鼠的Caspase3表达和Bax/Bcl-2比值明显增加,胰岛素样生长因子1(IGF-1)蛋白表达明显降低,而胰岛素样生长因子1受体(IGF-1R)蛋白表达出现代偿性上调;治疗后各组大鼠海马细胞凋亡现象有明显的抑制,Caspase3表达和Bax/Bcl-2比值明显降低,IGF-1蛋白表达明显增加;IGF-1R蛋白表达趋向恢复正常;其中以VCS组变化最明显。Real Time-PCR方法观察App和Caspase3的mRNA表达水平,结果表明:β-actin、Caspase3和App标准曲线斜率和R~2分别为:-2.768,0.99999;-3.060,0.99346;-3.028,0.99486。且所测样品的Ct值均在标准曲线内,溶解曲线为单一峰型,表明Real Time-PCR反应特异,标准曲线理想。与C组比较,D组大鼠海马神经细胞App和Caspase3的mRNA表达水平明显升高,提示糖尿病大鼠海马存在明显细胞凋亡和蛋白沉积现象,容易导致学习记忆障碍。治疗后各组大鼠海马细胞App和Caspase3的mRNA表达水平均有不同程度的降低;其中以VCS组降低最明显,且比V组明显减少,总体效果依次为:VCS＞V+CS＞CS＞V。4.实验8w后,相关分析显示,钒的降糖作用,如非空腹血糖(bg)、空腹血糖(fb)和空腹胰岛素(Ins0)与实验大鼠血液中的钒含量之间存在相关关系,即血液中钒浓度越高,则大鼠bg和fb水平越低,Ins0分泌量越多。而与钒在各脏器钒含量之间无相关关系。结论:1.首次利用生物转化方法,以鹰嘴豆为载体,通过种子发芽的过程成功地富入微量元素钒,得到富钒鹰嘴豆芽(VCS)——一种可能的生物活性钒,在协同两者降糖作用的同时,又避免了单独使用无机钒所引起的毒副作用。具有重要的临床意义。2.富钒鹰嘴豆芽能够明显地改善糖尿病大鼠糖脂代谢和学习记忆功能障碍;3.富钒鹰嘴豆芽改善糖尿病大鼠学习记忆功能障碍的作用可能与其减少海马细胞凋亡、抑制App表达和增加IGF-1表达有关。更多还原

【Abstract】 Diabetes mellitus(DM) is clinic syndromes caused by absolute or relative deficient of insulin,with the main manifestation of glycometabolism disorders. Chronic diabetes mellitus can result in structural and functional lesions of multiple organs in human.Even more,it will cause diabetic neuropathy,including peripheral and central neuropathy.And these complications may become the primary causes of mutilation or case fatality in diabetic patients.In present,the therapeutics of DM,such as alimentary control,insulin and oral antidiabetic drug,cannot achieve expected goals in tiuman—controlling blood glucose in normal level,preventing glycometabolism disorders and chronic complications,shieldingβ-cell function,and,in the final,delaying the progression of DM.For these reasons,it is significantly necessary to develop new therapeutic path and to investigate the corresponding mechanism of action.Vanadium compounds have demonstrated insulin-mimetic effects with in vitro and in vivo studies.Vanadium compounds improve glucose homeostasis in animal models of both Type 1 and Type 2 diabetes mellitus,but the acceptance of vanadium compounds as antidiabetic agents has been slowed due to the concern for short-term gastrointestinal stress and potential long-term toxicity with vanadium accumulation. Recently,some studies of vanadium permeability and toxicity suggested that some kinds of vanadium organic compounds,for example,the combination of vanadium with some botanic antioxidants,might be benefit in reducing vanadium toxicity and increasing vanadium hypoglycemic effects.Chickpea has been traditional hypoglycemic foodstuff,what more,its seeds and its sprout contain variety of antioxidants such as,isoflavone,biochanin A,C and Formoononetin B,and have been investigated their potential hypoglycemic effects in diabetic animal models by several groups.Diabetic cognitive disorder is one of the common complications in DM. Nevertheless,as the nerve center of learning -memory,hippocampus is also the structural foundation.In the present study,with streptozotocin-induced diabetes in Wistar rats as objects,the effects and side effects of vanadium-enriched chickpea sprout(VCS),a new possible vanadium organic compounds,on impaired glycolipids metabolism and learning-memory ability of diabetes rats in morphological,functional and molecular biology aspects have been investigated.Objectives1.To incubate chickpea sprouts(CS) and vanadium-enriched chickpea sprout (VCS).2.To determine the most effective and safe dosage VCS as the therapeutic animal food.3.To observe the changes of some parameters in experimental rats after 8 weeks VCS- treatment,the parameters including:diabetic symptoms,blood glucose and lipid metabolism,space learning-memory ability,and morphology and molecular biology aspects.Methods1.Based on the method of Burguillo PD,the CS was performed with tap water, and the VCS were incubated with 8 dosages of sodium orthovanadate(SOV) solution as following:50μg/mL,100μg/mL,200μg/mL,400μg/mL,600μg/mL,800μg/mL, 1600μg/mL and 2000μg/mL(VCS 50～2000),respectively.2(1).The vanadium concentrations of vanadium-enriched chickpea sprout powder,of blood specimen and tissues in diabetic,were determined using a biological specimen analysis method(GB/T5009—2003) by use of a plasma emission spectroscopy(IRIS IntrepidⅡ).Spearman analysis was used to correlate the serumⅤconcentration with clinical response.2(2).Male Wistar rats weighting between 188 and 220g were obtained from the Institute of Experimental Animals,Tongji Medical School,Huazhong Technology University(SCXK2004-0007),and housed in air-conditioned room(temperature: 23±10℃;relative humidity:55±5%;12-h/day photoperiod:) in the animal facility of Taishan Medical University.Diabetes was induced by a single intravenous tail vein injection of streptozotocin(STZ) 45 mg/kg.On day 14 following STZ administration, animals with a blood glucose level≧15mM were considered diabetic.2(3).Then,the rat food containing vanadium-enriched chickpea sprout (VCS-food) was prepared by adding 5%(w/w) the VCS powder in the standard rat food pellet.After 4-week’s intervention to diabetic rats with VCS-food as animal feeds,the most effective dosages of VCS-food(VCS100) was determined according to the changes of blood glucose,clinical manifestation,mental status and praxiology of the diabetic.3(1).On day 14 following STZ administration,diabetic animals were randomly divided into five groups.The control rats(C) and diabetic rats(D) received only standard pellet food and were given tap water ad libitum.Four treated groups(V group,treated with 0.5mg/mL SOV solution;CS group,treated with pellet food containing 5%of chickpea sprout powder;V+CS group,treated with 0.2mg/mL SOV solution combined with pellet food containing 5%of chickpea sprout powder;and VCS group,treated with pellet food containing 5%of vanadium-enriched chickpea sprout powder;) were received different therapeutics for eight weeks.At the end of 8-weeks,some diabetic parameters were measured as following: Plasma insulin(ins),and leptin(LEP) levels were measured using radioimmunoassay kits(Boster biological company,Wuhan,China) using the manufacturer protocol. Plasma total cholesterol(TC),triglyceride(TG),high density lipoprotein cholesterol (HDL-C),low density lipoprotein cholesterol(LDL-C),and function of alanine aminotransferase(ALT),aspartate aminotransferase(AST),blood urea nitrogen (BUN),and creatinine clearance rate(Cr) were measured using the diagnostic kits (Ortho-Clinical Diagnostic,Inc.USA) with manufacturer protocol.The plasma GHbA1 was measured by HPLC.The OGTT studies were then carried out in the morning after an overnight-fasting.The 0-hour(baseline) blood samples were drawn for glucose, insulin and lipid profile analysis.Then a glucose solution(1g/ml) was immediately given to the animal at a dose of 2g/kg body weight via gavages,and then the glucose levels were checked at the intervals of 30,60,90 and 120 min,respectively.And plasm specimens were prepared for OGRIT test.What’s more,before sacrifice,a part of experimental rats were trained to search for the escape platform under the pool water by using Morris water maze for four days to observe their space learning-memory ability.The testes were carried out in the fifth day,escape latencies and swimming paths were recorded using the video tracking system for each rat.3(2).In order to investigate the changes ofhippocampal neurons and the effect of VCS,electronmicroscope and immunohistochemistry were used.Then the hippocampus mRNA was then extracted,and corresponding cDNA was synthesized. Real Time-PCR was used to detect mRNA expressions of amyloid protein precursor (App),and Caspase3.At the same time,the brain was sliced,and immunoblotting (western-blotting) was used to detect the protein expressions of Caspase3,bax,bcl-2, IGF-1,and IGF-1R.4.Statistical analysisOne-way analysis of variance(ANOVA) was used to compared the Means±SEM.Correlation analysis was made using Spearman analysis.The significance levels were tested at P＜0.05.Results2(1).After 9d from the administration with STZ(45mg/kg,i.p.),the diabetic rats had diabetic symptoms such as polydipsia,polyuria,and hyperglycemia.And after 14d,the rats with blood glucose≧15mM/L were determined to be diabetic ones.This showed that the administration with STZ at dose of 45mg/kg,i.p.to induce diabetic model was a successful method,with achievement ratio:92%,death rate:4%.1.The growth of CS and VCS were well with little rotten sprout.But observation of sprout growth manifested that SOV might slightly inhibited the growth of chickpea sprout,but there were no significant difference among 8 different dosages.2(3).After 4-week preliminary experiment,the results showed that VCS 100 was the best select for the diabetic therapeutic,according to the changes of blood glucose, diabetic manifestation,mental status,and praxiology of the diabetic rats.2(1).The vanadium concentrations were related with the doses of the different SOV,and the vanadium concentrations in blood specimen and organs differentiated from intervention in different diabetes groups.3(1).Compared with the C group,the D group rats hadn’t only typically diabetic symptoms such as polydipsia,polyuria,and hyperglycemia,but also increased levels of plasma lipid and GHbA1,decreased levels of serum insulin(Ins),and leptin(LEP), impaired function ofALT,AST,BUN,and Cr,and,abnormal levels ofOGTT,OGIRT, and their AUCs.Otherwise,After 8-weeks therapeutics for diabetes,much expected changes of above parameters had appeared in four treated groups,and the most rational results happened in VCS group as following:the typical diabetic symptoms of polydipsia,polyuria,and emaciation disappeared,blood glucose level lowered significantly,the levels of plasma lipid and GHbA1 decreased,serum Ins,and LEP recovered,at the same time,the impaired function of hepar and kidney were ameliorated,and,the levels of OGTT,OGIRT,and their AUCs normalized,as well, without any side effects(gastrointestinal stress) during the experimental period caused by vanadium inorganic compounds as seen before.The effect of VCS food on spatial learning and memory was measured using a water maze test.After 8 weeks of diabetes,D group rats could swim for more than 3h, which means that the movements of diabetic rats were not harmed.Compared with the C group rats,the D ones exhibited significantly delayed latencies to locate the platform and decreased score of platform-searching in all four quadrants(P＜0.05). After treatment,the time for locating the platform and the score of platform-searching were improved significantly.The best effect was observed for VCS group.3(2).After 8 weeks of diabetes,morphological analysis of the hippocampal neurons by HE staining revealed that the neuronal cells were in the state ofraritas and disorder,accompanied with reduced neuronal density.The ultra structural analysis of the hippocampal pyramidal neurons by electronmicroscope revealed that the cell body and cell process were swelling obviously,endochylema was vacuolated,the cytoplasmic organoids of neurons were ambiguous,rough endoplasmic reticulum and free ribosome were significantly decreased,chondriosome was swelling,lyzing and demolishing,and cellular membrane was disaggregated.The ultrastructural analysis also showed that the nerve fiber and nerve plexus were ambiguous,synaptic cleft was widened.While,VCS food fed group had the improved morphological changes, obviously diminished synapse cleft and increased neuron density.Apoptosis was demonstrated after 8 weeks of diabetes by electronmicroscope observation.Compared with C group,increased expressions of positive cells were shown with increased Caspase3 and ratio of bax/bcl-2 in the D group hippocampus. And the same changes of protein expressions and mRNA expression with Caspase3 and bax/bcl-2 were shown by Western-blotting and Real Time-PCR in D group,as well.However,VCS food could ameliorate the changes above.The result of Real Time-PCR analysis was that,the mRNA expressions of App in D group were higher than that in C.But the treatment results showed that VCS could improve the impaired learning- memory ability of the diabetic rats,and inhibit the mRNA expressions of App.In this experiment,immunchistochemistry and western-blotting were used to detect the expressions of insulin-like growth factorl(IGF-1) and insulin-like growth factor 1 receptor(IGF-1R).Compared with the C group,the results of protein expression in IGF-1 were significantly decreased in D group.And IGF-1R protein expression was obviously up-regulated compensatedly in the diabetic rats with deficiency of learning and memory.This indicated that the deficiency of IGF-1 and up-regulation of IGF-1R might account for the impaired learning memory ability in diabetic rats.While VCS food could reverse the changes of IGF-1 and IGF-1R in diabetic rats,and produce its neuroprotective effect,by improving the deficiency of learning-memory in the diabetic rats.4.After 8w treatment,the results of relationship analysis showed that,the VCS antidiabetic effect significantly related with the vanadium intakes of VCS in diabetic rats,and the vanadium in blood,but not with the vanadium concentrations in vital organs.Conclusions1.It is the first time to enrich the vanadium into the chickpea sprout through the sprout process of seeds by biological method,so that to form vanadium-enriched chickpea sprout(VCS)-a new possible biological actived vanadium,which may have some new expected characteristics such as:not only enhancing the antidiabetic effect of both vanadium and chickpeas,but attenuating the adverse reaction resulted from the use of inorganic vanadium compound alone.2.VCS can significantly ameliorated the impaired glycolipids metabolism and learning-memory in diabetic rats.3.The possible mechanism of VCS in protecting the hippocampus injury from diabetes,maybe relate with the effects of VCS on lessening the apoptosis of hippocampal neurons,inhibiting the expression of App and IGF-1R,and increasing the expression of IGF-1 in diabetic hippocampus.更多还原