【作者】 刘萍；

【导师】 赵仲堂；

【作者基本信息】 山东大学 ， 流行病与卫生统计学， 2007， 博士

【摘要】 【目的】铝在环境中的广泛存在和在日常生活中的广泛应用使人们接触铝的机会大大增加。我国正常人摄铝主要来源于食品添加剂、含铝药物和铝制品等,铝暴露的问题比较严重且未引起足够重视。研究表明,铝过多进入体内会对人体健康造成危害。铝是一种慢性蓄积性神经毒素,铝在Alzheimer型老年性痴呆（AD）、透析性痴呆（Dialysis Encephalopathy,DE）等疾病中发挥的毒作用也已得到许多学者的肯定。流行病学调查结果表明,某些神经退行性疾病的发生与机体接触铝的量有关,发病率随机体接触铝量的增加而增加。目前已证明,铝是DE肯定的、唯一的致病因子。职业暴露的铝作业工人出现明显的神经功能性改变。目前临床上对于铝负荷过多引起的疾病倾向于使用铝螯合剂。国外学者主要使用去铁胺（DFO）,发现排铝效果显著。但是DFO为天然微生物生长素,不能合成;不仅成本高,且口服无效,只能经肌肉或静脉注射给药;此外,DFO具有耳毒性、视觉障碍和生殖毒性等毒副作用。因而寻求一种价格低、口服有效、毒副作用小的排铝螯合剂具有重要的临床意义。1,2-二甲基-3-羟基-4-P比啶酮（deferiprone,DFP）做为一种Fe³⁺螯合剂用于排铁。本课题利用Fe³⁺与Al³⁺均为三价金属离子,且半径相似的特点,把DFP作为一种新型铝螯合剂进行研究。通过研究拟达到以下目的;1.建立一种新的合成DFP的方法;2.完成DFP的一般毒理学实验;3.对DFP的排铝效果从多方面进行研究;4.探讨DFP对慢性荷铝动物的保护作用机制。为寻求一种价格低廉、安全有效的治疗铝中毒的药物提供理论依据。【方法】1.DFP的合成2-甲基-3-羟基-4-吡喃酮与甲胺水溶液于95～100℃加热回流,得到白色结晶即DFP。2.DFP的毒理学实验2.1 LD₅₀实验取健康昆明种小鼠50只,雌雄各半,按体重随机分为5个剂量组。以灌胃方式给药1次,观察1周（w）,记录动物死亡数。2.2外周血和骨髓微核实验取健康昆明种小鼠60只,雌雄各半,按体重随机分为5组;阴性对照组、环磷酰胺阳性对照组、1/8LD₅₀组、1/4LD₅₀组、1/2LD₅₀组,分别于第二次与第七次给药后24h进行DFP的微核实验。2.3精于畸变实验健康昆明种雄性小鼠150只,按体重随机分为5组;阴性对照组、环磷酰胺阳性对照组、1/8LD₅₀组、1/4LD₅₀组、1/2LD₅₀组,给药5天（d）,分别在给药后第14、28、35、42和70d进行DFP的精子畸变实验。3.DFP的排铝作用研究3.1小鼠的排铝实验健康昆明种小鼠84只,雌雄各半,22～24g,自由饮水和进食。随机分为6组;阴性对照组、荷铝对照组、预防组、低剂量组、中剂量组、高剂量组,以灌胃方式给药.将小鼠以颈椎脱臼法处死,取其血清、肝、肾、脑和骨骼组织。用原子吸收分光光度法测定小鼠血清和各组织中的铝、铜、锌、钙、铁、镁含量。3.2家兔排铝实验3.2.1急性排铝实验雄性新西兰大白兔24只,荷铝后随机分成荷铝对照组、低、中、高剂量组。将DFP的灭菌水溶液给低、中、高剂量组灌胃。收集尿液,待做元素铝及其他必需元素的测定。3.2.2与维生素C（Vc）、美司那（Mesan）协同排铝作用将24只新西兰大白兔随机分为荷铝组、DFP组、DFP+Vc组、DFP+美司那组。荷铝后依次灌胃给予灭菌无离子水、DFP、DFP+Vc、DFP+美司那。收集尿液,待做元素铝的测定。3.2.3不同给药方式的排铝作用比较18只新西兰大白兔随机分为荷铝对照组、灌胃组和注射组,荷铝后分别以灌胃和皮下注射方式给DFP。收集尿液,待做元素铝的测定。3.2.4亚慢性排铝实验新西兰大白兔16只,随机分为阴性对照组、荷铝对照组、DFP组。荷铝后DFP组用DFP溶液灌胃2周。分别于实验开始、染铝结束及给DFP结束时从兔耳静脉取血。第三次取血后,将兔处死,取肝、肾、脑、骨组织,待做血清及组织中铝及其他必需元素的测定。4.DFP对荷铝动物的保护作用机制研究4.1对荷铝小鼠抗氧化系统的恢复作用健康昆明种小鼠84只,雌雄各半,22～24g,自由饮水和进食。随机分为6组;阴性对照组、荷铝对照组、预防组、低、中、高剂量组,以灌胃方式给药。实验结束时,小鼠眼球取血及肝、肾、脑组织测定超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）活力和丙二醛（MDA）含量。4.2 DFP对慢性荷铝小鼠神经系统的保护作用健康昆明种小鼠98只,雌雄各半,22～24g,自由饮水和进食。按体重随机分为7组,以灌胃方式给药,分组情况如下;阴性对照组、预防组、低剂量组、中剂量组、高剂量组、荷铝组、盐酸吡硫醇对照组。4.2.1对荷铝小鼠学习记忆能力的影响实验结束前两天用跳台仪对小鼠进行跳台训练,24h后进行测试,记录小鼠跳台潜伏期和5min内的错误次数。4.2.2对中枢神经递质的恢复作用取各组小鼠测血清及脑组织匀浆中乙酰胆碱酯酶（AchE）活力。4.2.3对铝致海马损伤恢复的形态学观察小鼠海马苏木素-伊红染色（HE）染色,进行病理学观察,分析小鼠海马的形态学改变。4.2.4对荷铝小鼠细胞凋亡的保护作用给药结束后,小鼠心脏灌注进行固定,取海马组织,用免疫组织化学进行原位检测海马区Bax和Bcl-2蛋白的表达,检测DFP对铝致细胞凋亡的保护作用。4.3 DFP对铝诱发小鼠遗传物质损伤的抑制作用采用昆明种小鼠进行骨髓细胞微核实验和精子畸变实验。两种实验均随机分为5个剂量组;阴性对照组、阳性对照组、荷铝对照组、低剂量组和高剂量组。4.4 DFP对荷铝动物肝肾功能的影响Wistar大鼠AlCl₃染毒3周后分别给予不同剂量的DFP 1周,测定血清谷丙转氨酶（ALT）、谷草转氨酶（AST）、碱性磷酸酶（ALP）的活力及总蛋白、白蛋白、总胆红素和结合胆红素、肌酐、尿素氮、尿酸的含量。同时测定肝、肾组织中铝、锌、铜、铁、钙、镁等元素的含量。【结果】1.DFP的合成结果表明;熔点、红外与核磁波谱符合目标化合物的结构特征。收率为58.9%。2.DFP的毒理学实验2.1 DFP的LD₅₀实验DFP的LD₅₀为562.67mg/kg,其95%的可信限为501.19～630.96mg/kg。2.2外周血和骨髓微核实验外周血和骨髓PCE微核率1/8LD₅₀、1/4LD₅₀组微核率与阴性对照组相比均无显著性差异（P＞0.05）;1/2LD₅₀组微核率高于阴性对照组（P＜0.05）。2.3精子畸变实验1/8LD₅₀、1/4LD₅₀组与阴性对照组比较在第14、28、35、42、70d观察均无显著性差异;1/2LD₅₀组与阴性对照组比较在第14、28、35d观察均有显著性差异（P＜0.05）,而在第42、70d观察均无显著性差异。3.DFP的排铝作用研究3.1小鼠的排铝实验结果3.1.1排铝作用脑、骨骼和肾脏铝中,预防组、低、中、高剂量组铝含量均低于荷铝组（P＜0.05）。肝脏铝含量比较荷铝组高于其它各组,但无统计学意义。血清铝比较,预防组、低、中、高剂量组均低于荷铝组（P＜0.05）,但高于阴性对照组（P＜0.05）,低、中、高剂量组之间存在剂量反应关系。3.1.2对其它必需元素的影响脑、肝脏、肾脏和骨骼中的镁含量各组之间均无统计学意义。肾、肝、骨组织各组钙含量无统计学意义。脑和肝脏中的锌含量各组之间均无统计学意义。脑、肾脏和骨骼各组之间的铁含量相比,均无统计学意义。脑、骨骼和肝脏铜含量比较各组无统计学意义。3.2家兔排铝实验结果3.2.1急性排铝作用低、中、高剂量组在给DFP后每小时的排铝量较前1小时均有显著性增加（P＜0.01）,其排铝高峰在2～4小时范围内。6小时总排铝量低、中、高剂量组与对照组相比均有显著性增加（P＜0.01）。3.2.2与Vc、美司那（Mesan）协同排铝作用DFP组、DFP+Vc组和DFP+美司那组均显示了明显的排铝效果,但3组的排铝曲线不同,后二组曲线峰值前移,提示铝代谢受到了DFP与Vc、美司那的共同作用。3.2.3不同给药方式的排铝作用比较6小时排铝总量注射组与灌胃组比较有显著性增加（P＜0.05）,排铝高峰灌胃组位于给药后第3h,注射组位于第2h。3.2.4亚慢性排铝作用给DFP后DFP组血清铝浓度显著低于给DFP前,亦显著低于荷铝组（P＜0.05）。骨、脑、肝和肾组织中的铝含量DFP组均低于荷铝组（P＜0.05）。3.2.5对必需元素铜、锌、锰排出的影响3.2.5.1急性排铝给DFP前后每小时铜、锌、锰在尿中排出均无显著性差异。给DFP 6小时后铜、锌、锰在尿中排出的总量各组之间亦无显著性差异。3.2.5.2亚慢性排铝脑、肝、肾、骨组织中铜、锌、锰的含量荷铝组和给DFP组差异均无显著性,即DFP对微量元素铜、锌、锰的动员及排出不产生影响。4.DFP对荷铝动物的保护作用机制研究4.1对荷铝小鼠抗氧化系统的恢复作用肝脏、脑、血清SOD活力、GSH-PX活力、MDA含量阴性对照组与荷铝组比较均有显著性差异（p＜0.05）;用药各组SOD活力、GSH-PX活力、MDA含量与阴性对照组比较肝、肾、脑、血清均无显著性差异。4.2 DFP对慢性荷铝小鼠神经系统的保护作用4.2.1对荷铝小鼠学习记忆能力的影响小鼠跳台实验表明;与荷铝组相比,预防组、低、中、高剂量组潜伏期显著降低（P＜0.01,P＜0.05）,且各剂量组之间存在剂量效应关系;各剂量组与对照组相比均无统计学意义。荷铝组小鼠的错误次数显著高于阴性对照组（P＜0.01）,其它各组与阴性对照组相比无统计学意义（P＞0.05）。4.2.2对中枢神经递质的恢复作用AchE活力荷铝组高于阴性对照组（P＜0.05）,中、高剂量组和预防组与荷铝组比较有显著性差异（P＜0.05）,与阴性对照组比较均无显著性差异。4.2.3对铝致海马损伤恢复的形态学观察对组织切片光镜下进行观察,荷铝组海马有明显损伤,用药各组损伤的组织均有一定程度的恢复。4.2.4对荷铝小鼠细胞凋亡的保护作用低剂量组、盐酸吡硫醇对照组Bcl-2蛋白表达与荷铝组比较无统计学意义,中、高剂量组和预防组均高于荷铝组（P＜0.05）;预防组、低、中、高剂量组均高于盐酸吡硫醇对照组（P＜0.05）。荷铝组、预防组、低剂量组Bax蛋白表达均高于阴性对照组和盐酸吡硫醇对照组（P＜0.05）,中、高剂量组、盐酸吡硫醇对照组与阴性对照组比较无统计学意义;高剂量组低于盐酸吡硫醇对照组（P＜0.05）。4.3 DFP对铝诱发小鼠遗传物质损伤的抑制作用DFP低、高剂量组的微核率均低于荷铝组（P＜0.01）;低、高剂量组比较有显著性差异（P＜0.01）,微核率随DFP剂量增加而降低。低、高剂量组精子畸变率显著低于荷铝组（P＜0.05）,但仍高于阴性对照组（P＜0.05）。低、高剂量组之间比较亦有显著性差异（P＜0.05）,呈现剂量-效应关系。4.4 DFP对荷铝动物肝肾功能的影响4.4.1对肝功能的影响各组大鼠的ALT活力比较差异均无统计学意义;低、中、高剂量组的AST活力显著低于荷铝组（P＜0.01）;低剂量组ALP活力与荷铝组相比差异有统计学意义（P＜0.01）;荷铝组大鼠血清中总蛋白含量显著低于阴性对照组（P＜0.01）,预防组、中、高剂量组则显著高于荷铝组（P＜0.01）,且与阴性对照组相比无统计学意义;各组大鼠血清中白蛋白含量比较均无统计学意义;与阴性对照组相比,各组大鼠血清中总胆红素含量均显著增加（P＜0.05,P＜0.01）,而结合胆红素含量比较均无显著性差异。Al含量测定结果显示;低、中、高剂量组均显著低于荷铝组（P＜0.01）,且含量呈下降趋势;给药各组的大鼠肝脏中锌、铜、铁、钙、镁的含量与阴性对照组相比差异均无统计学意义（P＞0.05）。4.4.2对肾功能的影响荷铝组尿素氮含量显著高于阴性对照组（P＜0.05）,DFP各组与阴性对照组相比均无统计学意义;各组大鼠血清中肌酐、尿酸含量比较差异均无统计学意义。荷铝组肾脏中Al含量显著高于阴性对照组（P＜0.01）,DFP各组均显著低于荷铝组（P＜0.01）;荷铝组、低剂量组大鼠肾脏中铜含量均显著低于阴性对照组（P＜0.01,P＜0.05）,而中、高剂量组与阴性对照组相比差异无统计学意义;低、中、高剂量组的大鼠肾脏中锌、铁、钙、镁的含量与阴性对照组相比差异均无统计学意义。【结论】1.DFP一般合成分三步进行,步骤多,时间长,反应条件要求苛刻。本研究建立的一步合成法,缩短了时间,降低了成本,提高了收率。2.DFP毒性低、排铝效果显著,同时对其它必需元素锌、镁、钙、铜、锰均无促排作用。3.DFP能够改善荷铝小鼠的学习记忆能力。4.DFP能拮抗铝诱发的中枢神经递质功能减退。5.DFP对铝引起的凋亡相关蛋白改变有一定的调节作用。6.DFP对铝诱发小鼠遗传物质损伤具有抑制作用。7.DFP对荷铝动物肝、肾功能具有一定的保护作用。总之,本研究建立了DFP新的合成方法;对DFP的毒性进行了评价;首次系统、全面地对DFP的排铝效果以及对荷铝动物的保护作用机制进行了研究。为寻求一种价格低廉、安全有效的治疗铝中毒的药物提供了理论依据。更多还原

【Abstract】 Objective;Aluminum（Al）is ubiquitous in the environment,and is extensively used in contemporary life.Food additive,drugs and utensils comprising of aluminum are main sources of aluminum uptake in China.Aluminum is a known chronic neurotoxicant. There is evidence that aluminum has been linked to neurodegenerative disorders including Alzheimer’s Diseases（AD）,dialysis encephalopathy（DE）.In present,it can be affirmed that aluminium is the only factor associated with DE.Employees in aluminum refinery could obviously be caused mental dysfunction.Chelators have been useful to treat aluminium-overload diseases in clinical practice.So far,the only diagnosis of Al accumulation and treatment of Al toxicity available has been achieved with cbelater desferrioxamine（DFO）.It was found that DFO can reduce Al-induced mortality associated with the dialysis encephalopathy syndrome,the trabecular bone Al,and can improve bone histomorphometry and Al-related bone disease.Because of the poor gastrointestinal absorption,it can be administered by slow subcutaneous perfusion,resulting in a reduced compliance of the therapy.The use of DFO in dialysis patients induces major side effects.In some patients,ocular toxicity, disturbances of colour vision and loss of hearing have been described.So a cheap, orally active and non-toxic aluminum chelator is needed. 1,2-dimethyl-3-hydroxypyrid -4-one（deferiprone,DFP）originally treatment of iron overload.Aluminum and iron are hard acids with similar ionic radii（54 and 64 pro）. They bind to the same plasma proteins.In the following experiments,DFP is used as a new type of aluminum chelating agent.Fri st,the s ynthesis of DFP has been accomplished.Scond,toxicological experiment has been done by LD₅₀experiment, Peripheral blood and bone marrow cell micronucleus experiment,and Sperm aberration experiment.Third,The effect of DFP on aluminum mobilization and elimination from tissues and serum as well as the influence on the excretion of other trace elements in mice and rabbits was investigated.Fourth,the treatment mechanism of DFP in curing animals poisoned by aluminum is studied.Those studies can provide theoretical foundation for seeking a cheap,orally active and non-toxic aluminum chelator for aluminum overload.Methods1.The synthesis of DFPDFP is synthesized from 2-methyl-3-hydroxy-4-pyrones and Methylamine（CH₃NH₂） via heating at 95～100℃6.5h.2.Toxicological experiment2.1 LD₅₀experiment 50 healthy mice of Kunming strain（25 males and 25 females） were randomly divided into 5 groups according to their body weight.The animals were given the medicine solution with the lavage method and observed for one week, then the number of dead mice were recorded.2.2 Peripheral blood and bone marrow cell micronucleus experiment 120 healthy mice of Kunming strain（60 males and 60 females）were randomly divided into 5 groups according to their body weight;negative control group,Cyclophosphamide positive control group,1/8LD₅₀group,1/4LD₅₀group and 1/2LD₅₀group.Then DFP bone marrow cell micronucleus experiment 24 hours after the second and the seventh application of medicine were conducted.2.3 Sperm aberration experiment 150 healthy male mice of Kunming strain were randomly divided into 5 groups according to their weight;negative control group, Cyclophosphamide positive control group,1/8LD₅₀group,1/4LD₅₀group,1/2LD₅₀ group.The medicine was applied for 5 successive days.On the 14th,28th,35th,42nd and 70th day after the application of medicine,DFP Sperm aberration experiment was carried out.3.Effect of DFP removing aluminum3.1 Effect of DFP removing aluminum in mice 84 healthy Kunming mice,which were different in sex,were randomly divided into 6 groups by body weight;Negative control group,Al-exposed group,preventive group low-dose group,medium-dose group,and high-dose group.The mice were given food and water ad libitum.The mice were sacrificed by decapitation at the end of the treatment.The serum samples were obtained by collecting and centrafugating blood samples.The selected organs（liver,kidney,brain and bone）were removed,weighted,and frozen for later analysis of aluminum,copper,zinc,calcium,iron and magnesium contents by atomic absorption speetrophotometry.3.2 Effect of DFP removing aluminum in rabbits3.2.1 Acute removing aluminumTwenty four New Zealand white rabbits were treated with aluminum.Rabbits were randomly divided into four groups;Al-only,low-dose group,medium-dose group, and high-dose group.DFP（500μmol/kg）,DFP（750μmol/kg）,DFP（1000μmol/kg）were given intragastrically（ig）respectively.Urine was collected hourly for 6h and analyzed for aluminum and essential elements copper,zinc and manganese.3.2.2 Combination with Vc or Mesna on urinary aluminum excretion Twenty four New Zealand white rabbits were treated with aluminum.Rabbits were randomly assigned to four groups;Al-only,DFP,DFP +Vc,DFP +Mesna.The agents mentioned above were given intragastrically.Urine was collected for six hours and the aluminum concentration was determined by atomic absorption spectrophotometry.3.2.3 Comparison of the efficacy of removing aluminum with various administration of DFP Eighteen New Zealand white rabbits were treated with aluminum.Rabbits were randomly assigned to three groups;Al-only,injection group, intragastrically group.DFP was given by subcutaneous injection and intragastrically respectively.Urine was collected for six hours and the aluminum concentration was determined by atomic absorption spectrophotometry.3.2.4 Subchronic removing aluminum Sixteen New Zealand rabbits were randomly divided into three groups;control,Al-only and DFP+Al.The Al-only and DFP+Al animals received injections of aluminum for three weeks.One week after the last Al injection the DFP+Al rabbits were given DFP intragastfically for 2 weeks.At the 42th day the animals were sacrificed and the organs were taken and digested. Blood was taken from the ear artery 3 times（at the initiation of the experiment, before and after DFP administration）.The aluminum and copper,zinc,manganese were determined by atomic absorption spectrophotometry.4.Study on the mechanism of DFP protecting against chronic aluminum-exposed animals4.1 Effect of recovering the anti-oxidizing system 84 healthy Kunming mice, which were different in sex,were randomly divided into 6 groups by body weight; Negative control group,Al-exposed group,preventive group low-dose group, medium-dose group,and high-dose group.The mice were given food and water ad libitum.The mice were sacrificed by decapitation at the end of the treatment.The serum samples were obtained by collecting and centrafugating blood samples.The selected organs were removed.The activities of SOD,GSH-PX and the content of MDA in the blood serum and the homogenate of liver,kidney and brain tissues of the mice were determined in every group.4.2 Protecting effect of DFP on nervous system of aluminum -loaded mice 98 healthy Kunming mice,which were different in sex,were randomly divided into 7 groups by body weight;Negative control group,low-dose group,medium-dose group, high-dose rate group,Al-exposed group,preventive group and pydtinol -exposed group.the mice were given food and water ad libitum.4.2.1 The effects of DFP on learning and memory impairment caused by aluminum in mice Two days before the administration ended,step-down test was performed to investigate the effects of DFP on learning and memory impairment caused by aluminum in mice.Step-down latency（SDL）and error times in 5 minutes were noted.4.2.2 Effect of recovering the neurotransmitter in the central nervous system The activity of AchE in the blood serum and the homogenate of brain tissues of the mice were determined in every group.4.2.3 Pathomorphological observation of hippocampus T he hippocampus of the mice were fixed with 4% paraformaldehyde,and then did hematoxylin-eosin staining. Pathomorphological observations were carried out to investigate the morphological changes.4.2.4 DFP protect against the apoptosis caused by aluminum The expression of anti-apopotosis protein Bcl-2 and apopotosis protein Bax in chronic Al-exposed mice and DFP-treated hippocampus was measured by immnohistochemical method,aimed to detect if DFP could protect against the apoptosis induced by aluminum.4.3 Protective effect of DFP on damaged genetic material of mice induced by aluminum.The development of micronuclei（MN）in bone marrow cells and sperm abnormalities were used as mutagenic bioassay in Kun Ming mice.Mice were randomly divided into five groups;negative control group,cyclophosphamide positive control group,Al-exposed group,low-dose group and high-dose group.4.4 Effect of DFP on liver and renal function of aluminum-induced rats Wistar rats were administered with aluminum chloride for 3 weeks.Then they were treated with different doses of DFP.The activity of alanine aminotransferase（ALT）,glutamic oxalacetic transaminase（AST）,alkaline phosphatase（ALP）and the contents of total protein（TP）,albumin,total bilirubin（TB）and conjugated bilirubin,creatinine（Cr）, urea nitrogen（BUN）and uric acid（UA）in serum were detected.The concentrations of Al,Zn,Cu,Fe,Ca and Mg in liver and kidney tissues were measured simultaneously.Results1.The synthesis of DFPThe results showed that the melting point and the infrared spectrum and nuclear-magnetic spectrum corresponded with the structural characteristics of the target compound.DFP yield was 58.9%.2.Toxicological experiment2.1 The LD₅₀experiment of DFP Th e results showed that the LD₅₀of DFP was 562.34mg/kg.The 95% credibility range of DFP fell within 501.19～630.96mg/kg.2.2 Peripheral blood and bone marrow cell micronucleus experiment The results showed that there were no significant differences in the 1/8LD₅₀group and the 1/4LD₅₀group in comparison with the negative control group（P＞0.05）;The peripheral blood and the PCE micronucleus rote of bone marrow cell of the 1/2LD₅₀group were higher than that of the negative control group（P＜0.05）.2.3 Sperm aberration experiment The results showed that there were no significant differences in the 1/8LD₅₀group and the 1/4LD₅₀group in comparison with the negative control group in observations conducted in the 14th,28th,35th,42nd,and 70th day（P＞0.05）; While significant differences appeared between the 1/2LD₅₀group and the negative control group in observations conducted in the 14th,28th and 35th day（P＜0.05）; there were no significant differences between the 1/2LD₅₀group and the negative control group in observations conducted in the 42nd day and 70th day（P＞0.05）.3.Effect of DFP removing aluminum3.1 Effect of DFP removing aluminum in mice3.1.1 Effect of DFP removing aluminum Aluminum contents of preventive group, low-dose group,medium-dose group and high-dose group in brains and kidneys is lower than that of aluminum-exposed group（P＜0.05）.Aluminum contents of preventive group,low-dose group,medium -dose group is higher than that of the negative control group（P＜0.05）in brains and kidneys,yet the same condition could not be observed in high-dose group.No significant differences existed among aluminum contents of various groups in livers.As far as aluminum contents in bone be concerned,aluminum contents of preventive group,low-dose group,medium-dose group and high-dose group is lower than that of aluminum-exposed group （P＜0.05）and no significant differences exist between them and the negative control group.In comparison with aluminum contents in serum,preventive group,three doses groups are lower than aluminum-exposed group and higher than the negative control group.Dose-response relations exist among three doses groups.3.1.2 Effects of DFP removing other essential elements In comparison with magnesium contents,no significant differences existed among various groups in the livers,kidneys,brains and bones.In the contents of calcium,no significant differences existed among various groups in the livers,kidneys and bones.In livers and brains,no significant differences could be observed among zinc contents of various groups. There are no significant differences among various groups in the contents of copper contents in the livers,brains and bones.3.2 Effect of DFP removing aluminum in rabbits3.2.1 Acute removing aluminumComparison of hourly aluminum output after sterile water and DFP administration suggests that DFP could increase aluminum output above control beyond the 6h monitored after administration（P＜0.01）.The peak height appeared in 2-4 hours after administration.6h cumulative excretion of aluminum in the urine rose significantly in response to DFP administration when compared to control treatment（P＜0.001）.3.2.2 Combination with Vc or Mesna on urinary aluminum excretion DFP, DFP+ Vc and DFP+ Mcsna had a comparable effect on aluminum excretion,but the peak height in the aluminum excretion figure was changed.This indicates that DFP, Vc and Mcsna had a mutually interactive effect on urinary aluminum elimination.3.2.3 Comparison of the efficacy of removing aluminum with various administration of DFP Comparison of 6h cumulative excretion of aluminum in the urine,injection group could increase aluminum output above intragastrically group（P＜0.05）.The peak height appeared 3 hours and 2 hours after administration for injection group and intragastrically group respectively.3.2.4 Subchronic removing aluminum DFP could highly mobilize aluminum stores from tissues.At the end of experiment the aluminum contents of bone,kidney, liver and brain in DFP groups were significantly lower than that in Al-only group. 3.2.5 Effects of DFP removing essential elements copper,zinc and manganese3.2.5.1 Acute removing aluminum Urinary excretion of copper,zinc,and manganese before and after the administration of DFP varied slightly in each group, but there was no consistent increase or decrease when all the rabbit’s data were compared.3.2.5.2 Snbehronie removing aluminum Serum concentrations of copper,zinc and manganese before and after the administration of DFP varied slightly in each group,but there was no significant difference.With respect to the copper,zinc and manganese concentration,the obvious difference after DFP administration had not observed in the analyzed organs.4.Study on the mechanism of DFP protecting against chronic aluminum-exposed animals4.1 Effect of recovering the anti-oxidizing system The results showed that in comparison with the corresponding data of the model group（P＜0.05）,significant differences existed in the negative control group in terms of the activities of SOD, GSH-PX and the content of MDA in liver,brain and the blood serum.In comparison with the corresponding data of the negative control group（P＞0.05）,no significant differences existed among the medicine-treated groups in the terms of the activities of SOD,GSH-PX and the content of MDA in liver,kidney,brain and the blood serum.4.2 Protecting effect of DFP on nervous system of aluminum -loaded mice4.2.1 The effects of DFP on learning and memory impairment caused by aluminum in miceThe step-down test showed that the SDL of preventive group,low-dose group, medium-dose group and high-dose group is significantly lower than that of aluminium-exposed group（P＜0.01,P＜0.05）.At the same time,dose-response relation exists and there is no significance between three doses groups and the negative control group.The ET of negative control group is lower than that of aluminium-exposed group（P＜0.05）.There is no significance between three doses groups and the negative control group.4.2.2 Effect of recovering the neurotransmitter in the central nervous system The results showed that the activity of AchE in the model group was higher than that of the negative control group（P＜0.05）.In comparison with the model group, significant differences existed in the high-dose group,medium-dose group and the preventive group（P＜0.05）.No significant differences existed between the medicine-treated groups and the negative control group.4.2.3 Pathomorphological observation of the hippocampus The hippocampus damage had been found among aluminum-exposed mice in the pathological examination,significant recovering could be observed in the DFP-treated groups.4.2.4 DFP protect against the apoptosis caused by aluminum The immunohistochemical localization of Bcl-2 and Bax in the hippocampus of mice was examined.As far as Bcl-2 protein in hippocampus concerned,there is no significant differences observed in low-dose group and pyritinol-exposed group（P＞0.05）while significantly increased in medium-dose group,high-dose group and preventive group as compared with aluminium-exposed group（P＜0.05）.The number of Bax positive cells of aluminium-exposed group,preventive group and low-dose group is more than that of negative control group and pyritinol-treated group（P＜0.05）,and that of of the high-dose group is higher than that of pyfitinol-treated group（P＜0.05）.4.3 Protective effect of DFP on damaged genetic material of mice induced by aluminum In comparison to alum treated group,the micronucleus frequency of the lower and higher dose of DFP group was apparently lower than alum treated group （p＜0.01）.Significant difference could be observed between the lower and the higher DFP group（p＜0.01）and frequency of MN PCE decreased with increasing DFP doses. Of the two groups treated with DFP plus alum,the both incidences of abnormal sperms were significantly lower than that treated with alum alone（p＜0.05）,yet higher than the negative control（p＜0.05）respectively.Significant difference could be observed between the lower and the higher DFP group（p＜0.05）,and the incidence of abnormal sperms decreased with increasing DFP doses.4.4 Effect of DFP on liver and renal function of aluminum-induced rats4.4.1 Effect of aluminum chelating agent on liver function The result of ALT activity demonstrated that there was no statistical significance among all the groups （P＞0.05）.The AST activity of low,moderate and high DFP dose groups was lower than the positive control（P＜0.01）.The result of ALP activity indicated that the low DFP dose group was significantly different from the positive control（P＜0.01）.The TP content of the Al group was significantly lower than the negative control（P＜0.01）. The moderate and high DFP group were higher than the Al group（P＜0.01）,while the same condition hasn’t been exhibited between the moderate and high DFP dose groups and negative control.The result of albumin contents demonstrated that there was no statistical significance among all the groups.Compared with the negative control,the TB contents of the other 5 groups raised obviously（P＜0.05,P＜0.01）.The contents of the conjugated bilirubin in all groups were not significantly different. The aluminum concentration of the three different dose groups was significantly lower than the positive control（P＜0.01）.Compared with the negative control,the Zn, Cu,Fe,Ca,Mg concentrations of DFP dose groups were not significantly different （P＜0.05）.4.4.2 Effect of aluminum chelating agent on renal function The BUN content of the Al group was significantly lower than the negative control.The results of UA and Cr contents demonstrated that there was no statistical significance among all the groups.The Al level in kidney remarkably rose with the increase of Al intake.The aluminum concentration of the three different dose groups was significantly lower than that of the Al group（P＜0.01）.The aluminum concentration of the moderate and high DFP dose groups,as compared with the negative control,was no statistical significance.The Cu concentration of the Al group and the lower DFP dose group was significantly lower than that of the negative control,while there were no statistical significance between the moderate,high DFP dose groups and negative control. Compared with the negative control,the Zn,Fe,Ca,Mg concentrations of the lower, moderate and high DFP dose groups were not significantly different.Conclusions1.A new DFP synthesis method has been established.This method could make the synthetic time shortened and make DFP synthesis more easily and cheaper.2.DFP is a compound with low toxicity.DFP could chelated aluminum and reduces aluminum contents in animals and has no obvious effect on other essential elements such as zinc,magnesium,copper,calsium.3.DFP could improve the learning and memory ability induced by aluminum.4.DFP could counter the aluminum-induced aging of the neurotransmitter in the central nervous system.5.DFP could regulate the changes of apoptosis-related protein caused by aluminum.6.DFP has protective effect on damaged genetic material of mice induced by aluminum.7.DFP could protect liver and renal function of aluminum-induced ratsIn a word,a new DFP synthesis method has been established,the acute toxicity of DFP has been evaluated.The effect of DFP on aluminum mobilization and elimination from tissues and serum and the mechanism of DFP protecting against chronic aluminum-exposed animals has been studied first.Those studies can provide theoretical foundation for seeking a cheap,orally active and non-toxic aluminum chelator for aluminum overload.更多还原