【作者】 任娜；

【导师】 刘萍；

【作者基本信息】 山东大学 ， 卫生检验学， 2019， 硕士

【摘要】 [目的]铝广泛存在于自然界中,随着工业进程的发展,人们接触和摄入铝的途径也越来越多。铝可在人体中蓄积,并引起慢性毒性作用。研究发现,铝的毒性可能与铝可以引起组织氧化损伤有关。黄酮类化合物本身具有显著的清除自由基及抗氧化作用,可以改善脂质代谢,同时是一种金属螯合剂。本实验以Wistar大鼠为实验对象,通过六水氯化铝以灌胃方式建立大鼠铝中毒模型,再选取三类具有代表性的黄酮类化合物(芦丁、葛根素、水飞蓟素)进行解毒治疗,通过测定抗氧化指标、相关元素、三磷酸腺苷酶、单胺类神经递质以及氨基酸类神经递质,探究黄酮类化合物对铝染毒大鼠神经递质的调节及抗氧化系统的保护作用。[方法]1.实验动物将64只健康SPF级雄性Wistar大鼠按体重随机分为8组,分别为阴性对照组,铝染毒组,芦丁低、高剂量组,葛根素低、高剂量组及水飞蓟素低、高剂量组,每组8只。采用灌胃给药方式,每天1次,每周给药6天。实验共进行12周,前四周建立铝中毒模型,后八周使用黄酮类化合物进行治疗。阴性对照组连续12周给予1.0ml/d生理盐水;铝染毒组,芦丁低、高剂量组,葛根素低、高剂量组和水飞蓟素低、高剂量组前4周每天分别给予281.40mg/kg三氯化铝溶液,后8周每天分别给予1ml生理盐水,100mg/kg、200mg/kg芦丁,100mg/kg、200mg/kg葛根素,100mg/kg、200mg/kg水飞蓟素。给药阶段结束后,实验大鼠先禁食24小时,后断头处死,收集颈动脉血,低温下取出脑组织并于-80℃冰箱内保存待用。2.黄酮类化合物对铝染毒大鼠生长发育的影响每日记录大鼠的运动状况,并观察其日常饮食情况,每周称量体重三次。实验结束后,称量大鼠的最终体重并记录。3.黄酮类化合物对铝染毒大鼠大脑皮层和血清抗氧化系统的影响使用生理盐水制备10%大鼠脑组织匀浆液并将大鼠血清稀释到合适浓度。按试剂盒步骤测定SOD和GSH-Px的活力,以及MDA含量。4.黄酮类化合物对铝染毒大鼠脑组织中ATPase活力的影响使用生理盐水制备10%大鼠脑组织匀浆。按试剂盒步骤测定Na+K+-ATPase、Mg2+-ATPase和Ca2+-ATPase的活力。5.黄酮类化合物对铝染毒大鼠脑组织中钙、镁、铜、铁、锌元素的影响准确取0.2g脑组织,微波消解后使用火焰原子吸收光谱法测吸光度,计算Ca、、Mg、、Cu、Fe、Zn元素的含量。6.黄酮类化合物对铝染毒大鼠大脑皮层中单胺类神经递质的调节准确称量大鼠脑组织0.2g,按m(g):V(ml)=1:9的比例加入0.1mol/L的HC104溶液,制成10%组织匀浆液,使用高效液相色谱—荧光检测法检测各组大鼠脑组织中NE、E、DA、5-HT的含量。7.黄酮类化合物对铝染毒大鼠大脑皮层中氨基酸类神经递质的调节准确称量大鼠脑组织,使用乙腈水溶液(V:V=1:1)配成10%组织匀浆液,通过二硝基氟苯(DNFB)进行衍生化后使用高效液相色谱一二极管阵列检测法检测大鼠脑组织中Asp、Glu、Gly、Tau和GABA的含量。[结果]1.黄酮类化合物对铝染毒大鼠生长发育的影响实验期间,观察发现对照组大鼠毛发洁白发亮、精神状态良好。而铝染毒组大鼠随着染毒的进行,出现生长迟缓、反应迟钝,毛发稀疏、无光泽等现象。染毒组大鼠体重自第6周起明显低于对照组(P<0.05)。芦丁高剂量组、水飞蓟素高剂量组大鼠体重自第9周起明显高于铝染毒组(P<0.05)。2.黄酮类化合物对铝染毒大鼠大脑皮层抗氧化系统的影响染毒组的SOD活力比对照组明显降低(P<0.05);与染毒组相比,芦丁高剂量组和水飞蓟素低、高剂量组的SOD活力明显升高(P<0.05)。染毒组的GSH-Px活力明显低于对照组(P<0.05);与染毒组比较,芦丁高剂量组、水飞蓟素高剂量组的GSH-Px活力明显升高(P<0.05)。染毒组的MDA含量明显高于阴性对照组(P<0.05);芦丁高剂量组、葛根素高剂量组、水飞蓟素高剂量组的MDA含量与染毒组相比显著降低(P<0.05)。然而,三种黄酮类化合物的低剂量组和高剂量组相比三个指标均无明显差异(P>0.05)。3.黄酮类化合物对铝染毒大鼠血清抗氧化系统的影响铝染毒组大鼠血清中的GSH-Px活力较对照组明显下降(P<0.05);与铝染毒组相比,芦丁高剂量组和水飞蓟素高剂量组的GSH-Px活力明显提高(P<0.05)。铝染毒组大鼠血清中的MDA含量较对照组显著提高(P<0.05);芦丁高剂量组、葛根素高剂量组和水飞蓟素高剂量组MDA含量与铝染毒组相比明显降低(P<0.05)。4.黄酮类化合物对铝染毒大鼠脑组织中ATPase活力的影响随着铝的摄入,大鼠脑组织中的Na+K+-ATPase、Mg2+-ATPase、Ca2+-ATPase活性都有所降低。铝染毒组大鼠的脑组织中Na+K+-ATPase活力显著低于阴性对照组(P<0.05);而芦丁高剂量组,水飞蓟素低、高剂量组的Na+K+-ATPase活力明显高于染毒组(P<0.05)。铝染毒组大鼠的脑组织中Mg2+-ATPase活力明显低于阴性对照组(P<0.05);而芦丁高剂量组、葛根素高剂量组和水飞蓟素高剂量组的Mg2+-ATPase活力明显比染毒组高(P<0.05)。铝染毒组大鼠的脑组织中Ca2+-ATPase活力比阴性对照组有所降低,但差异无统计学意义(P>0.05);各解毒组的Ca2+-ATPase活力相对染毒组都有所提高,但差异无统计学意义(P>0.05)。5.黄酮类化合物对铝染毒大鼠脑组织中Ca,、Mg,、Cu,、Fe,、Zn元素的影响铝染毒组大鼠与阴性对照组相比,Mg.、Fe、Zn均明显降低(P<0.05);与染毒组相比,芦丁高剂量组,葛根素高剂量组,水飞蓟素低、高剂量组Mg含量明显提高(P<0.05);芦丁低、高剂量组,葛根素高剂量组和水飞蓟素高剂量组Fe含量均高于铝染毒组,差异具有统计学意义(P<0.05);葛根素高剂量组,水飞蓟素低、高剂量组Zn含量显著高于染毒组(P<0.05)。与铝染毒组相比各解毒组Ca和Cu含量无明显差别(P>0.05)。6.黄酮类化合物对铝染毒大鼠大脑皮层中单胺类神经递质的调节与阴性对照组相比,铝染毒组大鼠脑组织中的NE含量明显降低(P<0.05);与铝染毒组相比,芦丁高剂量组,葛根素高剂量组,水飞蓟素低、高剂量组的大鼠脑组织中的NE含量显著升高(P<0.05)。与阴性对照组相比,铝染毒组大鼠脑组织中的E含量明显下降(P<0.05);葛根素高剂量组和水飞蓟素高剂量组的大鼠脑组织中的E含量比铝染毒组明显升高(P<0.05)。铝染毒组大鼠脑组织中的DA含量比阴性对照组降低(P<0.05);与铝染毒组相比,芦丁高剂量组和水飞蓟素高剂量组的大鼠脑组织中的DA含量明显升高(P<0.05)。与阴性对照相比,铝染毒组大鼠脑组织中的5-HT含量明显降低(P><0.05);与铝染毒组相比,芦丁高剂量组和水飞蓟素高剂量组的大鼠脑组织中的5-HT含量显著升高(P<0.05)。7.黄酮类化合物对铝染毒大鼠大脑皮层中氨基酸类神经递质的调节与阴性对照组比较,铝染毒组大鼠脑组织中的Asp含量明显升高(P<0.05);与铝染毒组相比,葛根素高剂量组,水飞蓟素低、高剂量组的大鼠大脑皮层中的Asp含量明显下降(P<0.05)。铝染毒组大鼠大脑皮层中的Glu含量与阴性对照组相比明显上升(P<0.05);芦丁低、高剂量组和水飞蓟素低剂量组的大鼠脑组织中的Glu含量与铝染毒组对比明显下降(P<0.05)。与阴性对照组相比,铝染毒组大鼠脑组织中的GABA含量显著降低(P<0.05);与铝染毒组相比,芦丁高剂量组,葛根素高剂量组,水飞蓟素低、高剂量组的大鼠脑组织中的GABA含量显著升高(P<0.05)。铝染毒组大鼠大脑皮层中的Tau含量与阴性对照组相比明显降低(P<0.05);与铝染毒组相比,芦丁低剂量组和水飞蓟素低、高剂量组的大鼠大脑皮层中的Tau含量明显提升(P<0.05)。铝染毒组大鼠脑组织中的Gly含量较阴性组有所下降,但差异并无统计学意义(P>0.05);各解毒组的大鼠大脑组织中的Gly含量较铝染毒组有所提高,但差异并无统计学意义(P>0.05)。[结论]1.黄酮类化合物能够改善铝染毒大鼠生长缓慢的现象。2.一定剂量的黄酮类化合物对铝染毒大鼠的脑组织和血清抗氧化系统损伤有显著的改善作用。3.—定剂量的黄酮类化合物能够有效的提高铝染毒大鼠大脑皮层中Na+K+-ATP酶和Mg2+-ATP酶的活力,但对Ca2+-ATP酶的活力并未产生显著影响。4.一定剂量的黄酮类化合物对铝引起的大鼠脑组织中Mg.、Zn、Fe的稳态失衡起着调节作用,对Ca和Cu元素的含量并未产生明显影响。5.黄酮类化合物具有抑制和改善铝致大鼠氨基酸类神经递质和单胺类神经递质含量紊乱的作用。更多还原

【Abstract】 ObjectivesWith the development of industry,people have more and more ways to contact and absorb aluminum.Aluminum can accumulate in the body and cause chronic toxicity.Studies have shown that the accumulation of high-dose aluminum can lead to neurotoxicity and cause clinical symptoms such as memory loss,tremor,convulsion and ataxia.At present,the mechanism of aluminum acting on these symptoms and pathological conditions is not clear.Most studies believe that aluminum is related to the oxidative damage caused by the increase of free radical content in tissues.Flavonoid compounds having significant free radical scavenging and antioxidant effects,can improve lipid metabolism,and is a metal chelating agent.In this study,aluminum poisoning model of rats was established by aluminum chloride hexahydrate gavage.Then,the protective effects of flavonoids on the regulation of neurotransmitters and the antioxidant system in aluminum poisoned rats were investigated by measuring the peroxidation index,related trace elements,ATP activity,monoamine neurotransmitter and amino acid neurotransmitter content.Methods1.Experimental animals64 male rats of SPF grade were randomly divided into eight groups:control,Al-treated,low-dose rutin,high-dose rutin,low-dose silymarin,high-dose silymarin and low-dose puerarin,high-dose puerarin.The control group received 1 mL/(kg·day)saline solution for 12 weeks.The other groups were exposed to Al at a dose of 281.40 mg/kg/day orally for 4 weeks.Then,they were respectively received 1mL/kg/day saline solution,100mg/(kg·day)rutin,200 mg/(kg·day)rutin,100mg/(kg·day)silymarin,200mg/(kg·day)silymarin,100mg/(kg·day)puerarin,and 200 mg/(kg·day)puerarin for 8 weeks.The tested chemicals were given by gavage.After the detoxification phase,rats in each group fasted 24h after the last administration,then the rat was decapitated,the brains were quickly removed,cleaned of adhering matters and washed with normal saline.All tissue samples were stored at-80℃ until required analysis.2.Effects of flavonoids on general growth and development of aluminum exposed ratsThe mental state,food and water consumption of the experimental rats were observed daily.The rats were weighed twice a week,and the weight changes of the rats were observed and recorded.3.Effects of flavonoids on antioxidant system of cerebral cortex and serum of rats poisoned with aluminumThe cerebral cortex was taken out and weighed.Normal saline was added according to mass:volume =1:9 to prepare 10%homogenate.The serum was diluted with normal saline to the appropriate concentration,SOD,GSH-Px and MDA were determined according to the kit procedure4.Effects of flavonoids on ATPase activity in the cerebral cortex of rats exposed to aluminumThe cerebral cortex was taken out and weighed.Normal saline was added according to mass:volume =1:9 to prepare 10%homogenate.The serum was diluted with normal saline to the appropriate concentration.Na+K+-ATPase,Mg2+-ATPase and Ca2+-ATPase were determined according to the steps of the kit.5.Effects of flavonoids on some elements in brain tissue of rats poisoned with aluminumThe absorbance of 0.2g brain tissue was measured by flame atomic absorption spectrometry after microwave digestion,and the contents of Ca,Mg,Cu,Fe and Zn were calculated.6.Effects of flavonoids on monoamine neurotransmitters in rat brain tissue exposed to aluminumAn appropriate amount of brain tissue was accurately weighed and made into 10%brain homogenate of m(g):V(ml)=1:9 with perchloric acid solution(0.1mol/L).The contents of NE,E,DA and 5-HT in brain tissues of rats in each group were determined by HPLC and fluorescence detection.7.Effects of flavonoids on amino acid neurotransmitters in brain tissue of rats exposed to aluminumAn appropriate amount of brain tissue was accurately weighed and made into 10%brain homogenate by acetonitrile aqueous solution(50%).Then,amino acid neurotransmitters in tissue homogenate were derived by DNFB.The contents of Asp,Glu,Gly,Tau and GABA in rat brain tissue were determined by HPLC and diode array assay.Results1.Effects of flavonoids on growth and development of aluminum-poisoned ratsDuring the experiment,the observation found that the control group of rats in good health,good mental state,hair white shiny.With the increase of A1 exposure time,the rats in the aluminum exposure group showed growth retardation,mental malaise,sparse hair and lackluster.With the progress of the experiment,the weight of the rats in each group increased continuously.Compared to the negative control group,the body weight of rats in the aluminum poisoning group was lower from week 6(P<0.05).Compared to the aluminum group,the body weight of rats in the high-dose rutin and high-dose silymarin group was significantly higher from week 9(P<0.05).2.Effects of flavonoids on antioxidant system of cerebral cortex of rats exposed to aluminumCompared with the negative control group,the activity of SOD in the poisoned group was significantly decreased(P<0.05).Compared with the poisoned group,SOD activity was significantly increased in the rutin high-dose group,the silymarin low-dose group,and the silymarin high-dose group(P<0.05).,Compared with the negative control group,the activity of GSH-Px in the poisoned group was significantly decreased(P<0.05).Compared with the poisoned group,the activity of GSH-Px in the rutin high-dose group and the silymarin high-dose group was significantly increased(P<0.05).Compared with the negative control group,MDA content in the poisoned group was significantly increased(P<0.05).MDA content in rutin high dose group,puerarin high dose group and silymarin high dose group was lower than that in the poisoned group(P<0.05).Compared with that of the high-dose group,the three indexes of the three flavonoids fell into no significant difference(P>0.05).3.Effects of flavonoids on serum antioxidant system of aluminum-poisoned ratsThe activity of SOD in serum of rats exposed to aluminum was lower than that of control group.SOD activity increased in the serum of the rats of the detoxification groups,but the difference was not statistically significant(P>0.05).The serum GSH-Px activity in the aluminum exposed group decreased significantly compared with the control group(P<0.05).Compared with the aluminum exposure group,the activity of GSH-Px in the rutin high-dose group and the silymarin high-dose group was significantly increased(P<0.05).Compared with the negative control group,MDA content in serum of rats exposed to aluminum was significantly higher(P<0.05).Compared with the aluminum exposure group,MDA content in rutin high dose group,puerarin high dose group and silymarin high dose group was significantly lower(P<0.05).4.Effects of flavonoids on ATPase activity in the cerebral cortex of rats exposed to aluminumWith the intake of aluminum,Na+K+-ATPase,Mg2+-ATPase and Ca2+-ATPase activity in rat brain decreased.Compared with the control group,Na+K+-ATPase activity was significantly decreased in the brain tissues of rats exposed to aluminum(P<0.05).Compared with the aluminum exposure group,the Na+K+-ATPase activity of the high dose rutin group,the low dose silymarin group and the high dose silymarin group was significantly higher(P<0.05).The activity of Mg2+-ATPase in the brain tissue of rats exposed to aluminum decreased significantly compared with the control group(P<0.05).Compared with the exposed group,the activity of Mg2+-ATPase in rutin high dose group,puerarin high dose group and silymarin high dose group was significantly higher(P<0.05).Compared with that of the control group,the Ca2+-ATPase of the rat brain fell into a decline,but the difference was not statistically significant(P>0.05).The Ca2+-ATPase activity of the detoxification group increased compared with that of the collection group,but the difference showed no statistical significance(P>0.05).5.Effects of flavonoids on Ca,Mg,Cu,Fe and Zn in brain tissue of rats poisoned with aluminumCompared with the control group,the contents of Mg,Fe and Zn in rats exposed to aluminum were significantly decreased(P<0.05).Compared with the poisoned group,the Mg content in the high-dose rutin group,the high-dose puerarin group,the low-dose silymarin group and the high-dose silymarin group was significantly increased(P<0.05).Compared with the aluminum exposure group,Fe contents in low dose rutin group,high dose rutin group,high dose puerarin group and high dose silymarin group were all higher(P<0.05).The content of Zn in the high-dose puerarin group,low-dose silymarin group and high-dose silymarin group was significantly higher than that in the poisoned group(P<0.05).Compared with the aluminum poisoning group,the content of Ca and Cu in each detoxification group showed no statistical significance(P>0.05),but the content of Ca and Cu in the detoxification group was lower than that in the negative group and the detoxification group.6.Effects of flavonoids on monoamine neurotransmitters in rat brain tissue exposed to aluminumCompared with the control group,the content of NE in brain tissue of rats exposed to aluminum decreased significantly(P<0.05).Compared with the aluminum exposure group,the contents of NE in the brain tissues of rats in the rutin high-dose group,puerarin high-dose group,silymarin low-dose group and silymarin high-dose group were significantly increased(P<0.05).Compared with the negative control group,the content of E in the brain tissue of rats exposed to aluminum decreased significantly(P<0.05).Compared with the aluminum exposure group,the E content in the brain tissues of the rats in the puerarin high-dose group and the silymarin high-dose group was significantly increased(P<0.05).Compared with the control group,the content of DA in the brain tissue of rats exposed to aluminum decreased significantly(P<0.05).Compared with the aluminum exposure group,the DA content in the brain tissues of rats in the rutin high-dose group and the silymarin high-dose group was significantly increased(P<0.05).Compared with the control group,the content of 5-HT in the brain tissue of rats exposed to aluminum decreased significantly(P<0.05).Compared with the aluminum exposure group,the 5-HT content in the brain tissues of rats in the rutin high-dose group and the silymarin high-dose group was significantly increased(P<0.05).7.Effects of flavonoids on amino acid neurotransmitters in braintissue of rats exposed to aluminumCompared with the control group,the content of Asp in brain tissue of rats exposed to aluminum increased significantly(P<0.05).Compared with the aluminum exposure group,the content of Asp in the brain tissue of rats in the puerarin high-dose group,the silymarin low-dose group and the silymarin high-dose group decreased significantly(P<0.05).Compared with the control group,the content of Glu in brain tissue of rats exposed to aluminum increased significantly(P<0.05).Compared with the aluminum exposure group,the Glu content in the brain tissue of the rats in the low dose group,the high dose group and the low dose group of silymarin decreased significantly(P<0.05).Compared with the control group,the content of GABA in the brain tissue of rats exposed to aluminum decreased significantly(P<0.05).The contents of GABA in brain tissue of rats with high dose of rutin,high dose of puerarin,low dose of silymarin and high dose of silymarin were significantly higher than those of the aluminum group(P<0.05).Compared with the control group,Tau content in brain tissues of rats exposed to aluminum decreased significantly(P<0.05).Compared with the aluminum exposure group,Tau content in brain tissues of rats in the low dose group of rutin,the low dose group of silymarin and the high dose group of silymarin was significantly increased(P<0.05).Compared with that of the control group,the Gly content of the rat’s brain tissue fell after aluminum poisoning(P>0.05).Compared with that of the aluminum group,the Gly content of the brain tissues of the detoxification groups increased somewhat,but the difference showed no statistical significance(P>0.05).Conclusions1.Flavonoids can improve the growth retardation of aluminum poisoned rats.2.A certain dose of flavonoids can significantly improve the brain tissue and serum antioxidant system of rats exposed to aluminum.3.A certain dose of flavonoids can effectively increase the activity of Na+K+-ATPase and Mg2+-ATPase in the cerebral cortex of rats exposed to aluminum,but has no significant effect on the activity of Ca2+-ATPase.4.A certain dose of flavonoids play a role in improving and regulating the homeostasis imbalance of Mg,Zn and Fe in rat brain tissue caused by aluminum,but have no significant effect on the metabolism of Ca and Cu.5.Flavonoids can inhibit and improve the content disorder of amino acid neurotransmitter and monoamine neurotransmitter induced by aluminum in rats.更多还原