EGCG联合维生素E对铅暴露仔鼠海马氧化应激及相关蛋白的影响

发布时间：2018-06-07 10:35

本文选题：铅 + EGCG　；参考：《郑州大学》2017年硕士论文

【摘要】：目的研究表没食子儿茶素没食子酸酯(epigallocatechin gallate,EGCG)与维生素E(vitamin E,VitE)联用对不同时期母鼠铅暴露所致仔鼠阿尔茨海默病(Alzheimer’s disease,AD)淀粉样变的干预作用,探讨EGCG与VitE的联用对铅致AD样病变是否存在协同作用及其可能机制。方法将SPF级昆明小鼠33只(雌22只、雄11只),按照体重顺序随机分为3组:对照组(雌2只、雄1只)、孕前期染铅组(雌10只、雄5只)和哺乳期染铅组(雌10只、雄5只)。孕前期染铅组雌鼠饮0.2%醋酸铅溶液染铅21d,染铅结束后与该组雄鼠合笼以获得孕鼠,自由饮去离子水直至仔鼠断乳。哺乳期染铅组正常饮去离子水、合笼,待孕鼠分娩后饮0.2%醋酸铅溶液染铅21d直至小鼠断乳。对照组在实验过程中自由饮去离子水,合笼后获得孕鼠直至分娩后仔鼠断乳。断乳后,孕前期染铅组和哺乳期染铅组所产仔鼠再按体重顺序编号,分别随机分为7组,即生理盐水组、玉米油组、EGCG干预组(3.0 mg/kg)、VitE干预组(30 IU/kg)、联合干预组(1)(EGCG 3.0 mg/kg+VitE 30 IU/kg)、联合干预组(2)(EGCG 1.5 mg/kg+VitE 60 IU/kg)、联合干预组(3)(EGCG 6.0 mg/kg+Vit E 15 IU/kg),每组14只,从21 d断乳后开始连续灌胃28 d。对照组所产仔鼠按体重顺序随机选取14只自由饮去离子水。干预结束后,对所有小鼠进行Morris水迷宫实验;用极谱仪测定小鼠血铅含量;用相应试剂盒测定小鼠海马组织中总超氧化物歧化酶(superoxide dismutase,SOD)活性和丙二醛(malondialdehvde,MDA)含量;用ELISA试剂盒检测小鼠海马组织中Aβ_(1-40)、Aβ_(1-42)含量;用Western blot检测小鼠海马组织中β淀粉样前体蛋白(β-amyloid precursor protein,APP)及β-内分泌酶1(β-secreatase-1,BACE1)蛋白的表达水平。使用SPSS 21.0版本进行统计分析,检验水准α=0.05。结果1.体重结果显示:孕前期染铅组和哺乳期染铅组的小鼠,与对照组相比,第5周龄开始生理盐水组、玉米油组及不同干预组小鼠体重均明显降低(P0.05);第9周龄时孕前期染铅组中联合干预组(1)和联合干预组(3)的小鼠体重分别高于生理盐水组和玉米油组(P0.05),哺乳期染铅组中联合干预组(1)的小鼠体重高于玉米油组(P0.05)。2.水迷宫结果显示:孕前期染铅组和哺乳期染铅组中,生理盐水组和玉米油组的平均逃避潜伏期均高于对照组(P0.05);EGCG干预组和VitE干预组的平均逃避潜伏期分别低于生理盐水组和玉米油组(P0.05);与单独干预组相比,孕前期染铅组中联合干预组(1)的平均逃避潜伏期降低(P0.05);哺乳期染铅组中联合干预组(1)和(3)的平均逃避潜伏期降低(P0.05)。孕前期和哺乳期染铅组中,生理盐水组和玉米油组的穿越平台次数均明显低于对照组(P0.05);三个联合干预组分别与生理盐水组和玉米油组相比,穿越平台次数明显增加(P0.05);与单独干预组相比,联合干预组(1)和(3)的穿越平台次数明显增加(P0.05)。3.血铅含量结果显示:孕前期染铅组和哺乳期染铅组中,生理盐水组和玉米油组的血铅含量均明显高于对照组(P0.05);EGCG干预组和三个联合干预组血铅含量均低于生理盐水组(P0.05);三个联合干预组血铅含量均低于玉米油组(P0.05),而单独干预组与联合干预组的血铅含量差异无统计学意义(P0.05)。4.氧化应激指标结果显示:孕前期染铅组和哺乳期染铅组中,生理盐水组和玉米油组与对照组相比,SOD活性显著降低(P0.05),MDA含量明显升高(P0.05);分别与生理盐水组和玉米油组相比,单独干预组和三个联合干预组的SOD活性升高(P0.05),MDA含量降低(P0.05);联合干预组与单独干预组相比,联合干预组(1)、(3)的SOD活性升高(P0.05),MDA含量差异无统计学意义(P0.05)。5.Aβ_(1-40)、Aβ_(1-42)含量结果显示:孕前期染铅组和哺乳期染铅组中,生理盐水组和玉米油组Aβ_(1-40)、Aβ_(1-42)含量均明显高于对照组(P0.05);与生理盐水组和玉米油组相比,单独干预组和联合干预组的Aβ_(1-40)、Aβ_(1-42)含量降低(P0.05);与单独干预组相比,哺乳期染铅组中,联合干预组(3)Aβ_(1-42)含量比VitE干预组低(P0.05)。6.APP、BACE1蛋白表达水平结果显示:孕前期染铅组和哺乳期染铅组中,与对照组相比,生理盐水组、玉米油组及不同干预组APP、BACE1蛋白水平明显增多(P0.05);且不同干预组与生理盐水组和玉米油组相比,APP、BACE1蛋白水平明显减少(P0.05);联合干预组与单独干预组相比,APP、BACE1蛋白水平明显减少(P0.05)。结论1.母鼠孕前期或哺乳期的铅暴露,可导致小鼠体内血铅含量升高,影响小鼠生长发育,损伤小鼠空间学习记忆能力,并且可以升高海马组织中氧化应激水平,诱导海马中APP和BACE1蛋白的过度表达,从而增加海马中Aβ_(1-40)、Aβ_(1-42)的水平。2.EGCG、VitE以及EGCG和VitE联用均可以改善铅暴露引起的小鼠学习记忆能力下降情况,降低AD样变小鼠海马中Aβ_(1-40)、Aβ_(1-42)的含量,降低APP及BACE1蛋白的表达水平,并且在适当剂量下的联合干预效果要优于单独使用EGCG或VitE。
[Abstract]:Objective to investigate the effect of combined use of epigallocatechin gallate (EGCG) and vitamin E (vitamin E, VitE) on amyloidosis of Alzheimer's disease (Alzheimer 's disease, AD) induced by lead exposure in mice at different times. Methods 33 Kunming mice (22 females and 11 males) were randomly divided into 3 groups according to the weight sequence: the control group (2 female, 1 male), the prepregnancy lead group (10 female, male 5) and the lactating group (10, male 5). The female rats in the pre pregnancy group were exposed to lead 21d in the lead group, and the lead was closed with the male mice. In the cage, the pregnant rats were free to drink deionized water until the offspring were weaned. The lactating group normally drank deionized water and closed the cage. After childbirth, 0.2% lead acetate solution was used to dye lead 21d until the mice were weaned. The control group was free to drink deionized water during the experiment. The offspring rats in the group and lactation stage were numbered according to the weight sequence and were randomly divided into 7 groups, namely the saline group, the corn oil group, the EGCG intervention group (3 mg/kg), the VitE intervention group (30 IU/kg), the combined intervention group (1) (EGCG 3 mg/kg+VitE 30 IU/kg), the combined intervention group (2) (EGCG 1.5 mg/kg+VitE 60 IU/kg), and the joint intervention group (3) (EGCG 6 mg/kg+Vi). T E 15 IU/kg), 14 rats in each group, 14 free drinking deionized water were selected randomly from 28 d. control group after 21 d weaning. After the intervention, all mice were tested in the Morris water maze test; the blood lead content of mice was measured by polarography; and the total superoxide dismutase in the hippocampus of mice was measured by the phase strain kit. Superoxide dismutase (SOD) activity and the content of malondialdehyde (malondialdehvde, MDA), and the content of A beta (1-40) and A beta (1-42) in the hippocampus of mice were detected by ELISA kit, and the beta amyloid precursor protein (beta -amyloid precursor) and beta endocrine enzyme 1 protein in the hippocampus of mice were detected by Western blot. The level of expression of SPSS 21 was statistically analyzed, and the results of test level alpha =0.05. results showed that the results of 1. body weight showed that the mice in the prepregnancy group and the breastfeeding group, compared with the control group, began to start the physiological saline group at fifth weeks of age, and the weight of the corn oil group and the different intervention groups were significantly decreased (P0.05), and the lead exposure group at the early stage of pregnancy at the age of ninth weeks was the lead group. The weight of mice in the combined intervention group (1) and the combined intervention group (3) was higher than that of the saline group and the corn oil group (P0.05). The weight of the mice in the combined intervention group (1) in the lactating period (1) was higher than that of the corn oil group (P0.05).2. water maze. The incubation period was higher than that of the control group (P0.05); the average escape latency of the EGCG intervention group and the VitE intervention group was lower than that of the saline group and the corn oil group (P0.05). Compared with the single intervention group, the average escape latency decreased (P0.05) in the combined intervention group (1) in the prepregnancy group, and the average escape of the combined intervention group (1) and (3) in the breastfeeding lead dyed lead group. The number of crossing platforms in the saline group and the corn oil group were significantly lower than that of the control group (P0.05) in the preconception and lactation period (P0.05). The number of crossing platforms increased significantly (P0.05) compared with the saline group and the corn oil group (1) and (3) in the combined intervention group (P0.05). The blood lead content of the crossing platform (P0.05).3. showed that the blood lead content of the normal saline group and the corn oil group was significantly higher than that of the control group (P0.05), the blood lead content in the EGCG intervention group and the three combined intervention group was lower than that of the normal saline group (P0.05), and the blood lead content in the three combined intervention group was more than that of the control group. The blood lead content of the group was lower than that of the corn oil group (P0.05), but there was no significant difference in the blood lead content between the single intervention group and the combined intervention group (P0.05). The results of.4. oxidative stress showed that the activity of SOD was significantly decreased (P0.05) and the content of MDA significantly increased (P0.05) compared with the control group. Compared with the normal saline group and the corn oil group, the activity of SOD increased (P0.05) and the content of MDA decreased (P0.05) in the individual intervention group and the three combined intervention group. The combined intervention group was compared with the single intervention group, and the SOD activity in the combined intervention group (1) and (3) increased (P0.05), and the MDA content was not statistically significant (P0.05).5.A beta (1-40), and A beta (1-42) content results. The contents of A beta (1-40) and A beta (1-42) in the normal saline group and the corn oil group were significantly higher than those in the control group (P0.05). Compared with the normal saline group and the corn oil group, the content of A beta (1-40) and the A beta (1-42) in the single intervention group and the combined intervention group were lower (P0.05). Compared with the single intervention group, the lactation period lead dye group was compared with the single intervention group. In the combined intervention group (3), the content of A beta (1-42) was lower than that of the VitE intervention group (P0.05).6.APP, and the expression level of BACE1 protein showed that the level of BACE1 egg white in the normal saline group, the corn oil group and the intervention group APP, and the BACE1 egg white level increased significantly (P0.05), and the different intervention groups and the saline group and the jade were compared with the control group. Compared with the rice oil group, the level of APP and BACE1 protein decreased significantly (P0.05), and the level of APP and BACE1 protein decreased significantly in the combined intervention group compared with the single intervention group (P0.05). Conclusion the lead exposure in the prepregnancy or lactation period of the 1. female mice could lead to the increase of blood lead content in the mice, the growth and development of mice and the ability of learning and memory in mice. To increase the level of oxidative stress in hippocampus, induced overexpression of APP and BACE1 protein in hippocampus, thus increasing A beta (1-40) and A beta (1-42) level.2.EGCG, VitE and EGCG and VitE can improve the decline of learning and memory ability in mice induced by lead exposure, and reduce A beta (1-40) and A beta (1-42) in the hippocampus of the AD like mice. The level of APP and BACE1 protein was decreased, and the combined intervention at appropriate dose was better than using EGCG or VitE. alone.
【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R749.16

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