具有潜在治疗阿尔茨海默病作用的3种中药成分的药效学观察及其机制研究

发布时间：2018-05-14 23:22

本文选题：具有 + 潜在　；参考：《北京协和医学院》2016年硕士论文

【摘要】：阿尔茨海默病(Alzheimer's disease, AD)是一种常见的中枢神经系统退行性疾病,以记忆力减退、认知功能障碍为主要临床症状,以脑内β-淀粉样多肽(p-amyloid peptide, Ap)沉积形成的纤维斑块(即老年斑),并伴有细胞内的神经纤维缠结(neurofibrillary tangle, NFT)为主要病理改变。Aβ多肽有39-42个氨基酸,由β淀粉样前体蛋白(APP)经p-分泌酶和γ-分泌酶水解后产生的多肽。APP的裂解过程有二种途径：①非淀粉样途径：APP经α-分泌酶水解,产生非淀粉样可溶小分子蛋白sAPPa和CTFa (C端片段),CTFa经γ-分泌酶裂解产生3KD的小分子P3和APP胞内区域的羧基末端(APP intracellular cytoplasmic domain, AICD),该途径不形成Ap。②淀粉样途径： APP经p-分泌酶裂解,产生可溶性sAPPβ和CTFβ, CTFβ经γ-分泌酶裂解产生可聚集、具有神经毒性的Ap。研究表明,Ap聚集和沉积形成的纤维斑块可引发脑内氧化应激及炎症等反应,导致神经元的坏死和凋亡,最终可引起AD。目前国内外用于治疗AD的药物主要为化学合成类药物如胆碱酯酶抑制剂等,但其作用局限且治标不治本。中草药的应用布我国有着悠久的历史,其对一些慢性疾病有很好疗效且几乎无副作用等特点。近年来研究表明,中药成分天麻素、黄芪总皂苷和芍药苷具有抗氧化、增加脑血流量、改善记忆力、神经保护等作用。但其是否对AD的治疗具有特异性及其具体作用的途径和相关机制尚不明确。为探讨以上问题,本课题主要研究内容为三种中药成分天麻素、黄芪总皂苷和芍药苷对阿尔茨海默病模型小鼠的药效学作用,同时利用双转基因细胞模型研究其可能的作用途径及相关机制。本课题研究内容主要包括二个部分：1.三种中药成分在转基因模型鼠体内抑制Ap斑块的药效学作用；2.三种中药成分在体外抑制Ap的作用途径及其相关机制。第一部分：三种中药成分对AD转基因模型鼠体内的药效学作用的研究目的：研究3种中药成分(天麻素(GAS)、黄芪总皂苷(AST)、芍药苷(PAE))对AD转基因模型5XFAD鼠学习认知和记忆能力的影响和抑制脑内Aβ斑块的效果,并观察3种中药成分的干预是否对小鼠的生长发育有影响。方法：选用1.5月龄的APP/PS1双转基因模型小鼠,随机分为7组,另取同窝阴性小鼠作为正常对照,每组10只。 (1)阳性对照组(Tg)：转基因阳性小鼠给予不含药物的生理盐水；(2)阴性对照组(Control):同窝阴性小鼠给予不含药物的生理盐水； (3)天麻素高剂量组(GAS-H)转基因阳性小鼠给予含300mg/kg/d天麻素的生理盐水(4)天麻素低剂量组(GAS-L):转基因阳性小鼠给予含150mg/kg/d天麻素的生理盐水(5)黄芪总皂苷高剂量组(AST-H):转基因阳性小鼠给予含160mg/kg/d黄芪总皂苷的生理盐水(6)黄芪总皂苷低剂量组(AST-L):转基因阳性小鼠给予含80mg/kg/d黄芪总皂苷的生理盐水(7)芍药苷高剂量组(PAE-H):转基因阳性小鼠给予含60mg/kg/d芍药苷的生理盐水(8)芍药苷低剂量组(PAE-L):转基因阳性小鼠给予含30mg/kg/d芍药苷的生理盐水。每周测量各组小鼠体重及监测小鼠的日均饮水量,按照体重及日均饮水量调整给药剂量。给药4个月后,通过Morris水迷宫行为学实验检测小鼠学习记忆及认知能力的变化；免疫组织化学染色法观察脑组织内Aβ斑块的变化；ELISA法检测小鼠脑组织匀浆中Aβ含量的水平；另外为评价药物对小鼠的是否具有肝肾毒副作用,ELISA法检测小鼠血清中谷草转氨酶、谷丙转氨酶、肌酐的变化及小鼠脑组织匀浆中IFN-γ和IL-4水平的变化。结果：1Morris水迷宫结果显示,与阳性对照组(Tg)组相比,用药各组小鼠平台寻找时间明显缩短,在原平台象限停留时间明显延长：第5天时,天麻素高剂量组(GAS-H)和低剂量组(GAS-L)平台寻找时间分别缩短83.66%和46.72%(P0.01)；,黄芪总皂苷高剂量组(AST-H)和低剂量组(AST-L)平台寻找时间分别缩短41.60%和23.78%(P0.01)；芍药苷高剂量组(PAE-H)和低剂量组(PAE-L)平台寻找时间分别缩短48.23%和29.31%(P0.01)；天麻素高剂量组(GAS-H)和低剂量组(GAS-L)在原目标象限停留时间分别延长2.28倍和1.83倍；黄芪总皂苷高剂量组(AST-H)和低剂量组(AST-L)在原目标象限停留时间分别延长1.88倍和1.38倍(P0.01)；芍药苷高剂量组(PAE-H)和低剂量组(PAE-L)在原目标象限停留时间分别延长1.83倍和1.62倍(P0.01)。2.免疫组化结果显示：5XFAD鼠的Tg组脑内海马和皮质区可见明显的大小不一、棕黄色的Ap斑块沉积,呈弥漫分布。GAS和AST及PAE各组与№组相比,脑内海马和皮质区Ap斑块呈散在分布,斑块体积和数目显著减少、染色变浅。Control组脑内未见Aβ斑块。IPP软件分析结果显示：与阳性对照组相比,3种中药成分均能够降低模型小鼠脑部海马区和皮层区Aβ斑块阳性面积。在海马区,天麻素高剂量组(GAS-H)阳性面积降低最为显著达70.13%；天麻素低剂量组(GAS-L)降低达39.25%(P0.01)；黄芪高剂量组(AST-H)和黄芪低剂量组(AST-L) Aβ斑块阳性面积分别降低62.39%和31.51%(P0.05)；芍药苷高剂量组(PAE-H)和芍药苷低剂量组(PAE-L) Aβ斑块阳性面积分别降低57.58%和30.52%(P0.05)。在皮层区,天麻素高剂量组(GAS-H)降低最为显著达80.96%；天麻素低剂量组(GAS-L)降低达36.67%；黄芪高剂量组(AST-H)和黄芪低剂量组(AST-L) Aβ斑块阳性面积分别降低69.57%和37.90%(P0.01)；芍药苷高剂量组(PAE-H)和芍药苷低剂量组(PAE-L) Aβ斑块阳性面积分别降低66.62%不口27.62%(P0.05)。3. ELISA检测小鼠脑匀浆中Aβ含量的结果显示,与Tg组脑匀浆Aβ的平均OD值相比,AST、GAS和PAE各组均有不同程度降低。其中,天麻素高剂量组(GAS-H)降低效果最为显著达62.18%；天麻素低剂量组(GAS-L)降低达35.94%；黄芪高剂量组(AST-H)和黄芪低剂量组(AST-L)小鼠脑匀浆中Aβ分别降低51.58%和24.07%(P0.01)；芍药苷高剂量组(PAE-H)和芍药苷低剂量组(PAE-L)小鼠脑匀浆中Aβ分别降低51.02%和14.16%(P0.05)。4. ELISA法检测小鼠脑组织匀浆中细胞因子水平的变化的结果显示,用药各组小鼠(天麻组小鼠、黄芪总皂苷组小鼠及芍药苷组小鼠)与阳性对照组小鼠相比,其脑组织匀浆中IFN-γ和IL-4的含量均无明显变化(P0.05)。5. ELISA法检测小鼠血清中谷草转氨酶、谷丙转氨酶、肌酐的结果显示,用药各组小鼠(天麻组小鼠、黄芪总皂苷组小鼠及芍药苷小鼠)与阳性对照组小鼠相比,其血清中谷草转氨酶、谷丙转氨酶、肌酐的含量均无明显变化(P0.05)。6.测量各组小鼠的体重变化的结果显示,在不同的时间点(1,2,3,4月),阴性对照小鼠,阳性对照小鼠及用药各组小鼠的体重无明显变化(P0.05)。第二部分：三种中药成分抑制模型细胞中Ap的作用途径及相关机制的研究目的：利用双基因转染细胞模型117细胞(过表达Ap和p-分泌酶)和146细胞(过表达Ap和γ-分泌酶),研究三种中药成分天麻素、黄芪总皂苷和芍药苷抑制Ap的作用机制。方法：天麻素、黄芪总皂苷和芍药苷以不同的浓度(10、25、50、100μmol·L-1)作用于117模型细胞和146细胞,ELISA双抗夹心法分别检测细胞内外Ap含量变化,Q-PCR法检测117细胞BACE mRNA和146细胞PS1 mRNA转录水平,Wsetern-blot法检测p-分泌酶和γ-分泌酶蛋白水平的变化。同时,CCK-8试剂盒检测三种中药成分对细胞活力的影响,流式细胞术检测细胞凋亡情况。结果：1. ELISA法检测结果显示,与未干预对照组相比,三种中药成分天麻素、黄芪总皂苷、芍药苷对117细胞模型和146细胞模型内外Aβ水平均有降低,且降低效果呈药物浓度依赖性。天麻素、黄芪总皂苷、芍药苷抑制117细胞内Ap最高分别达69.2%、64.8%和47.9%(P0.01),抑制117细胞外Ap最高分别达65.8%、67.8%和25.2%(P0.05)；天麻素、黄芪总皂苷、芍药苷抑制146细胞内Aβ最高分别达67.9%、82.6%和49.9%(P0.01),天麻素、黄芪总皂苷、芍药苷抑制146细胞外Aβ最高分别达67.1%、65.4%和50.9%(P0.01)。2. Q-PCR检测实验结果显示,天麻素对117细胞p-分泌酶转录水平有降低作用。与天麻素未干预对照组相比,经天麻素处理24 h的117细胞,BACE mRNA转录水平降低,且降低效果呈浓度依赖性。其中,25μmol·L-1天麻素剂量组降低22.8%,50和100μmol·L-1天麻素剂量组分别降低30.7%和32.9%(P0.01)。未观察到芍药苷、黄芪总皂苷对117细胞BACE mRNA转录水平有影响；天麻素、黄芪总皂苷、芍药苷三种中药成分对146细胞内γ-分泌酶转录水平均无影响。3. Western Blotting检测实验结果显示,天麻素在25、50、100μmol·L-1浓度均可降低β-分泌酶蛋白的表达,且在50μmol·L-1时降低最明显为47.9%(P0.01)。黄芪总皂苷、芍药苷均对β-分泌酶蛋白的表达无影响。天麻素、黄芪总皂苷、芍药苷均对γ-分泌酶蛋白表达均无影响。4.CCK-8法测定结果均显示,天麻素、黄芪总皂苷和芍药苷各自干预117细胞和146细胞24h后,各药物干预组细胞活性相对于未干预对照组无明显变化；且流式细胞术检测结果显示,天麻素、黄芪总皂苷和芍药苷干预117细胞24h后,各干预组的凋亡率与未干预组相比,未有差异变化。总结：长期以来对中药治疗AD是否具有特异性或特效性,如有特异性其作用途径和作用机制是什么,一直都不清楚。本课题在这方面做了探索研究,初步结果显示天麻素、黄芪总皂苷和芍药苷对AD的治疗均具有特异性,其作用途径是通过抑制Ap产生并减少Ap斑块形成,进而减少老年斑对神经细胞的毒性作用,抑制AD的病程发展。其中天麻素的作用最显著,其作用机制可能与抑制β-分泌酶的表达有关。
[Abstract]:In recent years , it has been shown that there are two ways to treat AD : ( 1 ) the effect of three traditional Chinese medicinal components gastrodin , total saponins of astragalus root and peony glycoside on Alzheimer ' s disease model mice .
2 . The effect of three traditional Chinese medicine components on learning cognition and memory ability of AD transgenic model mice was studied . The effects of three traditional Chinese medicine components ( gastrodin ( GAS ) , total saponin ( AST ) , and penonitin ( PAE ) on learning cognition and memory ability of AD transgenic mice were studied .
( 2 ) negative control group ( control ) : the same socket - negative mice were given no drug - containing physiological saline ;
( 3 ) Normal saline ( 4 ) gastrodin low - dose group ( GAS - L ) containing 300 mg / kg / day gastrodin was administered to transgenic male mice .
Immunohistochemical staining was used to observe the changes of A尾 plaque in brain tissue .
The level of A尾in brain homogenate was determined by ELISA .
The results were as follows : Compared with the positive control group ( Tg ) group , the results showed that the time of finding the platform was significantly shortened compared with that of the positive control group ( Tg ) group , and the residence time of the mice in the original platform was obviously prolonged : the time of finding time of the GAS - H and GAS - L platforms was shortened by 83.66 % and 46.72 % ( P0.01 ) .
The finding time of AST - H and AST - L was 41.60 % and 23.78 % respectively ( P0.01 ) .
The finding time of high - dose group ( PAE - H ) and low - dose group ( PAE - L ) was 48.23 % and 29.31 % ( P0.01 ) .
The residence time of GAS - H and GAS - L was 2.28 - fold and 1.83 - fold longer than those in low - dose group ( GAS - L ) .
In the high dose group ( AST - H ) and low - dose group ( AST - L ) , the retention time of AST - H and AST - L in the original target quadrant increased by 1 . 88 - fold and 1.38 - fold respectively ( P0.01 ) .
Compared with the positive control group , there was a significant decrease in the size and the number of plaques in the hippocampus and cortex of the brain , and the positive area of A尾plaques in the hippocampus and cortex area of the model mice was reduced .
The level of GAS - L decreased by 39.25 % ( P0.01 ) .
The positive rates of AST - H and AST - L were 62.39 % and 31.51 % , respectively ( P0.05 ) .
The positive area of PAE - H and PAE - L was 57.58 % and 30.52 % , respectively ( P0.05 ) .
The level of GAS - L decreased by 36.67 % .
The positive rates of AST - H and AST - L were 69.57 % and 37.90 % , respectively ( P0.01 ) .
The positive rates of PAE - H and PAE - L were 66.62 % and 27.62 % respectively ( P0.05 ) . The results showed that the levels of A尾in brain homogenate of mice showed that AST , GAS and PAE decreased in different degrees compared with the average OD value of the rat brain homogenate A 尾 . The results showed that the decrease of GAS - H in the high dose group ( GAS - H ) was the most significant ( 62 . 18 % ) .
The level of GAS - L decreased by 35.94 % .
In high dose group ( AST - H ) and low dose of Astragalus membranaceus ( AST - L ) , the levels of A尾 in brain homogenate decreased by 51.58 % and 24.07 % ( P0.01 ) .
In the high dose group ( PAE - H ) and low dose group ( PAE - L ) , the concentration of A尾 in brain homogenate decreased by 51.02 % and 14.16 % ( P0.05 ) . The results showed that the levels of IFN - 纬 and IL - 4 in brain homogenate of mice were not significantly higher than those in control group ( P0.05 ) . The results showed that there were no significant changes in the contents of glutamic oxalacetic transaminase , alanine aminotransferase and creatinine in serum of mice ( P0.05 ) . The results showed that , compared with the control group , the levels of A尾in 117 cell models and 146 cell models were significantly lower than those of untreated control group , and the results showed drug concentration - dependent . The maximum of Ap in 117 cells was 69.2 % , 64.8 % and 47.9 % ( P0.01 ) .
The highest levels of A尾in 146 cells were 67.9 % , 82.6 % and 49.9 % ( P0.01 ) , gastrodin , astragalus root total saponin , and paeonia were up to 67.1 % , 65.4 % and 50.9 % ( P0.01 ) respectively . Compared with the control group , the level of BACE mRNA transcription was decreased by 30 . 7 % and 32.9 % ( P0.01 ) .
There were no significant effects on the levels of 纬 - secreted enzymes in 146 cells . The results of Western Blotting test showed that the expression of 尾 - secreted enzyme was decreased at 25 , 50 and 100 渭mol 路 L - 1 , and the most obvious decrease was 47.9 % at 50 渭mol 路 L - 1 ( P0.01 ) .
The results showed that the effect of gastrodin , astragalus root saponins and penonidatin on AD treatment was not clear . The results showed that the effect of gastrodin , astragalus root total saponin and penonidatin on AD was not clear . The effect of gastrodin was most significant by inhibiting Ap production and reducing Ap plaque formation , and the mechanism of action of gastrodin could be related to the inhibition of 尾 - secretion .

【学位授予单位】：北京协和医学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R749.16

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