淫羊藿、黄芪、葛根有效成分组方对阿尔茨海默病脑铁超载的干预作用及其机制研究
发布时间：2018-07-30 07:03
【摘要】：研究表示AD患者的脑内铁水平异常升高,这可能与脑组织内铁代谢及蛋白表达异常,脑内自由基和炎症因子水平升高,外周铁代谢失衡及脏器功能衰退有关。降低脑铁的含量是防治AD的一个方法之一。铁螯合剂如去铁胺可以明显改善AD的临床症状,但其副作用也十分的明显,且口服效果较差,因此吸收好,生物利用度高的中药成为治疗AD铁超载的首选,具有巨大的潜力。中医认为AD是本虚标实之症,其中肾虚精亏,气血不足为本,痰瘀互结,浊毒内生为标。因此,AD的中医治疗应以补肾健脾,益气养血为本。淫羊藿具补肾填精、生精养髓之功效,黄芪具补气健脾、行血化瘀之功效,葛根具有清热排毒之功效三药合用,共同作用,起滋补肝肾、补脾养血、活血化瘀、清热解毒、健脑益智的功效。从淫羊藿,黄芪,葛根提取的有效成分具有清除自由基,降低炎症反应等多个功效,可以从多方面抑制铁超载,扬长避短。因此本课题以淫羊藿,黄芪,葛根有效成分进行组方对AD脑铁超载的干预和作用切入,为中药防治AD提供科学的依据。现代药理对这三种成分治疗AD的机制进行了大量研究,但多数从神经递质含量,氧化应激水平,Aβ代谢等方面着手,对脑铁超载关注不足。因此,本课题以铁代谢为切入点,以补肾补精,祛痰化瘀,补血益气为原则,将淫羊藿,黄芪,葛根三种药物的有效成分进行组方,以APP/PS1双转基因小鼠为动物模型,观察药物对动物模型的治疗疗效,以及脑铁的干预效果,并深入探讨其可能的机制,为AD的药物治疗开辟一个新的方向,为中药防治AD提供科学的依据。第一部分淫羊藿、黄芪、葛根有效成分组方对阿尔茨海默病转基因小鼠脑铁含量的氧化应激、炎性因子水平影响的研究目的:观察淫羊藿,黄芪,葛根及其有效成分组方对APP/PS1双转基因小鼠脑铁水平以及氧化应激、炎性因子水平的影响。方法:六月龄雄性app/ps1双转基因小鼠60只,随机分为模型组,复方组,淫羊藿组,黄芪组,葛根组,dfo组,其中复方组灌胃给予淫羊藿苷,黄芪甲苷,葛根素组方,淫羊藿组灌胃给予淫羊藿苷,黄芪组灌胃给予黄芪甲苷,葛根组灌胃给予葛根素,dfo组灌胃给予dfo(去铁胺),以上灌胃时长均为三个月。c57bl/6j小鼠10只作为阴性对照组,模型组及对照组给与等量生理盐水灌胃3个月。灌胃结束后,灌流后快速取出大脑皮层及海马,一侧皮层用火焰原子吸收法测定铁元素含量,海马用等离子质谱法(icp-ms)测定铁元素含量,另一侧皮层用生化法测定氧化因子(sod、mda、gsh-px)及炎性因子(il-1β、il6、tnf-α)的含量。结果:火焰原子吸收法结果显示模型组小鼠与对阴性照组小鼠相比皮质铁含量明显增加(p0.05),复方组小鼠与模型组小鼠相比皮质铁含量明显下降(p0.05);等离子质谱法(icp-ms)显示模型组小鼠与对阴性照组小鼠相比海马铁含量明显增加(p0.05),复方组小鼠与模型组小鼠相比海马铁含量明显下降(p0.05);elisa法结果显示模型组小鼠与对阴性照组小鼠相比皮质il-1β、il6、tnf三者含量明显增加(p0.05),复方组小鼠与模型组小鼠相比皮质il-1β、il-6、tnf三者含量明显下降(p0.05);模型组小鼠与对阴性照组小鼠相比皮质mda含量明显增加(p0.05),复方组小鼠与模型组小鼠相比皮质mda含量明显下降(p0.05)。模型组小鼠与对阴性照组小鼠相比皮质sod、gsh-px两者含量明显下降(p0.05),复方组小鼠与模型组小鼠相比皮质sod、gsh-px两者含量明显增加(p0.05)。第二部分淫羊藿、黄芪、葛根有效成分组方对阿尔茨海默病转基因小鼠血清铁、外周组织器官铁水平及氧化应激水平的影响目的:本实验以app/ps1双转基因小鼠为动物模型,来观察淫羊藿,黄芪,葛根有效成分组方对小鼠血清铁、外周组织器官铁水平及氧化应激水平的影响,进而探讨对淫羊藿、黄芪、葛根有效成分组分对脑铁干预是否与改善外周铁代谢失衡及脏腑功能衰退相关。方法:雄性的六月龄雄性app/ps1双转基因小鼠60只,随机分为模型组,复方组,淫羊藿组,黄芪组,葛根组,dfo组,其中复方组灌胃给予淫羊藿苷,黄芪甲苷,葛根素组方,淫羊藿组灌胃给予淫羊藿苷,黄芪组灌胃给予黄芪甲苷,葛根组灌胃给予葛根素,dfo组灌胃给予dfo(去铁胺),以上灌胃操作1次/日,时长均为三个月。中药淫羊藿,黄芪,葛根3种药物的有效成分按照比例3:2:2,即淫羊藿苷120mg/kg,黄芪甲苷80mg/kg,,葛根素80mg/kg体重给予小鼠灌胃。c57bl/6j小鼠10只作为阴性对照组,模型组及对照组给与等量生理盐水灌胃3个月。采用火焰原子吸收光谱技术测定肝脏,肾脏,心脏,脾脏和肺脏的铁含量。酶联免疫吸附试验法(elisa)检测小鼠血清铁(si),铁蛋白(fn)和转铁蛋白(tf)水平。生化试剂盒检测小鼠肝脏组织,肾脏组织,心脏组织,肺脏组织丙二醛(mda)含量,超氧化物歧化酶(sod)活力,谷胱甘肽过氧化物酶(gsh-px)活力。结果:火焰原子吸收光谱技术显示,与对照组小鼠相比,模型组小鼠外周铁含量明显升高(p0.05);与ad模型组相比,黄芪组、葛根组、复方组和dfo组铁含量降低(p0.05);复方组与各单方组各组小鼠相比,铁含量明显降低(p0.05),dfo组与复方组无明显差异(p0.05)。结果提示app/ps1双转基因小鼠肾脏组织,脾脏组织,心脏组织和肺脏组织等外周组织内存在铁超载,复方组可以显著降低各组织器官的铁含量,其效果优于单方组。与对照组小鼠相比,ad模型组小鼠si和fn含量明显升高(p0.05),tf水平明显下降;与ad模型组相比,复方组和dfo组si和fn含量降低,tf水平升高;复方组与各单方组各组小鼠相比,si和fn含量降低,tf水平升高,表明复方效果优于单方;dfo组与复方组无明显差异(p0.05)。为了深入探讨淫羊藿、黄芪、葛根有效成分组方对ad双转基因模型小鼠外周组织抗氧化能力的影响,本部分实验分别检测了ad模型双转基因小鼠肝脏组织,肾脏组织,心脏组织和肺脏组织内的sod、mda、gsh-px含量。与对照组小鼠相比,ad模型组肝脏、肾脏、心脏sod、gsh-px活力均有所下降,mda含量有所上升(p0.05);与ad模型组相比,淫羊藿组、黄芪组、葛根组、复方组和dfo组肝脏、肾脏、心脏中sod、gsh-px活力均有所升高(p0.05),mda含量有所降低,复方组与各单方组各组小鼠相比,sod、gsh-px活力均有所升高(p0.05),mda含量明显降低(p0.05),复方组优于单方组;dfo组与复方组无明显差异(p0.05)。第三部分淫羊藿、黄芪、葛根有效成分组方对阿尔茨海默病转基因小鼠血清epo及红细胞生成的影响目的:本实验以app/ps1双转基因小鼠为动物模型,来观察淫羊藿,黄芪,葛根有效成分组方对小鼠血清epo生成及红细胞的影响,并对其引起脑铁超载的可能机制进行探讨。方法:给药处理同前部分实验。分为模型组,复方组,淫羊藿组,黄芪组,葛根组,dfo组,c57bl/6j小鼠作为阴性对照组。从各组10只小鼠中随机选取4只,20%乌拉坦腹腔麻醉后快速摘眼球取血约1ml,静置后,冰上静置后4℃,3000r/min,离心15min,得血清,分装后-80℃保存用于小鼠血清促红细胞生成素(epo)的检测。各组另取小鼠3只,麻醉后快速摘眼球取血至加有肝素抗凝的ep管中,上下颠倒混匀,低温保存,迅速进行血常规检测。结果:与对照组小鼠相比,ad模型组小鼠epo含量明显降低(p0.05),结果提示app/ps1双转基因小鼠血清中存在epo生成减少。与ad模型组相比,淫羊藿组,黄芪组、葛根组、复方组和dfo组脑epo含量明显升高(p0.05),复方组和dfo组与各单方组各组小鼠相比,epo含量上升明显(p0.05)。小鼠血常规红细胞(rbc)检测结果显示:与对照组小鼠相比,ad模型组小鼠红细胞数量明显减少(p0.05),与ad模型组相比,淫羊藿组,黄芪组、葛根组、复方组和dfo组红细胞数量有所增加(p0.05),复方组和dfo组与各单方组各组小鼠相比,红细胞数量有明显增加(p0.05);复方组和dfo组相比无明显差异(p0.05)。血常规血红蛋白(hb)检测结果显示:淫羊藿组,黄芪组、葛根组、复方组和dfo组与ad模型组相比血红蛋白含量有所增加(p0.05),其中复方组和dfo组明显增加(p0.05),与c57组小鼠相比,ad模型组小鼠血红蛋白含量明显减少(p0.05)。红细胞压积(hct)结果显示,相比对照组,模型组小鼠的hct/百分比减少(p0.05),与模型组相比,复方组hct水平明显上升(p0.05)。ad模型组小鼠白细胞(wbc)数量增加(p0.05),淫羊藿组,黄芪组、葛根组、复方组和dfo组白细胞数量有所降低(p0.05)。其中复方组和dfo组相比无明显差异,使白细胞数量下降的较为明显。结果提示,ad模型小鼠体内有炎症的存在,经药物治疗后体内炎性水平好转。第四部分淫羊藿、黄芪、葛根有效成分组方对铁超载阿尔茨海默病转基因小鼠血脂的影响目的:研究淫羊藿,黄芪,葛根三者有效成分组方对血脂的影响,并进一步探讨血脂代谢紊乱对脑铁升高的影响。方法:六月龄雄性app/ps1双转基因小鼠,随机分为模型组,复方组,淫羊藿组,黄芪组,葛根组,dfo组,其中复方组灌胃给予淫羊藿,黄芪,葛根组方,淫羊藿组灌胃给予淫羊藿,黄芪组灌胃给予黄芪,葛根组灌胃给予葛根,dfo组灌胃给予dfo(去铁胺),以上灌胃时长均为三个月。c57bl/6j小鼠作为阴性对照组,模型组及对照组给与等量生理盐水灌胃3个月。各组随机选取3只,末次给药结束后,禁食禁水16小时,20%乌拉坦麻醉摘眼球取血后,4℃,3000r/min,离心15分钟获得血清后,用全自动生化分析仪测得各组的甘油三酯(tg),总胆固醇(tc),高密度脂蛋白(hdl)低密度脂蛋白(ldl)的含量。采用rt-pcr对肝脏hepcidinmrna表达进行测定。结果:与对照组小鼠相比,模型组小鼠tg和ldl含量明显增加(p0.05),hdl含量有所下降(p0.05),tc含量无明显变化(p0.05);与模型组小鼠相比,复方组小鼠tg和ldl含量明显下降,hdl含量有所增加(p0.05),tc含量无明显变化(p0.05)。结论:app/ps1双转基因小鼠的海马及皮质的铁水平均显著升高,血清铁水平升高,血清铁转运能力降低,外周器官组织内存在铁超载,小鼠体内炎性因子水平及自由基含量均升高,血清epo水平下降,血常规异常,血脂异常。应用淫羊藿,黄芪,葛根有效成分组方可以有效的改善转基因小鼠脑铁超载现象,使其脑内的炎症水平降低,氧化反应减少,且降低了外周铁含量,有效改善血管及外周器官的氧化损伤,提高AD双转基因模型小鼠血清EPO的水平,增加RBC数量,Hb和HTC含量增加,促进铁利用,降低铁超载,降低血脂,从而达到抗动脉粥样硬化,改善血液循环,防止外周铁沉积进而改善脑铁超载的作用。
[Abstract]:The abnormal increase in iron levels in the brain of AD patients may be related to the abnormal iron metabolism and protein expression in the brain, the increase of free radicals and inflammatory factors in the brain, the imbalance of peripheral iron metabolism and the deterioration of organ function. The reduction of iron content in the brain is one of the ways to prevent and control AD. Iron chelating agent, such as iron amine, can obviously improve AD. The clinical symptom, but its side effect is also very obvious, and the oral effect is poor, so good absorption, high bioavailability of traditional Chinese medicine has become the first choice for the treatment of AD iron overload, with great potential. Traditional Chinese medicine thinks that AD is the symptom of this deficiency, among which the deficiency of kidney is deficient, Qi and blood is insufficient, phlegm and blood stasis, and the endogeny of turbidity and poison is the standard. Therefore, the TCM treatment of AD It should make up the kidney and invigorate the spleen, nourish the blood and nourish the blood as the basis. The herba Epimedium has the efficacy of nourishing the kidney and nourishing the marrow. The Astragalus membranaceus has the effect of enriching the spleen and invigorating the spleen, making the blood stasis and removing blood stasis. The Pueraria has three drugs combined with the efficacy of clearing heat and detoxification. It plays the function of nourishing the liver and kidney, nourishing the spleen and nourishing blood, promoting blood circulation and removing stasis, clearing heat and detoxifying the brain and improving the wisdom of the brain. Extract from Epimedium, Astragalus Astragalus and pueraria lobata The effective components of the active ingredients can remove the free radical and reduce the inflammatory reaction and so on. It can inhibit the overloading of iron from many aspects, so the effective components of epimedium, Astragalus membranaceus and pueraria lobata are used for the intervention and effect of the AD brain iron overload, which provides scientific basis for the prevention and treatment of AD by traditional Chinese medicine. The mechanism of AD treatment has been a great deal of research, but most from the content of neurotransmitters, the level of oxidative stress, A beta metabolism and so on, it is not enough to pay attention to the overloading of the brain iron. Therefore, this subject takes iron metabolism as the breakthrough point, with the principle of supplementing the kidney, removing phlegm and removing blood stasis, enriching blood and enriching qi, and organizing the effective components of the three kinds of drugs, such as epimedium, astragalus, and pueraria lobata. APP/PS1 double transgenic mice were used as animal models to observe the therapeutic effect of drugs on animal models and the effect of cerebral iron intervention, and to explore the possible mechanism in order to open a new direction for the drug treatment of AD and to provide scientific basis for the prevention and control of AD by traditional Chinese medicine. Study on the effects of oxidative stress on iron content in the brain of transgenic mice with Alzheimer's disease and the effect of inflammatory factors: the effects of epimedium, Astragalus membranaceus, pueraria root and its effective components on the level of iron brain, oxidative stress and inflammatory factors in APP/PS1 double transgenic mice. Methods: 6 month old male app/ps1 double transgenic mice were randomly divided into two groups. The model group, the compound group, the Epimedium group, the Astragalus group, the puerarin group and the DFO group, of which the compound group was given the icariin, astragaloside, puerarin group, the Epimedium group gavage with icariin, the Astragalus group gavage with Huang Qijia glycoside, the GAGG group gavage puerarin, and the group DFO was given DFO (de ferric amine), and the length of the gavage was three months.C. 10 57bl/6j mice were used as negative control group. The model group and the control group were given the same amount of saline for 3 months. After the end of the gavage, the cerebral cortex and hippocampus were quickly removed. The iron content was measured by flame atomic absorption spectrometry. The content of iron was determined by plasma mass spectrometry (ICP-MS) in the hippocampus, and the other side of the cortex was used for biochemistry. Results: the contents of SOD, MDA, GSH-Px and inflammatory factors (IL-1 beta, IL6, tnf- alpha) were measured. Results: the results of flame atomic absorption spectrometry showed that the content of iron in the model group was significantly increased compared with the negative group (P0.05), and the content of iron in the compound group was significantly lower than that in the model group (P0.05), and the plasma mass spectrometry (icp-m) (icp-m). S) showed that the iron content in the hippocampus of the model mice was significantly increased compared with the negative mice (P0.05). The content of iron in the hippocampus of the compound group mice was significantly lower than that in the model group (P0.05). The results of the ELISA method showed that the contents of the model mice and the negative group were significantly increased (P0.05) in the cortex of IL-1 beta, IL6 and TNF three (P0.05), and the mice in the compound group were compared with those in the negative group. Compared with the model group, the content of IL-1 beta, IL-6, TNF three decreased significantly (P0.05), and the cortical MDA content of the model mice was significantly increased (P0.05) compared with the negative group mice (P0.05). The content of the cortical MDA in the compound group mice was significantly lower than that in the model group (P0.05). The mice in the model group and the negative irradiated mice were compared with the cortical SOD and GSH-Px. The content of the compound group mice was significantly increased (P0.05). The contents of SOD and GSH-Px in the mice were significantly increased (P0.05). The effect of the second part of epimedium, Astragalus membranaceus and Radix Puerariae on the serum iron, the level of iron and oxidative stress in the peripheral tissues of transgenic mice with Alzheimer's disease The gene mice were animal models to observe the effect of the effective components of epimedium, Astragalus membranaceus and pueraria lobata on the iron levels and the level of iron and oxidative stress in the peripheral tissue, and then to explore whether the intervention of the effective components of epimedium, astragalus, and pueraria lobata on the imbalance of the peripheral iron metabolism and the function decline of the viscera was related to the effect of the effective components of epimedium, astragalus, and pueraria. 60 male app/ps1 male app/ps1 double transgenic mice were randomly divided into model group, compound group, Epimedium group, Astragalus group, puerarin group, group DFO, of which the compound group gavage was given icariin, astragaloside, puerarin group, Epimedium group gavage to icariin, Astragalus group gavage to astragaloside, puerarin group gavage to puerarin, D Group fo was given DFO (de ferric amine) for 1 times a day for three months. The effective components of the 3 drugs of epimedium herb, Astragalus membranaceus and pueraria lobata were in proportion to 3:2:2, namely, icariin 120mg/kg, astragaloside 80mg/kg, and 80mg/kg weight of puerarin to mice,.C57bl/6j mice were given as negative control group, model group and control The content of iron in liver, kidney, heart, spleen and lung was measured by flame atomic absorption spectroscopy for 3 months. The serum iron (SI), ferritin (FN) and transferrin (TF) level of mice were detected by enzyme linked immunosorbent assay (ELISA). The liver tissue, kidney tissue and heart tissue of mice were detected by biochemical kit. The content of malondialdehyde (MDA), superoxide dismutase (SOD) activity and glutathione peroxidase (GSH-Px) activity in lung tissues. Results: flame atomic absorption spectrometry showed that the content of peripheral iron in the model group was significantly higher than that of the control group (P0.05). Compared with the ad model group, the content of iron in the Astragalus group, the Pueraria group, the compound group and the DFO group decreased. The iron content of the compound group was significantly lower (P0.05) than the mice in each group (P0.05), and there was no significant difference between the DFO group and the compound group (P0.05). The results suggested that the renal tissue, the spleen tissue, the heart tissue and the lung tissues of the app/ps1 double transgenic mice were overloaded with iron, and the compound group could significantly reduce the iron content in the tissues and organs. Compared with the control group, the content of Si and FN in the ad model group increased significantly (P0.05) and the level of TF decreased obviously. Compared with the ad model group, the Si and FN content of the compound group and DFO group decreased and the TF level increased, and the content of Si and FN was lower and the level of FN in the compound group was compared with the mice in each group. The compound effect was superior to the compound group. There was no significant difference between the DFO group and the compound group (P0.05). In order to investigate the effect of epimedium, Astragalus membranaceus and Radix Puerariae on the antioxidant capacity of the peripheral tissue of ad double transgenic mice, the experiment was carried out to detect the SOD, MDA in the liver tissue, the renal tissue, the heart tissue and the lung tissues of the ad model double transgenic mice. GSH-Px content. Compared with the control group, the liver, kidney, SOD, GSH-Px activity of the heart of the ad model group decreased and the MDA content increased (P0.05). Compared with the ad model group, the livers of epimedium, astragalus, kudzu, the liver, the kidneys, the kidneys, the heart, the SOD and GSH-Px activity of the heart were all elevated (P0.05), the MDA content was reduced, and compound group was reduced. The activity of SOD and GSH-Px increased (P0.05) and the content of MDA decreased significantly (P0.05) in each group, and the compound group was superior to the unilateral group; there was no significant difference between the group DFO and the compound group (P0.05). The effect of the third part of epimedium, Astragalus membranaceus and Radix Puerariae on the formation of serum EPO and erythrocyte in the Alzheimer's disease transgenic mice: In this experiment, app/ps1 double transgenic mice were used as animal models to observe the effect of epimedium, Astragalus membranaceus and Radix Puerariae on the formation of EPO and red blood cells in the serum of mice, and to discuss the possible mechanism of the overloading of brain iron. Group, group DFO and c57bl/6j mice were selected as negative control group. 4 of 10 mice were randomly selected from each group. 20% uran abdominal anesthesia was used to pick up the blood for 1ml. After statically placed, the serum was placed at 4, 3000r/min, centrifugation and 15min. The serum was stored at -80 C for the detection of erythropoietin (EPO) in mice serum. Another 3 mice were taken from each group. Only after anaesthesia, the blood was picked up quickly in the EP tube with heparin anticoagulant, mixed up and down and mixed up and down, preserved at low temperature, and carried out the blood test quickly. Results: compared with the control group, the content of EPO in the ad model mice decreased significantly (P0.05). The results suggested that the EPO generation in the serum of the app/ps1 double transgenic mice decreased. Compared with the ad model group, the prostitution of the mice was more than that of the ad model group. The content of EPO in the brain of the group of Astragalus membranaceus, the radix puerariae, the compound group and the DFO group increased significantly (P0.05). The content of EPO in the compound group and the DFO group increased significantly (P0.05). The test results of the blood routine red blood cell (RBC) in mice showed that the number of red blood cells in the mice of the ad model group decreased significantly (P0.05), and the ad model was compared with the control group. The number of red blood cells in the herba Epimedium group, the Astragalus group, the Puerariae group, the compound group and the DFO group increased (P0.05). The number of red blood cells in the compound group and the DFO group was significantly increased (P0.05) compared with the mice in each group (P0.05). The blood routine hemoglobin (HB) detection results showed that the Epimedium group and the Astragalus group were the result of the blood routine hemoglobin (HB) test. The hemoglobin content of the Pueraria group, the compound group and the DFO group increased (P0.05) compared with the ad model group, and the compound group and the DFO group increased significantly (P0.05). Compared with the C57 group, the hemoglobin content of the ad model mice decreased significantly (P0.05). The result of the hematocrit (HCT) showed that the percentage of hct/ in the model group decreased (p0.0) compared with the control group (p0.0). 5), compared with the model group, the HCT level of the compound group increased significantly (P0.05) in the.Ad model group, the number of white blood cells (WBC) increased (P0.05). The number of white blood cells in the herba Epimedium group, the Astragalus group, the Pueraria group, the compound group and the DFO group decreased (P0.05). The number of white cells in the compound group and the DFO group was not significantly different, and the number of leukocytes decreased obviously. The results suggested ad. The effect of the fourth part of epimedium, Astragalus membranaceus and Radix Puerariae on the blood lipid of transgenic mice with Alzheimer's disease: the effect of the effective components of epimedium, Astragalus and kudzu on the blood lipid, and to further explore the blood lipid generation. Methods: 6 month old male app/ps1 double transgenic mice were randomly divided into model group, compound group, Epimedium group, Astragalus group, Pueraria group and DFO group. The compound group was given Epimedium with epimedium, Radix Astragali, Puerariae group, Epimedium gavage to Herba epimedium, Astragalus group gavage to Astragalus membranaceus, Pueraria group gavage to pueraria. The root, group DFO was given DFO (deferamine), the length of.C57bl/6j mice was three months as the negative control group. The model group and the control group were given the same amount of saline for 3 months. 3 rats were randomly selected. After the last administration, the fasting was 16 hours, and 20% uranan was taken from the eyeball, 4, 3000r/min, and 15 minutes. After the serum was obtained, the content of triglyceride (TG), total cholesterol (TC) and high density lipoprotein (HDL) low density lipoprotein (LDL) were measured by the automatic biochemical analyzer. The expression of hepcidinmrna in the liver was measured by RT-PCR. Results: compared with the control group, the content of TG and LDL in the model group increased significantly (P0.05), and the HDL content was lower. There was no significant change in the content of TC (P0.05) in P0.05 (P0.05). Compared with the model group, the content of TG and LDL in the compound group decreased significantly, and the HDL content increased (p0.0).
【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R749.16
本文编号：2154218
[Abstract]:The abnormal increase in iron levels in the brain of AD patients may be related to the abnormal iron metabolism and protein expression in the brain, the increase of free radicals and inflammatory factors in the brain, the imbalance of peripheral iron metabolism and the deterioration of organ function. The reduction of iron content in the brain is one of the ways to prevent and control AD. Iron chelating agent, such as iron amine, can obviously improve AD. The clinical symptom, but its side effect is also very obvious, and the oral effect is poor, so good absorption, high bioavailability of traditional Chinese medicine has become the first choice for the treatment of AD iron overload, with great potential. Traditional Chinese medicine thinks that AD is the symptom of this deficiency, among which the deficiency of kidney is deficient, Qi and blood is insufficient, phlegm and blood stasis, and the endogeny of turbidity and poison is the standard. Therefore, the TCM treatment of AD It should make up the kidney and invigorate the spleen, nourish the blood and nourish the blood as the basis. The herba Epimedium has the efficacy of nourishing the kidney and nourishing the marrow. The Astragalus membranaceus has the effect of enriching the spleen and invigorating the spleen, making the blood stasis and removing blood stasis. The Pueraria has three drugs combined with the efficacy of clearing heat and detoxification. It plays the function of nourishing the liver and kidney, nourishing the spleen and nourishing blood, promoting blood circulation and removing stasis, clearing heat and detoxifying the brain and improving the wisdom of the brain. Extract from Epimedium, Astragalus Astragalus and pueraria lobata The effective components of the active ingredients can remove the free radical and reduce the inflammatory reaction and so on. It can inhibit the overloading of iron from many aspects, so the effective components of epimedium, Astragalus membranaceus and pueraria lobata are used for the intervention and effect of the AD brain iron overload, which provides scientific basis for the prevention and treatment of AD by traditional Chinese medicine. The mechanism of AD treatment has been a great deal of research, but most from the content of neurotransmitters, the level of oxidative stress, A beta metabolism and so on, it is not enough to pay attention to the overloading of the brain iron. Therefore, this subject takes iron metabolism as the breakthrough point, with the principle of supplementing the kidney, removing phlegm and removing blood stasis, enriching blood and enriching qi, and organizing the effective components of the three kinds of drugs, such as epimedium, astragalus, and pueraria lobata. APP/PS1 double transgenic mice were used as animal models to observe the therapeutic effect of drugs on animal models and the effect of cerebral iron intervention, and to explore the possible mechanism in order to open a new direction for the drug treatment of AD and to provide scientific basis for the prevention and control of AD by traditional Chinese medicine. Study on the effects of oxidative stress on iron content in the brain of transgenic mice with Alzheimer's disease and the effect of inflammatory factors: the effects of epimedium, Astragalus membranaceus, pueraria root and its effective components on the level of iron brain, oxidative stress and inflammatory factors in APP/PS1 double transgenic mice. Methods: 6 month old male app/ps1 double transgenic mice were randomly divided into two groups. The model group, the compound group, the Epimedium group, the Astragalus group, the puerarin group and the DFO group, of which the compound group was given the icariin, astragaloside, puerarin group, the Epimedium group gavage with icariin, the Astragalus group gavage with Huang Qijia glycoside, the GAGG group gavage puerarin, and the group DFO was given DFO (de ferric amine), and the length of the gavage was three months.C. 10 57bl/6j mice were used as negative control group. The model group and the control group were given the same amount of saline for 3 months. After the end of the gavage, the cerebral cortex and hippocampus were quickly removed. The iron content was measured by flame atomic absorption spectrometry. The content of iron was determined by plasma mass spectrometry (ICP-MS) in the hippocampus, and the other side of the cortex was used for biochemistry. Results: the contents of SOD, MDA, GSH-Px and inflammatory factors (IL-1 beta, IL6, tnf- alpha) were measured. Results: the results of flame atomic absorption spectrometry showed that the content of iron in the model group was significantly increased compared with the negative group (P0.05), and the content of iron in the compound group was significantly lower than that in the model group (P0.05), and the plasma mass spectrometry (icp-m) (icp-m). S) showed that the iron content in the hippocampus of the model mice was significantly increased compared with the negative mice (P0.05). The content of iron in the hippocampus of the compound group mice was significantly lower than that in the model group (P0.05). The results of the ELISA method showed that the contents of the model mice and the negative group were significantly increased (P0.05) in the cortex of IL-1 beta, IL6 and TNF three (P0.05), and the mice in the compound group were compared with those in the negative group. Compared with the model group, the content of IL-1 beta, IL-6, TNF three decreased significantly (P0.05), and the cortical MDA content of the model mice was significantly increased (P0.05) compared with the negative group mice (P0.05). The content of the cortical MDA in the compound group mice was significantly lower than that in the model group (P0.05). The mice in the model group and the negative irradiated mice were compared with the cortical SOD and GSH-Px. The content of the compound group mice was significantly increased (P0.05). The contents of SOD and GSH-Px in the mice were significantly increased (P0.05). The effect of the second part of epimedium, Astragalus membranaceus and Radix Puerariae on the serum iron, the level of iron and oxidative stress in the peripheral tissues of transgenic mice with Alzheimer's disease The gene mice were animal models to observe the effect of the effective components of epimedium, Astragalus membranaceus and pueraria lobata on the iron levels and the level of iron and oxidative stress in the peripheral tissue, and then to explore whether the intervention of the effective components of epimedium, astragalus, and pueraria lobata on the imbalance of the peripheral iron metabolism and the function decline of the viscera was related to the effect of the effective components of epimedium, astragalus, and pueraria. 60 male app/ps1 male app/ps1 double transgenic mice were randomly divided into model group, compound group, Epimedium group, Astragalus group, puerarin group, group DFO, of which the compound group gavage was given icariin, astragaloside, puerarin group, Epimedium group gavage to icariin, Astragalus group gavage to astragaloside, puerarin group gavage to puerarin, D Group fo was given DFO (de ferric amine) for 1 times a day for three months. The effective components of the 3 drugs of epimedium herb, Astragalus membranaceus and pueraria lobata were in proportion to 3:2:2, namely, icariin 120mg/kg, astragaloside 80mg/kg, and 80mg/kg weight of puerarin to mice,.C57bl/6j mice were given as negative control group, model group and control The content of iron in liver, kidney, heart, spleen and lung was measured by flame atomic absorption spectroscopy for 3 months. The serum iron (SI), ferritin (FN) and transferrin (TF) level of mice were detected by enzyme linked immunosorbent assay (ELISA). The liver tissue, kidney tissue and heart tissue of mice were detected by biochemical kit. The content of malondialdehyde (MDA), superoxide dismutase (SOD) activity and glutathione peroxidase (GSH-Px) activity in lung tissues. Results: flame atomic absorption spectrometry showed that the content of peripheral iron in the model group was significantly higher than that of the control group (P0.05). Compared with the ad model group, the content of iron in the Astragalus group, the Pueraria group, the compound group and the DFO group decreased. The iron content of the compound group was significantly lower (P0.05) than the mice in each group (P0.05), and there was no significant difference between the DFO group and the compound group (P0.05). The results suggested that the renal tissue, the spleen tissue, the heart tissue and the lung tissues of the app/ps1 double transgenic mice were overloaded with iron, and the compound group could significantly reduce the iron content in the tissues and organs. Compared with the control group, the content of Si and FN in the ad model group increased significantly (P0.05) and the level of TF decreased obviously. Compared with the ad model group, the Si and FN content of the compound group and DFO group decreased and the TF level increased, and the content of Si and FN was lower and the level of FN in the compound group was compared with the mice in each group. The compound effect was superior to the compound group. There was no significant difference between the DFO group and the compound group (P0.05). In order to investigate the effect of epimedium, Astragalus membranaceus and Radix Puerariae on the antioxidant capacity of the peripheral tissue of ad double transgenic mice, the experiment was carried out to detect the SOD, MDA in the liver tissue, the renal tissue, the heart tissue and the lung tissues of the ad model double transgenic mice. GSH-Px content. Compared with the control group, the liver, kidney, SOD, GSH-Px activity of the heart of the ad model group decreased and the MDA content increased (P0.05). Compared with the ad model group, the livers of epimedium, astragalus, kudzu, the liver, the kidneys, the kidneys, the heart, the SOD and GSH-Px activity of the heart were all elevated (P0.05), the MDA content was reduced, and compound group was reduced. The activity of SOD and GSH-Px increased (P0.05) and the content of MDA decreased significantly (P0.05) in each group, and the compound group was superior to the unilateral group; there was no significant difference between the group DFO and the compound group (P0.05). The effect of the third part of epimedium, Astragalus membranaceus and Radix Puerariae on the formation of serum EPO and erythrocyte in the Alzheimer's disease transgenic mice: In this experiment, app/ps1 double transgenic mice were used as animal models to observe the effect of epimedium, Astragalus membranaceus and Radix Puerariae on the formation of EPO and red blood cells in the serum of mice, and to discuss the possible mechanism of the overloading of brain iron. Group, group DFO and c57bl/6j mice were selected as negative control group. 4 of 10 mice were randomly selected from each group. 20% uran abdominal anesthesia was used to pick up the blood for 1ml. After statically placed, the serum was placed at 4, 3000r/min, centrifugation and 15min. The serum was stored at -80 C for the detection of erythropoietin (EPO) in mice serum. Another 3 mice were taken from each group. Only after anaesthesia, the blood was picked up quickly in the EP tube with heparin anticoagulant, mixed up and down and mixed up and down, preserved at low temperature, and carried out the blood test quickly. Results: compared with the control group, the content of EPO in the ad model mice decreased significantly (P0.05). The results suggested that the EPO generation in the serum of the app/ps1 double transgenic mice decreased. Compared with the ad model group, the prostitution of the mice was more than that of the ad model group. The content of EPO in the brain of the group of Astragalus membranaceus, the radix puerariae, the compound group and the DFO group increased significantly (P0.05). The content of EPO in the compound group and the DFO group increased significantly (P0.05). The test results of the blood routine red blood cell (RBC) in mice showed that the number of red blood cells in the mice of the ad model group decreased significantly (P0.05), and the ad model was compared with the control group. The number of red blood cells in the herba Epimedium group, the Astragalus group, the Puerariae group, the compound group and the DFO group increased (P0.05). The number of red blood cells in the compound group and the DFO group was significantly increased (P0.05) compared with the mice in each group (P0.05). The blood routine hemoglobin (HB) detection results showed that the Epimedium group and the Astragalus group were the result of the blood routine hemoglobin (HB) test. The hemoglobin content of the Pueraria group, the compound group and the DFO group increased (P0.05) compared with the ad model group, and the compound group and the DFO group increased significantly (P0.05). Compared with the C57 group, the hemoglobin content of the ad model mice decreased significantly (P0.05). The result of the hematocrit (HCT) showed that the percentage of hct/ in the model group decreased (p0.0) compared with the control group (p0.0). 5), compared with the model group, the HCT level of the compound group increased significantly (P0.05) in the.Ad model group, the number of white blood cells (WBC) increased (P0.05). The number of white blood cells in the herba Epimedium group, the Astragalus group, the Pueraria group, the compound group and the DFO group decreased (P0.05). The number of white cells in the compound group and the DFO group was not significantly different, and the number of leukocytes decreased obviously. The results suggested ad. The effect of the fourth part of epimedium, Astragalus membranaceus and Radix Puerariae on the blood lipid of transgenic mice with Alzheimer's disease: the effect of the effective components of epimedium, Astragalus and kudzu on the blood lipid, and to further explore the blood lipid generation. Methods: 6 month old male app/ps1 double transgenic mice were randomly divided into model group, compound group, Epimedium group, Astragalus group, Pueraria group and DFO group. The compound group was given Epimedium with epimedium, Radix Astragali, Puerariae group, Epimedium gavage to Herba epimedium, Astragalus group gavage to Astragalus membranaceus, Pueraria group gavage to pueraria. The root, group DFO was given DFO (deferamine), the length of.C57bl/6j mice was three months as the negative control group. The model group and the control group were given the same amount of saline for 3 months. 3 rats were randomly selected. After the last administration, the fasting was 16 hours, and 20% uranan was taken from the eyeball, 4, 3000r/min, and 15 minutes. After the serum was obtained, the content of triglyceride (TG), total cholesterol (TC) and high density lipoprotein (HDL) low density lipoprotein (LDL) were measured by the automatic biochemical analyzer. The expression of hepcidinmrna in the liver was measured by RT-PCR. Results: compared with the control group, the content of TG and LDL in the model group increased significantly (P0.05), and the HDL content was lower. There was no significant change in the content of TC (P0.05) in P0.05 (P0.05). Compared with the model group, the content of TG and LDL in the compound group decreased significantly, and the HDL content increased (p0.0).
【学位授予单位】：河北医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R749.16
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