WO2023045460A1 - 乳铁蛋白在预防治疗阿尔兹海默症中的应用 - Google Patents

乳铁蛋白在预防治疗阿尔兹海默症中的应用 Download PDF

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WO2023045460A1
WO2023045460A1 PCT/CN2022/101542 CN2022101542W WO2023045460A1 WO 2023045460 A1 WO2023045460 A1 WO 2023045460A1 CN 2022101542 W CN2022101542 W CN 2022101542W WO 2023045460 A1 WO2023045460 A1 WO 2023045460A1
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disease
lactoferrin
alzheimer
treatment
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成果
张�林
张伶俐
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四川大学
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/40—Transferrins, e.g. lactoferrins, ovotransferrins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

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  • the invention relates to the field of drugs for preventing and treating Alzheimer's disease, in particular to the application of lactoferrin in the preparation of drugs for preventing or treating Alzheimer's disease.
  • AD Alzheimer's disease
  • SPs senile plaques
  • NFTs Neurofibrillary tangles
  • AD patients directly or indirectly acts on neurons and glial cells through a series of cascade reactions such as free radical reaction, mitochondrial oxidative damage and inflammatory response, and finally leads to neuronal dysfunction. Or die, causing cognitive impairment and memory loss.
  • Alzheimer's disease is not only a disease that seriously affects the patient's own health and quality of life, but also seriously affects the happiness of the patient's entire family.
  • Clinically, Alzheimer's disease is characterized by comprehensive dementia such as memory impairment, aphasia, apraxia, agnosia, visual-spatial skill impairment, executive dysfunction, and personality and behavior changes.
  • Early mild patients mainly present with short-term memory loss , that is, forgetting the most recent events; time disorientation, that is, being unable to distinguish time and place direction, etc.; although still able to do some familiar daily tasks, it is difficult to understand new things and cannot deal with complex problems ;Emotional indifference, easy to irritate, talk less, sometimes can't call some things by name.
  • Moderate patients mainly show severe damage to near and far memory, that is, they cannot remember things that happened a long time ago; time and place disorientation, dyscalculia, aphasia, apraxia and agnosia; they can no longer independently move outdoors, dress, Needs help with personal hygiene; often restless, restless, urinary incontinence visible. Severe patients will have severe memory loss, only some fragments of memory remain; they are completely unable to take care of themselves in daily life, incontinence of urine and feces, need to be completely dependent on others for care, limbs are stiff, silent, and eventually coma.
  • the object of the present invention is to provide an effective active substance for preventing and treating Alzheimer's disease, which is used for preparing the medicine for preventing and treating Alzheimer's disease.
  • the present invention uses lactoferrin as an effective active substance for the preparation of a drug for the prevention and treatment of Alzheimer's disease, and the specific technical scheme is:
  • lactoferrin in the preparation of drugs for preventing Alzheimer's disease.
  • lactoferrin in preparation of medicine for treating Alzheimer's disease.
  • the concentration of lactoferrin is generally 2.5-120 ⁇ M Lf/g, preferably 20-80 ⁇ M Lf/g.
  • Lactoferrin (Lactoferrin, Lf) is an iron-binding glycoprotein composed of amino acids, mainly present in mammalian milk. Studies have shown that Lf has various biological functions such as immune regulation, anti-inflammatory response, and iron chelation, and studies have found that only a few neurons express Lf in the young brain, but there are a large number of neurons and glia in the aged brain Cells express Lf, suggesting that Lf may play an important role in the process of brain aging. However, whether Lf can protect neurons from Alzheimer's disease by inhibiting the inflammatory response, the inventors conducted a systematic study.
  • the present invention By designing an in vitro Alzheimer's disease model, the present invention proves for the first time that lactoferrin can reduce cell damage caused by A ⁇ , and by improving anti-inflammation ability, inhibiting inflammation level and regulating apoptosis, thereby exerting anti-Alzheimer's disease effect.
  • the present invention confirms that lactoferrin can reduce the phosphorylation level of Tau protein, shorten the time for mice to escape from the water maze, and improve learning and cognitive functions. Therefore, the present invention provides the application of lactoferrin in preventing and/or treating neurodegenerative diseases, and provides a basis for developing and expanding the drug effect of lactoferrin.
  • the present invention analyzes the effect of lactoferrin on cells, analyzes the effect of lactoferrin on cellular inflammatory factors (TNF- ⁇ , IL-6, IL-1 ⁇ , etc.), p38, JNK, ERK protein expression and The effect of phosphorylation level; analyze the effect of lactoferrin on the AD-related protein Tau and its phosphorylation level; at the same time, use different concentrations of lactoferrin to act on the cell model, and study the dependence between its neuroprotective effect and its concentration , to screen effective drug concentrations for the prevention and/or treatment of Alzheimer's disease.
  • Alzheimer's disease model APP/PS1 gene mouse analyze the effect of lactoferrin on the inflammatory response, learning ability and cognitive function of the mouse brain, and provide a basis for the prevention and/or treatment of Alzheimer's disease new research ideas.
  • Figure 1 is the CCK8 method to detect the toxic effect of Lf on normal N2a cells
  • Figure 2 is the CCK8 method to detect the effect of Lf on the viability of N2a cells induced by A ⁇ 25-35 ;
  • Fig. 3 is the effect of Lf on the apoptosis of N2a cells induced by ⁇ 25-35 ;
  • Figure 4 is the effect of Lf on the inflammatory factors of N2a cells induced by A ⁇ 25-35 ;
  • Figure 5 is the effect of Lf on the activation of TLR4/NF ⁇ B/I ⁇ B ⁇ signaling pathway
  • Figure 6 is the effect of Lf on the AD-related proteins in N2a cells induced by A ⁇ 25-35 ;
  • Figure 7 is the effect of Lf on the cognitive function learning ability of APP/PS1 transgenic mice
  • Figure 8 is the effect of Lf on A ⁇ protein, Tau protein and their phosphorylation in APP/PS1 transgenic mice;
  • the present invention uses lactoferrin as a medicinal substance to carry out an applied experiment for the prevention and/or treatment of Alzheimer's disease, and the experiment proves that lactoferrin has potential drug value against neurodegenerative diseases.
  • Example 1 CCK8 method detects the toxic effect of Lf on normal N2a cells
  • N2a cells in the logarithmic phase were inoculated in a 96-well plate with a cell density of 5 ⁇ 10 3 cells/well, and 6 replicate wells were set for each group.
  • Intervention treatment the blank group is DMEM complete medium without any cells; the control group is the cell culture medium without Lf; the experimental group is the cell culture medium with 2.5, 5, 10, 20, 40, 80 and 160 ⁇ M Lf added respectively .
  • Cell survival rate (A experimental group-A blank group)/(A control group-A blank group) ⁇ 100%, A is the absorbance at a detection wavelength of 450 nm.
  • Example 2 CCK8 method detects the effect of Lf on the viability of N2a cells induced by A ⁇ 25-35
  • N2a cells in the logarithmic phase were inoculated in a 96-well plate with a cell density of 5 ⁇ 10 3 cells/well, and 6 replicate wells were set for each group. Intervention treatment. Set up 20 ⁇ M A ⁇ 25-35 treatment group and set up 20 ⁇ M A ⁇ 25-35 +10 ⁇ M Lf treatment group, 20 ⁇ M A ⁇ 25-35 +20 ⁇ M Lf treatment group, 20 ⁇ M A ⁇ 25-35 +40 ⁇ M Lf treatment group, 20 ⁇ M A ⁇ 25-35 +80 ⁇ M Lf treatment group Treatment group, 20 ⁇ M A ⁇ 25-35 + 160 ⁇ M Lf treatment group. In the experimental group, they were treated with different concentrations of Lf for 24 hours, and then added 20 ⁇ M A ⁇ 25-35 for 24 hours. The cell viability of each group was detected by the CCK8 method, and the specific steps were as described in Example 1.
  • N2a cells in the logarithmic phase were seeded in 6-well plates at 2 ⁇ 10 5 cells/well, and after the cells adhered to the wall, corresponding intervention treatments were given according to the requirements of different experimental groups.
  • Set up blank control group A ⁇ 25-35 group, Lf group, A ⁇ 25-35 +Lf group.
  • the blank control group used complete cell culture medium without any intervention.
  • the A ⁇ 25-35 group was treated with 20 ⁇ M A ⁇ 25-35 for 24 hours.
  • the N2a cells were treated with 40 ⁇ g/ml Lf for 24 hours, and then treated with 20 ⁇ M A ⁇ 25-35 for 24 hours.
  • the cells in each well were trypsinized, centrifuged at 1500 rpm for 3 min at room temperature, and the supernatant was removed to collect N2a cells. Wash twice with PBS, according to the instructions in the Annexin V-FITC/PI cell apoptosis detection kit, add an appropriate amount of binding buffer to suspend the cells, add AnnexinV-FITC staining solution at room temperature and incubate for 5 min, then add PI staining solution, mix well The cell apoptosis rate was detected and calculated by flow cytometry, and each sample was repeated 3 times. Finally, it was processed using flow cytometry analysis software.
  • Example 4 The effect of Lf on the inflammatory factors of N2a cells induced by A ⁇ 25-35
  • Implementation steps Set up experimental groups according to Example 3, use RNA extraction kits to extract the total RNA of each group of cells after the intervention of each group of cells, measure its concentration, reverse transcribe it into cDNA, and dilute the cDNA by 5 to 10 times PCR amplification was performed, and the mRNA expression levels of IL-4, IL-6, IL-13, IL-1 ⁇ , and TNF- ⁇ were detected using GAPDH as an internal reference. Each sample was repeatedly detected three times, and the 2- ⁇ Ct method was used to calculate the gene expression level. The relative expression levels and primer sequences are shown in the table below.
  • the TLR4/NF ⁇ B/I ⁇ B ⁇ pathway can be activated under various adverse stimuli, inducing a cascade reaction mediated by various inflammatory factors.
  • Experimental grouping and drug treatment were set up according to Example 3. After the cell treatment, nuclear protein was extracted using a nuclear protein/cytoplasmic protein extraction kit, the protein was quantified by Bradford method, and TLR4 protein, nuclear protein NF- ⁇ B and plasma protein I ⁇ B ⁇ and their phosphorylation levels were detected by western blot.
  • the A ⁇ 25-35 group can induce a decrease in the level of plasma protein NF ⁇ B, an increase in the expression levels of TLR4 protein and nuclear protein NF ⁇ B, and an increase in the ratio of p-I ⁇ B ⁇ /I ⁇ B ⁇ ; After pretreatment with Lf, the expression of plasma protein NF ⁇ B increased, the expression of TLR4 and nuclear protein NF ⁇ B decreased, and the ratio of p-I ⁇ B ⁇ /I ⁇ B ⁇ decreased.
  • Experimental steps Set up experimental groups according to Example 3, perform drug treatment, extract protein, and detect the expression levels of Tau protein and p-Tau.
  • Embodiment 7 The influence of Lf on the cognitive function learning ability of APP/PS1 gene mice
  • mice were fed adaptively for 1 week, the APP/PS1 gene mice were randomly divided into 3 groups, the intervention control group given normal saline, the Lf low-dose group, and the Lf high-dose group, with 8 mice in each group. Eight wild-type mice were used as the model control group. Lf low-dose group and Lf high-dose group were given 2mg/kg/d and 6mg/kg/d exogenous lactoferrin, respectively. The intervention control group and the model control group were given the same volume of normal saline for intervention.
  • the water maze experiment includes two items: positioning navigation test and space exploration test.
  • the positioning navigation test repeated training of mice, enhances the memory of the mice to the surrounding environment, and can find the underwater escape platform in a short time.
  • This experiment is used to test the spatial memory function of the mice.
  • the animals participating in the experiment spent more time and energy to find the quadrant of the escape platform according to the prompt of spatial memory.
  • the quadrants are arranged clockwise, and the order is divided into the first, second, third, and fourth quadrants.
  • the area where the platform is located is the third quadrant, which is on the opposite side of the first quadrant.
  • the experiment period is five days, four days before the positioning navigation experiment, and the space exploration experiment on the fifth day. This procedure is a classic Morris water maze test procedure.
  • mice in the control group were 120s, while the mice in the model control group only took 20s, indicating that the learning ability and cognitive function of the mice in the intervention control group were significantly reduced; the Lf low-dose group
  • the water maze escape time of mice in the high-dose and high-dose groups was shortened to 70s and 59s, respectively, indicating that Lf can effectively improve the cognitive dysfunction of mice with neurodegenerative diseases and improve their learning ability.
  • Example 8 Effect of Lf on A ⁇ protein, Tau protein and phosphorylation thereof in SAMP-8 mice
  • Lf can inhibit the phosphorylation levels of ERK, JNK, and P38 proteins, reduce the expression of NF- ⁇ B protein, increase the level of anti-inflammatory factors, reduce the level of pro-inflammatory factors, and enhance the ability of cells to resist inflammation.
  • Lf can also reduce the phosphorylation level of Tau protein in nerve cells and brain tissue of mice with neurodegenerative diseases, and improve the learning ability and cognitive function of mice. Therefore, the present invention confirms the potential application value of Lf in Alzheimer's disease, and also provides a theoretical basis for the application of Lf in the prevention and/or treatment of Alzheimer's disease.

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Abstract

提供了乳铁蛋白在预防或治疗阿尔兹海默症中的应用。本申请证实了乳铁蛋白能够降低Aβ造成的细胞损伤，通过提高抗炎症能力，抑制炎症水平以及调控凋亡；还能够降低Tau蛋白磷酸化水平，提高学习和认知功能。

Description

乳铁蛋白在预防治疗阿尔兹海默症中的应用 技术领域
本发明涉及预防治疗阿尔兹海默症的药物领域，具体而言，涉及乳铁蛋白在制备阿尔兹海默症预防或治疗药物中的应用。
背景技术
阿尔茨海默症(Alzheimer’s disease，AD)是一种最为常见的神经退行性疾病，其主要病理特征是β淀粉样蛋白(Aβ)在神经元外过度沉积形成的老年斑(senile plaques，SPs)和神经元内Tau蛋白过度磷酸化聚集而成的神经纤维缠结(NFTs)，从而导致渐进性记忆丧失和认知功能障碍等一系列症状。但是，由于AD病因复杂，该病的发病机制至今仍不明确，“Aβ级联假说”和“炎症假说”均为当前科学界广泛公认的机制之一。这两种学说认为AD患者大脑中异常沉积的Aβ通过自由基反应、线粒体氧化损伤和炎症反应等一系列级联反应，直接或间接作用于神经元和神经胶质细胞，最终导致神经元功能异常或者死亡，引发认知障碍和记忆衰退。
阿尔茨海默病不仅是严重影响患者本人身体健康和生活质量的疾病,也严重影响患者整个家庭的幸福。阿尔茨海默病临床上以记忆障碍、失语、失用、失认、视空间技能损害、执行功能障碍以及人格和行为改变等全面性痴呆表现为特征，早期轻度患者主要表现为近期记忆减退，即会忘记最近最新发生的事；时间定向障碍，即分不清时间跟地点方向等；虽然仍能做些已熟悉的日常工作，但对新的事物却难以理解，也处理不了复杂的问题；情感淡漠，容易激惹生气，说话少，有时会叫不上一些东西的名字。中度患者主要表现为远近记忆严重受损，即记不住很久之前发生的事儿；时间、地点定向障碍，计算障碍，失语、失用和失认；已经不能独立在室外活动，穿衣、个人卫生方面都需要帮助；常常急躁不安，走动不停，可见尿失禁。重度患者则会严重记忆力丧失，仅仅存留有一些片段的记忆；日常生活已完全不能自理，大小便失禁，需要完全依赖别人照护，肢体僵直、缄默，最终昏迷。
研究开发能够预防或治疗阿尔茨海默症的药物，具有非常重大的社会意义。 有鉴于此，特提出本发明。
发明内容
本发明的目的在于提供一种预防阿尔兹海默症和治疗阿尔兹海默症的有效活性物质，以用于制备预防和治疗阿尔兹海默症的药物。
针对本发明的上述目的，本发明以乳铁蛋白为有效活性物质，用于制备预防治疗阿尔兹海默症的药物，具体技术方案为：
乳铁蛋白在制备预防阿尔兹海默症药物中的应用。
乳铁蛋白在制备治疗阿尔兹海默症药物中的应用。
在预防和治疗阿尔兹海默症药物中，乳铁蛋白浓度一般为2.5～120μM Lf/g，优选地为20～80μM Lf/g。
乳铁蛋白(Lactoferrin，Lf))是一种由氨基酸组成的铁结合糖蛋白，主要存在于哺乳动物的乳汁中。研究表明，Lf具有免疫调节、抗炎症反应、铁螯合等多种生物学作用，且研究则发现青年人脑中仅有少数神经元表达Lf，但在老龄大脑中有大量神经元和胶质细胞表达Lf，提示Lf可能在脑衰老过程中发挥着重要作用。但是Lf是否可以通过抑制炎症反应对阿尔兹海默症的神经元损伤具有保护作用，对此，发明人进行了系统的研究。
本发明通过设计体外阿尔兹海默症模型，首次证实了乳铁蛋白能够降低Aβ造成的细胞损伤，通过提高抗炎症能力，抑制炎症水平以及调控凋亡，从而发挥抗阿尔兹海默症作用。同时，通过APP/PS1转基因小鼠模型，本发明证实了乳铁蛋白能够降低Tau蛋白磷酸化水平，缩短小鼠逃离水迷宫时间，提高学习和认知功能。因此，本发明提供了将乳铁蛋白在预防和/或治疗神经退行性疾病中的应用，为开发和扩大乳铁蛋白的药物作用提供了基础。
目前阿尔兹海默症发病机制尚不明确，本发明重点研究炎症反应在阿尔兹海默症中的影响。本发明在以N2a细胞为模型的基础上，分析乳铁蛋白对细胞分析乳铁蛋白对细胞炎症因子(TNF-α、IL-6、IL-1β等)，p38、JNK、ERK蛋白表达量及磷酸化水平的影响；分析乳铁蛋白对AD相关蛋白Tau及其磷酸化水平的影响；同时采用不同浓度的乳铁蛋白作用于细胞模型上，研究其神经保护作用与其浓度之间的依赖性关系，为阿尔兹海默症的预防和/或治疗筛选有效的药物 浓度。在阿尔兹海默症模型APP/PS1基因小鼠的基础上，分析乳铁蛋白对小鼠大脑炎症反应、学习能力和认知功能的影响，为阿尔兹海默症的预防和/或治疗提供新的研究思路。
附图说明
为了更清楚地说明本发明实施例或现有技术中的方案，以下将对实施例或现有技术描述中所需要使用的附图作简单介绍。
图1为CCK8法检测Lf对于正常N2a细胞的毒性作用；
图2为CCK8法检测Lf对Aβ 25-35诱导的N2a细胞活力的影响；
图3为Lf对β 25-35诱导的N2a细胞凋亡的影响；
图4为Lf对Aβ 25-35诱导的N2a细胞炎症因子的影响；
图5为Lf对TLR4/NFκB/IκBα信号通路活化的影响；
图6为Lf对Aβ 25-35诱导的N2a细胞AD相关蛋白的影响；
图7为Lf对APP/PS1转基因小鼠认知功能学习能力的影响；
图8为Lf对APP/PS1转基因小鼠Aβ蛋白、Tau蛋白及其磷酸化的影响；
具体实施方式
下面将结合实施例对本发明的实施方案进行详细描述，但是下列实施例仅为了更好地说明和理解本发明，而不应视为限制本发明的范围或实施原则，本发明的实施方式不限于以下内容。
本发明是以乳铁蛋白为药效物质，进行阿尔兹海默症预防和/或治疗的应用性实验，并通过实验证明乳铁蛋白具有抗神经退行性疾病的潜在药物价值。
实施例1—CCK8法检测Lf对于正常N2a细胞的毒性作用
实验材料：小鼠神经母细胞瘤N2a，购于上海中科院典藏细胞库
实验步骤：将处于对数期的N2a细胞接种于96孔板中，细胞密度为5×10 3个/孔，每组设置6个重复孔，待细胞贴壁后按照不同实验分组要求给予相应的干预处理，空白组是不含任何细胞的DMEM完全培养基；对照组是不含Lf的细胞培养液；实验组是分别加入2.5、5、10、20、40、80和160μM Lf的细胞培养液。
分别干预24h后去除原有培养液，每孔按照CCK8与培养基1：9的体积比加入混合溶液，避光孵育2h后，采用酶标仪于450nm波长处测定各孔吸光度值， 并按公式计算出细胞存活率。计算公式为：
细胞存活率＝(A实验组-A空白组)/(A对照组-A空白组)×100％，A为检测波长为450nm出的吸光度。
实验结果如图1所示，2.5～160μM的Lf处理24h后，N2a细胞存活率呈现先增加后下降的趋势，表明Lf与N2a细胞存在低浓度促进细胞存活率，高浓度抑制细胞存活率的剂量反应关系。
实施例2—CCK8法检测Lf对Aβ 25-35诱导的N2a细胞活力的影响
实验步骤：将处于对数期的N2a细胞接种于96孔板中，细胞密度为5×10 3个/孔，每组设置6个重复孔，待细胞贴壁后按照不同实验分组要求给予相应的干预处理。设立20μMAβ 25-35处理组和设立20μM Aβ 25-35+10μM Lf处理组，20μM Aβ 25-35+20μM Lf处理组，20μM Aβ 25-35+40μM Lf处理组，20μM Aβ 25-35+80μM Lf处理组，20μM Aβ 25-35+160μM Lf处理组。在实验组中先用不同浓度Lf处理24h，再分别加入20μM Aβ 25-35处理24h。采用CCK8法检测每组的细胞存活率，具体步骤如实施例1所述。
实验结果如图2所示，与Aβ 25-35处理组相比，经过不同浓度Lf处理后的N2a细胞存活率显著提高，且与Lf浓度存在剂量反应关系。由此可见，Lf处理可以显著减少Aβ 25-35导致的细胞死亡。
实施例3—Lf对Aβ 25-35诱导的N2a细胞凋亡的影响
实施步骤：将处于对数期的N2a细胞以2×10 5个/孔接种于6孔板中，待细胞贴壁后按照不同实验分组要求给予相应的干预处理。设立空白对照组，Aβ 25-35组，Lf组，Aβ 25-35+Lf组。空白对照组使用完全细胞培养基，不做任何干预处理。Aβ 25-35组使用20μM Aβ 25-35处理24h。Lf组使用40μg/ml的Lf处理N2a细胞24h后再使用20μM Aβ 25-35处理24h。药物处理后，将每孔的细胞进行胰酶消化，室温下以1500rpm离心3min，去上清收集N2a细胞。PBS洗涤2次，按照Annexin V-FITC/PI细胞凋亡检测试剂盒中的说明书，加入适量结合缓冲液悬浮细胞，室温条件下加入AnnexinV-FITC染液孵育5min后，加入PI染液，充分混匀，用流式细胞仪检测并计算细胞凋亡率，每个样本重复3次。最后使用流式细胞分析软件处理。
实验结果如图3所示，空白对照组细胞凋亡率为8.2％，Lf组细胞凋亡率为 7.4％，与空白对照组无显著差异；而Aβ 25-35组与空白对照组相比，细胞凋亡率显著上升，凋亡率为20.7％；使用Lf干预后，Lf+Aβ 25-35组细胞凋亡率下降至13.9％。结果说明Lf可以降低MPP +和Aβ 25-35导致的细胞凋亡，缓解MPP +和Aβ 25-35造成的细胞毒性。
实施例4—Lf对Aβ25-35诱导的N2a细胞炎症因子的影响
实施步骤：按照实施例3设置实验分组，分别对各组细胞干预后使用RNA提取试剂盒提取各组细胞总RNA，测定其浓度后，将其反转录为cDNA，cDNA稀释5～10倍后进行PCR扩增，以GAPDH为内参检测IL-4、IL-6、IL-13、IL-1β、TNF-α的mRNA表达水平，每个样本重复检测三次，采用2 -△△Ct法计算基因相对表达量，引物序列见下表。
引物名称和序列
Figure PCTCN2022101542-appb-000001
实验结果如图4所示，与空白对照组相比Aβ 25-35组细胞促炎因子TNF-α、IL-6、IL-1βmRNA表达显著升高，抑炎因子IL-4、IL-13mRNA表达显著降低；与Aβ 25-35组相比，经Lf处理后可显著下调促炎因子mRNA表达，上调抑炎因子mRNA表达。同样，由图10B可知，与空白对照组相比，MPP +组细胞促炎因子TNF-α、IL-6、IL-1βmRNA表达显著升高，抑炎因子IL-4、IL-13mRNA表达显著降低；与MPP +组相比，经Lf处理可下调促炎因子mRNA表达，上调抑炎因子mRNA表达。结果表明Lf可减轻Aβ 25-35诱导的神经炎症。
实施例5—Lf对TLR4/NFκB/IκBα信号通路活化的影响
实施步骤：TLR4/NFκB/IκBα通路可在各种不良刺激因素下被活化，诱导多种炎症因子介导的瀑布式级联反应。按照实施例3设置实验分组和药物处理。 细胞处理结束后，使用核蛋白/胞浆蛋白提取试剂盒提取细胞核蛋白后，Bradford法对蛋白进行定量，westernblot检测TLR4蛋白、核蛋白NF-κB和浆蛋白IκBα及其磷酸化水平。
实验结果如图5所示，Aβ 25-35组与空白对照组相比，Aβ 25-35可以诱导浆蛋白NFκB水平下降，TLR4蛋白和核蛋白NFκB表达水平提高，p-IκBα/IκBα比值增加；而经过Lf预处理后，浆蛋白NFκB表达量增加，TLR4和核蛋白NFκB表达量降低，p-IκBα/IκBα比值减小。结果表明，Lf可降低Aβ 25-35诱导的N2a细胞内TLR4蛋白的高表达和IκBα高磷酸化水平，抑制NFκB的核转移，从而有效抑制TLR4/NFκB/IκBα信号通路的活化，从而提高细胞抗炎症能力，降低神经炎症水平。
实施例6—Lf对Aβ 25-35诱导的N2a细胞AD相关蛋白的影响
实验步骤：按照实施例3设置实验分组，进行药物处理，提取蛋白和检测Tau蛋白及p-Tau表达量。
实验结果如图6所示，各组Tau蛋白表达水平无明显差异；与空白对照组相比，Aβ 25-35组p-Tau蛋白表达明显增加；相较于Aβ 25-35组，Lf预处理后，p-Tau蛋白表达水平显著下降，p-Tau/Tau比值降低，结果表明Lf可抑制Aβ 25-35所致N2a细胞Tau蛋白的过度磷酸化，降低神经原纤维缠结。
实施例7—Lf对APP/PS1基因小鼠认知功能学习能力的影响
实验步骤：所有小鼠适应性喂养1周后，将APP/PS1基因小鼠随机分为3组，给予生理盐水的干预对照组，Lf低剂量组、Lf高剂量组，每组8只。以8只野生型小鼠作为模型对照组。Lf低剂量组和Lf高剂量组分别给予2mg/kg/d和6mg/kg/d外源性乳铁蛋白。干预对照组和模型对照组给予同体积生理盐水干预。水迷宫实验包括两项：定位航行试验和空间探索试验。
定位航行试验，重复训练小鼠，增强小鼠对周围环境的记忆，可在短时间内找到水下逃生平台，此实验用来检测小鼠的空间记忆功能。空间探索试验，参与实验的动物根据空间记忆的提示，要花更多的时间和精力去寻找逃生平台的象限。象限按顺时针排列，其顺序分为第一、二、三、四象限。平台所在的区域是第三象限，第三象限在第一象限的对侧。该实验周期为五天，定位航行实验前四天进行，第五天进行空间探索实验，此程序为经典的实验Morris水迷宫测试程序。
定位航行试验：开始试验之前，将APP/PS1基因和模型对照组的小鼠放在平台上，先让小鼠适应环境，适应的时间设定为20s。然后在第一象限面壁放在池内；开始计时，停止记录的时间为模型对照组的小鼠攀上平台并逗留5s。60s是本实验的最长记录，若小鼠不能在规定时刻内登台，则主动指引小鼠登上平台并停留10s，最终清洁小鼠，试验结束，将小鼠放入鼠笼。后面按照此流程，第一、二、三、四象限的顺序，每天按照顺时针方向走一圈，持续4天。记录每次实验的4个象限小鼠在平台上的潜伏时间，估算几个象限的平均值，以评估小鼠的空间学习能力。
空间探索实验：实验最后一天，去除水迷宫下的站台，水迷宫的试验环境、水温和定位航行试验均保持不变，于第1象限面壁池内放置试验小鼠，然后记录并观察小鼠60s内所经过的泅水路线，清理小鼠身上水渍，放入鼠笼。其他象限的实验则无需再进行。小鼠空间记忆能力的评估，通过记录小鼠穿越平台次数，以及小鼠到达第三象限所需的时间与总时间的比率等完成。
实验结果如图7所示，对照组小鼠逃离水迷宫的时间为120s，而模型对照组的小鼠仅为20s，说明干预对照组小鼠学习能力和认知功能显著降低；Lf低剂量组和高剂量组小鼠逃离水迷宫时间分别缩短至70s和59s，表明Lf可以有效改善神经退行性疾病小鼠认知功能障碍，提高其学习能力。
实施例8—Lf对SAMP-8小鼠Aβ蛋白、Tau蛋白及其磷酸化的影响
实施步骤：按照实施例7设置实验分组和干预处理。小鼠干预结束后每组小鼠取3只解剖并收集大脑组织，使用组织匀浆机破碎脑组织，提取脑组织总蛋白。按照实施例5检测小鼠脑组织Aβ蛋白、Tau蛋白及其磷酸化
实验结果如图8所示，与干预对照组相比，Lf低剂量组和Lf高剂量组Tau蛋白磷酸化水平显著降低，且两者比值显著减少，结果说明Lf可减轻神经退行性小鼠大脑神经原纤维磷酸化导致的神经原纤维缠结。
由如上实施例1-8可知，Lf可以抑制ERK、JNK、P38蛋白磷酸化水平，降低NF-κB蛋白表达，提高抑炎因子的水平，降低促炎因子水平，增强细胞抗炎症反应的能力。Lf还可以降低神经细胞和神经退行性疾病小鼠脑组织Tau蛋白磷酸化水平，提高小鼠学习能力和认知功能。因此本发明证实了Lf在阿尔兹海默症的潜在应用价值，也为Lf应用于预防和/或治疗阿尔兹海默症提供了理论依 据。
尽管已用具体实施例来说明和描述了本发明，然而应意识到，在不背离本发明的精神和范围的情况下可以作出许多其它的更改和修改。因此，这意味着在所附权利要求中包括属于本发明范围内的所有这些变化和修改。

Claims (4)

  1. 乳铁蛋白在预防阿尔兹海默症药物中的应用。
  2. 乳铁蛋白在治疗阿尔兹海默症药物中的应用。
  3. 根据权利要求1或2所述的应用，其特征在于乳铁蛋白在预防或治疗阿尔兹海默症药物中的浓度为2.5～120μM Lf/g。
  4. 根据权利要求3所述的应用，其特征在于乳铁蛋白在预防或治疗阿尔兹海默症药物中的浓度为20～80μM Lf/g。
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