CN115212205A

CN115212205A - Application of N-acetyl serotonin in preventing or preparing medicament for treating Alzheimer disease

Info

Publication number: CN115212205A
Application number: CN202210890827.9A
Authority: CN
Inventors: 苏强; 杨铭萱; 王卓然; 张艳丽; 李甜
Original assignee: First Hospital of Shanxi Medical University
Current assignee: First Hospital of Shanxi Medical University
Priority date: 2022-07-27
Filing date: 2022-07-27
Publication date: 2022-10-21

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of N-acetyl serotonin in preventing or preparing a medicine for treating Alzheimer disease. Experiments prove that the N-acetyl serotonin (NAS) provided by the invention has a good effect of inhibiting abnormal aggregation of beta-like amyloid protein in the prevention or preparation and treatment of Alzheimer's disease.

Description

Application of N-acetyl serotonin in preventing or preparing medicament for treating Alzheimer disease

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to application of N-acetyl serotonin in preventing or preparing a medicine for treating Alzheimer disease.

Alzheimer's Disease (AD) is the most common form of dementia, accounting for approximately 50% -75% of the population with dementia, has become an increasingly serious global health problem, placing a heavy burden on families and society, and has attracted general attention from governments and medical circles of various countries.

AD is a chronic progressive neurodegenerative disease that can be divided into early-onset AD and late-onset AD, depending on the age of onset of AD. The current research shows that late-onset AD is probably induced by complex genetic factors and environmental factors, wherein the genetic factors account for about 70 percent. In the etiological studies of AD, there is a lot of evidence that β -amyloid (Α β) protein is the major pathological link that induces the development of AD. Firstly, the A beta is a main component of senile plaques in AD brains and is also an important pathological characteristic of AD, and the A beta content in cerebrospinal fluid and blood is one of common biomarkers for differential diagnosis of AD; secondly, the aggregation of A beta can cause neurotoxicity, promote apoptosis of neuron cells, induce neuroinflammation reaction in brain, aggravate damage of neuron, and further cause AD hypomnesis and cognitive dysfunction [5]. Therefore, a β remains an important target currently recognized for screening anti-AD drugs.

A β is mainly produced by cleavage of a transmembrane Amyloid Precursor Protein (APP) having an extracellular domain, and APP is highly expressed in the brain. Under normal conditions, APP is involved in non-starch-like pathway processing, is cleaved by α -secretase and γ -secretase, and exists in the brain in soluble form. In the disease state, APP is abnormally cut by beta-secretase and gamma-secretase to form shorter soluble A beta polypeptide, and A beta is further deposited in the brain to form insoluble plaques. At present, it is widely believed that a β aggregation in large amounts in AD brain is one of the main causes of neurotoxicity. The brain A beta is mainly A beta ₄₀ And Abeta ₄₂ Two forms, abeta ₄₀ About 90% of A beta protein, A beta ₄₂ 5-10% of total Abeta, and Abeta ₄₂ The high insolubility and the stronger neurotoxicity are main toxic components of A beta aggregation to form insoluble plaques, can cause ion channel closure, calcium homeostasis change, mitochondrial oxidative stress increase and the like, and finally cause neuron death. The pathogenesis of AD is not clear, either, because of its complex etiology. To date, there is no effective therapeutic agent. Currently, acetylcholinesterase inhibitors and N-methyl-D aspartate receptor antagonists are mainly used clinically, but these drugs cannot reverse the progression of AD. Therefore, drug development aimed at inhibiting the aggregation of a β in AD brain may become a potential therapeutic approach for AD. N-acetyl serotonin (NAS) is an intermediate in the endogenous synthesis reaction from 5-hydroxytryptamine to melatonin. It is prepared from 5-hydroxytryptamineProduced by reaction with acetyl-coa catalyzed by N-acetyltransferase, followed by methylation of NAS by S-adenosylmethionine to melatonin catalyzed by acetyl serotonin O-methyltransferase. Like melatonin, NAS is also an agonist of melatonin receptors (MT 1, MT2 and MT 3) and is considered to be a neurotransmitter. However, the application of NAS in preventing or treating AD drugs or health care products is not yet seen.

Disclosure of Invention

Based on the background, the invention provides the application of N-acetyl serotonin in preventing or preparing the medicament for treating the Alzheimer disease. Aiming at solving the technical problems of unsatisfactory drug effect and large toxic and side effect of the existing clinical treatment drugs for inhibiting the aggregation of Abeta and resisting AD.

The technical scheme of the invention provides application of N-acetyl serotonin in preventing or preparing medicaments for treating Alzheimer disease.

In the above application, the Alzheimer disease is abnormal aggregation of beta-amyloid protein.

The N-acetyl serotonin is used as an inhibitor of A beta protein aggregation.

The abnormal aggregation of the A beta protein comprises inhibiting the oligomerization or fibrosis of the A beta protein caused by gene mutation or environmental factors to form A beta oligomers and insoluble fibers and plaques.

The N-acetyl serotonin is applied to functional foods and health products.

Also includes an effective amount of N-acetyl serotonin.

The effective antagonistic A beta neurotoxicity dose of N-acetyl serotonin is 5-50 mu M.

Also comprises a carrier of the N-acetyl serotonin which is accepted in pharmacy.

The carrier comprises at least one of an adjuvant, a compound or a composition for stabilizing the N-acetyl serotonin, which imparts to the pharmaceutical dosage form.

The invention has the beneficial effects that: experiments prove that the NAS has relatively strong inhibition effect on A beta aggregation, and the NAS effectively antagonizes SH-SY5Y neurotoxicity induced by the A beta under a proper dosage, so that the NAS can possibly prevent or treat AD neurotoxicity caused by abnormal A beta protein aggregation. In addition, the invention provides the application possibility of NAS in preparing drugs for preventing/treating AD, and the result shows that the NAS acts on classical 3 × Tg-AD pathological model mice: the NAS disclosed by the invention can obviously improve the learning and memory ability of 3 × Tg-AD and the pathological characteristics of brain A beta, and shows that the NAS has an obvious therapeutic effect on 3 × Tg-AD mice, and the NAS provided by the invention has an effect of treating AD and can be applied to preparation of drugs for preventing or treating AD.

N-acetyl serotonin (NAS): is an intermediate endogenously produced by 5-hydroxytryptamine to produce melatonin, is an agonist of melatonin receptors (MT 1, MT2 and MT 3), and is considered to be an intracerebral neurotransmitter.

Beta-like amyloid protein (a β): a polypeptide containing 39 to 43 amino acid residues, produced by cleavage of APP by beta-secretase and gamma-secretase in pathological conditions, in which A beta ₄₀ And Abeta ₄₂ The protein is the major component of amyloid plaques, abeta ₄₂ The decrease in levels in cerebrospinal fluid may precede the appearance of amyloid plaques and is one of the common biomarkers for diagnosing AD. Due to soluble A beta ₄₂ The reduction is highly correlated with amyloid plaque formation and the occurrence of AD symptoms and is considered neurotoxic. Therefore, the study mainly used a β ₄₂ To simulate the related experiments of in vitro A beta aggregation, neuron toxicity induction and the like.

Alzheimer's Disease (AD): is the most common type of dementia, accounting for about 50% to 75% of the population with dementia. AD is a chronic progressive neurodegenerative disease characterized primarily by aggregation of the brain ap protein to form insoluble plaques, neuronal Tau protein aggregation, and synaptic loss. According to the age of onset of AD, it can be classified into early-onset AD and late-onset AD. The current research shows that late-onset AD is probably induced by complex genetic factors and environmental factors, wherein the genetic factors account for about 70 percent. There is currently no effective treatment.

Based on a large number of researches, the NAS has a relatively strong effect of inhibiting the aggregation of the Abeta protein, and the effect of inhibiting the toxicity of the Abeta is realized, so that the activity of neurons is improved, and the AD brain pathological characteristics and the learning and memory ability are improved. Based on this, the present invention provides the use of NAS in the following related aspects.

In one aspect, embodiments of the present invention provide the use of NAS as an inhibitor of a β protein aggregation. Relevant experiments show that the NAS is an active ingredient, can effectively inhibit abnormal aggregation of Abeta protein, is expected to realize the function of inhibiting the deposition of amyloid plaques in AD brain, is an endogenous substance, and has the characteristics of small toxic and side effects, high tolerance and the like. In addition, in vitro experiments show that the effective dose of the NAS for inhibiting SH-SY5Y neuron toxicity induced by Abeta is low, the neuroprotective effect of higher-concentration NAS is more obvious, and the drug effect has good dose dependence relation with the concentration thereof in a certain range.

On the other hand, based on the above-described effect of the NAS on inhibiting the aggregation of a β, it is suggested that the NAS can be used for the preparation of a medicament for the prevention/treatment of AD. Therefore, the embodiment of the invention provides the application of NAS as a medicine or health-care product for preventing/treating AD, wherein the NAS is an effective component for inhibiting the aggregation of the A beta protein, so that the effects of treating the A beta deposition in AD brain and learning and memory function damage are achieved, and the effect of preventing/treating AD is achieved. The inventor proves that the NAS is used as a medicine for treating a 3 × Tg-AD model mouse through related experiments, and in-vivo experiments show that the NAS remarkably improves the learning and memory capacity of the 3 × Tg-AD mouse and obviously reduces the level of Abeta in a 3 × Tg-AD brain, so that the NAS provided by the invention has the potential of treating AD and can be applied to preparation of medicines or health care products for preventing or treating AD.

Also, based on the above, NAS has the functions and functions of inhibiting A beta aggregation, improving the learning and memory ability of 3 × Tg-AD mice and improving the pathological features of A beta in brain. The medicament comprises an effective dose of active ingredients for preventing/treating AD. Of course, the active ingredient is NAS and the "effective" is the ingredient that is therapeutically effective alone in the prevention or treatment of AD. The "effective dose" refers to an effective amount capable of preventing or treating AD, and refers to an amount of NAS sufficient to show benefit or clinical significance to an individual. One skilled in the art will appreciate that the actual amount or dose administered and the time course of administration will depend on the nature and severity of the disease being prevented or treated, the age and general condition of the subject being prevented or treated, and the mode of administration, among other factors.

In addition, the medicament for preventing/treating AD may further include a carrier component of a pharmaceutically acceptable NAS. The pharmaceutically acceptable carrier component of the NAS can be a corresponding carrier of a corresponding dosage form prepared according to the AD drug administration mode. The carrier includes, but is not limited to, at least one of an adjuvant, a compound or a composition for imparting stability to the NAS. Wherein the adjuvants can be conventional liquid adjuvants and powder adjuvants. The adjuvant can be gelatin, pregelatinized starch, etc. Any pharmaceutically acceptable carrier that can support the NAS or further facilitate the stabilization and absorption of the NAS is within the scope of the disclosure of the embodiments of the present invention. Therefore, the drugs for preventing/treating AD can be selected according to the type of the carrier required for clinical administration, so as to present corresponding dosage forms.

Therefore, the medicament for preventing/treating AD contains the NAS, so that the medicament can effectively inhibit A beta protein aggregation, improve the learning and memory capacity of 3 x Tg-AD mice and the function and effect of the pathological features of the brain A beta, and endow the medicament for preventing/treating AD with effective preventing/treating effect. In addition, the NAS is an endogenous protein substance, so the NAS has small toxic and side effects and is safer to use clinically.

Drawings

FIG. 1 ThT fluorescence intensity assay for A β protein aggregation;

FIG. 2 is an electron microscope for observing the aggregation of A beta protein;

FIG. 3 immunoblotting to reflect aggregation of A.beta.protein;

FIG. 4 detection of NAS antagonism of A β 42 induced SH-SY5Y neuronal cytotoxicity;

FIG. 5NAS inhibition of A beta 42 induced SH-SY5Y neuronal apoptosis-related protein expression;

FIG. 6 shows that the intraperitoneal injection of NAS improves the results of the behavioral experiments of 3 × Tg-AD mice and WT mice;

FIG. 7 the expression of A beta pathologically-related protein in the brain of 3 × Tg-AD mice improved by the NAS intraperitoneal injection.

Detailed Description

Now, the use of NAS for inhibiting a β protein aggregation, antagonizing a β -induced neuronal toxicity, and improving learning and memory ability and intracerebral a β pathology in 3 × Tg-AD mice will be further elaborated with reference to specific examples.

As mentioned above, due to A beta ₄₂ Has effects in aggregating and inducing neuronal toxicity. Therefore, hereinafter, A β is used ₄₂ A beta protein model was designed and studied as a representative to verify the effects of NAS in inhibiting A beta protein aggregation, antagonizing A beta-induced neuronal toxicity, and improving learning and memory ability and intracerebral A beta pathological characteristics in 3 × Tg-AD mice.

The experimental raw materials and the related reagents in the following examples:

main experimental apparatus: full-wavelength multifunctional microplate readers (Biotek, USA), electrophoresis (BIO-RAD, USA), membrane transfer devices (BIO-RAD, USA), western blotting gel imaging devices (BIO-RAD, USA), electron microscopes (JEOL, JEM-1011), electronic balances (Aohaus instruments, inc.), pipettors (Eppendorf, germany), open field experiment devices, morris Water maze experiment devices, video tracking systems (Ethovision 3.0, noldus Information technology, netherlands).

Main experimental materials and experimental animals: abeta (beta) ₄₂ Protein (Hubei Qiangyao Biotechnology Co., ltd.), NAS (Hubei Qiangyao Biotechnology Co., ltd.), thioflavin T (Shanghai Ebo anti-trade Co., ltd.), 6E10 rabbit antibody (U.S., cell Signaling Technology), beta-actin mouse antibody (Wuhan Border bioengineering Co., ltd.), caspase 3/p17/p19 polyclonal rabbit antibody (U.S., proteintech), clear caspase-3 rabbit antibody (U.S., cell Signaling Technology), goat anti-rabbit IgG (Wuhan Border bioengineering Co., ltd.), goat anti-mouse IgG (Wuhan Border bioengineering Co., ltd.), ECL chemo-hypersensitive luminescent liquid (Wuhan Border bioengineering Co., ltd.), CCK-8 Cell proliferation and cytotoxicity detection kit (Wuhan Border bioengineering Co., ltd.), wild Type (WT) control mouse, 3 × Tg-AD mice (purchased from jackson laboratories).

Example 1: NAS inhibition of Abeta ₄₂ ThT fluorescence monitoring experiment of protein fibrosis.

The method comprises the following steps: thioflavin T (ThT) and Abeta ₄₂ The beta-sheet layer in the aggregated fiber filament interacts with each other to generate fluorescence, and the fluorescence intensity of the fluorescence is in positive correlation with the amount of the fiber, so that the fluorescence can be used for quantitative analysis of the amount of the protein fiber. Precision weighing of Abeta ₄₂ The lyophilized protein was dissolved in DMSO to prepare a stock solution having a concentration of 1mM, and diluted to 20. Mu.M with PBS at the time of use. NAS was weighed accurately, made up into high concentration drug stock (200. Mu.M) in DMSO, and diluted to 20. Mu.M in PBS for use. The respective reagents were added to 96-well plates, with 12 replicates per sample, grouped according to the experiments in table 2. The samples were incubated at 37 ℃ for 108 hours, and the fluorescence values of the samples in each well were monitored dynamically every 12 hours using a microplate reader (Biotek Synergy H1). Deriving data from A beta ₄₂ Group A and A beta ₄₂ The relative fluorescence intensities of the + NAS groups were plotted for kinetic response curves. Wherein, the key parameters of the microplate reader are set according to the parameters shown in the table 1.

Table 1: setting of key parameters of microplate reader

Table 2: NAS for Abeta ₄₂ Experimental grouping of protein fibrosis

Grouping	NAS	Aβ ₄₂	ThT	PBS
					Aβ ₄₂ Group(s)	0μM	20μM	20μM	200 μ L/well
Aβ ₄₂ + NAS group	20μM	20μM	20μM							200 μ L/well

As a result: the response curve of the relative fluorescence intensity with time is shown in FIG. 1. The relative fluorescence intensity gradually increased with the passage of time, which means that A.beta. ₄₂ The number of aggregates of (a) gradually increases. After addition of NAS, the relative fluorescence intensity reached a maximum approximately up to 12 hours, then remained essentially stationary, and at 96 hours with A β ₄₂ The group differences are the most significant, which shows that the drug concentration is 20 mu M, the A beta can be effectively inhibited ₄₂ And (4) performing fibrosis aggregation. As is clear from the experimental results of FIG. 1, the NAS indicates the A.beta. ₄₂ The aggregation of the protein was inhibited, and the inhibition was already evident at a concentration of 20. Mu.M (. P.ltoreq.0.05,. P.ltoreq.0.01).

Example 2: NAS inhibition of Abeta ₄₂ Transmission electron microscopy experiments on protein fibrosis.

The method comprises the following steps: the NAS-treated A beta can be visually observed by a Transmission Electron Microscope (TEM) ₄₂ Aggregation fibrosis was used as a strong evidence for ThT fluorescence experiments. Separately diluting A.beta.with PBS ₄₂ And NAS stock to 20 μ M, divided into Abeta ₄₂ Group A and A beta ₄₂ + NAS group, incubating at 37 deg.C for 96 hr, taking 5 μ L samples, dripping on copper net, standing at room temperature for 2min, and removing much by filter paperAnd (3) adding 1% phosphotungstic acid dropwise into the rest samples, carrying out negative dyeing for 1 minute, and standing at room temperature for 20min. The plate was placed in a transmission electron microscope (JEOL, JEM-1011), observed at an accelerating voltage of 80kV, photographed and the image was saved.

As a result: the transmission electron micrograph is shown in FIG. 2, in which FIG. 2-A is a control group, and Abeta is observed by electron microscopy ₄₂ Protein fibers, which show that the addition of PBS does not cause A beta ₄₂ Solubilization of protein fiber, and Abeta ₄₂ Forming uniform and slender fiber filaments with higher fiber density. As can be seen from FIG. 2-B, there was no obvious Abeta in the electron microscope picture after the NAS was added ₄₂ Fiber entanglement formation. Indicating that NAS effectively blocks A beta ₄₂ The protein aggregates to form fibers.

Example 3: NAS inhibition of Abeta ₄₂ Immunoblot assay for protein aggregation

The method comprises the following steps: the 6E10 monoclonal antibody can be used for detecting the content of the Abeta oligomers in a protein immunoblotting experiment, and can further prove the ThT fluorescence experiment in example 1. Two groups of samples in example 2 are added into SDS-PAGE gel with 10 holes, the sample loading amount of each hole is 20 mu L, each group is provided with 6 multiple holes, electrophoresis is carried out at constant voltage of 80V, then the PVDF membrane is used for membrane rotation at 15V for 15 minutes, the skimmed milk powder is used for sealing for 3 hours, and 6E10 monoclonal antibody is used for overnight incubation. The following day the strips were washed three times for 15 minutes each, followed by incubation of the strips with goat anti-rabbit secondary antibody at room temperature for 120 minutes and washing of the secondary antibody five times for 10 minutes each. Finally, the strip is exposed in an exposure instrument by using an ECL chemiluminescence reagent, an exposed image is reserved, and the developed image is quantitatively analyzed.

As a result: the results of the immunoblotting experiment are shown in FIG. 3, in which A.beta.is shown in FIG. 3-A ₄₂ Representative immunoblot strips incubated alone or with NAS and A β after addition of NAS ₄₂ The content of toxic oligomers formed by aggregation is significantly reduced. FIG. 3-B shows A.beta.oligomer content/A.beta. ₄₂ As a result of quantitative analysis of the monomer content, it was found from the graph that A.beta.was added after adding NAS ₄₂ The increase of the monomer content and the decrease of the oligomer content indicate that the NAS can inhibit the A beta ₄₂ The monomers aggregate to form a β oligomers with neurotoxicity (. About.p < 0.05).

Example 4: NAS antagonizing A beta ₄₂ Induction ofToxicity test of SH-SY5Y neuron

The method comprises the following steps: the CCK-8 reagent is reduced by dehydrogenases in the mitochondria of living cells to a highly water-soluble orange-yellow formazan product, the amount of which is proportional to the number of living cells. The absorbance of the cell is measured at the wavelength of 450nm by using a microplate reader, and the cell activity can be indirectly reflected. SH-SY5Y cells are human neuroblastoma cells, and are commonly used in cytological study of AD and other neurodegenerative diseases, and are prepared by collecting logarithmic phase SH-SY5Y cells, adding 100 μ L cell suspension to each well of 96-well plate, adjusting the cell number to 2000, and making 5% CO at 37 deg.C ₂ After 24 hours of culture in a cell incubator, adding medicines according to the experiment groups in the table 4, setting 8 multiple holes in each group, continuously culturing for 24 hours in the cell incubator, sucking culture solution, adding 90 muL of complete culture medium and 10 muL of CCK-8 reagent into each hole, measuring the absorbance OD value of each hole at 450nm by using an enzyme-labeling instrument after 2 hours, setting key parameters of the enzyme-labeling instrument in the table 3, calculating the relative absorbance of the experiment group and drawing a relative OD value histogram depending on NAS concentration.

Table 3: setting of key parameters of microplate reader

Table 4: NAS inhibition of Abeta ₄₂ Experimental groups for Induction of SH-SY5Y cytotoxicity

As a result: CCK-8 results are shown in FIG. 4, A.beta. ₄₂ Relative OD was decreased, while co-treatment with 5 μ M, 20 μ M and 50 μ M NAS increased relative OD, with the most significant increase in OD with 20 μ M NAS (P < 0.05, P < 0.01). The results indicate that A.beta. ₄₂ Has cytotoxicity, can remarkably inhibit SH-SY5Y neuron activity, and has low concentrationNAS processing of degree can reverse Abeta ₄₂ The effective concentration of the induced neuron damage is 5-50 mu M, and the ability of promoting the neuron activity is most remarkable when the concentration is 20 mu M.

Example 5: NAS inhibition of Abeta ₄₂ Induced apoptosis of SH-SY5Y neurons

The method comprises the following steps: caspase-3 is the most important terminal cutter in apoptosis, has a relative molecular weight of about 32kDa, and generates a caspase-3 cleavage fragment (cleared caspase-3) with a molecular weight of about 17kDa during activation. The ratio of cleared caspase-3 to caspase-3 is believed to reflect the degree of apoptosis, with higher ratios leading to more severe apoptosis. In this example, SH-SY5Y cells were cultured in 6-well plates and administered in groups according to the experiment of Table 5, and the optimum NAS concentration of 20 μ M was selected for administration intervention according to the results of example 4, with 5 replicate samples per group. The cells after administration were at 37 ℃,5% CO ₂ After 24 hours of incubation in a cell incubator, cellular proteins were extracted and heat denatured. And adding each group of samples into 10-well SDS-PAGE gel, loading 15 mu L of sample to each well, performing 80V constant pressure electrophoresis, then performing membrane rotation for 25 minutes at 15V by using a PVDF membrane, sealing for 3 hours by using skim milk powder, and respectively performing overnight incubation by using beta-actin, caspase-3 and cleared caspase-3 primary antibody. The following day the strips were washed once three times for 15 minutes each, after which the strips were incubated with either goat anti-mouse secondary antibody or goat anti-rabbit secondary antibody for 120 minutes at room temperature and the secondary antibodies were washed five times for 10 minutes each. Finally, the strip is exposed in an exposure instrument by using an ECL chemiluminescence reagent, an exposed image is reserved, and the developed image is subjected to quantitative analysis.

Table 5: NAS inhibition of Abeta ₄₂ Experimental grouping of induced SH-SY5Y neuron apoptosis

Grouping	NAS	Aβ ₄₂
			Blank control group	0μM	0μM
NAS group	20μM	0μM
			Aβ ₄₂ Group of	0μM	5μM
Aβ ₄₂ + NAS group	20μM	5μM

As a result: FIG. 5 shows the results of example 5, and FIG. 5-A shows representative immunoblot bands of three proteins, beta-actin, caspase-3, and cleaned caspase-3, of groups of SH-SY5Y neurons passing through A beta ₄₂ After single treatment, the total caspase-3 content is reduced, the clear caspase-3 content is increased, and NAS and A beta are added ₄₂ After the neurons are treated together, the total caspase-3 level of the neurons rises, and the clear caspase-3 content falls, which indicates that the NAS can inhibit the Abeta ₄₂ Mediated neuronal apoptosis is exacerbated. FIG. 5-B is the quantitative analysis result of the cleaned caspase-3/caspase-3 ratio, from which it can be understood that A.beta.was added separately ₄₂ There was a trend towards promotion of neuronal apoptosis (P = 0.098), whereas the clear caspase-3/caspase-3 ratio decreased with statistical differences (. About.P < 0.01) after addition of NAS, indicating that 20. Mu.M NAS was effective in inhibiting A.beta. ₄₂ Function of induced SH-SY5Y neuronal apoptosis.

Example 6: behavioral experiment for improving learning and memory abilities of 3 XTG-AD mice by intraperitoneal injection of NAS (non-aqueous medium)

The method comprises the following steps: open field experiments, new object identification experiments and Morris water maze experiments are commonly used animal behavior experiments reflecting emotion and cognitive memory ability. Six-month-old 3 × Tg-AD mice and WT mice were selected and randomly divided into a WT group, a WT + NAS group, a 3 × Tg-AD group, and a 3 × Tg-AD + NAS group, with 7 mice per group. Mice were given NAS (20 mg/kg) or saline (0.9%) intraperitoneally for 28 consecutive days, once daily. After the intraperitoneal injection is finished, the open field experiment, the new object identification experiment, the Morris water maze experiment and other behavioral experiments are carried out on each group of mice. (1) open field experiment: mice were placed in the center of a 40cm x 40cm open field device, allowed to explore freely for 5 minutes, and the trajectory was captured using a trajectory tracking system, one mouse at a time, and 75% alcohol was used to remove odors and remaining feces after each mouse was completed. And (3) defining the area of 1/4 of the center as a central area, acquiring the total movement path of the mouse and the relative time ratio of the central area, counting, and deriving a track route map of the mouse during the free exploration. The longer the mouse stays in the central zone indicates the lower the anxiety depression degree of the mouse. (2) New object identification experiment: the experiment divides the two rounds to go on, and the first round experiment uses open field experiment for the adaptation period open field device, put into two colours, the identical object A of shape and object B in one side in open field for two object centres are apart from wall 10cm, and two centre of a circle intervals 20cm make the mouse freely move about 5 minutes in open field, record mouse movement track, think it is exploring the object when the mouse gets into the object for the region that centre of a circle radius is 7.5 cm. And (3) entering a second round of experiment, namely a testing period, 6 hours later, replacing the object B of the first round of experiment with an object C with completely different shape and color, enabling the mice to freely move for 5 minutes in an open field, recording the time for exploring the object A and the object C by using a video tracking system, and removing odor and leaving excrement by using 75% of alcohol after each mouse is finished. The new object recognition index (NOI) of each mouse is calculated as the total length of time for exploring object C/(total length of time for exploring object a + total length of time for exploring object C) × 100%, and the larger the NOI value is, the better the cognition and memory ability of the new things in the mouse is. (3) Morris Water maze experiment: a pool with the diameter of 1.2 meters is placed in the center of a room and is divided into 4 quadrants. The pool was filled with opaque water at 23 ℃ and the platform was 1 cm below the water surface in the fourth quadrant (target quadrant). Each time the mouse was placed in the water back to the center of the pool at the center point of each quadrant. The time was started when the mouse was placed in the water. If the animal does not find the platform within 60s, it can be guided to the bench. The positional navigation phase lasted 5 days, with 4 experiments per mouse per day. The quadrant order was randomly changed for each mouse per experiment. The 6 th day of the experiment is a space exploration stage, the underwater platform is removed, the animals are released from any two quadrants except the target quadrant, and the swimming tracks of the animals and the swimming time of the four quadrants are recorded. The mouse swimming trajectory and escape latency were recorded using a video tracking system. The shorter the escape latency during the positioning navigation, the better the learning ability of the mouse is reflected; the greater the percentage of time spent in the target quadrant during the space exploration phase, the better the memory of the mouse is reflected.

As a result: the results of the mouse behavioural experiments are shown in figure 6. Fig. 6-a is the total movement path in the open field experiment for each group of mice, suggesting that NAS significantly improved the locomotor capacity of WT mice (. Star.p < 0.01). FIG. 6-B is a graph of the ratio of the residence time in the central zone of each group of mice in the open field experiment, and the results show that the anxious-depressed level of 3 × Tg-AD mice is higher than that of WT mice (. About.P < 0.05), while the intraperitoneal injection of NAS improves the anxious-depressed state of WT mice (. About.P < 0.05) and 3 × Tg-AD mice (P = 0.09), respectively. FIG. 6-C is a graph of representative movement traces for each group of mice in the open field experiment, with the total area of movement of the mice in the blue box and the area of the central zone in the red box. Fig. 6-D is a histogram of the new object recognition index (NOI) of each group of mice at the test stage of the new object recognition experiment, the NOI of the 3 × Tg-AD mouse is significantly reduced (P < 0.05) compared with the WT mouse, which indicates that the spatial recognition memory capacity of the 3 × Tg-AD mouse of the AD model mouse is impaired, and the NAS injected into the abdominal cavity can significantly improve the NOI of the 3 × Tg-AD mouse (P < 0.05), which indicates that the NAS has the function of improving the spatial recognition memory capacity of the 3 × Tg-AD mouse. Fig. 6-E are line graphs of escape latency versus time for each group of mice during the Morris water maze positioning navigation phase. * Denotes 3 × Tg-AD + Saline vs WT + Saline, ^## represents 3 XTg-AD + NAS vs 3 XTg-AD + Saline. FIG. 6-E results demonstrate that 3 × Tg-AD mice injected intraperitoneally with NAS escape latency is lower than their control 3 × Tg-AD mice throughout the localized voyage phase and have statistical statistics in the day 5 localized voyage experimentThe difference between the two theories ( ^## P is less than 0.01). FIG. 6-F is a schematic representation of representative swim trajectories for each group of mice at day 5 of the positional voyage phase. Fig. 6-G is a statistical histogram of the percent of target quadrant residence time for each group of mice during the spatial exploration phase, with 3 × Tg-AD mice having a reduced percent of target quadrant residence time (./P < 0.05) compared to WT mice, but an increased percent after intraperitoneal NAS injection (P = 0.13). The Morris water maze result shows that the learning and memory behaviors of the 3 × Tg-AD mice are damaged, and the learning and memory abilities of the 3 × Tg-AD mice can be improved by the intraperitoneal injection of the NAS.

Example 7: improvement of 3 XTG-AD mouse intracerebral Abeta pathology by NAS intraperitoneal injection

The method comprises the following steps: the 3 × Tg-AD mouse can express APP protein in brain, and the APP protein is cracked to generate A beta protein and CTF protein. Mice in four groups, WT group, WT + NAS group, 3 × Tg-AD group and 3 × Tg-AD + NAS group in example 6 were sacrificed, 4 mice per group. After the left ventricle of each mouse is perfused with physiological saline, the head is cut off, and the tissue proteins are extracted and thermally denatured after the bilateral cerebral cortex tissues are taken on ice. Adding each group of samples into SDS-PAGE gel with 10 pores, loading 12 mu L of each pore, performing 80V constant voltage electrophoresis, and then performing membrane conversion by using a PVDF membrane under the condition of 15V, wherein the membrane conversion time of APP protein is 45 minutes, the membrane conversion time of beta-actin protein is 25 minutes, and the membrane conversion time of A beta and CTF protein is 7 minutes. Skim milk was blocked for 3 hours and incubated overnight with beta-actin, abeta, and CTF primary antibody, respectively. The following day the strips were washed once three times for 15 minutes each, followed by incubation of the strips at room temperature for 120 minutes using either goat anti-mouse secondary antibody or goat anti-rabbit secondary antibody, and washing the secondary antibody five times for 10 minutes each. Finally, the strip is exposed in an exposure instrument by using an ECL chemiluminescence reagent, an exposed image is reserved, and the developed image is subjected to quantitative analysis.

As a result: FIG. 7 shows the results of example 7, and FIG. 7-A shows four representative immunoblot bands of proteins such as APP, CTF, abeta, and β -actin in cortical tissues of mice in each group, from which it can be seen that the expression levels of APP, CTF, and Abeta in cortical tissues of WT mice are very low, the expression levels of APP, CTF, and Abeta in cortical tissues of 3 × Tg-AD mice are significantly increased, and the intraperitoneal injection of NAS significantly reduces the contents of CTF and Abeta proteins in cortical tissues of 3 × Tg-AD mice, which indicates that the intraperitoneal injection of NAS can reduce the production of Abeta in the cortical tissues of 3 × Tg-AD mice and alleviate the pathology of Abeta in the brain. Fig. 7-B, C, D are the results of quantitative analysis of the relative amounts of APP, CTF, and Α β proteins in mouse cortical tissue (P < 0.05, P < 0.005), respectively.

In one aspect of the invention, there is provided the use of NAS as an inhibitor of a β protein aggregation. The abnormal aggregation of a β protein includes oligomerization or fibrosis of a β protein caused by gene mutation or environmental factors and formation of toxic a β oligomers and insoluble a β fibers and plaques. The NAS of the invention has the following beneficial effects: experiments prove that the NAS has a relatively strong function of inhibiting abnormal aggregation of the A beta protein, so that the NAS can play a role in effectively inhibiting the abnormal aggregation of the A beta protein after being used as an inhibitor of the abnormal aggregation of the A beta protein, thereby preventing or treating AD neurotoxicity caused by the abnormal aggregation of the A beta protein. On the other hand, the invention provides the application of the NAS in the medicines and health products for preventing/treating the AD, and in-vivo experiments show that the NAS remarkably improves the learning and memory capacity of a 3 x Tg-AD mouse and obviously reduces the level of Abeta in a 3 x Tg-AD brain, which indicates that the NAS provided by the invention has the potential of treating the AD and can be applied to the preparation of the medicines or health products for preventing or treating the AD. In addition, the NAS as an endogenous substance of an organism has no toxicity, good tolerance and small side effect, and is expected to become a potential medicament for treating AD and other diseases related to the main pathological characteristics of abnormal aggregation of Abeta.

Claims

1.N-acetyl serotonin can be used for preventing or preparing medicine for treating Alzheimer disease.

2. Use according to claim 1, characterized in that: the Alzheimer's disease is abnormal aggregation of beta-like amyloid.

3. Use according to claim 1, characterized in that: the use of said N-acetyl serotonin as an inhibitor of the aggregation of A beta proteins.

4. Use according to claim 3, characterized in that: the abnormal aggregation of the A beta protein comprises inhibiting the oligomerization or fibrosis of the A beta protein caused by gene mutation or environmental factors to form A beta oligomers and insoluble fibers and plaques.

5. Use according to claim 1, characterized in that: the N-acetyl serotonin is applied to functional foods and health products.

6. Use according to claim 1, characterized in that: comprising an effective amount of N-acetyl serotonin.

7. Use according to claim 6, characterized in that: the dose of the N-acetyl serotonin for effectively antagonizing A beta neurotoxicity is 5 mu M-50 mu M.

8. Use according to claim 6 or 7, characterized in that: also comprises a carrier of the N-acetyl serotonin which is accepted in pharmacy.

9. Use according to claim 8, characterized in that: the carrier comprises at least one of an adjuvant, a compound or a composition for stabilizing the N-acetyl serotonin, which imparts to the pharmaceutical dosage form.