CN113230239B

CN113230239B - Application of learning and memory capacity medicine for treating Alzheimer's disease

Info

Publication number: CN113230239B
Application number: CN202110129474.6A
Authority: CN
Inventors: 张海燕; 袁增强
Original assignee: Jiangxi Yisen Aoyou Pharmaceutical Technology Co ltd
Current assignee: Jiangxi Yisen Aoyou Pharmaceutical Technology Co ltd
Priority date: 2021-01-29
Filing date: 2021-01-29
Publication date: 2023-08-29
Anticipated expiration: 2041-01-29
Also published as: CN113230239A

Abstract

The invention provides 1,2, 4-trimethoxybenzene or alpha-terpineol shown in the following formula 1 or 2 for the use of a learning and memory drug for treating Alzheimer's disease;pavlov's olfactory memory test of Abeta transgenic drosophila model prepared by elav-Gal4 promoter shows that after 1,2, 4-trimethoxybenzene or alpha-terpineol is given to drosophila in AD model, the ability of avoiding electric shock according to odor information is also obviously improved, and the learning and cognition ability of drosophila in AD model can be improved by 1,2, 4-trimethoxybenzene or alpha-terpineol, and the application of drosophila in AD model is disclosed.

Description

Application of learning and memory capacity medicine for treating Alzheimer's disease

Technical Field

The invention relates to a medicine, in particular to a medicine for treating Alzheimer's disease learning and memory defects.

Background

With aging of people, the incidence of aging-related diseases increases year by year. Among them, dementia diseases mainly including Alzheimer's Disease (AD) are dominant in neurodegenerative diseases. Alzheimer's disease is a neurodegenerative disease characterized by generalized dementia such as dysmnesia, aphasia, disuse, disrecognition, impairment of visual space skills, dysfunction in execution, personality and behavioral changes, commonly known as senile dementia.

According to the latest statistics, the existing dementia patients in China are 5000 tens of thousands. The incidence of AD was confirmed to be 3.94% in people over 60 years old. The high incidence of AD and the self-care ability of AD patients decrease year by year, bringing a tremendous burden to society and home. The existing medicines for clinical use can only partially relieve symptoms (such as memantine) and cannot delay the disease progression. The lack of AD control drugs has caused families and society to spend a great deal of time taking care of the daily life of AD patients and other demented patients, etc. This will undoubtedly become a potential social problem in the year-by-year expensive future. The increasing number of AD patients and the rising cost of personnel are increasingly accentuating the need to provide a drug that can delay the progression of AD disease.

The invention comprises the following steps:

AD differs from other demented diseases in that its pathology is mainly represented by β -amyloid (aβ) plaque deposition, tau entanglement and brain atrophy. Based on these pathological changes and related molecular mechanism studies, the Aβ hypothesis and the Tau hypothesis were proposed for the pathogenesis of AD. The Abeta hypothesis is a short peptide formed by N-terminal 1-42 amino acid residues formed by abnormal cleavage of the precursor protein APP by secretase, also called Abeta _1-42 (hereinafter, abbreviated as Abeta). Aβ can self-assemble in the extracellular space to form oligomers and deposit, producing neurotoxicity and inducing glial activation, ultimately inducing apoptosis. Based on the Abeta hypothesis, there are a variety of transgenic animal models of Abeta and its precursor proteins, such as a 5xFAD transgenic mouse model, an APP/PSEN1 transgenic mouse model, and an Abeta transgenic drosophila model. The animal models can well simulate pathological manifestations of Abeta deposition of AD patients with age, and reproduce clinical characteristics of cognitive impairment and hypolearning and memory capacity of AD patients.

The invention aims to provide a compound for the use of a learning and memory drug for treating Alzheimer's disease and a compound for the use of the drug for preparing the learning and memory drug for Alzheimer's disease, wherein the compound is 1,2, 4-trimethoxybenzene shown in the following formula:

and capsules, tablets or granules prepared by pharmaceutically acceptable auxiliary agents.

The Abeta transgenic drosophila model of the invention is prepared by expressing human Abeta in the whole brain neurons through elav-Gal4 promoter _1-42 . Human Abeta can be detected in brain on the first day after the drosophila emergence _1-42 Protein expression and brain with ageThe total amount of aβ in the sections increased, and extensive deposition of aβ was detected at 45 days of age. In Drosophila brain tissue, as Abeta is deposited, the levels of various inflammatory factors are up-regulated, and these pathological changes are consistent with clinical patients. Besides consistent pathological manifestations, the Drosophila model can also simulate clinical symptoms of AD patients, and is mainly characterized by increasing age, and the Drosophila model sequentially shows phenotypes such as learning and memory defects, crawling ability defects, neuron degeneration, life shortening and the like. Thus, the model is one of the better models for simulating various phenotypes in AD and for preclinical studies, and is also the preferred animal model for screening AD therapeutic drugs based on Abeta hypothesis.

Drawings

Fig. 1:1,2, 4-trimethoxybenzene (B4) improves the learning and cognition ability detection result of the AD model drosophila;

fig. 2: alpha-terpineol (A4) improves the learning and cognition ability detection result of the AD model drosophila;

fig. 3: genetic map of WT drosophila: P35 x 2U;

fig. 4: genetic map of AD drosophila P35H 29.3.

Examples

Materials and methods

Drosophila strain

All Drosophila used in this experiment were backcrossed for at least 5 generations to make them all at w ¹¹¹⁸ (isoCJ 1) in order to avoid the influence of different genetic backgrounds on the results. Thus, w ¹¹¹⁸ The drosophila melanogaster strain (abbreviated as "2U") was the control group for all experiments at this time. To avoid the potential adverse effect of long-term expression of Abeta 42 protein on drosophila strains, drosophila (H29.3) carrying regulatable human Abeta 42 gene is only used for regulating and starting drosophila elav-GAL4 expressed by Abeta 42 gene ^c155 (abbreviated as "P35") and its progeny express the Aβ42 protein in the nervous system. P35 was crossed with the genetic background Drosophila 2U, and the progeny Drosophila did not express Aβ42 protein. Learning tests were performed on the offspring described above. See fig. 3 and 4 for genetic map.

Drosophila feeding

Parent Drosophila was raised at 24℃and 40-60% relative humidity, and in a standard environment with 12 hours of light per day. From the second day after the emergence of the offspring drosophilse:Sup>A (day after eclosion, DAE 2), the control drosophilse:Sup>A (elav/Y;+/+) was selected under the microscope with the experimental group of drosophilse:Sup>A (elav/Y; UAS-A. Betse:Sup>A. 42/+). The selected drosophila melanogaster was transferred to 29 ℃ and subjected to subsequent feeding and drug treatment at 40±15% relative humidity.

Sample treatment and drosophila feeding

Using the samples, 50uL volumes were measured and weighed.

Based on its actual weight, a mother liquor of 5mg/ml was prepared using DMSO and the dissolution of the sample mother liquor is shown in the above table. The mother liquor was diluted to 0.05mg/ml working solution with 4% sucrose water and stored in a-20 ℃ refrigerator. Stability tests show that the working solution still has no obvious phase separation phenomenon after being stood for one week at room temperature.

Control compound Memantine (MEM) stock (100 mM) was stored in a-20℃refrigerator. The day before the start of dosing MEM was diluted to 100uM with 4% sucrose working solution. The test shows that the working liquid has no influence on the learning and memory ability of the drosophila.

From day 3 to day 9 (7 days total), each group was fed a drug for 4 hours per day, with a rest of 20 hours in fresh food. The drug was fed through filter paper, and 50ul of drug was fed per tube of Drosophila per day. Meanwhile, the control group drosophila was fed with an equal amount of sugar water or 100uM MEM. In this experiment, 8 data points were measured for each drug, each data point being an average of 2 Drosophila olfactory memories.

Detecting pavlov's olfactory memory:

study memory was tested in a constant temperature and humidity laboratory at 25 ℃,70% relative humidity.

During the training phase, about one hundred fruit flies are loaded into a training tube with copper mesh laid inside, and the flies smell two odors (usually OCT and MCH) successively. Each smell lasted 1min and 45s of fresh air was introduced immediately after the end. While the first smell was smelled by the drosophila, a shock penalty of 1 minute duration was also received. Such a procedure is performed successively, i.e. one training period. In order to avoid natural preference of Drosophila for smell, 1 tube of Drosophila will smell OCT first and receive electric shock, and another 1 tube of Drosophila smell MCH first and receive electric shock, so that the average value of 2 tube of Drosophila detection values is one data point.

During the detection phase, the fruit flies after training are immediately transferred to the selection point of the T-shaped maze for two minutes, and are selected in two odor containers. After 2 minutes, drosophila in different containers were counted separately and their behavior index PI was calculated. If the flies in the two containers are distributed in a 50:50 mode, pi=0, the flies cannot memorize the relation between electric shock and smell; pi=100 indicates that 100% of drosophila will memorize the connection between smell and electric shock, and avoid electric shock according to smell information.

Example 1:1,2, 4-trimethoxybenzene (B4) improves learning and cognition ability of AD model drosophila

The detection results are shown in fig. 1:

as shown in fig. 1, the aβ transgenic AD model drosophila had significantly reduced ability to avoid electric shock according to the odor information, compared to wild type drosophila, indicating that its olfactory memory ability was lost. After memantine is given as a positive control drug, the ability of Abeta transgenic AD model drosophila to avoid electric shock is remarkably improved according to smell information, which shows that memantine partially relieves the memory ability of AD model drosophila. Meanwhile, after the other group of AD model drosophila is given with 1,2, 4-trimethoxybenzene, the electric shock avoiding capability of the drosophila is also obviously improved according to the smell information, which indicates that the 1,2, 4-trimethoxybenzene can improve the learning and cognition capability of the AD model drosophila and prompts that the drosophila can be used for preparing medicines for treating AD.

Example 2: alpha-terpineol (A4) improves learning and cognition ability of AD model drosophila

The detection results are shown in fig. 2:

as shown in fig. 2, aβ transgenic AD model drosophila had significantly reduced ability to avoid electric shock according to odor information compared to wild type drosophila, indicating that these drosophila had lost olfactory memory. Whereas, after the positive control drug memantine was given, aβ transgenic AD model drosophila had a significant return in the ability to avoid electric shock according to the odor information, indicating that memantine partially alleviated the memory of AD model drosophila. Meanwhile, after the alpha-terpineol is given to the fruit flies of another group of AD models, the ability of avoiding electric shock according to smell information is also obviously improved, which shows that the alpha-terpineol can improve learning and cognition ability of the fruit flies of the AD models, and shows that the alpha-terpineol can be used for preparing medicines for treating AD.

Finally, it should be noted that: the above embodiments are only for illustrating the technical aspects of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the above embodiments, it should be understood by those of ordinary skill in the art that: modifications and equivalents may be made to the specific embodiments of the invention without departing from the spirit and scope of the invention, which is intended to be covered by the claims.

Claims

1. The application of the medicine for preparing the medicine for learning and memorizing the Alzheimer's disease is characterized in that the medicine is 1,2, 4-trimethoxybenzene shown in the following formula

2. The use according to claim 1, wherein the medicament is in the form of a capsule, tablet or granule made with pharmaceutically acceptable auxiliaries.