CN114344302B

CN114344302B - Luo Shasi use of a cerebellar acetaldehyde dehydrogenase inhibitor for the preparation of a sober-up product

Info

Publication number: CN114344302B
Application number: CN202210171891.1A
Authority: CN
Inventors: 蒋晓萌; 杨潇潇; 江宏; 高永耀
Original assignee: Zhejiang Jianfeng Pharmaceutical Co ltd
Current assignee: Zhejiang Jianfeng Pharmaceutical Co ltd
Priority date: 2022-02-24
Filing date: 2022-02-24
Publication date: 2023-06-20
Anticipated expiration: 2042-02-24
Also published as: CN114344302A

Abstract

The invention discloses an application of Luo Shasi in preparing cerebellar acetaldehyde dehydrogenase inhibitors and sobering products, wherein different behaviours are adopted to evaluate the influence of roflumilast on a mouse drunk model, and a western blotting method is adopted to explore the influence of the roflumilast on mouse ALDH2 protein. The result shows that Luo Shasi has obvious effect of improving the drunk mice and is expected to become sobering-up agent for scientific research and medicine clinic.

Description

Luo Shasi use of a cerebellar acetaldehyde dehydrogenase inhibitor for the preparation of a sober-up product

Technical Field

The invention belongs to the application field of roflumilast and explores a mechanism of action through a behavioural test and western blotting.

Background

Alcohol is one of the most commonly abused drugs, and when the alcohol concentration in blood exceeds 1000mg/L, humans have obvious symptoms of alcoholism, such as excitement, walking instability, sleepiness, anesthesia, etc. Long-term, repeated alcoholism, such that both physical and mental aspects are significantly altered, results in serious impairment of social functions, professional functions and social fitness. The consequences of excessive alcohol consumption and even alcohol abuse are very serious and may even be life threatening. High consumption of wine may increase the risk of developing certain tumors and may also cause impaired immune function and damage to the central nervous system.

The cerebellum is involved in the control of motor coordination, planning, fine regulation of voluntary movements, and cognitive functions in animals. Recently, it has been found that the cerebellum area has an alcohol metabolic pathway independent of the liver, indicating that alcohol intake into the body will cross the blood brain barrier directly into the cerebellum for metabolism, which provides theoretical support for drunk behavior. It was further found that blocking the expression of cerebellar de-acetaldehyde dehydrogenase (ALDH 2) eliminates the alcoholic response in mice, and that overexpression of this enzyme aggravates the alcoholic behaviour of mice. Luo Shasi As a low oxygen induction factor prolyl hydroxylase (HIF-PH) inhibitor, the inhibitor can inhibit ubiquitination degradation of HIF, and has a certain treatment effect on alcoholic fatty liver disease. However, it has not been reported whether roflumilast has an ameliorating effect on drunk and whether the regulatory mechanism involves the ALDH2 gene in the cerebellar region.

Disclosure of Invention

The present invention has been made to solve the above problems, and an object of the present invention is to provide Luo Shasi for its new use, which is as follows:

luo Shasi he is used for preparing cerebellar acetaldehyde dehydrogenase inhibitors.

Luo Shasi its use in the preparation of sobering-up products.

The invention has the beneficial effects that:

the regulation of the roflumilast in the drunk reaction of the mice is evaluated by a behavioral test and a western blotting method, and the result shows that Luo Shasi can inhibit the expression of the ALDH2 gene to a certain extent, has obvious regulation effect on drunk and is expected to become sober-up agent for scientific research and pharmaceutical clinic.

Drawings

FIG. 1 is a field behavioral test of a roflumilast controlled sobering experiment;

fig. 2 is a pole climbing behavioral test of a roflumilast controlled sobering experiment;

FIG. 3 shows protein expression and gray scale statistics of ALDH2 in the brain regions of mice of each group.

Detailed Description

The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1

The experiment was performed using a mouse drunk behavioural test and a western blot method.

1. Experimental animals: male C57BL/6J mice 24, 8 weeks old, 25-28g pre-experimental body weight. The animals used were kept in the animal house for at least 1 week to accommodate the new environment before being used in the experiment. Mice were randomly divided into 3 groups, namely, a control group, an alcohol group and an alcohol plus roflumilast group, 8 mice per group were marked by cutting the toe. Luo Shasi he was dissolved in DMSO and diluted with PBS, and the dose of 10mg/Kg/day was used in this experiment, which was administered three days in advance.

2. Pole climbing training: 3 days before the experiment, the mice are placed on a smooth round rod for pole climbing training, so that the mice can independently climb down from the rod once and train three times a day.

3. Sobering-up functional behavioural index determination

Ethanol (31.25%, 2.0g/kg intraperitoneal injection) was administered to the experimental group, and the control group was intraperitoneal-injected with the same volume of sterilized water. Drunkenness determination is started 10min after ethanol is added: the mice are placed in open field to measure the activity track, and after the test is finished, the pole climbing training is performed, and the crawling condition is recorded.

Pole climbing experiment: the limb muscle strength and the coordination movement ability of the mice are mainly studied. Holding the tail of the mouse, placing the tail of the mouse downwards at the top of the rod, allowing the mouse to naturally climb down, recording the time of the mouse climbing from the top of the rod to (or sliding down) the box by using a stopwatch, continuously measuring 3 times to obtain an average value, recording the falling time of the mouse as 1s, recording the falling time as 0s if the muscles of the hind limbs are stiff, and recording the falling time as 0s.

Open field experiments: the field of the experiment was a square open box (length x width x height=40 cm x 40cm x 35 cm), with 25 cells equally divided at the bottom. The mice of the control group are put into the open field one by one, observed for 10min, and recorded within 5 min: (1) number of animal climbing cells (over 1/3 of animal body is over line and over line in different cells); (2) number of animal stands (with the animal's two front legs lifted, the mass is all standing on the rear leg); (3) number of times of moving physical hair. What was done before the laboratory: time of immobility; average speed; total crawling distance.

4. Western immunoblotting

At the end of the mouse behavioural test, euthanasia was performed, the mouse brain region was removed, the protein lysate was added, and the mixture was centrifuged at 12000rpm/min at 4℃and centrifuged for 10 minutes. Protein concentration was measured by BCA method, fixed concentration was set, and SDS-PAGE gel was run. After the transfer of the membrane is completed, cutting strips of corresponding molecular weights of internal reference and ALDH2, cleaning residual milk by PBS after 5% of skimmed milk is closed, applying corresponding primary antibodies, and incubating overnight at 4 ℃. After recovery of primary antibody, the strips were washed three times with 8 minutes each with PBST. And applying a secondary antibody corresponding to the primary antibody, recovering the secondary antibody after 1 hour, and cleaning with PBST for three times, wherein each time is 8 minutes later. Finally, the exposure operation is carried out on a chemical exposure instrument.

5. Analysis of results

When the mice were intraperitoneally injected with alcohol, the alcohol intoxicated feature was first exhibited by 10 minutes, which was manifested in a slow movement, in the open field experiment (fig. 1), the mice in the alcohol group had a reduced mobility, and the range of movement was reduced, in contrast to the recovery of the mobility of the roflumilast treatment group, which also increased the range of movement in the open field. In the pole-climbing test (fig. 2), it was also found that the mice of the alcohol group had greatly reduced grip ability and fallen all down. The roflumilast treatment group has the advantages that the grabbing capability of part of mice is enhanced, and the mice can climb up and down smoothly on the rod. Further, we found that alcohol stimulated expression of ALDH2, increased alcoholic response, and that roflumilast regulated to some extent was also achieved by inhibiting expression of this gene (figure 3).

According to the application of the roflumilast as the novel sober-up agent, the regulation of the drunk reaction of the roflumilast mice is evaluated through a behavioral test and a western immunoblotting method. The results show that Luo Shasi he has obvious regulating effect on drunk. Is expected to become sobering-up agent for scientific research and medicine clinic.

It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.

Furthermore, it should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and that this description is provided for clarity only, and that the disclosure is not limited to the embodiments described in detail below, and that the embodiments described in the examples may be combined as appropriate to form other embodiments that will be apparent to those skilled in the art.

Claims

1. Luo Shasi its use in the preparation of sobering-up products.