阅读说明：本技术 哌啶-1-二硫代甲酸-3-甲基-1，4-二氧-1，4-二氢萘-2-甲基酯的用途 (Use of piperidine-1-dithiocarboxylic acid-3-methyl-1,4-dioxo-1, 4-dihydronaphthalene-2-methyl ester ) 是由 孙诚 王玥珺 于 2021-01-25 设计创作，主要内容包括：本发明公开了一种哌啶-1-二硫代甲酸-3-甲基-1,4-二氧-1,4-二氢萘-2-甲基酯的用途,是在治疗帕金森疾病中的应用。本发明实验结果发现,帕金森模型(PD)小鼠采取灌胃方式给予PKM2-IN-1,发现该药物可显著缓解PD相关症状,如恢复黑质部位多巴胺能神经元数量,提高中脑部位酪氨酸羟化酶的表达,提高小鼠运动能力和嗅觉功能。(The invention discloses an application of piperidine-1-dithioformic acid-3-methyl-1,4-dioxo-1, 4-dihydronaphthalene-2-methyl ester in treating Parkinson's disease. The experimental result shows that the Parkinson model (PD) mouse adopts the gastric lavage mode to administer PKM2-IN-1, and the drug can obviously relieve PD related symptoms, such as the recovery of the number of dopaminergic neurons at the substantia nigra part, the improvement of the expression of tyrosine hydroxylase at the midbrain part and the improvement of the motor capacity and the olfactory function of the mouse.)

1. An application of piperidine-1-dithioformic acid-3-methyl-1,4-dioxo-1, 4-dihydronaphthalene-2-methyl ester in preparing a preparation for treating Parkinson's disease.

Technical Field

The invention relates to application of a compound piperidine-1-dithioformic acid-3-methyl-1,4-dioxo-1, 4-dihydronaphthalene-2-methyl ester.

Background

Parkinson's Disease (PD), also known as parkinsonism tremor, is the second largest neurodegenerative disease after alzheimer's disease that severely impacts human quality of life. Statistically, 1 out of every 800 people worldwide will have parkinson, and by 2030, the prevalence of parkinson will double, with over 900 million patients expected due to the accelerated aging process. The direct medical costs to the patient per year are expected to exceed $ 10,000, placing a heavy burden on the home and society. Parkinson's disease is characterized by progressive, multiple and occult onset of disease, and mainly manifests as bradykinesia, myotonia, resting tremor and postural instability. The main pathological features of parkinson are mainly represented by the decrease of dopaminergic neuronal cells in the substantia nigra site. The dopamine-producing cells in the brain gradually lose function affecting the nervous system, limiting the ability of the patient to control the muscles. Clinical treatment of Parkinson is mainly based on dopamine replacement therapy. The long-term use of the therapy can cause various adverse reactions, such as insane, insomnia, hallucination and other mental symptoms. Therefore, the search for new PD therapeutic drugs has great economic and social value.

Disclosure of Invention

The invention aims to provide application of piperidine-1-dithioformic acid-3-methyl-1,4-dioxo-1, 4-dihydronaphthalene-2-methyl ester.

The technical solution of the invention is as follows:

an application of piperidine-1-dithioformic acid-3-methyl-1,4-dioxo-1, 4-dihydronaphthalene-2-methyl ester in preparing a preparation for treating Parkinson's disease.

The invention provides application of piperidine-1-dithioformic acid-3-methyl-1,4-dioxo-1, 4-dihydronaphthalene-2-methyl ester in treating Parkinson's disease. According to the experimental result, a Parkinson model (PD) mouse adopts a gastric lavage mode to administer PKM2-IN-1, and the drug is found to be capable of remarkably relieving PD related symptoms, such as the number of dopaminergic neurons at the substantia nigra part is recovered, the expression of tyrosine hydroxylase at the midbrain part is improved, and the motor capacity and the olfactory function of the mouse are improved.

Drawings

The invention is further illustrated by the following figures and examples.

FIG. 1 is a structural formula of a compound piperidine-1-dithioformic acid-3-methyl-1,4-dioxo-1, 4-dihydronaphthalene-2-methyl ester (piperidine-1-carbodithioic acid-3-methyl-1,4-dioxo-1, 4-dihydronaphthalene-2-ylmethylether).

FIG. 2 is a schematic representation of piperidine-1-dithiocarboxylic acid-3-methyl-1,4-dioxo-1, 4-dihydronaphthalene-2-methyl ester (PKM2-IN-1) reducing parkinsonian model mouse motor dysfunction. Wherein: a: an experimental process; b: tail suspension experiment; c: climbing rod experiment; and D, olfactory test. PKM 2-IN-1: piperidine-1-dithiocarboxylic acid-3-methyl-1,4-dioxo-1, 4-dihydronaphthalene-2-methyl ester; MPTP: n-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (N-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine). P <0.05, p <0.01, one-way ANOVA assay.

FIG. 3 is a schematic representation of piperidine-1-dithiocarboxylic acid-3-methyl-1,4-dioxo-1, 4-dihydronaphthalene-2-methyl ester (PKM2-IN-1) alleviating the loss of dopaminergic neurons at the substantia nigra locus IN the mouse model Parkinson. Wherein: a: morphological analysis of dopaminergic neurons in substantia nigra. Immunohistochemical analysis was performed using the TH antibody. B: and analyzing the expression quantity of TH protein in the midbrain part. The protein expression was analyzed by a western blot method. TH: tyrosine hydroxylase (tyrosine hydroxylase); vehicle: a control group; PKM 2-IN-1: piperidine-1-dithiocarboxylic acid-3-methyl-1,4-dioxo-1, 4-dihydronaphthalene-2-methyl ester; MPTP: n-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (N-methyl-4-phenyl-1,2,3, 6-tetrahydropyridine). P <0.01, # p <0.001, # p <0.01, one-way ANOVA assay.

Detailed Description

An application of piperidine-1-dithioformic acid-3-methyl-1,4-dioxo-1, 4-dihydronaphthalene-2-methyl ester in preparing a preparation for treating Parkinson's disease.

Experimental procedure

1. Preparation of Parkinson's animal model

12 weeks old C57BL/6J male mice, PD model mice were induced by intraperitoneal injection of neurotoxin MPTP (N-methyl-4-phenyl-l,2,3, 6-tetrahydropyridine). Three days before administration, a series of behavioural training such as rod rotating, rod climbing and the like is performed on the mice every day. Mice with the same motor ability are selected as much as possible, so that the influence caused by individual difference is avoided, and the mice are divided into two groups. One group was saline group, and the other group was MPTP group. Then, MPTP (20mg/kg/day) was intraperitoneally administered for one week, and the behavioral assessment was performed again. Mice with a significant decrease in behavioural performance compared to control mice (injected with an equivalent volume of saline) were considered to be successfully induced PD model mice for subsequent experiments.

2. Drug administration treatment

Piperidine-1-dithiocarboxylic acid-3-methyl-1,4-dioxo-1, 4-dihydronaphthalene-2-methyl ester is dissolved in corn oil. Mice were treated with intragastric administration at a dose of 50mg/kg/day once a day for 7 consecutive days. Control mice were given an equivalent volume of corn oil.

3. Tail suspension experiment of mice

The immobility time in the tail suspension experiment of the mouse can reflect the depression state, and the specific method comprises the steps of fixing 1/3 behind the tail of the mouse by using an adhesive tape, suspending the mouse on a foam box with the height of 50cm, enabling the head of the mouse to be 15cm away from a table top, then carrying out 10min video recording, and observing the immobility time of the mouse in the period of time.

4. Pole climbing experiment

A pole of 1cm diameter, 50cm length and 1.5cm diameter cork pellets fixed at the top was made and gauze was wrapped to increase friction, the pole was placed upright, the mouse was placed on the pellet at the top of the home made climbing pole, then the time from the beginning of the animal's movement to the complete turn to head down until it returned to the bottom of the pole was recorded, 5 minutes apart for each test, 3 tests were averaged. Three days before tail vein injection, the mice are trained by climbing poles at the same time every day, and the mice with larger differences are removed to reduce experimental errors.

5. Olfaction function test

Feeding mouse cheese in advance before olfactory sensation test detection, fasting for 20h before olfactory sensation test, putting the mouse into a cage box of clean bedding materials in the test process, burying the cheese under the bedding materials by 2cm respectively in the middle, the upper left, the upper right, the lower left and the lower right, and detecting the time when the mouse finds the cheese.

6. Morphological analysis of dopaminergic neurons in substantia nigra

A histochemical method is used for staining tyrosine hydroxylase positive cells at the substantia nigra part of an experimental mouse, and the main operation steps are as follows: 1. placing the mouse brain tissue obtained from the materials in 4% paraformaldehyde, and placing in a refrigerator at 4 ℃ for 24 hours; 2.1 preparing 20% of sucrose by XPB, and dehydrating for 24 hours in sequence, wherein the time is prolonged if the tissue does not sink to the bottom; 3. treating dehydrated brain tissue slice, adjusting thickness to 12 μm, oven standing at 37 deg.C overnight, and storing at-20 deg.C; 4. baking the slices at 60 ℃ for 2 hours before dyeing, and washing the slices with PBS for 3 times, wherein each time is 5 minutes; 6. sealing with sealing liquid at 37 deg.C for 30 min; washing with PBS for 2 times, and blocking endogenous catalase for 5 minutes; washing with PBS for 2 times, using a nonspecific staining blocking agent for 30 minutes, and drying in a 37 ℃ oven; 8. incubated with TH antibody (1:200) overnight in a refrigerator at 4 deg.C; washing with PBS for 2 times, adding biotin-labeled goat anti-mouse/rabbit IgG, and washing at 37 deg.C for 1 hr; washing with PBS for 2 times, adding streptavidin-peroxidase, and washing at 37 deg.C for 1 hr; washing with PBS for 2 times, and performing DAB color development; 12. dehydrating with alcohol, sequentially adding 50% alcohol, 70% alcohol, 80% alcohol, 95% alcohol and 100% alcohol, each for 5 min; 13. absolute ethyl alcohol, dimethylbenzene and dimethylbenzene are transparent, each channel is 5 minutes, and the neutral resin is sealed and then is observed by a microscope to take a picture. The number of TH positive cells was statistically analyzed using Stereo investigerator software.

7. Preparation of protein sample at substantia nigra part and detection of expression level

The formula of the tissue lysate comprises: 25mM Tris-HCl, pH 7.4; 10mM NaF; 10mM Na₄P₂O₇；2mM Na₃VO₄(ii) a 1mM EGTA; 1mM EDTA; 1% NP-40; 10 mu g/ml Leuppeptin; 10 μ g/ml Aprotinin; 2mM PMSF; 20nM Okadaic acid. The homogenate was homogenized with a bench homogenizer (Polytron, PT2100), the sample was spun to lyse at 4 ℃ for 1 hour and centrifuged (13000rpm, 4 ℃) for 20 minutes after which the supernatant was carefully removed and the remaining supernatant was transferred to another centrifuge tube and centrifuged again. This process was repeated 2 times to completely remove the lipids from the protein sample. And (3) determining the protein content of the sample by using a protein determination kit, adjusting the protein concentration of all samples to the same level according to the obtained result, adding a loading buffer solution, uniformly mixing, boiling for 5 minutes at 100 ℃, and cooling to room temperature for western blot analysis.

The samples obtained in the above steps were separated by polyacrylamide gel electrophoresis (SDS-PAGE), and the proteins on the gel were transferred to a PVDF membrane. The PVDF membrane after the completion of the membrane transfer was blocked with 5% bovine serum albumin in TBST (Tris-buffered saline solution/Tween) buffer at room temperature for 1 hour. The blocked PVDF was incubated with primary antibody overnight (4 ℃). After the primary antibody had been treated, the PVDF membrane was washed three times with TBST and then treated with a secondary antibody at room temperature for 1 hour. After the secondary antibody effect was completed, the sample was washed three times with TBST. Finally, the PVDF membrane was reacted with a chemiluminescent reaction system (Roche) and exposed to X film (Kodak). Quantification of the expression level of each protein was analyzed by the software Quantity-One (Bio-Rad).