Application of MAO-B inhibitor in preparation of drugs for treating neurodegenerative diseases

Abstract

The invention relates to application of an MAO-B inhibitor in preparation of a medicine for treating neurodegenerative diseases, and belongs to the technical field of medicines. The tussilagone is found to have a monoamine oxidase B inhibition effect for the first time, and can be used for preventing and treating neurodegenerative diseases caused by overexpression of monoamine oxidase B. According to the invention, zebra fish is used as a model animal for detection to find that tussilagone can achieve the protection effect of a second-generation MAO-B inhibitor rasagiline (Rasagiline), and tussilagone is a potential compound for preventing and treating the Parkinson's disease, has important significance in research on treatment and prevention of the Parkinson's disease, and has a wide market prospect.

Description

Technical Field

[0001] The invention relates to application of a MAO-B inhibitor in preparing a drug for treating neurodegenerative diseases, and belongs to the technical field of medicine. Background Art

[0002] Neurodegenerative diseases are diseases characterized by the progressive loss of neurons in the central nervous system or peripheral nervous system. The degenerative process may involve the progressive loss of neuronal structure and function or progressive neuronal cell death. This progressive neurodegeneration often leads to physical disability and mental deterioration, affecting the lives of millions of people worldwide. Parkinson's disease (PD) is a major neurodegenerative disease.

[0003] Parkinson's disease is a neurological disease characterized by a significant decrease in striatal dopamine (DA) content as the main pathological change caused by the degeneration and death of dopaminergic neurons in the substantia nigra of the midbrain. Its symptoms include resting tremor, muscle rigidity, abnormal posture and gait, and decreased movement. Further development of PD may also cause patients to be completely unable to take care of themselves and require long-term care, bringing significant economic and social burdens to families and society. According to statistics, the global incidence of PD is about 1%, mainly affecting people over 65 years old. With the aging of the global population, the incidence of PD is showing a gradual upward trend. It is estimated that by 2030, the number of PD patients in my country will be close to 5 million.

[0004] Monoamine oxidase B (MAO-B) is a mitochondrial membrane complex enzyme that plays an important role in the DA metabolic pathway. The reduction of DA content is closely related to the overexpression of MAO-B. MAO-B can decompose DA into homovanillic acid, accompanied by the generation of free radicals H2O2, which has toxic effects on nerve cells. In addition, MAO-B can oxidize 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and promote MPP + Generation of MPP + It can inhibit the activity of DA neuron mitochondrial complex enzymes and promote the death of DA neurons, thereby inducing neurodegeneration. Studies have shown that the polymorphism and abnormal expression of the MAO-B gene are related to neurological diseases such as Parkinson's disease, Alzheimer's disease, schizophrenia, and depression. In addition, the content of MAO-B increases with age and is considered to be an enzyme associated with aging. Inhibiting the overactivity of MAO-B can prevent some dopamine decomposition that occurs with aging, reduce cognitive decline, and reduce the risk of neurodegenerative diseases. It can be seen that MAO-B is closely related to neurological diseases such as PD. Inhibiting the overexpression of MAO-B can promote DA metabolism, increase the DA level of neurons, and reduce the formation of MPTP. It is an effective treatment for PD.

[0005] At present, there are few types of monoamine oxidase inhibitors used in clinical practice, and most of them are chemically synthesized. Chinese patent document CN105153195A (application number 201510548883.4) discloses the use of a tricyclic pyrazole [1,5-d][1,4]benzoxazepine derivative as a reversible and selective monoamine oxidase inhibitor drug; Chinese patent document CN114149417A (application number 202111238909.7) synthesized a class of monoamine oxidase inhibitors with iron ion chelation activity, which has significant advantages for neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease with complex pathogenesis. Rasagiline is a second-generation monoamine oxidase inhibitor, a common drug used to treat Parkinson's disease. Rasagiline binds to the MAO-B enzyme, inhibits its activity, reduces the degradation and clearance of dopamine, thereby increasing the concentration of dopamine in the synaptic cleft and relieving the symptoms of Parkinson's disease. Since most chemically synthesized drugs have many side effects such as hepatotoxicity and nephrotoxicity in disease treatment, it is of great significance to develop novel monoamine oxidase inhibitors from natural sources with low side effects.

[0006] Tussilagone is a sesquiterpenoid component isolated from the traditional Chinese medicine Tussilagon. Its chemical structure is as follows:

[0007] Studies have shown that coltsfoot ketone inhibits lipopolysaccharide (LPS)-induced neuronal apoptosis by scavenging reactive oxygen species (ROS), suggesting that coltsfoot ketone is expected to be a lead compound in drug research for neurodegenerative diseases. However, the effect of coltsfoot ketone on monoamine oxidase has not been explored and deserves further study. Summary of the invention

[0008] In view of the deficiencies of the prior art, the present invention provides an application of a MAO-B inhibitor in the preparation of a drug for treating neurodegenerative diseases.

[0009] The technical solution of the present invention is as follows: Application of coltsfoot ketone as a monoamine oxidase B inhibitor in the preparation of drugs for the treatment of neurodegenerative diseases.

[0010] Preferably, the use of coltsfoot ketone as a monoamine oxidase B inhibitor in the preparation of a drug for treating Parkinson's disease.

[0011] Further preferably, the coltsfoot ketone can inhibit the expression of monoamine oxidase B, protect dopaminergic neurons, reduce nerve cell apoptosis, improve behavioral disorders, and thus alleviate Parkinson's disease symptoms.

[0012] Application of coltsfoot ketone as a monoamine oxidase B inhibitor in the preparation of monoamine oxidase B inhibitory products.

[0013] A drug for treating Parkinson's disease, comprising an effective amount of coltsfoot ketone.

[0014] Preferably, the Parkinson's disease therapeutic drug contains one or more pharmaceutically acceptable carriers or adjuvants.

[0015] More preferably, the auxiliary agent is at least one of a sustained-release agent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption enhancer, a surfactant or a lubricant.

[0016] Preferably, the Parkinson's disease therapeutic drug can inhibit the expression of monoamine oxidase B, protect dopaminergic neurons, reduce nerve cell apoptosis, improve behavioral disorders, and thus alleviate Parkinson's disease symptoms.

[0017] Preferably, the Parkinson's disease therapeutic drug is in the form of tablets, pills, capsules, oral solutions or injections.

[0018] Beneficial effects: The present invention discovers for the first time that coltsfoot ketone has a monoamine oxidase B inhibitory effect and can be used to prevent and treat neurodegenerative diseases caused by overexpression of monoamine oxidase B. The present invention uses zebrafish as a model animal for detection and finds that coltsfoot ketone can achieve the protective effect of the second-generation MAO-B inhibitor Rasagiline, and is a potential compound for preventing and treating Parkinson's disease. It is of great significance for the research on the treatment and prevention of Parkinson's disease and has broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 This is the simulated conformation diagram of coltsfoot ketone binding to MAO-B protein; Figure 2 This is the simulated conformation diagram of rasagiline binding to MAO-B protein; Figure 3 The Western Blot electrophoresis diagram of the inhibitory effect of coltsfoot ketone concentration on MAO-B; Figure 4 The swimming trajectories of zebrafish in each group under different treatment conditions; Figure 5 It is a bar graph of the total swimming distance of zebrafish in each group under different treatment conditions; Figure 6 This is a line graph showing the change in swimming speed of zebrafish in each group under different treatment conditions over time; Figure 7 Images were collected for dopaminergic neurons in each group of zebrafish under different treatment conditions; Figure 8The bar graphs of dopaminergic neuron lengths in zebrafish of each group under different treatment conditions; Fig. 9 Images were collected for apoptosis staining of PC12 cells in each group under different treatment conditions; Fig.10 It is a bar graph showing the percentage of PC12 cell apoptosis in each group under different treatment conditions; Fig.11 Images were collected for the ROS fluorescence intensity of PC12 cells in each group under different treatment conditions; Fig.12 This is a bar graph of the mean fluorescence intensity of ROS in PC12 cells in each group under different treatment conditions. DETAILED DESCRIPTION

[0020] The technical scheme of the present invention is further described below in conjunction with the embodiments and the drawings of the specification, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the embodiments are conventional technical means well known to those skilled in the art, and the reagents used can be obtained through commercial or commercial channels. Among them, the composition ratio of DMEM complete medium is: DMEM basal medium: fetal bovine serum: double antibody = 100:10:1.

[0021] Example 1 Molecular docking The protein amino acid sequence of MAO-B (human) was obtained from the PDB (Protein Data Bank) database. The amino acid sequences of the protein A chain and B chain were the same, as shown in SEQ ID NO.1. The 3D structures of the small molecules coltsfoot ketone and rasagiline were constructed using Chem Draw software. Molecular docking was performed using Autodock Vina software, with binding energy (BE) as the evaluation index. The lowest energy was the most stable conformation. The molecular docking diagram was visualized using Pymol software, as shown in Figure 1. Figure 1 As shown in Figure 2, the binding energy of coltsfoot ketone to MAO-B protein is -7.629 kcal / mol. Figure 2 As shown in the figure, the binding energy of rasagiline to MAO-B protein is -7.3 kcal / mol, which indicates that rasagiline has a better binding ability to MAO-B protein, providing a theoretical basis for rasagiline to be used as a MAO-B inhibitor.

[0022] Example 2 Semi-qualitative analysis of the inhibitory effect of coltsfoot ketone on MAO-B protein by Western Blot PC12 cells were cultured and resuspended in DMEM complete medium and counted to prepare PC12 cell suspension at 1.5 × 10 52 mL of PC12 cell suspension was evenly inoculated into a 6-well plate at a density of cells / mL and cultured in a 37°C, 5% CO2 incubator. When the cell density reached 60%, DMEM complete medium containing different concentrations of coltsfoot ketone was added to each group, with final concentrations of 0, 2.5, 5, 10, 15, and 20 μM, respectively. After 24 h of drug treatment, cells from each group were collected, and total cell protein was extracted and subjected to Western Blot electrophoresis. The specific operations were as follows: (1) 300 μL of RIPA lysis buffer containing 10% protease inhibitor and 10% phosphorylated protease inhibitor was added to each well. After standing on ice for 5 min, the cells were scraped off with a cell scraper, broken with an ultrasonic disruptor, centrifuged at 4°C and 11,000 rpm for 30 min, and the supernatant was collected into a new EP tube; (2) The protein concentration was detected using a BCA protein detection kit, and the protein of each group was adjusted to 4 μg / μL using RIPA lysis buffer; (3) 8 μL of protein sample was taken, 20% volume of 5× Loading Buffer was added, and the sample was heated in a metal bath at 100°C for 10 min for denaturation; (4) Electrophoresis: 10% SDS-PAGE gel was prepared, 32 μg of protein was loaded, and then electrophoresis was performed at 80 V constant voltage for 2 min. h; (5) Transfer: remove the gel block and transfer it to a methanol-activated PVDF membrane. The transfer conditions are constant current 250 mA, 60 min, and ice bath; (6) Blocking: place the PVDF membrane in 5% skim milk blocking solution and block it on a shaker at room temperature for 2 h; (7) Incubate with primary antibody diluent at 4°C overnight, and wash the membrane three times with TBST on the next day, each time for 10 min; (8) Incubate with secondary antibody at room temperature for 1 h, and wash the membrane three times with TBST, each time for 10 min; (9) Development: Evenly spread the prepared ECL color development working solution on the PVDF membrane, expose and image it with a gel imager, and analyze the grayscale value of the protein bands with Image J software.

[0023] The experimental results are as follows Figure 3 As shown in the figure, GAPDH (glyceraldehyde-3-phosphate dehydrogenase) is an internal reference. As the concentration of coltsfoot ketone increases, the expression of MAO-B protein shows a downward trend. This shows that coltsfoot ketone can inhibit the expression of MAO-B protein.

[0024] Example 3 Effects of coltsfoot ketone on PD-like behavior in zebrafish (1) Obtaining zebrafish embryos: Healthy adult wild-type AB zebrafish were placed in a spawning tank at a ratio of 2:2 between male and female, with a partition placed in the middle, and placed in a dark environment. The partition was removed at 8:30 am the next day, and the eggs were collected 2 hours later, rinsed with fish water, and transferred to fish water containing 0.2 ppm methylene blue. The eggs were cultured in a 28°C constant temperature incubator with controlled light (14 h light / 10 h dark), and dead eggs were removed promptly every day.

[0025] The method for preparing the above-mentioned fish water is as follows: 20 mL of fish water jelly solution, dilute to 1 L with purified water; The components of the above-mentioned fish culture jellyfish liquid are: NaCl 14.658 g, KCl 0.633 g, CaCl22.425 g, MgSO4·7H2O 4.06 g, and the volume is adjusted to 1 L with pure water.

[0026] (2) Experimental groups and zebrafish embryo pretreatment: Normal 1 dpf (day past fertilization) zebrafish larvae were selected, demembraned with 1 mg / mL protease, and then transferred into a six-well plate. 30 fish were placed in each well, and two replicate wells were set for each group. A control group (control, Ctl), MPTP model group, Rasagiline positive control group, and schizophylline group (0.5, 1, 2, 3 μM) were set. The control group was diluted to 5 mL with fish water, the MPTP model group was diluted to 5 mL with fish water containing 60 μM MPTP, the Rasagiline positive control group was diluted to 5 mL with fish water containing 1 μM Rasagiline and 60 μM MPTP, and the schizophylline group was diluted to 5 mL with fish water containing 0.5, 1, 2, 3 μM schizophylline and 60 μM MPTP, respectively. Drugs were administered from the first day to the fourth day, during which the fish water containing the corresponding drugs was replaced every day and dead fish were sucked out.

[0027] (3) Behavioral testing After 4 days of drug treatment, 24 larvae were randomly selected from each group, washed three times with fish water, and transferred to a 48-well plate, with 1 mL of fish water added to each well. The movement trajectory of each larvae was recorded using a Viewpoint zebrafish behavior analyzer. The entire experiment lasted 30 minutes, including 10 minutes of adaptation time. The swimming trajectory, distance, and speed of zebrafish were recorded and analyzed using Zeblab software.

[0028] The swimming trajectory of zebrafish Figure 4 As shown in Figure 2, the total swimming distance and swimming speed of zebrafish change as follows: Figure 5 , Figure 6 As shown in the results, MPTP treatment caused a significant decrease in the swimming trajectory and total swimming distance of zebrafish, and a significant decrease in swimming speed. After treatment with different concentrations of coltsfoot ketone, the swimming trajectory of zebrafish increased significantly, the total swimming distance increased, and the swimming speed increased in a concentration-dependent manner. This suggests that coltsfoot ketone can improve the abnormal behavior of zebrafish caused by MPTP, help restore the motor ability of zebrafish, and have a certain neuroprotective effect.

[0029] Example 4 Protective effect of coltsfoot ketone on dopaminergic neurons in zebrafish with PD (1) Healthy adult zebrafish Vmat2:GFP The transgenic strain was placed in a spawning tank to obtain zebrafish embryos, the specific method was the same as step (1) of Example 3.

[0030] (2) Select 1 dpf (day past fertilization) zebrafish fry with normal development for decapping and drug treatment. The decapping method and grouping and drug administration method are the same as step (2) of Example 3. After 3 days of treatment, wash the fry 2-3 times with fish water. Add anesthetics to each group 1 min before taking pictures. Place the anesthetized zebrafish on a slide and observe and collect images using a Zeiss fluorescence microscope. Based on the collected pictures, the length of dopaminergic neurons was statistically analyzed using ImageJ software. Ten zebrafish pictures were randomly selected from each group.

[0031] The experimental results are as follows Figure 7 , Figure 8 As shown in the figure, after MPTP-induced zebrafish, the length of dopaminergic neurons was shortened compared with the Ctl group, indicating that the model was successful. Compared with the MPTP model group, the length of dopaminergic neurons in the Rasagiline positive control group increased significantly, indicating that the positive drug alleviated the damage of dopaminergic neurons and had a neuroprotective effect. Compared with the MPTP model group, the length of dopaminergic neurons in the 1, 2, and 3 μM coltsfoot ketone groups increased significantly in a concentration-dependent manner, and 3 μM coltsfoot ketone had a better therapeutic effect than Rasagiline. This shows that coltsfoot ketone has a protective effect on dopaminergic neurons.

[0032] Example 5 Hoechst 33342 / PI double staining to detect cell apoptosis (1) PC12 cells were cultured and resuspended in DMEM complete medium and counted to prepare PC12 cell suspension. The PC12 cell suspension was evenly inoculated in a 96-well plate at a density of 8000 cells / well. Four replicate wells were set up in each group, namely Ctl group, MPP group, and + (1-methyl-4-phenylpyridinium ion) model group, coltsfoot ketone (1.25, 2.5, 5, 10, 15, 20 μM) groups, among which, no drug was added to the Ctl group, and MPP + The model group was added with a final concentration of 2 mM MPP + , the coltsfoot ketone group was added with a final concentration of 2mM MPP + and 1.25, 2.5, 5, 10, 15, 20 μM coltsfoot ketone; and then cultured in a 37°C, 5% CO2 cell culture incubator.

[0033] (2) When the cell density reaches about 60%, the drug is administered according to the above grouping.

[0034] (3) After 24 h of drug treatment, the original culture medium was removed. Hoechst 33342 and PI stain were added to the DMEM complete culture medium and mixed thoroughly to obtain the staining solution. The final concentration of both in this system was 50 μg / mL. The prepared staining solution was added to completely cover the cells. The cells were incubated in a 37°C cell culture incubator for 20 min. Images were collected using a high-content imaging system and the proportion of PI-positive cells was counted. The experimental results are shown in Figure 2. Fig. 9 , Fig.10 As shown, in MPP + Under the action of oxaliplatin, the number of apoptotic cells increased, and under the action of oxaliplatin, the number of apoptotic cells decreased significantly in a concentration-dependent manner, indicating that oxaliplatin can reduce MPP + Induces cell apoptosis and protects nerve cells.

[0035] Example 6 ROS reactive oxygen species level detection (1) Cell plating and administration are the same as step (1) and step (2) of Example 5.

[0036] (2) After 24 h of drug treatment, the original culture medium was removed, DCFH-DA (2,7-dichlorofluorescein diacetate) was diluted with DMEM basal culture medium to a final concentration of 10 μM, 100 μL of the diluted DCFH-DA solution was added to each well, and the cells were incubated in a 37°C cell culture incubator for 20 min. The dye was aspirated, PBS buffer (pH 7.2-7.4) was added, and images were collected using a high-content imaging system. The fluorescence intensity and fluorescence area were calculated using Image-Pro Plus software, and the average fluorescence intensity between the groups was compared. The average fluorescence intensity reflects the generation of ROS.

[0037] Average fluorescence intensity = fluorescence intensity / fluorescence area The experimental results are as follows Fig.11 , Fig.12 As shown, in MPP + Under the action of ketone, the level of reactive oxygen species in cells increased significantly, and with the increase of the concentration of ketone, the level of ROS reactive oxygen species in cells showed a downward trend. This shows that ketone plays a neuroprotective role by reducing cell apoptosis and inhibiting the production of reactive oxygen species.

Claims

1. The application of coltsfoot ketone as a monoamine oxidase B inhibitor in the preparation of drugs for the treatment of neurodegenerative diseases.

2. The use according to claim 1, characterized in that Application of coltsfoot ketone as a monoamine oxidase B inhibitor in the preparation of drugs for the treatment of Parkinson's disease.

3. The use according to claim 2, characterized in that The coltsfoot ketone can inhibit the expression of monoamine oxidase B, protect dopaminergic neurons, reduce nerve cell apoptosis, improve behavioral disorders, and thus alleviate Parkinson's disease symptoms.

4. The use of coltsfoot ketone as a monoamine oxidase B inhibitor in the preparation of monoamine oxidase B inhibitory products.

5. A drug for treating Parkinson's disease, characterized in that: The medicine comprises an effective amount of coltsfoot ketone.

6. The Parkinson's disease therapeutic drug according to claim 5, characterized in that: The medicine contains one or more pharmaceutically acceptable carriers or adjuvants.

7. The Parkinson's disease therapeutic drug according to claim 6, characterized in that: The auxiliary agent is at least one of a sustained-release agent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant or a lubricant.

8. The Parkinson's disease therapeutic drug according to claim 5, characterized in that: The drug can inhibit the expression of monoamine oxidase B, protect dopaminergic neurons, reduce nerve cell apoptosis, improve behavioral disorders, and thus alleviate Parkinson's disease symptoms.

9. The Parkinson's disease therapeutic drug according to claim 5, characterized in that: The dosage form of the medicine is tablet, pill, capsule, oral solution or injection.

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