Application of aspirin or derivative thereof in treating alpha-synuclein abnormal aggregation related diseases

Abstract

The invention discloses an application of aspirin or a pharmaceutically acceptable salt thereof, or a hydrate thereof in preparation of a pharmaceutical composition for treating diseases related to abnormal aggregation of alpha-synuclein. It is found that aspirin can significantly reduce aggregation of alpha-synuclein and protein in a phosphorylated form of alpha-synuclein, increase the number of dopaminergic neurons and improve symptoms of mice suffering from Parkinson's disease. The results indicate that aspirin can be used as a medicine for treating diseases related to alpha-synuclein aggregation. The invention provides a new drug choice for clinical treatment of alpha-synuclein aggregation related diseases.

Description

Technical Field

[0001] The present invention relates to the field of medicines, and in particular, provides use of aspirin or a pharmaceutically acceptable salt thereof, or a hydrate thereof in preparing a pharmaceutical composition for treating diseases associated with abnormal aggregation of alpha-synuclein. Background Art

[0002] Aspirin, also known as acetylsalicylic acid, is a widely used nonsteroidal anti-inflammatory drug used to relieve symptoms such as pain, fever and inflammation. Many other health benefits have also been reported, including improvements in neurodegenerative diseases, cancer and other aging-related chronic diseases, which has also made aspirin known as a "magic" drug molecule. The most well-known anti-inflammatory mechanism of aspirin is to acetylate the 516th serine of the cyclooxygenase COX-2, thereby inhibiting the production of prostaglandins to exert antipyretic, analgesic and anti-inflammatory effects. In addition, the DAcMS method based on stable isotope deuterium labeling of aspirin and large-scale acetylation proteome analysis has demonstrated that aspirin can acetylate thousands of protein substrates, revealing aspirin's many potential substrates and related mechanisms. However, the effects and mechanisms of aspirin on neurodegenerative diseases (such as Alzheimer's disease, Parkinson's disease, etc.) are not fully understood. So far, it is unknown whether aspirin can promote the clearance of α-synuclein aggregates, the main pathological protein in Parkinson's disease.

[0003] α-Synuclein is a protein widely expressed in neurons, mainly located in the vesicles at the synaptic terminals. Under normal circumstances, it may be involved in regulating the release of neurotransmitters and regulating synaptic plasticity. However, abnormal aggregation of α-synuclein can lead to the occurrence of α-synucleinopathy. α-Synucleinopathy is a type of neurodegenerative disease, which is mainly divided into three types: Parkinson's Disease (PD), Dementia with Lewy Bodies (DLB) and Multiple System Atrophy (MSA). In addition, various axonal atrophies, Alzheimer's disease with amygdalar restricted Lewy bodies (AD / ALB), REM sleep behavior disorder (RBD), and pure autonomic failure (PAF) also have abnormal aggregation of α-synuclein. Abnormal aggregation of α-synuclein has a negative impact on the function and survival of nerve cells, leading to the damage or death of nerve cells and ultimately the development of the disease.

[0004] In addition, α-synuclein is also involved in the occurrence and progression of a variety of tumors, such as melanoma, brain tumors, pancreatic ductal adenocarcinoma, bladder cancer, etc. The role of α-synuclein in melanoma has attracted attention. Studies have shown that α-synuclein can affect the susceptibility of cells to ultraviolet radiation-induced damage by regulating the synthesis of melanin in melanoma. In addition, α-synuclein has also been identified in a variety of brain tumors, such as ganglioglioma, medulloblastoma, neuroblastoma, primitive neuroectodermal tumor, pinealocyte tumor / pinealoblastoma, central neurocytoma and meningioma. α-synuclein may be related to the enhanced proliferation, invasion and metastasis of tumor cells.

[0005] Given the complex relationship between α-synuclein and neurodegenerative diseases and tumors, research on the clearance of α-synuclein and its phosphorylated aggregates by aspirin can provide important guidance for the development of new treatments and interventions, bringing new hope for the treatment and management of related diseases. Summary of the invention

[0006] The present invention discloses a new use of aspirin or a pharmaceutically acceptable salt thereof, or a hydrate thereof, which can be used to prepare a medicine or a pharmaceutical composition for treating diseases associated with alpha-synuclein aggregation, thereby providing a new option for clinical drug treatment of diseases associated with alpha-synuclein aggregation.

[0007] A use of aspirin or a pharmaceutically acceptable salt thereof, or a hydrate thereof in preparing a pharmaceutical composition for treating diseases associated with abnormal aggregation of alpha-synuclein.

[0008] In another preferred embodiment, the pharmaceutical composition is used to reduce the aggregation of α-synuclein.

[0009] In another preferred embodiment, the pharmaceutical composition is also used to increase the number of neurons.

[0010] In another preferred embodiment, the disease is a neurodegenerative disease.

[0011] In another preferred embodiment, the neurodegenerative disease is selected from the group consisting of Parkinson's disease, Alzheimer's disease, Lewy body dementia, multiple system atrophy, axonal atrophy, rapid eye movement sleep behavior disorder, and pure autonomic failure.

[0012] In another preferred embodiment, the pharmaceutical composition is also used to improve decreased spontaneous activity and motor coordination disorders caused by neurodegenerative diseases.

[0013] In another preferred embodiment, the disease is a tumor.

[0014] In another preferred embodiment, the tumor is selected from the following group: melanoma, brain tumor, pancreatic ductal adenocarcinoma, bladder cancer.

[0015] In another preferred embodiment, the brain tumor includes but is not limited to ganglioglioma, medulloblastoma, neuroblastoma, primitive neuroectodermal tumor, pinealocyte tumor / pineoblastoma, central neurocytoma and meningioma.

[0016] In another preferred embodiment, the drug or pharmaceutical composition is an oral preparation.

[0017] In another preferred embodiment, the oral preparation is a tablet, capsule, pill, granule, decoction, oral liquid, drop pill or syrup.

[0018] In another preferred embodiment, the pharmaceutical composition contains 0.01%-99% by weight of aspirin or a pharmaceutically acceptable salt thereof as an active ingredient.

[0019] In another preferred embodiment, the pharmaceutical composition contains 0.1%-90% by weight of aspirin or a pharmaceutically acceptable salt thereof as an active ingredient.

[0020] In another preferred embodiment, the pharmaceutical composition contains a therapeutically effective amount of aspirin or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers.

[0021] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Aspirin was shown to promote the clearance of α-synuclein aggregates in neurons and glioma H4 cells, microglia BV2, and cervical cancer HeLa cells.

[0023] in, Figure 1 A is a representative immunofluorescence image of primary neurons (scale bar, 10 μm). Figure 1 B is the quantification result of immunofluorescence.

[0024] Figure 1 C is a representative immunoblot result (top) and quantitative result (bottom) of the reduction of α-synuclein aggregates in glioma H4 cells after aspirin treatment.

[0025] Figure 1 D: Aspirin reduces the abundance of α-synuclein in microglial BV2 cells (top) and cervical cancer HeLa cells (bottom).

[0026] Figure 2 Aspirin was shown to promote the clearance of α-synuclein aggregates in a mouse model of PD.

[0027] in, Figure 2 A shows the experimental scheme for establishing the PD mouse model and aspirin treatment.

[0028] Figure 2 B is the result of the open field test of PD mice, each point represents a mouse.

[0029] Figure 2 C is the result of the rotarod test of PD mice, and each point represents one mouse.

[0030] Figure 2 D is a representative immunofluorescence image showing that aspirin treatment reduced α-synuclein aggregates in the striatum (scale bar, 100 μm), and the rightmost side shows an enlarged and merged image (scale bar, 10 μm).

[0031] Figure 2E: Aspirin treatment reduced the abundance of p-α-syn in the striatum, where each dot represents one hemibrain of a mouse.

[0032] Figure 2 F: Aspirin treatment reduced the density of p-α-syn in the striatum, where each dot represents one hemibrain of a mouse.

[0033] Figure 2 G shows the changes in DA neuron abundance in the striatum after aspirin treatment, where each point represents one hemibrain of a mouse.

[0034] Figure 2 H is a representative immunofluorescence image showing that aspirin treatment increased the number of DA neurons in the substantia nigra (scale bar, 200 μm), and the rightmost side shows an enlarged and merged image (scale bar, 20 μm).

[0035] Figure 2 I represents the abundance of p-α-syn in the substantia nigra after aspirin treatment, where each dot represents one hemibrain of a mouse.

[0036] Figure 2 J is the density of p-α-syn in the substantia nigra after aspirin treatment, where each dot represents one hemibrain of a mouse.

[0037] Figure 2 K represents the changes in DA neuron abundance in the substantia nigra after aspirin treatment, where each point represents one hemibrain of a mouse.

[0038] Figure 2 L: Aspirin treatment increased the number of DA neurons in the substantia nigra, where each dot represents one hemibrain of a mouse. DETAILED DESCRIPTION

[0039] After long-term and in-depth research, the inventors found that aspirin, a conventional antipyretic, analgesic and anti-inflammatory drug on the market, can reduce α-synuclein aggregates in cultured primary neurons and Parkinson's disease model mice, and improve Parkinson's-like symptoms in Parkinson's disease model mice, and has a neuroprotective effect. Based on the above findings, the inventors completed the present invention.

[0040] the term

[0041] As used herein, the term "pharmaceutically acceptable salt" refers to salts formed by the compounds of the present invention and pharmaceutically acceptable inorganic acids and organic acids. Inorganic acids include: hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid; organic acids include: formic acid, acetic acid, propionic acid, succinic acid, naphthalene disulfonic acid (1,5), oxalic acid, tartaric acid, lactic acid, salicylic acid, benzoic acid, valeric acid, diethylacetic acid, malonic acid, succinic acid, fumaric acid, pimelic acid, adipic acid, maleic acid, malic acid, aminosulfonic acid, phenylpropionic acid, gluconic acid, ascorbic acid, nicotinic acid, isonicotinic acid, methanesulfonic acid, p-toluenesulfonic acid, citric acid, and amino acids.

[0042] The term "hydrate" refers to a complex formed by coordination of a compound of the present invention with water.

[0043] The term "α-syn" refers to α-synuclein.

[0044] The term "DA" refers to dopamine.

[0045] The term "DAT" is dopamine transporter.

[0046] The term "TEM" refers to transmission electron microscopy.

[0047] The term "PBS" refers to phosphate buffered saline.

[0048] The term "PFA" refers to paraformaldehyde.

[0049] The term "MAP2" refers to microtubule-associated protein 2.

[0050] The term "α-syn PFFs" refers to α-synuclein fibrils.

[0051] The term "PD" refers to Parkinson's disease.

[0052] The term "p-α-syn" refers to phosphorylated α-synuclein.

[0053] Pharmaceutical composition

[0054] The present invention provides a pharmaceutical composition, which has a significant effect of reducing alpha-synuclein aggregates, and contains a therapeutically effective amount of aspirin or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers.

[0055] The compound itself or a mixture of a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient, diluent, etc. can be orally administered in the form of tablets, capsules, pills, granules, decoctions, oral liquids, drop pills or syrups. The pharmaceutical composition preferably contains 0.01% to 99% by weight of aspirin or a pharmaceutically acceptable salt thereof as an active ingredient, more preferably 0.1% to 90% by weight of the active ingredient.

[0056] The above-mentioned preparations can be prepared by conventional pharmaceutical methods. Examples of available pharmaceutical adjuvants include excipients (e.g., sugar derivatives such as lactose, sucrose, glucose, mannitol and sorbitol; starch derivatives such as corn starch, potato starch, dextrin and carboxymethyl starch; cellulose derivatives such as crystalline cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose calcium, carboxymethyl cellulose sodium; gum arabic; dextran; silicate derivatives such as magnesium aluminum metasilicate; phosphate derivatives such as calcium phosphate; carbonate derivatives such as calcium carbonate; sulfate derivatives such as calcium sulfate, etc.), binders (e.g., gelatin, polyvinyl pyrrolidone and polyethylene glycol), disintegrants (e.g., cellulose derivatives such as carboxymethyl cellulose sodium, polyvinyl pyrrolidone), lubricants (e.g., talc, calcium stearate, magnesium stearate, spermaceti, boric acid, sodium benzoate, leucine), stabilizers (methyl parahydroxybenzoate, propyl parahydroxybenzoate, etc.), flavoring agents (e.g., commonly used sweeteners, acidulants and spices, etc.), and diluents.

[0057] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. In the following examples, all experimental methods without specifying specific conditions are usually carried out under normal conditions or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0058] Example 1. Effect of aspirin on mouse and human α-synuclein fibers

[0059] 1. Materials and Methods

[0060] 1.1 Drugs and reagents

[0061] Aspirin was purchased from Sigma.

[0062] The antibodies used include: α-synuclein monoclonal rabbit antibody (1:1000, Abcam, ab138501), β-actin monoclonal mouse antibody (1:1000, Sigma, A1978), HRP-labeled polyclonal goat anti-rabbit antibody (1:1000, Beyotime, A0208), HRP-labeled polyclonal goat anti-mouse antibody (1:1000, Beyotime, A0216), DAT monoclonal rat antibody (1:500, Abcam, ab5990), phosphorylated α-synuclein (S129) monoclonal rabbit antibody (1:250, Abcam, ab51253), MAP2 polyclonal chicken antibody (1:1000, Abcam, ab5392). Goat anti-rabbit Alexa Fluor 568 (1:1000, Abcam, ab175471), goat anti-rat Alexa Fluor 647 (1:1500, Abcam, ab150159) and goat anti-chicken Alexa Fluor 488 (1:1000, Thermo Fisher, A-11039).

[0063] 1.2 Cell lines and cell culture

[0064] Cell lines source Organization Source Culture medium H4 Laboratory storage Human glioma cells High glucose DMEM HeLa Laboratory storage Human cervical cancer epithelial cells High glucose DMEM BV2 Laboratory storage Mouse microglia High glucose DMEM

[0065] 1.3 Animals

[0066] Male C57BL / 6J mice (4 weeks old) were provided by the animal laboratory (Shanghai Lingchang Biotechnology Co., Ltd.). Mice were group-housed and maintained under standard conditions with a light-dark cycle (12 h-12 h), a room temperature of 25 ± 2 °C, and free access to food and water.

[0067] All animal procedures were reviewed and approved by the Animal Care and Use Committee of the Center for Interdisciplinary Research of Biochemistry, Chinese Academy of Sciences, and were in accordance with the Guide for the Care and Use of Laboratory Animals of the Chinese Academy of Sciences.

[0068] 1.4 Preparation of recombinant mouse and human α-synuclein fibrils (α-syn PFFs)

[0069] The gene encoding mouse or human full-length α-synuclein was inserted into the pET22 vector and then expressed in Escherichia coli BL21 (DE3) cells together with yeast N-acetyltransferase complex B57. α-synuclein was purified using a strong cation exchange column (GE Healthcare, 17-5156-01) and preparative gel filtration chromatography Superdex 75 (GE Healthcare, 28-9893-33). The α-synuclein was purified in a 50 mM Tris, pH 7.5, 150 mM KCl and 0.05% NaN3 100 μM α-synuclein monomer and 0.5% (mol / mol) α-syn PFFs were added to the buffer and incubated at 37°C with constant stirring (900 rpm) for 7 days. α-synuclein fiber particles were collected by centrifugation at 14,462g for 45 minutes. Mouse or human α-synuclein fiber particle pellets were washed with phosphate buffered saline (PBS) and suspended in PBS and concentrated to 2 μg / μL and 500 mM, respectively. α-syn PFFs were prepared by ultrasound at 20% power for 15 cycles (1 second on / off) on ice. The physical state of the fibers was examined using a transmission electron microscope (TEM).

[0070] 1.5 Rat primary neurons

[0071] Primary cortical neurons were isolated from embryonic day 16-18 Sprague-Dawley (SD) rats purchased from Shanghai SIPPR BK Laboratory Animal Company. 2 Under the conditions of , primary neurons were cultured in Neurobasal medium containing B-27 supplement, 0.5mM L-glutamine and 1% (v / v) penicillin / streptomycin for 7 days. PBS or 100nM human α-syn PFFs were then added for 5 days. Then 0.1mM or 1mM aspirin was added and co-treated with α-syn PFFs, and culture was continued for 14 days, using 1‰ DMSO as a control. Finally, primary neurons were collected and used for immunofluorescence staining experiments.

[0072] 1.6 Aspirin treatment of cells loaded with α-synuclein aggregates

[0073] 3 μM α-syn PFFs were added to BV2, HeLa and H4 cells and incubated for 4 hours. The cells were then washed twice with PBS and fresh medium containing different concentrations of aspirin was added and incubated for 18 hours. After the cells were collected, they were lysed in a loading buffer containing 2% SDS. The proteins were boiled at 95°C for 30 minutes and then separated by 10% SDS-PAGE for immunoblotting experiments.

[0074] 1.7PD model mice

[0075] Mice were anesthetized by blowing 0.5% isoflurane into the anesthesia mask with 1% air flow. α-syn PFFs (2μg / μL) were stereotaxically injected into the dorsal striatum of both hemispheres of the brain at a dose of 0.2μg / g (body weight). The control group of mice was injected with sterile PBS at a dose of 0.1μL / g (body weight). The injection speed was controlled using a microsyringe, and the speed was controlled at 0.5μL / min when 0.2μL was injected before injection, and the speed was controlled at 0.2μL / min when the remaining volume was injected. After the mice recovered from anesthesia, they were transferred to the previous cage until the next experiment.

[0076] The numbers of mice in the PBS group, α-syn PFFs-control group, and α-syn PFFs-aspirin group were 10, 11, and 12, respectively.

[0077] 1.8 Oral administration of aspirin to mice

[0078] Aspirin was mixed into strawberry-flavored jelly and fed to mice. 15.2 mg of aspirin was dissolved in 20 mL of Splenda solution containing 0.1% methylcellulose. Only jelly was used as a vehicle. Since mice showed avoidance behavior to new types of diets, 3-4 days of pre-training were introduced until they could finish eating jelly within 1 minute. Next, Parkinson's disease model mice were divided into two groups, one group received the vehicle (jelly without aspirin) and the other group received aspirin jelly. The mice were fed once a day at a dose of 6 mg / kg (body weight). In order to carry out precise dosage control, the amount of jelly required for each mouse was carefully calculated according to its weight.

[0079] 1.9 Behavioral Testing

[0080] All behavioral training and testing were performed between 4 and 8 hours after lights on. To ensure that mice were fully acclimated to the testing environment, they were moved to the behavioral room at least 1 hour before testing.

[0081] For the open field test, mice were placed in a 40×40 cm experimental box, which was placed in a light / sound isolation box and monitored by a camera. The light in the experimental box was kept at a brightness of 30 to 35 lux. After the mouse adapted to the environment, it was placed in the center of the experimental box and allowed to explore for 10 minutes. The mouse activity was monitored and recorded using a camera, and data analysis was performed using EthoVision XT (Noldus11.5). Between each round of testing, the experimental box was thoroughly cleaned with 75% alcohol and allowed to dry naturally.

[0082] For the rotarod test, all mice were first trained on the rotarod (Sansbio, YLS-4C) for three days. The daily training consisted of two constant speed adaptations (4 rpm / min) and one acceleration training (0-40 rpm / min, with an acceleration of 24 rpm / min). 2 ). Each training session lasted 2 minutes, and between each session, the mice were returned to their original cages to rest for 3 minutes. After resting for 1 day, the mice were subjected to two acceleration tests on the rotating rod, each time with a speed from 0 to 40 rpm / min and a speed of 40 rpm / min maintained for 3.5 minutes. The total duration of the mice on the rotating rod was recorded and used for further analysis. Between each round of testing, the rotating rod was thoroughly cleaned with 75% alcohol and allowed to dry naturally.

[0083] 1.10 Immunofluorescence staining and confocal imaging

[0084] For the treatment of brain slices: mice were anesthetized with isoflurane and then perfused with 10 mL of ice-cold PBS and 40 mL of 4% paraformaldehyde (PFA). The whole brain was removed and fixed in 4% PFA overnight. After fixation, the brain was dehydrated in PBS containing 30% sucrose for 3 days, and then it was quickly frozen in an embedding medium (OCT, tissue freezing medium, Leica) and stored at -80°C, and then 30 μm coronal sections were cut using a Leica cryostat (model: CM3050s-1-1-1). The slices were placed on slides treated with poly-lysine and stored at -20°C. Before staining, the slices were rinsed with PBS and then incubated at room temperature for 2 hours in a PBS solution containing 5% normal goat serum and 0.1% Triton X-100. For cell samples: the slices were rinsed three times with PBS and then fixed with PBS containing 4% PFA and 4% sucrose for 10 minutes at room temperature. The cells were permeabilized with PBS containing 0.15% Triton X-100 for 15 minutes and subsequently incubated with PBS solution containing 5% bovine serum for 30 minutes at room temperature.

[0085] Antibodies were added to brain slices or cell samples, incubated overnight at 4°C, and then washed three times with PBST buffer. Secondary antibodies were added in the dark and incubated at room temperature for 2 hours in the dark. The slices were mounted with mounting medium (Invitrogen, ProLong Gold antifade reagent with DAPI), and fluorescence was observed using an SP8 laser confocal microscope (Lesia) or a spinning disk microscope (Ador). DAT staining was used to mark DA neurons and their neurites; p-α-syn staining was used to mark α-synuclein aggregates; MAP2 staining was used to mark the cell bodies and dendrites of primary neurons.

[0086] 1.11 Image Analysis

[0087] Six mice were used for immunofluorescence imaging in the PBS group, α-syn PFFs-control group, and α-syn PFFs-aspirin group. Two brain slices were obtained from each mouse, and each dot represented one hemibrain of the mouse. Images were batch analyzed using Image J and Imaris (Oxford Instruments). First, all images were projected in the z direction by maximum intensity, followed by a uniform background subtraction. For all brain regions except the substantia nigra, the signal intensity of all channels in the entire field of view was analyzed. To visualize and analyze the substantia nigra, the images were integrated, and the substantia nigra region was manually selected by referring to the mouse brain atlas. The same background signal subtraction and consistent contrast adjustment were used for the data of each brain region, and the mean fluorescence intensity of p-α-syn and DAT was measured. The particle analysis module in Image J was used to automatically select p-α-syn aggregates (automatic black and white contrast, particle diameter between 2 and 80 μm), and the aggregate density (# / 1mm 2 ) and size. The Spot module of Imaris was used to automatically select and count DAT-positive DA neurons in the substantia nigra. In brief, after manually selecting the cell body of a typical DAT-positive cell as a template, Imaris automatically selected all spherical structures with a diameter of 12 μm, which avoided most non-cell body DAT-positive neurite signals. Then "Quality" was applied to filter the data. Through subjective observation, the lower threshold was set between 36.4 and 872 to include all DAT-positive cell bodies and exclude noise signals. The distribution and mean of the lower threshold used in each group were similar. To display the immunofluorescence results, the average signal intensity of p-α-syn and the average signal intensity of DAT were normalized according to the PBS group. For experiments in different batches, they were normalized according to their respective control groups before combination. All data are presented as mean ± standard error of the mean (SEM).

[0088] 1.12 Statistical analysis

[0089] Statistical analysis was performed using GraphPad (version 8.0.2). One-way ANOVA was used for pairwise multiple comparisons between groups, and two-tailed Student's t-test was used for comparisons between two independent samples. The p value was corrected using the Benjamini-Hochberg method. The significance level was set at p<0.05, as shown below: * p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001.

[0090] 2. Experimental results

[0091] 2.1 Aspirin reduces α-synuclein aggregates in cultured primary neurons and cells

[0092] Aggregate forms of α-synuclein include Lewy bodies and Lewy neurites, which are highly prevalent in PD brains. Previous studies have shown that aspirin has potential benefits for PD patients. To explore whether aspirin regulates the clearance of α-synuclein aggregates, we used α-syn PFFs to trigger aggregation of endogenous α-synuclein. First, we isolated and cultured primary rat neurons and added α-syn PFFs for 5 days after 7 days in vitro culture, followed by aspirin treatment for 14 days. We found that aspirin significantly reduced α-synuclein phosphorylated at serine 129 (p-α-syn), a pathological marker of α-synuclein aggregates ( Figure 1 A and 1B). In addition, through immunoblotting experiments on H4, HeLa, and BV2 cells, we found that aspirin can also reduce the abundance of α-synuclein in a concentration-dependent manner, and the higher the aspirin concentration, the more significant the effect ( Figure 1 C and Figure 1 D).

[0093] 2.2 Aspirin reduces α-synuclein aggregates and improves Parkinson-like behavior in PD model mice

[0094] Synthetic mouse α-syn PFFs were directly injected into the striatum of both sides of the mouse brain to construct PD model mice (see Method 1.7, Figure 2 A), treated with aspirin and investigated the effects.

[0095] Four weeks after model surgery, when the mice had developed some synuclein pathology and began to show mild PD-like behavioral defects, oral aspirin was started once a day. Aspirin treatment lasted for 8 weeks, and behavioral experiments were performed at the 4th and 8th weeks of treatment to evaluate its effects. The results of the open field test showed that in the 4th week of treatment, there was no significant difference in the spontaneous activity behavior of mice, regardless of whether they were injected with α-syn PFFs or whether they were treated with aspirin. However, in the 8th week of treatment, the spontaneous activity behavior of mice injected only with α-syn PFFs was significantly reduced, while the spontaneous activity behavior of α-syn PFFs mice was significantly increased by aspirin treatment ( Figure 2 B). In addition, the results of the rotarod test showed that aspirin treatment greatly improved the motor coordination disorder of α-syn PFFs mice in the fourth week of treatment, and this improvement persisted after 8 weeks of treatment, and the effect was more significant ( Figure 2C). In conclusion, the results of behavioral experiments indicate that aspirin has a positive effect in improving PD-like symptoms, which provides strong support for its application in the treatment of neurodegenerative diseases.

[0096] Synucleinopathy and loss of DA neurons are two common pathological features of PD. To reveal the cellular mechanism of aspirin, we compared these two pathological features in PD model mice treated with aspirin for 8 weeks and those not treated with aspirin by immunohistochemistry. Notably, the abundance and density of p-α-syn were significantly reduced in the striatum of mice treated with aspirin ( Figure 2 Furthermore, in the substantia nigra pars compacta, despite the presence of similar synuclein pathology, aspirin significantly reduced the loss of DA neurons caused by α-syn PFFs ( Figure 2 These results suggest that the beneficial effects of aspirin in PD are at least in part achieved by modulating the progression of synucleinopathy and protecting DA neurons in the substantia nigra pars compacta.

[0097] In summary, the data obtained from PD model mice strongly support the following conclusion: aspirin treatment can regulate the progression of α-synuclein pathology, protect DA neurons in the substantia nigra, and improve PD-like behavioral manifestations, thereby showing significant neuroprotective effects.

[0098] 3. Discussion

[0099] Abnormal aggregation of α-synuclein plays an important role in the pathological process of neurodegenerative diseases and tumors. Therefore, studying the clearance of α-synuclein aggregates is expected to alleviate the symptoms of related diseases, prevent disease progression, and provide potential therapeutic strategies and targets. The inventors unexpectedly discovered and confirmed for the first time that aspirin, as an "all-purpose drug", can promote the clearance of α-synuclein and its phosphorylated form of protein aggregates and improve PD-like symptoms in Parkinson's disease mice. This discovery provides a new dimension for our understanding of the pharmacology of aspirin and also provides favorable support for its treatment of diseases related to α-synuclein aggregation.

[0100] All documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference individually. In addition, it should be understood that after reading the above teachings of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the claims attached to this application.

Claims

1. Use of aspirin or a pharmaceutically acceptable salt thereof, or a hydrate thereof in preparing a pharmaceutical composition for treating a disease associated with abnormal aggregation of α-synuclein.

2. The use according to claim 1, characterized in that The pharmaceutical composition is used for reducing the aggregation of α-synuclein.

3. The use according to claim 1, characterized in that The pharmaceutical composition is also used to increase the number of neurons.

4. The use according to claim 1, characterized in that The disease is a neurodegenerative disease.

5. The disease according to claim 4, characterized in that The neurodegenerative disease is selected from the group consisting of Parkinson's disease, Alzheimer's disease, Lewy body dementia, multiple system atrophy, axonal atrophy, rapid eye movement sleep behavior disorder, and pure autonomic failure.

6. The use according to claim 1, characterized in that: The pharmaceutical composition is also used to improve decreased spontaneous activity and motor coordination disorders caused by neurodegenerative diseases.

7. The use according to claim 1, characterized in that The disease is a tumor.

8. The disease according to claim 7, characterized in that The tumor is selected from the group consisting of melanoma, brain tumor, pancreatic ductal adenocarcinoma, and bladder cancer.

9. The use according to claim 1, characterized in that The medicine or pharmaceutical composition is an oral preparation.

10. The preparation according to claim 9, characterized in that The oral preparation is a tablet, capsule, pill, granule, decoction, oral liquid, drop pill or syrup.

Images