Application of psoralen in prevention and treatment of Parkinson's disease

Abstract

The invention discloses an application of psoralen in prevention and treatment of Parkinson's disease. The invention discloses application of psoralen in preparation of a medicine for preventing and / or treating Parkinson's disease. The psoralen screened by the invention can be combined with NLRP3, so that the activation of NLRP3 inflammasomes is inhibited, and the expression level of caspase-1 and IL-1beta after LPS / ATP induction is reduced. The psoralen can improve the athletic ability of a subacute MPTP Parkinson's disease model mouse, reverse mouse striatum dopaminergic neuron loss and inhibit excessive activation of microglial cells so as to inhibit neuroinflammation and play a role in neuroprotection. The results indicate that the psoralen can be used as a treatment medicine for preventing and treating the Parkinson's disease.

Description

Technical Field

[0001] The invention belongs to the field of medicine and provides a new application of psoralen, and particularly relates to the application of psoralen in the prevention and treatment of Parkinson's disease. Background Art

[0002] Parkinson's disease is the world's second largest neurodegenerative disease after Alzheimer's disease. Its typical motor symptoms are resting tremor, muscle rigidity, bradykinesia, and postural disorders. In addition, patients may also have other non-motor symptoms, such as sensory impairment, autonomic dysfunction, anxiety, depression, cognitive dysfunction, etc. At present, drug or surgical treatment can only improve the symptoms of Parkinson's disease, but cannot prevent the progressive loss of neurons, which brings great psychological pressure and economic burden to patients and their families.

[0003] The pathogenesis of Parkinson's disease involves Lewy body formation, mitochondrial dysfunction, protein clearance disorders, neuroinflammation, and oxidative stress. Among them, the involvement of neuroinflammation in the course of Parkinson's disease has been confirmed in many human samples and animal models, including the involvement of NLRP3 inflammasome in the inflammatory response of glial cells. By recognizing a variety of pathogen-associated molecular patterns and damage-associated molecular patterns, such as bacterial endotoxins, ATP, and endogenous proteins released by damaged cells, the NLRP3 inflammasome is activated and then self-cleaves caspase-1. Activated caspase-1 further cleaves downstream GSDMD, pro-IL-1β, and pro-IL-18. The GSDMD-N terminus formed by cleavage can be transported to the cell membrane to form pores, resulting in increased cell membrane permeability, cell swelling, and membrane rupture, ultimately leading to cell apoptosis. Therefore, inhibiting the excessive activation of NLRP3 inflammasomes in microglia is crucial for delaying the pathological progression of Parkinson's disease.

[0004] Psoralea corylifolia L. is a dried mature fruit of the leguminous plant Psoralea corylifolia L. It has a fragrant aroma, spicy and slightly bitter taste. It contains coumarins, mainly psoralen and isopsoralen (angelica root), as well as chalcones, flavonoids, monoterpenoids, psoralen toxins, isopsoralen dihydroflavonoids and other ingredients. Among them, the main active ingredient psoralen has multiple pharmacological effects such as anti-rheumatic arthritis, anti-asthmatic and anti-tumor, and has a relatively broad development prospect. Existing technologies show that psoralen mainly acts peripherally, and it is unclear whether it can pass through the blood-brain barrier and act in the central nervous system. The drugs we screened out through computer virtual screening and SPR experiments can bind to NLRP3 and inhibit the activation of NLRP3 inflammasomes, but it is not clear whether psoralen plays a neuroprotective role in Parkinson's disease and its mechanism of action. At the same time, whether psoralen can prevent or treat Parkinson's disease has not been reported. Summary of the invention

[0005] The invention provides a medical use of psoralen, which can be used for preventing and treating Parkinson's disease.

[0006] The purpose of the present invention can be achieved through the following technical solutions:

[0007] Use of psoralen in preparing medicine for preventing and / or treating Parkinson's disease.

[0008] The present invention screens out that psoralen can bind to NLRP3 through computer virtual screening.

[0009] Psoralen in the preparation inhibits NLRP3 inflammasome-associated inflammatory diseases.

[0010] The NLRP3 inflammasome-related inflammatory diseases described in the present invention include, but are not limited to, neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease.

[0011] As a preferred embodiment of the present invention, psoralen is used in the preparation of a drug for inhibiting inflammatory response mediated by NLRP3 in macrophages.

[0012] As another preferred embodiment of the invention, psoralen is used in the preparation of a drug for inhibiting inflammatory response mediated by NLRP3 in microglia.

[0013] Beneficial effects:

[0014] The psoralen screened by the present invention can bind to NLRP3. Applying LPS / ATP to induce NLRP3-mediated inflammatory response in mouse bone marrow-derived macrophages, administration of psoralen can reduce the expression of caspase-1 and IL-1β in cells, and reduce the secretion of IL-1β to the supernatant. Similarly, psoralen can inhibit the activation of mouse microglia and reduce the expression levels of caspase-1 and IL-1β after LPS / ATP induction. Psoralen can improve the motor ability of mice with subacute MPTP Parkinson's disease model, reverse the loss of dopaminergic neurons in the mouse striatum, and inhibit the excessive proliferation and activation of microglia. The above results suggest that psoralen has the effect of inhibiting microglial NLRP3-mediated neuroinflammation and can be used as a therapeutic drug for the prevention and treatment of Parkinson's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 It is the chemical structural formula of psoralen.

[0016] Figure 2 Psoralen is a compound that can bind to NLRP3 that was discovered through computer virtual screening.

[0017] Figure 3The binding ability of psoralen to NLRP3 was determined by SPR.

[0018] Figure 4 After mouse primary bone marrow-derived macrophages were stimulated with LPS and ATP and protected with psoralen, the supernatant was collected and the expression of IL-1β secreted into the supernatant was detected by enzyme-linked immunosorbent assay (ELISA). ### p<0.005, compared with the control group; *p<0.05, **p<0.01, ***p<0.005, compared with the model group.

[0019] Figure 5 After mouse primary microglia were stimulated with LPS and ATP and protected with psoralen, total cell protein and supernatant protein were collected, and the expressions of cytoplasmic pro-IL-1β, IL-1β, pro-caspase-1 and caspase-1 were detected by Western Blot.

[0020] Figure 6 After biotin and biotinylated psoralen were administered to HEK-293T cells, total cell proteins were collected and the binding of NLRP3 and psoralen was detected by CO-IP (Co-Immunoprecipitation) experiment.

[0021] Figure 7 This is the mass spectrometry spectrum of the drug concentration detected in the brain tissue homogenate after psoralen was treated with mice.

[0022] Figure 8 This is the effect of psoralen on the behavior of mice with subacute MPTP Parkinson's disease model. *p<0.05, **p<0.01, compared with the control group; &p<0.05, &&p<0.01, &&&p<0.005, compared with the model group.

[0023] Fig. 9 To investigate the effect of psoralen on tyrosine hydroxylase (TH)-positive neurons in the striatum of mice with subacute MPTP Parkinson's disease model.

[0024] Fig.10 The effect of psoralen on IBA-1 positive microglia in the midbrain of mice with subacute MPTP Parkinson's disease model. DETAILED DESCRIPTION

[0025] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used in the present application have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0026] The following examples will enable those skilled in the art to fully understand the present invention, but are not intended to limit the present invention in any way.

[0027] Example 1: Psoralens that can bind to NLRP3 were screened out through computer virtual screening and SPR.

[0028] 1. Materials

[0029] 1. Reagents and consumables

[0030] His-tagged NLRP3 protein was purchased from Huamei Biotechnology Co., Ltd.; CM5 chip was purchased from GE.

[0031] 2. Experimental Methods

[0032] 1. Computer virtual screening

[0033] The Alphafold2 database model was used to predict the possible binding regions of the target protein NLRP3 (PDB: 6NPY), and the small molecules binding to NLRP3 were screened according to the Gibbs free energy score using the Pymol software.

[0034] 2. Protein Binding Experiments Using SPR

[0035] Different concentrations of psoralen (25 μM to 200 μM) were run using a CM5 chip under the conditions of running buffer, and the binding rate and dissociation rate were measured at a flow rate of 25 μl / min. The solution was injected for 1.5 min, and then the ligand-free buffer was run to analyze the dissociation for 2.5 min. The nonspecific ligand binding curve was corrected by subtracting the signal of the negative control well. The equilibrium dissociation constant (Kd) was derived using the Reichert software interaction model.

[0036] 3. Experimental Results

[0037] One of the substances that can bind to NLRP3 screened by computer virtual screening is psoralen. SPR experiments confirmed that psoralen can bind to NLRP3 with a dissociation constant Kd of 209μM.

[0038] Example 2: Psoralen inhibits NLRP3-mediated inflammatory response in mouse bone marrow-derived macrophages.

[0039] 1. Materials

[0040] 1. Reagents and consumables

[0041] The cell culture medium DMEM and fetal bovine serum were purchased from Invitrogen; GM-CSF was purchased from Peprotech; penicillin, streptomycin, and antibody diluent were purchased from Synsemi; LPS, ATP, DMSO, and anti-IL-1β were purchased from Sigma; IL-1β ELISA detection kit was purchased from ExCell Bio; BCA protein quantification kit was purchased from Keygen; anti-NLRP3 and anti-caspase-1 were purchased from AidpoGen; anti-GAPDH was purchased from Proteintech; drug psoralen was purchased from Selleck; protein lysis buffer was purchased from Solebao; protease and phosphatase inhibitors were purchased from Thermo; loading buffer was purchased from Beyotime; and ECL chemiluminescent substrate kit was purchased from Tanon.

[0042] 2. Isolation and Culture of Primary Mouse Bone Marrow-derived Macrophages

[0043] The mice were euthanized by carbon dioxide, and the hind limbs of the mice were separated after disinfection with 75% ethanol, the skin and muscle tissue were removed, and the tibia was taken out. After the bones at both ends were removed, the bone marrow cells were flushed out with DMEM supplemented with 100U / ml penicillin and 100μg / ml streptomycin using a syringe. The cells were mixed by pipetting and collected in a centrifuge tube, centrifuged at 1000g for 5 minutes at room temperature, the supernatant was discarded, and complete culture medium was added to the cell pellet. After resuspending, the cells were centrifuged at 6×10 6 The cells were seeded at a density of 100 μg / ml in 12-well plates and cultured in a 37°C, 5% CO2 incubator. The complete culture medium contained 90% DMEM, 10% fetal bovine serum, 100 U / ml penicillin, 100 μg / ml streptomycin, and 20 ng / ml GM-CSF. Mouse primary bone marrow-derived macrophages in the logarithmic growth phase were used for the experiment.

[0044] 2. Experimental Methods

[0045] 1. Treatment of primary mouse bone marrow-derived macrophages

[0046] After the cells were treated with 100ng / ml LPS for 4h, the culture medium was discarded, and 10μM psoralen was given for pretreatment for 1h, and then 5mM ATP was given to stimulate and the supernatant and cells were collected. DMEM supplemented with 100U / ml penicillin and 100μg / ml streptomycin was used for drug treatment. Cells without psoralen and LPS / ATP were used as blank controls, and the results of more than three experiments were used for statistical testing.

[0047] 2. Western Blot

[0048] (1) Preparation of total cell protein: Collect cell supernatant, rinse cells twice with phosphate buffer, add protein lysis buffer, quickly scrape and collect cells, place on ice for 40 minutes, centrifuge at 16000g for 15 minutes at 4℃, and aspirate the supernatant as total cell protein. Take 1μl for BCA method to determine protein concentration, add loading buffer according to volume to the remaining protein stock solution, denature in a metal bath at 95℃ for 5 minutes, and store at -20℃. Phosphate buffer contains 2.9g / L Na2HPO4·12H2O, 0.3g / L NaH2PO4·2H2O, 8.5g / L NaCl, pH 7.2-7.6.

[0049] (2) Preparation of cell supernatant protein: Collect cell supernatant, centrifuge at 1000 g for 5 min at 4°C, then centrifuge at 13000 g for 5 min to remove dead cells, aspirate supernatant, add pre-cooled equal volume of methanol and pre-cooled 1 / 4 volume of chloroform, invert several times for extraction, centrifuge at 13000 g for 10 min at 4°C, aspirate supernatant, add 500 μl of methanol, vortex to wash away chloroform, centrifuge at 13000 g for 5 min at 4°C, aspirate supernatant, place in oven at 37°C for 5 min to evaporate methanol, add 50 μl of 2.5× loading buffer, denature in a metal bath at 95°C for 5 min, and store at -20°C.

[0050] (3) Polyacrylamide gel electrophoresis: Prepare separation gel and stacking gel of different concentrations according to the molecular weight of the target protein. The amount of sample loaded per well is about 30 μg. Electrophoresis is performed at a constant voltage of 80V to 120V for about 2 hours until bromophenol blue runs to the bottom of the gel. The electrophoresis buffer contains 3g / L Tris, 14.4g / L glycine, and 1g / L SDS.

[0051] Polyacrylamide gel:

[0052] Element 8% separation gel 10% separation gel 15% separation gel Concentrated Glue Deionized water 2.55ml 1.95ml 0.75ml 2.8ml 30% AA stock solution 1.95ml 2.55ml 3.75ml 660μl 1.5M Tris-HCl, pH 8.8 2.85ml 2.85ml 2.85ml --- 1M Tris-HCl, pH 6.8 --- --- --- 500μl 10% SDS 75μl 75μl 75μl 40μl 10% ammonium persulfate 75μl 75μl 75μl 40μl TEMED 3μl 3μl 3μl 4μl Total volume 7.5ml 7.5ml 7.5ml 4ml

[0053] (4) Transfer: After electrophoresis, remove the concentrated gel and excess bromophenol blue at the bottom, and transfer the protein bands to the PVDF membrane using the wet transfer method. Transfer at 300 mA constant current for 120 min. The transfer buffer contains 3 g / L Tris, 14.4 g / L glycine, and 20% methanol, and is precooled before use.

[0054] (5) Blocking: After transfer, remove the PVDF membrane, pass through TBST, and then immerse the membrane in TBST containing 5% skimmed milk powder for 2 h.

[0055] (6) Primary antibody incubation: After blocking, wash the membrane with TBST for 5 min × 3 times, transfer the PVDF membrane to a sealed plastic film, add the antibody diluted in proportion, and shake at 4°C overnight.

[0056] (7) Secondary antibody binding: Wash the membrane with TBST for 10 min × 3 times, add the secondary antibody labeled with horseradish peroxidase, and incubate at room temperature for 2 h. After incubation, wash the membrane with TBST for 10 min × 3 times. TBST contains 2.42 g / L Tris, 8 g / L NaCl, 1‰ Tween-20, pH 7.2-7.6.

[0057] (8) ECL color development: Prepare fresh ECL chemiluminescent substrate and evenly add it to the membrane surface. Expose the membrane to color in a dark environment. Compare the gray value of the target protein with the gray value of the internal reference GAPDH for semi-quantitative analysis.

[0058] 3.ELISA experiment

[0059] The cell supernatant was tested by double antibody sandwich ELISA. After centrifugation at 4℃1500g for 10min, the supernatant was collected and used for the experiment. The standard and sample were added to the antibody-coated microplate, and after adding the biotinylated antibody, it was placed in a microplate constant temperature oscillator at 37℃ for 1h and washed 5 times. The enzyme conjugate working solution was added, incubated at 37℃ in the dark for 30min, and the plate was washed 5 times. The colorimetric substrate was added and incubated at 37℃ in the dark for 15min. The stop solution was added, and the absorbance value was measured at a wavelength of 450nm within 10min, and the sample concentration was calculated according to the standard curve.

[0060] 3. Experimental Results

[0061] LPS / ATP stimulation caused an increase in the levels of IL-1β and caspase-1 in the cytoplasm of mouse bone marrow-derived macrophages, and IL-1β secreted into the supernatant. Psoralen could significantly reduce the levels of the above indicators, indicating that psoralen inhibited the NLRP3-mediated inflammatory response of mouse bone marrow-derived macrophages.

[0062] Example 3: Psoralen inhibits NLRP3-mediated inflammatory response in mouse microglia.

[0063] 1. Materials

[0064] 1. Reagents and consumables

[0065] The cell culture medium DMEM and fetal bovine serum were purchased from Invitrogen; penicillin, streptomycin, and antibody diluent were purchased from Synthesize; LPS, ATP, DMSO, and anti-IL-1β were purchased from Sigma; the BCA protein quantification kit was purchased from Keygen; anti-NLRP3 and anti-caspase-1 were purchased from AidpoGen; anti-GAPDH was purchased from Proteintech; the drug psoralen was purchased from Selleck; protein lysis buffer was purchased from Solebao; protease and phosphatase inhibitors were purchased from Thermo; loading buffer was purchased from Beyotime; and the ECL chemiluminescent substrate kit was purchased from Tanon.

[0066] 2. Isolation and Culture of Mouse Microglia

[0067] After disinfecting newborn mice 1 to 3 days old with 75% ethanol, remove the whole brain and place it in pre-cooled DMEM supplemented with 100U / ml penicillin and 100μg / ml streptomycin. Under a microscope, remove the meninges and blood vessels, separate the cortical tissue, rinse the tissue with DMEM, change to DMEM containing 0.25% pancreatic enzyme, and place it in a 37℃, 5% CO2 incubator for digestion for 5 minutes. Aspirate and discard the digestion solution, add complete culture medium to terminate digestion, gently blow the tissue into a single cell suspension, filter through a 200-mesh sieve, collect the filtrate, centrifuge at 1000g for 5 minutes at room temperature, discard the supernatant, add complete culture medium to the cell pellet, resuspend and inoculate in a T75 bottle, and place it in a 37℃, 5% CO2 incubator for culture. After that, the medium was changed every 3.5 days. On the 7th day of culture, microglia were obtained by beating, blown off and collected, centrifuged at 1000g for 5 minutes at room temperature, and the supernatant was discarded. Complete medium was added to the cell pellet, and after resuspending, it was evenly inoculated in a six-well plate or a 24-well plate. After the cells grew for 2 to 3 days and expanded into a continuous monolayer, they could be used for experiments. The complete medium contained 90% DMEM, 10% fetal bovine serum, 100U / ml penicillin and 100μg / ml streptomycin.

[0068] 2. Experimental Methods

[0069] 1. Treatment of mouse microglia

[0070] The same treatment method as in Example 2 for mouse bone marrow-derived macrophages.

[0071] 2. Western Blot operation steps are the same as in Example 2.

[0072] 3. Experimental Results

[0073] LPS / ATP stimulation caused an increase in the levels of IL-1β and caspase-1 in primary microglia, as well as IL-1β secreted into the supernatant. Psoralen could significantly reduce the levels of the above indicators, indicating that psoralen inhibited the NLRP3-mediated inflammatory response in primary microglia.

[0074] Example 4: Psoralen can bind to NLRP3.

[0075] 1. Materials

[0076] 1. Reagents and consumables

[0077] The biotinylated psoralen was commissioned to be synthesized by Nanjing Ruizhi Biopharmaceutical Co., Ltd.

[0078] 2. Experimental Methods

[0079] After HEK293 cells were treated with biotin and biotinylated psoralen, the cells were collected in a 1.5 mL EP tube, lysate was added, lysed on ice for 1 h, centrifuged at 4°C 12000g for 15 min, the cell supernatant was collected, 20 μL was taken as input, 5× loading buffer was added, boiled at 95°C for 5 minutes, and stored at -20°C. Antibody (Flag-NLRP3) was added to the remaining 280 μL protein lysate, shaken overnight at 4°C, 25 μL biotin breads (Thermo) were added and shaken for 4 hours. The precipitate was collected by centrifugation at 4°C 2500g, the precipitate was washed with RIPA lysate, the supernatant was discarded, 5× loading buffer was added, boiled at 95°C for 5 minutes, and Western Blot operation was performed, the steps were the same as in Example 2.

[0080] 3. Experimental Results

[0081] Psoralen can bind to NLRP3.

[0082] Example 5: Study on the penetration of psoralen through the blood-brain barrier

[0083] 1. Materials

[0084] 1. Reagents and consumables

[0085] Chromatographic grade methanol, formic acid and other mobile phases were purchased from MERCK.

[0086] 2. Experimental Methods

[0087] (1) Scheme and grouping

[0088] C57 mice were injected intraperitoneally with 20 mg / kg once at 6 hr, 12 hr and 48 hr, with n=4 at each time point, half of them were male and half were female.

[0089] (2) Preparation of mouse plasma and brain tissue homogenate:

[0090] Plasma: After intraperitoneal injection of 20mg / kg psoralen, mice were intraperitoneally injected with 4% chloral hydrate at a dose of 10ml / kg at 6hr, 12hr and 48hr. After the whole body muscles of the mice relaxed, the whiskers were trimmed, one eyeball was removed, and the head was turned down, and the blood was suspended and dripped into a heparin-coated blood collection tube. It was left to stand at 4℃ overnight, and centrifuged at 4℃ 3000g for 10min on the second day, and the upper plasma was collected and stored in aliquots at -80℃ for later use.

[0091] Brain tissue homogenate: At each time point, the whole brain tissue of the mouse was taken, and the left and right hemispheres were weighed and divided into a doffer tube. One side of the brain was frozen at -80℃ for later use, and the other half of the brain was homogenized with 2ml / kg normal saline, centrifuged at 12000rpm at 4℃ for 15min, and the supernatant was collected and divided and frozen at -80℃ for later use.

[0092] (4) Sample processing

[0093] Add 10 μl of internal standard to every 200 μl of plasma sample or brain tissue homogenate, add 600 μl of ethyl acetate, vortex to mix, centrifuge at 16000g at 4℃ for 20 min, and collect the supernatant. Evaporate the solvent under a gentle and stable flow of nitrogen, and then redissolve the residue with 125 μl of chromatographic pure methanol. This step is used to concentrate the sample. Ultrasound at 250W power and 40kHz frequency for 5 min, centrifuge at 16000g at 4℃ for 20 min, collect 120 μl of supernatant and place it in a clean vial, and inject 5 μl from each vial into the UPLC-MS system.

[0094] (5) UPLC-MS analysis

[0095] Psoralen in mouse plasma and brain tissue was quantified using UPLC-MS. The system was equipped with an electrospray ionization (ESI) interface and Analyzer 1.6 software.

[0096] Chromatographic conditions: Compounds were separated on the chromatographic column, mobile phase gradient elution, eluent A was 0.1% (V / V) formic acid aqueous solution, B was acetonitrile, elution program was as follows: 0.0-1.0min maintained at 10% B; 1.0-3.0min mobile phase increased from 10% B to 50% B; 3.0-5.0min mobile phase increased from 50% B to 100% B; 5.0-6.0min maintained at 100% B; 6.0-6.1min mobile phase decreased from 100% B to 10% B; 6.1-8.0min maintained at 10% B, returned to the initial state, and waited for the next injection. Flow rate was 0.3ml / min, temperature was 40°C.

[0097] Mass spectrometry conditions: using positive ion mode, H-ESI source operating parameters are: positive ion spray voltage 3500V; negative ion spray voltage 2500V; sheath gas pressure 50Arb; auxiliary gas pressure 20Arb; sweep gas pressure 1Arb; ion transfer tube temperature 300℃; vaporization temperature 300℃. The peak area ratio internal standard method was used to quantify psoralen ions by multiple reaction monitoring.

[0098] 3. Experimental Results

[0099] The chromatographic peak graph of the psoralen standard showed that the retention time of the compound was 4.36min. 6 hours after administration, the chromatographic peak graph of brain tissue homogenate showed that compared with the chromatographic peak graph of blank mouse brain tissue homogenate, there was an obvious single peak at 4.36min, and after comparison with the peak graph of the standard, it was determined to be the peak graph of psoralen. The above results show that after mice were gavaged with 20mg / kg psoralen, the drug was distributed in the brain tissue, suggesting that psoralen can pass through the blood-brain barrier.

[0100] Example 6: Application of psoralen in preventing and treating Parkinson's disease in mice.

[0101] 1. Materials

[0102] 1. Animals: The wild-type mice with C57BL / 6 genetic background were provided by the Experimental Animal Center of Nanjing Medical University. They were male, 2-3 months old, weighing 20-25 g. They were fed with standard feed and had free access to drinking water. The room temperature was 24±2℃, the humidity was 50-60%, and the ventilation was good. The light and dark time were 12 h each per day.

[0103] 2. Reagents and consumables

[0104] The antibody diluent was purchased from Synsemi; MPTP and DMSO were purchased from Sigma; DAB colorimetric kit was purchased from Keygen; anti-TH and anti-IBA-1 were purchased from Millipore; and psoralen was purchased from Selleck.

[0105] 2. Experimental Methods

[0106] 1. Preparation of Subacute MPTP Parkinson's Disease Model in Mice and Psoralen Injection

[0107] MPTP was diluted with saline to a working solution of 2 mg / ml. 20 mg / kg MPTP was injected subcutaneously at the back of the neck of mice once a day for 5 consecutive days. The mice in the control group were treated with sterile saline.

[0108] Psoralen was diluted with 0.5% sodium carboxymethylcellulose to a working solution of 5 mg / ml. Mice were gavaged with 20 mg / kg psoralen once a day, 3 days before MPTP administration, and continued for 11 days. The mice in the control group were treated with sterile saline. Behavioral testing was performed on the second day after the last administration, and the mice were killed on the third day for specimen collection.

[0109] 2. Mouse behavioral testing

[0110] (1) Rotarod test: used to evaluate the motor coordination ability of mice. Mice were trained at a rotation speed of 15 rpm for 5 min every day for 3 consecutive days. On the 4th day, the mice's staying time was recorded at a rotation speed of 5-25 rpm.

[0111] (2) Pole climbing test: used to evaluate the autonomous movement behavior of mice. The mouse is placed head up on the top of a 1 cm diameter, 50 cm high pole with a 1.2 cm diameter wooden ball on the top of the pole. The time from the start of the movement to the complete turn of the head down (T-Turn) and the total time to crawl to the bottom of the pole (T-TLA) are recorded. The shortest time is used during the experiment. Failure to completely turn over, fall or slide is counted as 120 seconds. Training is performed twice a day for 3 consecutive days, and the formal experiment is carried out on the 4th day.

[0112] (3) Open-field test: used to evaluate the crawling speed and autonomous movement behavior of mice. After the mice were adapted to the 50 cm × 50 cm × 50 cm box for 15 min, the software observed and recorded the movement trajectory and movement speed of the mice within 5 min.

[0113] 3. Western Blot

[0114] (1) Preparation of tissue protein: Rinse the tissue twice with phosphate buffer, add 30 μl of protein lysis buffer for every 1 mg of tissue, grind the tissue using a frozen ball mill, place it on ice for lysis for 40 min, centrifuge it at 16000g for 15 min at 4°C, and aspirate the supernatant as tissue protein. Take 1 μl for BCA method to determine protein concentration, add loading buffer to the remaining protein stock solution according to volume, denature it in a metal bath at 95°C for 5 min, and store it at -20°C.

[0115] (2) The steps of electrophoresis to ECL color development are the same as those in Example 1.

[0116] 4. Immunohistochemistry

[0117] After the mice were perfused with saline for 5 minutes through the left ventricle, they were followed by 4% paraformaldehyde for 10 minutes, and the brains were removed and fixed in 4% paraformaldehyde for 24 hours. The brains were dehydrated in 20% sucrose solution (phosphate buffer) for 3 days and 30% sucrose solution (phosphate buffer) for 3 days. The tissues were embedded in OCT glue and frozen sections were made with a thickness of 20 μm. The brain slices in the substantia nigra were collected and placed in 50% phosphate buffer + 50% glycerol and stored at -20°C. Before use, brain slices were selected according to the method of taking one every three or six brain slices, and immunohistochemistry was performed by the bleaching method. The specific steps are as follows: rinse the brain slices with phosphate buffer for 10 minutes × 3 times, remove horseradish peroxidase with 3% hydrogen peroxide for 15 minutes, wash the brain slices with phosphate buffer for 10 minutes × 3 times, and block and permeabilize the membrane with phosphate buffer containing 10% goat serum and 0.3% Triton X-100 at room temperature for 2 hours. Anti-TH and anti-IBA-1 were diluted with phosphate buffer at a titer of 1:1000, and brain slices were incubated with them at 4°C overnight. The next day, the antibodies were discarded and the brain slices were rinsed with phosphate buffer for 10 min × 3 times. The horseradish peroxidase-labeled secondary antibody was diluted with phosphate buffer at a titer of 1:1000 and incubated at room temperature for 2 h. The antibodies were discarded and the brain slices were rinsed with phosphate buffer for 10 min × 3 times. DAB color developing solution was used for color development in the dark, the slices were mounted, dehydrated, and sealed, and photographed and counted using a stereological microscope.

[0118] 3. Experimental Results

[0119] Compared with the control group, the motor function of the subacute MPTP Parkinson's disease model mice was impaired, the number of striatal TH neurons decreased, and the number of IBA-1 positive microglia increased. After psoralen was administered, the motor function impairment of the Parkinson's disease model mice was alleviated, the number of striatal TH neurons was reduced, and the proliferation and activation of IBA-1 positive microglia decreased, indicating that psoralen has a therapeutic effect on Parkinson's disease.

[0120] The above examples prove that psoralen can bind to NLRP3, inhibit the NLRP3-mediated inflammatory response in bone marrow-derived macrophages and microglia, and has a significant therapeutic effect on the subacute MPTP Parkinson's disease pathological model in mice, indicating that the psoralen of the present invention has the potential to treat Parkinson's disease and can be used in the prevention and treatment of Parkinson's disease.

Claims

1. Use of psoralen in the preparation of a drug for preventing and / or treating Parkinson's disease.

2. Application of psoralen in the preparation of drugs for inhibiting NLRP3 inflammasome-related inflammatory diseases.

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