JP5820127B2 - Preventive / ameliorating agent for neurodegenerative diseases with amyloid fibril formation - Google Patents

Description

  The present invention relates to a preventive / ameliorating agent for neurodegenerative diseases accompanied by the formation of amyloid fibrils, and specifically relates to a prophylactic / ameliorating agent for neurodegenerative diseases containing nucleoprotein as an active ingredient.

With an aging society, the number of patients with neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease is increasing year by year. Symptoms of these diseases are various such as learning / memory disorder, exercise, and language disorder, but these disorders not only impair the patient's own social life, but also a great burden for caregivers including their families. Furthermore, the increase in the number of patients with neurodegenerative diseases that require such long-term treatment is pressing not only in Japan but also in developed countries. Therefore, in addition to new treatment methods using drugs, there is a need for the development of preventive medical approaches based on alternative medical methods that suppress (improve) the progression of symptoms of neurodegenerative diseases and prevent their onset.
In many neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, multiple sclerosis and the like, amyloid fibril formation (formation of poorly soluble protein aggregates) has been observed. The formation of this hardly soluble protein aggregate is considered as a factor deeply involved in the development of neurodegenerative diseases. Senile plaques that are characteristic pathological structures in Alzheimer's disease are protein inclusion bodies deposited in tissues, and the constituent components are amyloid fibrils formed by amyloid β peptide (hereinafter also referred to as Aβ). Although pathogenic mutations to various causative genes have been found in familial Alzheimer's disease families, all have been reported to promote the accumulation of Aβ in the brain (see, for example, Non-Patent Documents 1 and 2). .) It has also been reported that aggregates of Aβ showed neurocytotoxicity in an experimental system using cultured cells (see, for example, Non-Patent Documents 3 and 4). Furthermore, animals introduced with Aβ gene of human Alzheimer's disease family and animals injected with Aβ in the brain showed accumulation of amyloid fibrils, showing human Alzheimer's disease-like symptoms such as cognitive / learning disorders, and these It has also been reported that Alzheimer's disease-like symptoms have been improved by suppressing the accumulation of amyloid fibrils (see, for example, Non-Patent Document 5). These findings suggest that amyloid fibril formation is deeply involved in the onset and progression of Alzheimer's disease, and therefore, by controlling amyloid fibril formation, many neurodegenerative diseases including Alzheimer's disease can be treated. This is thought to lead to the development of law.

  On the other hand, white silkworms contain nucleoprotein, which is a complex of deoxyribonucleic acid (DNA) and protamine, and are used as functional foods. The nucleoprotein has an immunomodulatory action (for example, see Non-Patent Document 6), an antioxidant action (for example, see Non-Patent Document 7), a vasodilatory action (for example, see Non-Patent Document 8), and the like. It has been shown to have a preventive / improving usefulness against atopic disease, cerebral infarction, and rheumatoid arthritis. However, the prophylactic and / or ameliorating effects of nucleoprotein on neurodegenerative diseases have not been fully clarified, and in particular what effect nucleoprotein has on amyloid fibril formation. No reports have been made.

Hardy, J. (1997). Amyloid, the presenilins and Alzheimer's disease. Trends Neurosci 20, 154-9. Hardy, J. and Selkoe, D.J. (2002). The amyloid hypothesis of Alzheimer's disease: progress and problems on the road to therapeutics. Science 297, 353-6. Lambert, M.P. et al. (1998) .Diffusible, nonfibrillar ligands derived from Abeta1-42 are potent central nervous system neurotoxins.Proc Natl Acad Sci U S A 95, 6448-53. Klein, W.L., Krafft, G.A. and Finch, C.E. (2001) .Targeting small Abeta oligomers: the solution to an Alzheimer's disease conundrum? Trends Neurosci 24, 219-24. Walsh, DM, Klyubin, I., Fadeeva, JV, Cullen, WK, Anwyl, R., Wolfe, MS, Rowan, MJ and Selkoe, DJ (2002) .Naturally secreted oligomers of amyloid beta protein potently inhibit hippocampal long-term potentiation in vivo.Nature 416, 535-9. Sudo, N., Aiba, Y., Takaki, A., Tanaka, K., Yu, XN, Oyama, N., Koga, Y. and Kubo, C. (2000). Dietary nucleic acids promote a shift in Th1 / Th2 balance toward Th1-dominant immunity. Clin Exp Allergy 30, 979-87. Ohtaki, H. et al. (2010) Nucleoprotein Diet Ameliorates Arthritis Symptoms in Mice Transgenic for Human T-Cell Leukemia Virus Type I (HTLV-1). J Clin Biochem Nutr 46, 93-104. Matsunaga, M. et al. (2003) .Nucleoprotamine diet derived from salmon soft roe protects mouse hippocampal neurons from delayed cell death after transient forebrain ischemia.Neurosci Res 47, 269-76.

  An object of the present invention is to provide a preventive / ameliorating agent for neurodegenerative diseases accompanied by amyloid fibril formation.

  As a result of intensive studies to solve the above problems, the present inventors have found that the nucleoprotein inhibits the formation of amyloid β peptide fibers, and suppresses the loss of midbrain substantia nigra dopamine nerves, Thus, the present invention was completed by confirming that a drug containing a nucleoprotein as an active ingredient is effective in the prevention and improvement of neurodegenerative diseases accompanied by the formation of amyloid fibrils.

That is, the present invention
(1) A preventive / ameliorating agent for neurodegenerative diseases associated with amyloid fibril formation, comprising a nucleoprotein as an active ingredient,
(2) The prevention / improvement according to (1) above, wherein the prevention / amelioration of the neurodegenerative disease is due to inhibition of amyloid β peptide fiber formation by the nuclear protein and / or suppression of dopamine neurons in the midbrain substantia nigra. Remedy,
(3) The preventive / ameliorating drug according to (1) or (2), wherein the neurodegenerative disease is Alzheimer's disease,
(4) The preventive / ameliorating agent according to (1) or (2), wherein the neurodegenerative disease is Parkinson's disease,
(5) The prophylactic / ameliorating agent according to any one of (1) to (4), wherein the nucleoprotein is nucleoprotamine,
(6) The prophylactic / ameliorating agent according to (5) above, wherein the nucleoprotamine is derived from anther white moth.
About.

The present invention provides a preventive / ameliorating agent for neurodegenerative diseases accompanied by amyloid fibril formation.
The present inventors have now found that nucleoprotein reduces amyloid fibril formation in in vitro studies. Amyloid fibrils are protein aggregates formed by regularly associating proteins, and are characterized by forming a regular repeating structure. Amyloid fibrils are thought to be formed by regular associations due to sequence-specific interactions of proteins. Suppression of amyloid fibril formation by this study can be broadly divided into two mechanisms: delay / suppression of aggregation or promotion of dissociation (degradation) of aggregated amyloid fibrils.

  Aβ, which is an amyloidogenic protein used in this study, has been subjected to detailed structural analysis of the amyloid fibrils formed (for example, Luhrs, T., Ritter, C., Adrian, M., Riek-Loher, D. , Bohrmann, B., Dobeli, H., Schubert, D. and Riek, R. (2005) .See 3D structure of Alzheimer's amyloid-beta (1-42) fibrils.Proc Natl Acad Sci USA 102, 17342-7. .) One interaction believed to be important for Aβ fibril formation is the association by electrostatic interaction between the 23rd aspartic acid residue and the 28th lysine residue of the adjacent Aβ molecule . The protein constituting the nucleoprotein has abundant positive amino acid residues similar to the 28th lysine residue of the Aβ molecule, and the DNA further includes a base exhibiting electrical polarity. Therefore, it is considered that the nucleoprotein has such a unique structure, so that the structure (partial structure) enters the association part between Aβ molecules and competes or inhibits it. As a result, it is considered that Aβ cannot construct a regular amyloid fibril structure, and amyloid fibril formation is suppressed.

How much amyloid fibril formation (○: No additive) by adding 10 μM Aβ is suppressed by addition of 0.6 μg / mL (▲), 6 μg / mL (▼) and 60 μg / mL (■) of nucleoprotein (NP) It is the graph which showed whether it is done. It is a graph which shows the concentration dependence of the inhibitory effect of a nucleoprotein (NP: ●) with respect to amyloid fibril formation of 10 micromol A (beta). It is a figure which shows the outline of the experimental schedule of motor function evaluation and histological evaluation of Parkinson's disease-like model mouse. It is a graph which shows the evaluation result of the motor function by the foot-fault test of a mouse | mouth on the 7th day after administration of physiological saline (Saline: (circle)) or MPTP ((circle)). A group in which Parkinson's disease model mice created by MPTP administration were ingested with a non-nucleic acid-free diet (non-NP: ○) and a diet containing 1.2% nucleoprotein (NP: ●) It is a graph which shows the foot-fault rate in the 7th day after administration of MPTP. It is a photograph (saline (Saline: upper figure), MPTP (lower figure)) of TH immunostaining of dopamine nerve near the mouse midbrain substantia nigra on the 8th day after administration of physiological saline (Saline) or MPTP. . A graph showing the number of TH-positive cells (TH ⁺ cells) when TH immunostaining was performed on dopamine neurons in the vicinity of the substantia nigra of mice 8 days after administration of saline (Saline: ○) or MPTP (●). is there. It is a photograph showing a typical stained image of FJB (Fluoro Jade B) staining in the vicinity of the substantia nigra of mice on the 8th day after administration of physiological saline (Saline: upper diagram) or MPTP (lower diagram). After MPTP administration of Parkinson's disease-like model mice created by MPTP administration, a group ingested a non-nucleic acid-free diet (non-NP) and a diet in which a diet containing nucleoprotein 1.2% (NP) was ingested Photo of TH immunostaining of dopamine nerve near mouse midbrain substantia nigra on day 8 (group receiving non-nucleic acid diet (non-NP: upper figure), diet containing nucleoprotein 1.2% (NP: the figure below)). A group in which Parkinson's disease model mice created by MPTP administration were ingested with a non-nucleic acid-free diet (non-NP: ○) and a diet containing 1.2% nucleoprotein (NP: ●) 8 is a graph showing the number of TH positive cells (TH ⁺ cells) when TH immunostaining is performed on dopamine neurons in the vicinity of mouse midbrain substantia nigra on day 8 after administration of MPTP.

The present invention will be described in more detail.
The preventive / ameliorating agent for neurodegenerative diseases associated with the formation of amyloid fibrils according to the present invention is characterized by containing a nucleoprotein as an active ingredient.
Many diseases with the formation of poorly soluble protein aggregates (amyloid fibril formation) at the time of pathology have been reported, and more than 30 types of proteins involved in amyloid fibril formation have been reported. The proteins formed and the diseases caused by amyloid fibril formation by the proteins are summarized in Table 1 and Table 2 below.

Among the diseases shown above, Parkinson's disease, Alzheimer's disease, Huntington's chorea, etc. are many neurodegenerative diseases with amyloid fibril formation in the nervous system and neuronal cell death, but amyotrophic lateral sclerosis Diseases in which amyloid fibril formation is observed in muscles, blood vessels and systemic organs / tissues such as organ amyloidosis have also been reported.
Therefore, the diseases that are the targets of the preventive / ameliorating agent of the present invention include all the diseases shown above.
In addition, examples of the disease that is the target of the preventive / ameliorating agent of the present invention include Alzheimer's disease and Parkinson's disease.

The nucleoprotein contained as an active ingredient in the preventive / ameliorating agent of the present invention means a complex in which the protein is bound to ribonucleic acid, and examples of the protein include proteins with high positive charge that can bind to ribonucleic acid. .
Specific examples of the nucleoprotein include a complex in which a positively charged protein such as polylysine, polyarginine, polyornithine, histone, avidin, and protamine is bound to ribonucleic acid.
Preferably, nucleoprotamine in which protamine is bound to ribonucleic acid is used.
As said nucleoprotamine, the nucleoprotamine obtained from the testis (white child) of a fish shellfish is preferable, and the nucleoprotamine obtained from the white child of a salmon is more preferable.

The nucleoprotein can be obtained, for example, by performing operations such as extraction / purification from the testes (white eggs) of fish and shellfish containing a large amount of nucleoprotein.
For example, nucleoprotamine, a nucleoprotein derived from koji, is obtained by washing kojiko with ethanol and crushing, solid-liquid separation, collecting solids, sterilizing, drying, crushing, and then sieving Can be obtained as a powdery substance.
Moreover, it can manufacture not only by the above-mentioned naturally-derived thing but also by making the protein and ribonucleic acid rich in the above-mentioned positive charge couple | bonded by a well-known method, and making it a composite_body | complex.
Moreover, the nucleoprotein can also use a commercial item.

Examples of the administration form of the preventive / ameliorating agent for neurodegenerative diseases accompanied by amyloid fibril formation according to the present invention include non-injection (subcutaneous, intravenous, intramuscular, intraperitoneal injection), ointment, suppository, aerosol, etc. Oral administration or oral administration by tablets, capsules, granules, pills, syrups, solutions, emulsions, suspensions and the like can be mentioned.
The prophylactic / ameliorating agent for neurodegenerative diseases accompanied by amyloid fibril formation according to the present invention is about 0.01 to 99.5% by mass, preferably about 0.1 to 30% by mass, of the nucleoprotein as an active ingredient with respect to the mass of the whole composition. %.

The preventive / ameliorating agent for neurodegenerative diseases accompanied by the formation of amyloid fibrils according to the present invention may contain other pharmaceutically or veterinary active compounds in addition to the nucleoprotein which is an active ingredient.
The clinical dose of nucleoprotein contained in the preventive / ameliorating agent for neurodegenerative diseases accompanied by amyloid fibril formation of the present invention varies depending on age, body weight, patient sensitivity, symptom level, etc., but is usually effective. The dosage is about 0.001 to 5.0 g, preferably about 0.5 to 2.5 g per day for an adult. However, if necessary, an amount outside the above range can be used.
The preventive / ameliorating agent for neurodegenerative diseases accompanied by the formation of amyloid fibrils of the present invention is formulated for administration by conventional pharmaceutical means.

That is, tablets, capsules, granules, pills for oral administration are excipients such as sucrose, lactose, glucose, starch, mannit; binders such as hydroxypropylcellulose, syrup, gum arabic, gelatin, sorbit , Tragacanth, methylcellulose, polyvinylpyrrolidone; disintegrating agents such as starch, carboxymethylcellulose or calcium salts thereof, microcrystalline cellulose, polyethylene glycol; lubricants such as talc, magnesium or calcium stearate, silica; lubricants such as sodium laurate It is prepared using glycerol or the like.
Injections, solutions, emulsions, suspensions, syrups and aerosols are active ingredient solvents such as water, ethyl alcohol, isopropyl alcohol, propylene glycol, 1,3-butylene glycol, polyethylene glycol; surfactants such as Sorbitan fatty acid ester, polyoxyethylene sorbitan fatty acid ester, polyoxyethylene fatty acid ester, polyoxyethylene ether of hydrogenated castor oil, lecithin; suspending agents, for example, carboxymethyl sodium salt, cellulose derivatives such as methylcellulose, tragacanth, gum arabic, etc. Natural rubbers; prepared using preservatives such as esters of paraoxybenzoic acid, benzalkonium chloride, sorbate and the like.
Examples of ointments that are transdermal preparations include white petrolatum, liquid paraffin, higher alcohol, macrogol ointment, hydrophilic ointment, and aqueous gel base.
Suppositories are prepared using, for example, cacao butter, polyethylene glycol, lanolin, fatty acid triglyceride, coconut oil, polysorbate and the like.

Formulation examples of the preventive / ameliorating agent for neurodegenerative diseases accompanied by amyloid fibril formation according to the present invention are shown below.
Formulation Example 1
Tablet Nucleoprotein 20g
Lactose 260g
600g microcrystalline cellulose
Corn starch 350g
Hydroxypropylcellulose 100g
CMC-Ca 140g
Magnesium stearate 30g
Total amount 1,500g
After the above ingredients are mixed by a conventional method, 10,000 sugar-coated tablets containing 1 mg of the active ingredient are produced.
Formulation Example 2
Capsule Nucleoprotein 20g
430 g of lactose
1,000g microcrystalline cellulose
Magnesium stearate 50g
Total amount 1,500g
The above ingredients are mixed by a conventional method and then filled into gelatin capsules to produce 10,000 capsules containing 1 mg of active ingredient in one capsule.
Formulation Example 3
Soft capsule nucleoprotein 25g
PEG400 464g
Saturated fatty acid triglyceride 1,500 g
Peppermint oil 1g
Polysorbate 80 10g
Total amount 2,000g
After mixing the above ingredients, a No. 3 soft gelatin capsule is filled by a conventional method to produce 10,000 capsules containing 1 mg of the active ingredient in one capsule.
Formulation Example 4
Ointment Nucleoprotein 1.0g
Liquid paraffin 10.0g
Cetanol 20.0g
White petrolatum 68.4g
Ethylparaben 0.1g
l-Menthol 0.5g
Total amount 100.0g
The above ingredients are mixed by a conventional method to make a 1% ointment.
Formulation Example 5
Suppository nuclear protein 1g
Whittepsol H15 * 478g
Whittepsol W35 * 520g
Polysorbate 80 1g
Total amount 1,000g
“Trademark name of triglyceride compound Witepsol = Witepsol”
The above ingredients are melt-mixed in a conventional manner, poured into a suppository container and solidified by cooling to produce 1,000 1 g suppositories containing 1 mg of the active ingredient.
Formulation Example 6
Injection nucleoprotein 1mg
5mL distilled water for injection
When used, dissolve.

Production Example 1: Production of Nucleoprotein After washing coconut milk with ethanol, it was pulverized with a blender. The solid content was collected by solid-liquid separation, then sterilized (70 ° C., 30 minutes) and dried (water content 7% or less).
The obtained dried product was pulverized and then passed through a 40 mesh screen to obtain a nucleoprotein (nucleoprotamine).
The characteristics of the obtained nucleoprotein were as follows.
・ Appearance Pale yellow or light brown powder ・ Particle size 40 mesh pass ・ Odor with peculiar odor ・ Taste Peculiar taste ・ DNA content 27% or more ・ Protein content 50-52%
・ Moisture content: 7% or less

Test Example 1: Evaluation of amyloid β peptide fiber formation inhibitory activity 1) Preparation of amyloid β peptide (Aβ) solution Lyophilized powder of amyloid β peptide (Aβ) (manufactured by Peptide Institute, Inc.) was dissolved in dimethyl sulfoxide. The peptide aggregates were pulverized by sonication. The obtained Aβ solution was diluted with a phosphate buffer (10 mM PBS) to a final concentration of 10 μM and subjected to the subsequent reaction.
2) Evaluation of amyloid β peptide (Aβ) fibril formation inhibitory activity
The amount of Aβ fibril formation was measured using specific binding between amyloid fibrils and thioflavin T. It is known that thioflavin T binds to the cross β structure, which is a quaternary structure characteristic of amyloid fibrils, and the fluorescence intensity increases. Thioflavin T (final concentration 10 μM) and various concentrations of nucleoprotein (NP) solution or solvent (control group) were added to and mixed in the solution containing Aβ, and added to a 96-well microplate (final volume 200 μL) ). After incubating at 37 ° C., thioflavin T-derived fluorescence intensity (Ex: 440 nm, Em: 480 nm) was measured every 20 minutes until 30 hours, and changes in Aβ fibril formation with time were examined.
The measured fluorescence intensity of each sample was expressed as a relative value when the fluorescence intensity of the control group 30 hours after the start of incubation was taken as 100%. During the experiment, the microplate was covered with film to prevent evaporation of the solution during incubation.
The data is shown as an average value ± standard deviation.
FIG. 1 shows how much amyloid fibril formation is caused by the addition of Aβ (10 μM) when nucleoprotein (NP) (0.6 μg / mL (▲), 6 μg / mL (▼) and 60 μg / mL (■)) is added. A graph showing whether (◯: No additive) is suppressed (n = 5) is shown in FIG. 2, and the concentration dependence of the inhibitory effect of nucleoprotein (NP: ●) on amyloid fibril formation of Aβ (10 μM) is shown in FIG. The graph which shows sex was shown.

3) Results As a result of incubating Aβ and thioflavin T at a final concentration of 10 μM, thioflavin T-derived fluorescence intensity was observed to increase over time. Therefore, it was considered that Aβ formed amyloid fibrils under these experimental conditions. The fluorescence characteristics of thioflavin T were not significantly affected by adding nucleoprotein alone to the reaction solution.
Next, the effect of nuclear proteins on Aβ fibril formation was examined (FIG. 1). When nucleoproteins with final concentrations of 0.6, 6, and 60 μg / mL were added to the reaction solution, the fluorescence intensity derived from thioflavin T 30 hours after the start of incubation was 93.0 ± 6.8, 83.8, respectively, compared to the control group.
± 12.5 and 23.1 ± 7.9% (FIG. 1). The concentration dependence of the Aβ fibril formation inhibitory effect of nuclear proteins is summarized in FIG. Aβ fibril formation was significantly suppressed when a final concentration of 60 μg / mL nucleoprotein was added (* p <0.05). The results showed that nucleoprotein suppressed Aβ fibril formation.

Test Example 2: Motor function evaluation in Parkinson's disease-like model mouse 1) Preparation of Parkinson's disease-like model mouse
MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine HCl, manufactured by Sigma, St. Louis, MO) was dissolved in physiological saline at a concentration of 5 mg / ml.
This solution was injected intraperitoneally 4 times every 2 hours to give a dose of MPTP 20 mg / kg body weight to male wild-type C57BL / 6 mice (body weight 20-30 g, 8-10 weeks old). After administration, Parkinson's disease-like model mice were prepared.
2) Motor function evaluation (foot-fault test)
The motor function of the mice was evaluated by foot-fault test on the 7th day after MPTP administration.
In the foot-fault test, a 31 cm x 47 cm wire mesh (about 1.5 cm square size) is placed on a transparent case, and the mouse is placed on this wire mesh and allowed to walk freely for 5 minutes. It was. The observation was performed from below the wire mesh, and the number of times the hind limb was removed and the total number of steps in the hind limb were counted. The ratio of the number of times the wire net was stepped over the total number of steps was calculated as the foot-fault rate (number of times the wire net was stepped off / total number of steps).
3) Experiment schedule of motor function evaluation Motor function evaluation was performed according to the following schedule shown in FIG.
Mice were randomly divided into two groups and fed with a nucleic acid-free diet (non-NP) or a diet containing 1.2% nucleoprotein (NP) from 7 days before MPTP (-7 days). Motor function evaluation was performed 7 days after MPTP administration. Each period during the experiment is shown in FIG. Based on MPTP administration (Day 0), intake of various foods was started 7 days before (-7 days). Seven days after MPTP administration (7 days), the motor function of the mice was evaluated by a foot-fault test.
All data were expressed as mean ± standard deviation. The test for significant difference from the control group was performed by Student T test. The difference was considered significant when p <0.05.
Fig. 4 shows a graph showing the results of evaluation of motor function by mouse foot-fault test on day 7 after administration of physiological saline (Saline: ◯) or MPTP (●), and Fig. 5 is prepared by administration of MPTP. After administration of MPTP in a group that received a non-nucleic acid-free diet (non-NP: ○) and a diet that contained 1.2% nucleoprotein (NP: ●) The graph which shows the foot-fault rate in the 7th day was shown.

4) Results
About the mouse | mouth which administered MPTP (MPTP administration group) or the physiological saline (control group), the foot-fault rate 7 days after administration was compared. As a result, the foot-fault rate in the control group was 0.035 ± 0.009, whereas the foot-fault rate in the MPTP-administered group was 0.088 ± 0.025, and the foot-fault rate in the MPTP-administered group was higher than that in the control group. Significantly higher (p <0.05 (Student's t-test)) (FIG. 4). Therefore, it was shown that the motor function (Parkinson's disease-like model mouse) to which MPTP was administered was reduced in motor function.
Next, the effect of nuclear proteins on Parkinson's disease-like symptoms was examined. Mice randomly divided into two groups were fed a nucleic acid-free diet (non-NP group) or a diet containing 1.2% nucleoprotein added to the nucleic acid-free diet (NP group) from 7 days before MPTP administration. Reared in These animals were subjected to a foot-fault test 7 days after MPTP administration to calculate the foot-fault rate (FIG. 5). 0.066 ± for non-NP group
0.017 was exhibited, which was substantially the same as that of the MPTP group. However, the foot-fault rate of the NP group was 0.038 ± 0.015, which was significantly lower than the foot-fault rate of the non-NP group [p <0.05 (Student's t-test)].
From the above results, it was shown that the ingestion of nucleoprotein suppressed the decrease in motor function, which is a Parkinson's disease-like symptom.

Test Example 3: Histological Evaluation in the Midbrain Substantia of Parkinson's Disease-Like Model Mice 1) Test Method The mice used in Test Example 2 were administered intraperitoneally (50 mg / kg body weight) on the 8th day after MPTP administration. ), And physiological saline was refluxed from the right ventricle to remove blood, and then 10% neutral buffered formalin solution was refluxed to fix the tissue. The brain was immediately removed, and the brain was postfixed with 10% neutral buffered formalin solution for 3 days. The brain was trimmed in the forehead direction around Bregma -1 mm and embedded in paraffin according to the usual method. The embedded brain was sliced to a thickness of 4 μm using a microtome. In the midbrain substantia nigra region, 6 slices were taken at 200 μm intervals from around Bregma -2 mm according to the brain map and used for tissue staining.
Dopamine neurons in the midbrain substantia nigra were identified by TH (Tyrosine Hydroxylase) immunostaining. The paraffin section prepared by the above method was subjected to immunohistochemical staining after deparaffinization. The sections were immersed in PBS containing 0.3% hydrogen peroxide for 30 min to inactivate endogenous peroxidase in the tissues. After washing with PBS, it was incubated with 2.5% normal horse serum (NHS) for 1 hour to block nonspecific reactions. Thereafter, the sections were added with rabbit anti-TH antibody (Millipore) diluted 2000 times with 2.5% NHS and incubated overnight at 4 ° C. After washing with PBS, the sections were added with biotin-modified goat anti-rabbit IgG antibody (Vector) diluted 200-fold with 2.5% NHS and incubated for 2 hours. Then, after incubation for 60 minutes with Avidin-Biotin solution (Vector) (Avidin-Biotin-Complex method), antigen-antibody reaction was detected by DAB method with 3,3′-Diaminobenzidine (DAB). Finally, the tissue was stained with a hematoxylin solution and observed with an optical microscope.
The number of TH positive cells was counted as the number of TH positive cells by counting cells that co-stained the positive reaction by TH immunostaining and the nucleus by hematoxylin staining.
Here, all data were expressed as mean ± standard deviation. The test for significant difference from the control group was performed by Student T test. The difference was considered significant when p <0.05.
Neuronal degeneration was identified by tissue staining with FJB (Fluoro Jade B), a fluorescent dye that binds to degenerated neurons. The deparaffinized section was immersed in a potassium permanganate solution for 15 min. After washing with distilled water, it was immersed in FJB staining solution for 30 min. After washing with distilled water, the sections were air-dried in the dark, immersed in xylene, and sealed. Stained sections were observed under a fluorescence microscope.
Fig. 6 shows a photograph (saline (Saline: upper figure), MPTP (lower figure) of dopamine neurons in the vicinity of the substantia nigra of the mouse 8 days after administration of physiological saline (Saline) or MPTP. )), FIG. 7 shows graphs showing the number of TH positive cells (saline (Saline: ◯), MPTP (●)), and FIG. 8 shows saline (Saline: upper diagram). Alternatively, a photograph showing a typical stained image of FJB (Fluoro Jade B) staining in the vicinity of the substantia nigra of mice 8 days after administration of MPTP (below) is shown, and FIG. 9 shows Parkinson's disease-like state produced by MPTP administration. Mice on the 8th day after MPTP administration in a group in which a model mouse was fed a non-nucleic acid diet (non-NP: ○) and a diet in which a diet containing 1.2% nucleoprotein (NP: ●) was ingested Photograph of TH immunostaining of dopamine nerve near the midbrain substantia nigra (non-NP: upper figure) Group were fed a diet was contained 1.2% nucleoprotein: indicates (NP below)), FIG. 10 shows a chart showing the TH-positive cell numbers at that time.

2) Results The number of dopamine neurons in the midbrain substantia nigra was compared between the MPTP administration group and the control group. FIG. 6 shows a typical TH immunohistochemical staining image, and FIG. 7 shows a comparison of the number of TH positive cells. TH-positive cells stained in brown decreased in the MPTP administration group compared to the control group. As a result of counting the number of TH positive cells in both groups, it was 698.4 ± 144.5 in the MPTP administration group compared with the TH positive cell count in the control group (1262.0 ± 361.3), and the number of TH positive cells in the MPTP administration group was significantly less [P <0.05 (Student's t-test)].
Degenerated neurons in the midbrain substantia nigra were examined by FJB staining (FIG. 8). As a result, no FJB positive cells were observed in the control group, but FJB positive cells were observed in the MPTP administration group.
Next, the number of dopamine neurons in the midbrain substantia nigra was compared between the non-NP group and the NP group. The immunohistological staining image of TH is shown in FIG. 9, and the comparison of the number of TH positive cells is shown in FIG. Many TH positive cells were observed strongly and strongly in the NP group. As a result of counting the number of TH positive cells in both groups, the non-NP group was 452 ± 122, while the NP group counted 704 ± 214. The number of TH positive cells in the NP group was significantly higher than that in the non-NP group [p <0.05 (Student's t-test)].
From the above results, it was shown that ingestion of nucleoprotein suppressed not only the motor function, which is a Parkinson's disease-like symptom, but also the fallout of the midbrain substantia nigra.

Claims (3)

A prophylactic / ameliorating agent for neurodegenerative diseases associated with the formation of amyloid β peptide fibers and / or the loss of midbrain dopamine neurons , comprising nucleoprotamine derived from a white moth as an active ingredient. Preventing and improving agents of claim 1 Symbol mounting said neurodegenerative disease is Alzheimer's disease. Preventing and improving agents of claim 1 Symbol mounting said neurodegenerative disease is Parkinson's disease.