Application of nattokinase in preparation of medicine for preventing and / or treating Parkinson's disease

Abstract

The invention belongs to the technical field of novel application of nattokinase, and provides application of nattokinase in preparation of a medicine for preventing and / or treating Parkinson's disease. According to the invention, an MPTP / MPP + induced Parkinson's disease (PD) in-vivo and in-vitro model is established, and a neuroprotection mechanism of nattokinase is disclosed by adopting a systematic experiment method. Results show that the nattokinase can significantly improve dyskinesia and intestinal dysfunction of MPTP-induced PD mice; the damage of dopaminergic neurons of the middle brain is effectively relieved, and the density of tyrosine hydroxylase positive neurons is maintained; the expression of TLR4 and NLRP3 inflammasome pathways and related proinflammatory cytokines is inhibited, and the neuroinflammatory response is relieved; pINK1 / Parkin expression is up-regulated, mitochondrial autophagy is activated, and damaged neurons are cleared. Direct association between nattokinase and Parkinson's disease prevention and treatment is established for the first time, an innovative solution is provided for developing a novel anti-Parkinson's disease medicine with dual effects of motor function improvement and neuroprotection, and the application has important development value in the field of treatment of neurodegenerative diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of new uses of nattokinase, and specifically relates to the application of nattokinase in the preparation of drugs for preventing and / or treating Parkinson's disease. Background Art

[0002] Parkinson's disease (PD) is currently the second most common neurodegenerative disease. The initial symptoms of the disease mainly manifest as unilateral hand tremors. As the disease progresses, tremors occur in the lower limbs and other parts. Patients gradually become slow in movement, have muscle stiffness, poor body balance ability, and then are accompanied by a decline in cognitive ability, and even cause many psychological problems, seriously threatening the health of middle-aged and elderly people. Since the 1980s, the prevalence of Parkinson's disease has been on the rise worldwide, and the trend has been particularly obvious in the past two decades. With the aggravation of the global population aging, the economic and social burden brought by Parkinson's disease will become heavier and heavier.

[0003] The main pathological feature of PD is the degeneration and death of dopaminergic neurons in the substantia nigra. The pathological change process of Parkinson's disease involves multiple mechanisms, such as the aggregation of α-syn, neuroinflammation, mitochondrial dysfunction, immune response, and the bidirectional regulation of the gut-brain axis. The complexity and uncertainty of the pathogenic factors pose great difficulties for the treatment of PD. More than 80% of patients worldwide still use traditional drugs such as levodopa (L-DOPA) that act on the dopamine replacement principle as the first-choice drugs to maintain normal motor function, but its long-term use will cause serious complications (such as end-of-dose phenomena and dyskinesia). Therefore, it is urgent to explore new targets and treatment paradigms. Finding drugs with good disease-modifying effects is of great significance for protecting the physical health of middle-aged and elderly people.

[0004] Nattokinase (NK) is derived from the traditional fermented food natto and is an alkaline serine protease produced during the natto fermentation process. It was initially famous for its thrombolytic effect and has significant advantages such as higher safety and fewer side effects. Recent studies have shown that NK has a variety of pharmacological effects: anti-inflammatory, antioxidant, improving mitochondrial function, regulating the gut microbiota, etc. At present, there is no report on the role of nattokinase in preventing or treating Parkinson's disease. Summary of the Invention

[0005] The purpose of the present invention is to provide a new use of nattokinase.

[0006] In the first aspect, the present invention provides the application of nattokinase in the preparation of products for preventing and / or treating Parkinson's disease.

[0007] The nattokinase described in the present invention is a serine protease produced during the natto fermentation process, which has functions such as anti-inflammatory, antioxidant stress, antithrombotic, improving blood lipid levels, and neuroprotection. Nattokinase can be obtained commercially or by fermentation through various methods, and the fermentation methods are mature.

[0008] Furthermore, the prevention and / or treatment of Parkinson's disease is reflected in at least one of the following aspects:

[0009] 1) Alleviating the motor dysfunction caused by Parkinson's disease;

[0010] 2) Alleviating the dopaminergic neuron damage caused by Parkinson's disease;

[0011] 3) Inhibiting the neuroinflammation caused by Parkinson's disease

[0012] 4) Alleviating the intestinal dysfunction caused by Parkinson's disease;

[0013] 5) Inhibiting the secretion of inflammatory factors and the expression of proteins related to the TLR4 / NLRP3 signaling pathway in PC12 cells induced by MPP + ;

[0014] 6) Restoring the expression of proteins related to the PINK1 / Parkin signaling pathway in PC12 cells induced by MPP + ;

[0015] Furthermore, the alleviation of the motor dysfunction caused by Parkinson's disease specifically means significantly improving the decline in spatial exploration ability and motor coordination ability caused by Parkinson's disease.

[0016] Furthermore, the alleviation of the dopaminergic neuron damage caused by Parkinson's disease specifically can be significantly improving the reduction of Nissl bodies and the loss of tyrosine hydroxylase in the brain tissue.

[0017] Furthermore, the inhibition of the neuroinflammation caused by Parkinson's disease specifically can be significantly inhibiting the release of inflammatory factors in the brain tissue.

[0018] Furthermore, the alleviation of the intestinal dysfunction caused by Parkinson's disease specifically can be improving the reduction of fecal water content and the shortening of colon length caused by Parkinson's disease.

[0019] The product includes drugs, pharmaceutical compositions or health products.

[0020] The drugs, pharmaceutical compositions or health products use the nattokinase as the active ingredient.

[0021] Optionally, the preparation forms of the drugs include injections, powders, granules, powders, pills, oral liquids or tablets.

[0022] In the above application, when preparing a drug, nattokinase can be used as one of the active ingredients or as the sole active ingredient.

[0023] In the above application, when preparing a drug, pharmaceutically acceptable excipients can also be added.

[0024] In a second aspect, the present invention provides the use of nattokinase in the preparation of a product for alleviating motor dysfunction caused by Parkinson's disease.

[0025] In a third aspect, the present invention provides the use of nattokinase in the preparation of a product for alleviating dopaminergic neuron damage caused by Parkinson's disease.

[0026] In a fourth aspect, the present invention provides the use of nattokinase in the preparation of a product for alleviating neuroinflammation caused by Parkinson's disease.

[0027] In a fifth aspect, the present invention provides the use of nattokinase in the preparation of a product for alleviating intestinal dysfunction caused by Parkinson's disease.

[0028] In a sixth aspect, the present invention also provides a method for preventing and / or treating Parkinson's disease, which includes administering an effective amount of nattokinase or a pharmaceutical composition of nattokinase to a subject with Parkinson's disease.

[0029] In the present invention, the term "effective amount" refers to a dose that can achieve treatment, prevention, alleviation, and / or remission of the diseases or disorders described in the present invention in a subject.

[0030] In the present invention, the term "subject" refers to an individual or animal individual suffering from or likely to suffer from Parkinson's disease or Parkinson's-related disorders described in the present invention, and can also refer to an individual or animal individual used for a certain purpose, such as for scientific research purposes. Specifically, the individual is, for example, an animal individual, especially a mammalian individual, such as a human, pig, dog, cat, cow, sheep, horse, rat, mouse, rabbit, guinea pig, monkey, etc.

[0031] The present invention first discloses that nattokinase can alleviate the motor dysfunction of MPTP-induced Parkinson's mice; alleviate the damage of dopaminergic neurons in the substantia nigra of the midbrain; reduce the expression levels of TLR4, NLRP3, Caspase-1, and IL-6 in the brain tissue of Parkinson's mice, and promote the expression of Parkin in the brain tissue; alleviate intestinal dysfunction, and has potential protective and therapeutic effects on Parkinson's disease through multiple mechanisms. These findings lay a scientific foundation for considering nattokinase as a potential drug for treating Parkinson's disease. Description of the Drawings

[0032] Figure 1Effects of nattokinase on motor function of MPTP-induced Parkinson's disease mice. (A) Movement trajectories of mice in each group in the open field test; (2) Exploration time of the central area of mice in each group in the open field test; (B) Pole climbing time of mice in each group.

[0033] Figure 2 Effects of nattokinase on intestinal function of MPTP-induced Parkinson's disease mice. (A) Fecal water content of mice in each group; (B) Colon length of mice in each group; (C) Colon length of mice in each group.

[0034] Figure 3 Effects of nattokinase on organ indexes of MPTP-induced Parkinson's disease mice. (A) Heart; (B) Liver; (C) Lung; (D) Kidney.

[0035] Figure 4 Effects of nattokinase on dopamine neuron damage induced by MPTP in Parkinson's disease mice.

[0036] Figure 5 Effects of nattokinase on the expression of tyrosine hydroxylase in the substantia nigra of MPTP-induced Parkinson's disease mice. (A) Immunohistochemical images of tyrosine hydroxylase in the substantia nigra of mice in each group; (B) Quantification of immunohistochemical images of tyrosine hydroxylase in the substantia nigra of mice in each group.

[0037] Figure 6 Effects of nattokinase on the expression of TLR4, NLRP3, and IL-6 in the brains of MPTP-induced Parkinson's disease mice. (A) mRNA expression of TLR4, NLRP3, and IL-6 in the brains of mice in each group; (B) protein expression of TLR4, NLRP3, and IL-6 in the brains of mice in each group.

[0038] Figure 7 Effects of nattokinase on the expression of Parkin in the brain of MPTP-induced Parkinson's disease mice. (A) The mRNA expression of Parkin in the brain of each group of mice; (B) The protein expression of Parkin in the brain of each group of mice.

[0039] Figure 8 Nattokinase and MPP + Effects of induced tyrosine hydroxylase expression in PC12 cells.

[0040] Figure 9 Nattokinase and MPP + Effects of induced PINK1 / Parkin expression in PC12 cells.

[0041] Figure 10 Nattokinase and MPP +Effect of induced expression of TLR4 / NLRP3 and inflammatory factors in PC12 cells. (A) mRNA expression of TLR4, NLRP3, Caspase-1, and IL-6 in PC12 cells of each group; (B) Content of IL-1β in the culture supernatant of PC12 cells of each group; (C) Protein expression of TLR4, NLRP3, Caspase-1, and IL-6 in PC12 cells of each group.

[0042] (Compared with the control group, #p < 0.05, ##p < 0.01, p < 0.001, #p < 0.0001; compared with the model group, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.) Detailed implementation manners

[0043] The following examples will further illustrate the present invention, but do not limit the present invention thereto.

[0044] Example 1: Therapeutic effect of nattokinase on MPTP-induced Parkinson's disease mouse model

[0045] In the present invention, a Parkinson's disease mouse model was induced by MPTP to explore the neuroprotective effect of nattokinase. The motor function was evaluated by open field / rod climbing experiments, and the safety and intestinal protective effects were analyzed by detecting intestinal function indexes and main organ indexes; the protective effect on dopamine neurons was observed by Nissl staining and TH immunohistochemistry, and the expression changes of TLR4, NLRP3, IL-6 inflammatory pathways and the mitochondrial autophagy-related factor Parkin in the brain were detected by qPCR / Western Blot to reveal its potential mechanism of action.

[0046] (1) Effect of nattokinase on MPTP-induced motor dysfunction in mice

[0047] 1. Experimental materials and instruments

[0048] (1) Main reagents: Nattokinase (enzyme activity ≥ 300,000 FU / g, Shuangjun Biotechnology Co., Ltd.), 1-methyl-4

[0049] phenylpyridinium ion (MPTP, purity ≥ 98%, Shanghai Macklin Biochemical Co., Ltd.), levodopa (L-

[0050] DOPA, Shanghai Fuda Pharmaceutical Co., Ltd.).

[0051] (2) Antibodies: Anti-NLRP3 Rabbit pAb, Anti-TH Rabbit pAb, Anti-TLR4 RabbitpAb,

[0052] Anti-Pink1 Rabbit pAb, Anti-Caspase-1 Rabbit pAb, Anti-Parkin Rabbit pAb, Anti-IL-6

[0053] Rabbit pAb, HRP-labeled goat anti-rabbit IgG, Marker, 5×SDS-PAGE protein loading buffer, PVDF transfer membrane, ECL luminescent solution.

[0054] (3) Instruments: Open field test chamber (50×50×50 cm, self-made), pole climbing device (self-made), fluorescence quantitative PCR instrument (QuantStudio 5, ABI)

[0055] 2. Animal experiment design

[0056] (1) Experimental animals: 36 SPF-grade male C57BL / 6 mice (8 weeks old, body weight 20±2 g, Australia Cili Biotechnology Co., Ltd.).

[0057] (2) Experimental grouping: After 1 week of adaptive feeding, they were randomly divided into:

[0058] Control group (n = 9): Intraperitoneal injection of 0.9% normal saline

[0059] Model group (n = 9): MPTP 25 mg / kg / d (dissolved in normal saline)

[0060] Nattokinase group (n = 9): MPTP + NK 10,000 FU / kg / d

[0061] Positive drug group (n = 9): MPTP + L-DOPA 150 mg / kg / d

[0062] (3) Administration method: MPTP was injected from 9:30 to 10:30 every morning for 7 days. Nattokinase and L-DOPA were injected from 4:30 to 5:30 every afternoon for 14 days. The control group was injected with an equal volume of normal saline.

[0063] 3. Experimental methods

[0064] (1) Open field test: The mice were slowly placed into the open field chamber (50 cm×50 cm×50 cm, the bottom was divided into 25 equal large squares, and the central 9 squares were divided into the central area) facing the corner, allowed to freely explore the inside of the open field chamber for 5 min, and the time the mice were located in the central area was recorded. Their feces and urine were cleaned, the inside was wiped with alcohol, and after evaporation, the next experiment was carried out.

[0065] (2) Pole climbing experiment: A metal pole with a diameter of 1 cm and a height of 50 cm was used. A small ball with a diameter of 2 cm was fixed at the top. The pole was evenly wrapped with bandages to prevent slipping. Before the formal pole climbing experiment, certain training was carried out. The mouse's tail was lifted to make its two front paws grasp the small ball. Timing started when its two hind legs left the top of the device and stopped when its two hind legs left the pole of the device. The time was recorded.

[0066] 4. Experimental results

[0067] See Figure 1 A and 1B. Compared with the control group, the exploration time of the mice in the model group in the central area was significantly reduced, and their autonomous exploration ability decreased; compared with the model group, the exploration time of the mice in the nattokinase group in the central area was significantly increased, indicating that nattokinase improved the autonomous exploration ability of the model mice. See Figure 1 C. Compared with the control group, the time taken by the mice in the model group to climb the pole was significantly increased, and their motor coordination ability was poor; compared with the model group, the climbing time of the mice in the nattokinase group was slightly improved, and their motor coordination ability was slightly improved. The above shows that nattokinase can improve the motor dysfunction of PD mice.

[0068] (2) Effects of nattokinase on MPTP-induced intestinal dysfunction in mice

[0069] 1. Experimental methods

[0070] Determination of fecal water content: The experimental mice were fasted but allowed to drink water for 12 hours. They were fed appropriately 2 hours before the start of the experiment and the experiment started 2 hours later. One mouse was placed in each cage. After 1 hour of free activity, the feces were collected, weighed wet, recorded, dried in an oven at 65 °C, weighed dry, recorded, and the fecal water content was calculated.

[0071] Water content (%) = (wet weight - dry weight) / wet weight × 100%

[0072] Detection of colon length: The intestinal tissues of the mice were quickly separated, straightened, and the colon length (from the root of the ileocecal junction to the root of the rectum) was measured.

[0073] 2. Experimental results

[0074] See Figure 2 . Compared with the control group, the fecal water content of the mice in the model group was significantly reduced, and the colon length was significantly shortened; while the fecal water content of the mice in the nattokinase group was significantly increased, and the colon length was significantly increased, basically returning to normal, indicating that nattokinase improved the intestinal function of the model mice.

[0075] (3) Effects of nattokinase on the organ indices of MPTP-induced mice

[0076] 1. Experimental methods

[0077] Sample collection and organ index determination: Weigh the mice and record their weights. Decapitate the mice by cervical dislocation, quickly cut off the mouse heads, dissect and remove the brains on ice. Use a scalpel to divide the brain tissue into two parts along the mid-sagittal plane. Fix one half in tissue fixative for making tissue sections. Isolate the striatum from the other half, put it into a centrifuge tube, quickly freeze it in liquid nitrogen, and immediately store it at -80 °C for later use. Dissect the abdominal cavity of the mice, take out the liver, kidneys, lungs, and heart, label and weigh them, and calculate the organ index. Organ index = organ weight / mouse weight × 100%

[0078] 2. Experimental results

[0079] See Figure 3 , compared with the control group, there were no significant differences in the organ indices of the heart, liver, lungs, and kidneys of the mice in each group, indicating that nattokinase has relatively high safety.

[0080] (IV) Effects of nattokinase on MPTP-induced dopamine neuron damage in mice

[0081] 1. Experimental method

[0082] Nissl staining: The brain tissue sections were successively dewaxed and hydrated, stained with cresyl violet, differentiated, dehydrated, and sealed. Images were collected under a microscope.

[0083] Immunohistochemical staining: The brain tissue sections were successively dewaxed and hydrated, antigen repaired, endogenous peroxidase removed, blocked, incubated with primary antibody, secondary antibody conjugated, enzyme-catalyzed color development, counterstained, and sealed. Images were collected under a microscope.

[0084] 2. Experimental results

[0085] See Figure 4 , compared with the control group, the number of Nissl bodies in the substantia nigra of the model group mice decreased; the number of Nissl bodies in the nattokinase protection group was relatively increased compared with the model group, indicating that NK can protect against dopaminergic neuron damage.

[0086] See Figure 5 , compared with the control group, the density of tyrosine hydroxylase-positive neurons in the substantia nigra of the model group mice was significantly reduced; compared with the model group, the density of tyrosine hydroxylase-positive neurons in the substantia nigra of the nattokinase protection group mice was significantly increased, indicating that nattokinase can reverse the loss of tyrosine hydroxylase in the model mice.

[0087] (V) Effects of nattokinase on the expression of TLR4, NLRP3, and IL-6 in the brains of MPTP-induced mice

[0088] 1. Experimental method

[0089] (1) Real-time fluorescence quantitative polymerase chain reaction: Take the brain tissues of mice in each group, extract RNA, measure the total RNA concentration using Nano Drop2000, and detect the expression of related mRNAs by RT-qPCR.

[0090] (2) Immunoblotting experiment: Take the brain tissues of mice in each group, add RIPA lysis buffer to extract the protein of brain tissues of mice in each group, detect the protein concentration of the samples by BCA method, and detect the expression of related proteins by Western Blot.

[0091] 2. Experimental results

[0092] See Figure 6 , compared with the control group, the mRNA and protein expressions of TLR4, NLRP3, and IL-6 in the brain tissues of mice in the model group were significantly increased; while compared with the model group, the mRNA expressions of TLR4 and IL-6 in the natto kinase protection group were significantly decreased, and the protein expression levels of TLR4, NLRP3, and IL-6 were all significantly decreased, indicating that natto kinase can reduce neuroinflammation in the brains of model mice.

[0093] (VI) Effect of natto kinase on the expression of Parkin in the brain of mice induced by MPTP

[0094] See Figure 7 , compared with the control group, the mRNA and protein expressions of Parkin in the brain tissues of mice in the model group were significantly decreased; compared with the model group, the expression of Parkin in the natto kinase protection group of mice was significantly increased.

[0095] Example 2: Effect of natto kinase on MPP + -induced PC12 cells

[0096] Based on the neuroprotective effect verified by the animal model in Example 1, Example 2 used an MPP + -induced Parkinson's disease model of PC12 cells (with the same neurotoxic metabolic pathway as MPTP) to reveal the mechanism of action of natto kinase at the cellular level. Through gradient concentration settings, it was confirmed that natto kinase exerts a protective effect through a triple synergistic mechanism: ① regulating the expression of TH to maintain dopaminergic characteristics (corresponding to the TH immunohistochemical results in Example 1); ② activating the PINK1 / Parkin mitophagy pathway (deeply analyzing the molecular basis of the change in Parkin expression in Example 1); ③ inhibiting the TLR4 / NLRP3 inflammatory cascade reaction (forming a cross-model verification with the brain tissue detection in Example 1). The experimental design continued the detection system in Example 1 (qPCR / WB / ELISA) to construct a complete evidence chain from whole animals to cell molecules, from behavioral phenotypes to signal pathways.

[0097] (I) Effect of natto kinase on MPP +Regulation of tyrosine hydroxylase expression in PC12 cells induced by

[0098] 1. Experimental Materials

[0099] PC12 cells, DMEM medium, anti-penicillin / streptomycin solution, cell freezing solution, special horse serum, fetal bovine serum, PBS buffer, 1-methyl-4-phenylpyridinium ion (MPP + ), and the rest of the experimental materials are the same as those in Experimental Example 1.

[0100] 2. Experimental Grouping

[0101] Control group, MPP + (500mM) induction group, 0.2FU / mL, 0.4FU / mL, 0.8FU / mL nattokinase protection group.

[0102] 3. Experimental Methods

[0103] (1) Real-time fluorescence quantitative polymerase chain reaction: Cells from each group were taken, RNA was extracted, and the total

[0104] RNA concentration, RT-qPCR detection of related mRNA expression.

[0105] (2) Immunoblotting experiment: Cells from each group were added with RIPA lysis buffer to extract cell proteins, and the protein concentration of samples was detected by BCA method.

[0106] Western Blot was used to detect the expression of related proteins.

[0107] 4. Experimental Results

[0108] like Figure 8 As shown, MPP + Induction led to significant changes in the expression of tyrosine hydroxylase (TH) in PC12 cells: Western blot analysis showed that the expression of TH protein decreased to 32.7±5.1% of the control group (p<0.001). After intervention with 0.2FU / mL nattokinase, the expression of TH protein recovered to 66.8±7.3% of the normal level (p<0.001vs the control group).

[0109] (II) Effect of Nattokinase on MPP + Regulation of mitophagy-related proteins in PC12 cells induced by

[0110] See also Figure 9 Compared with the control group, MPP +The expression levels of PINK1 and Parkin in induced PC12 cells were significantly decreased compared with those in the control group, and the process of mitophagy was inhibited. Nattokinase intervention up-regulated the expression of PINK1 and Parkin and promoted the process of mitophagy.

[0111] (III) Effects of Nattokinase on MPP + Effects of Nattokinase on the Expression of TLR4 / NLRP3 and Inflammatory Factors in MPP

[0112] The content of IL-1β in the cell supernatant was determined by enzyme-linked immunosorbent assay: The treated cells in each group in the 6-well plate were taken, and the culture medium was aspirated into a sterilized centrifuge tube, centrifuged at 4°C and 1000 rpm for 10 min, and the supernatant was taken and aliquoted for standby. The standard product was diluted, samples were added, color was developed, and the absorbance value was measured according to the requirements. The standard curve was drawn based on the OD value, and the sample concentration was calculated.

[0113] 2. Experimental Results

[0114] See Figure 10 , and the expression levels of neuroinflammatory-related factors TLR4, NLRP3, Caspase-1, IL-6, and IL-1β were detected by RT-qPCR, Western blot, and ELISA experiments. Compared with the control group, the expression levels of the above genes in MPP + -induced PC12 cells were significantly increased, suggesting that MPP + triggered an inflammatory response in PC12 cells. The expression levels of the above genes in PC12 cells treated with nattokinase were significantly lower than those in the model group, indicating that nattokinase played a protective role in reducing the inflammatory response of MPP + -induced PC12 cells.

Claims

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

2. The application according to claim 1, characterized in that, The product described is a product for alleviating the motor dysfunction caused by Parkinson's disease.

3. The application according to claim 1, characterized in that The product described is a product for alleviating the dopaminergic neuron damage caused by Parkinson's disease.

4. The application according to claim 1, characterized in that The product described is a product for alleviating the neuroinflammation caused by Parkinson's disease.

5. The application according to claim 1, wherein The product described is a product for alleviating the intestinal dysfunction caused by Parkinson's disease.

6. The application according to any one of claims 1-5, characterized in that: The product includes a drug, a pharmaceutical composition or a health product.

7. The application according to claim 6, characterized in that, The preparation form of the drug includes an injection, a powder, a granule, a powder, a pill, an oral liquid or a tablet.

8. A product for preventing and / or treating Parkinson's disease, characterized in that, The active ingredient of the product described includes nattokinase.

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