Application of Dubosiella and pharmaceutical composition containing Dubosiella

Abstract

The invention discloses an application of Dubosiella and a pharmaceutical composition containing the Dubosiella. The invention provides novel application of Dubosiella, Dubosiella can regulate and control gastrointestinal functions to relieve constipation phenotypes of PD mice, and existing researches show that the risk of constipation patients suffering from PD is remarkably increased, so that Dubosiella is expected to further relieve the disease risk and disease symptoms of PD while relieving constipation by regulating the diversity of intestinal flora. According to the application, it is clear that dyskinesia of PD nematodes can be remarkably relieved by supplementing Dubosiella at the nematode level, and it is prompted that Dubosiella is expected to become potential probiotics for cooperatively regulating and controlling symptoms of PD peripheral and central diseases in the future.

Description

Technical Field

[0001] The invention relates to the fields of biomedicine and food health care, and in particular to use of Dubosiella and a pharmaceutical composition containing the same. Background Art

[0002] Parkinson's disease (PD) is the second most common neurodegenerative disease in clinical practice. Typical clinical movement disorders of PD include resting tremor, muscle rigidity, etc. Non-motor clinical manifestations include hypotension, sleep disorders, depression and gastrointestinal dysfunction. Non-motor disorders generally occur earlier than motor dysfunction. Therefore, non-motor features combined with additional biochemical and / or imaging examinations may provide an effective way to identify prodromal PD symptoms and intervene. PD is characterized by abnormal aggregation of α-synuclein (α-Syn), and its lesions involve all areas of the brain-gut axis, including the central nervous system, autonomic nervous system and enteric nervous system. The highly bidirectional communication between the brain and the intestine is transmitted through immunological, neuroendocrine and other processes, which are significantly affected by intestinal microorganisms. The intestinal microbiota and its related metabolites interact with the central nervous system through a series of biochemical and functional inputs, thereby affecting the development of the disease. The inventors found in their research that PD patients generally have an imbalance of intestinal flora, gastrointestinal dysfunction is one of the most common and disabling non-motor symptoms of PD patients, and constipation is the most common and most painful prodromal symptom of PD, with an incidence rate of up to 70%-80%. This suggests that constipation may be related to the occurrence and development of PD. However, there are few studies in the prior art on this aspect, and there are few studies in the prior art on the correlation between constipation and PD, and the corresponding mechanism is not clear. Therefore, studying the relationship between gastrointestinal dysfunction and the progression of PD disease and clarifying the corresponding mechanism is expected to provide new selection targets for early drug treatment of PD. Summary of the invention

[0003] The purpose of the present invention is to provide a use of Dubosiella.

[0004] Another object of the present invention is to provide a use of a pharmaceutical composition.

[0005] Another object of the present invention is to provide a method for preventing and / or treating PD.

[0006] In order to solve the above technical problems, the first aspect of the present invention provides the use of Dubosiella for:

[0007] (i) prevention and / or treatment of PD;

[0008] (ii) preparing a medicament for preventing and / or treating PD;

[0009] (iii) prevention and / or treatment of constipation; and / or

[0010] (iv) preparing a medicament for preventing and / or treating constipation.

[0011] The second aspect of the present invention provides a pharmaceutical composition, comprising: Dubosiella; and a pharmaceutically acceptable carrier or excipient.

[0012] In a third aspect of the present invention, a pharmaceutical composition is provided for (i) preventing and / or treating PD; (ii) preparing a drug for preventing and / or treating PD; (iii) preventing and / or treating constipation; and / or (iv) preparing a drug for preventing and / or treating constipation, wherein the pharmaceutical composition comprises: Dubosiella; and a pharmaceutically acceptable carrier or excipient.

[0013] In some preferred embodiments, each milliliter of the pharmaceutical composition contains no less than 1x10 Dubosiella 6 CFU, more preferably not less than 1x10 8 CFU.

[0014] In some preferred embodiments, the pharmaceutical composition is a solid preparation, and the solid preparation is selected from at least one of tablets, lozenges, candies, chewable tablets, chewing gum, capsules, powders, granules, coated microparticles, coated tablets, enteric-coated tablets and capsules.

[0015] In some preferred embodiments, the pharmaceutical composition is a liquid preparation, and the liquid preparation is selected from at least one of an oral solution, a suspension, an emulsion and a syrup.

[0016] In a fourth aspect of the present invention, a health care product composition is provided, wherein the health care product composition is used for (i) relieving and / or improving constipation; (ii) relieving and / or improving PD; (iii) preparing food or health care products for relieving and / or improving constipation; (iv) preparing health care products for relieving and / or improving PD, wherein the health care product composition comprises Dubosiella; and a carrier or excipient acceptable to the health care product.

[0017] The fifth aspect of the present invention provides the use of a health care product composition for (i) relieving and / or improving constipation; (ii) relieving and / or improving PD; (iii) preparing food or health care products for relieving and / or improving constipation; (iv) preparing health care products for relieving and / or improving PD.

[0018] In some preferred embodiments, the health product is selected from at least one of beverages, yogurt vinegar, juice, ice cream, bread, biscuits, cereals, health bars, paste, seasonings, milk, cheese and fermented milk.

[0019] A sixth aspect of the present invention provides a method for preventing and / or treating PD, the method comprising the steps of:

[0020] administering to the subject a therapeutically effective amount of Dubosiella; or,

[0021] A therapeutically effective amount of the pharmaceutical composition according to the second aspect of the present invention is administered to a subject.

[0022] A seventh aspect of the present invention provides a method for preventing and / or treating constipation, the method comprising the steps of:

[0023] administering to the subject a therapeutically effective amount of Dubosiella; or,

[0024] A therapeutically effective amount of the pharmaceutical composition according to the second aspect of the present invention is administered to a subject.

[0025] Compared with the prior art, the present invention has at least the following advantages:

[0026] (1) The present invention provides a new use of Dubosiella, which can regulate gastrointestinal flora disorders to further alleviate the constipation phenotype of PD mice. Previous studies have shown that patients with constipation have a significantly increased risk of developing PD. Therefore, Dubosiella is expected to alleviate constipation by regulating the diversity of intestinal flora, while further reducing the risk and symptoms of PD.

[0027] (2) The present invention clearly shows that replenishing Dubosiella at the PD nematode model level can significantly reduce the motor dysfunction of PD nematodes, suggesting that Dubosiella is expected to become a potential probiotic for synergistically regulating peripheral and central disease symptoms of PD in the future.

[0028] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described below (such as embodiments) can be combined with each other to form a new or preferred technical solution. Due to space limitations, they will not be described one by one here. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] One or more embodiments are exemplarily described by the pictures in the corresponding drawings, and these exemplary descriptions do not constitute limitations on the embodiments.

[0030] Figure 1ASchematic diagram of electron microscope for preparing PFF according to an embodiment of the present invention, magnification: left (68000×) / right (98000×). Scale bar = 200 nm.

[0031] Figure 1B The length distribution of PFF at different length stages after ultrasonic treatment according to the embodiment of the present invention is shown in FIG.

[0032] Figure 1C This is the PFF length distribution in the α-syn stage according to an embodiment of the present invention.

[0033] Figure 1D It is a schematic diagram of the spread of PFF in various brain regions 60 days and 270 days after being injected into the striatum according to an embodiment of the present invention (the red dots represent the PFF fluorescence signal).

[0034] Figure 1E This is the distribution of pSer129-α-Syn in various brain regions of mice 270 days after PFF was injected into the striatum according to an embodiment of the present invention. Scale bar = 50 μm.

[0035] Figure 2A Statistical graph of body weight of mice in each group at the ninth month according to the embodiment of the present invention.

[0036] Figure 2B It is a statistical graph of the head turning time results of each group of pole climbing experiments according to an embodiment of the present invention.

[0037] Figure 2C It is a statistical chart of the total time results of each group of pole climbing experiments according to an embodiment of the present invention.

[0038] Figure 2D It is a statistical diagram of the results of the rotarod test of each group of mice according to the embodiment of the present invention.

[0039] Figure 2E is the SNc brain region TH of each group of mice in the embodiment of the present invention + Representative immunofluorescence images of neurons. Scale bar, 200 μm.

[0040] Figure 2F is the TH in each group of SNc according to the embodiment of the present invention. + Statistics of unbiased stereological counting data of neurons.

[0041] Figure 2G This is a statistical diagram of the relative content of DA neurotransmitters detected by striatal HPLC-MS according to an embodiment of the present invention. The data are expressed as mean ± SEM; **p < 0.01, ***p < 0.001 and ****p < 0.0001.

[0042] Figure 3 3 is a species analysis and diversity assessment diagram of the intestinal flora differences between the PD group and the control group according to an embodiment of the present invention.

[0043] Figure 4 The cluster heat map and VIP bar graph according to the embodiment of the present invention show the changes in neurotransmitters between WT-Vehicle and WT-PFF groups.

[0044] Figure 5A is a Sepeaman correlation heat map according to an embodiment of the present invention.

[0045] Figure 5B This is a correlation diagram between Serotonin and intestinal flora diversity index Ace, Shannon, and MDS1 according to an embodiment of the present invention.

[0046] Figure 5C It is a correlation diagram between PE and intestinal flora diversity index Ace, Shannon, and MDS1 according to an embodiment of the present invention.

[0047] Fig. 6A 1 is a schematic diagram of the mouse behavior detection process according to an embodiment of the present invention.

[0048] Figure 6B It is the number of fecal particles per unit time of 24 hours in WT aged mice according to the embodiment of the present invention. Analysis method Student's t test, *p<0.05, data are expressed as mean±SEM. (WT: n=10; WT-Dubosiella: n=9).

[0049] Figure 6C It is the number of fecal particles per unit time of 2 hours in WT aged mice according to the embodiment of the present invention. Analysis method Student's t test, *p<0.05, data are expressed as mean±SEM. (WT: n=10; WT-Dubosiella: n=9).

[0050] Fig.6D This is a statistical chart of the weight of the unnested materials in the nesting experiment according to the embodiment of the present invention (WT: n=10; WT-Dubosiella: n=9; hA53T: n=6; hA53T-Dubosiella: n=7).

[0051] Fig. 6E This is a statistical diagram of the nesting score results of the nesting experiment according to an embodiment of the present invention (WT: n=10; WT-Dubosiella: n=9; hA53T: n=6; hA53T-Dubosiella: n=7).

[0052] Fig. 6F This is a statistical graph of the colon length results of mice according to an embodiment of the present invention. (WT: n = 7; WT-Dubosiella: n = 5; hA53T: n = 6; hA53T-Dubosiella: n = 6).

[0053] Figure 6G This is a survival curve of the NL5901 strain (PD nematodes) according to an embodiment of the present invention.

[0054] Figure 6H It is a survival curve diagram of the N2 strain nematode (WT) according to an embodiment of the present invention.

[0055] Fig.6I The number of eggs stored in the abdomen of the NL5901 strain according to the embodiment of the present invention (OP50 group: n=70; 1:1 group: n=31; 1:2 group: n=67; 1:4 group: n=38).

[0056] Figure 6J This is a statistical graph of the number of swings of the NL5901 strain within 30 seconds of swimming according to an embodiment of the present invention. (OP50: n=30; 1:2: n=30; 1:4: n=30).

[0057] Figure 6K The figure is a statistical graph of the number of swings of the N2 strain within 30 seconds of swimming according to the embodiment of the present invention. (OP50: n = 31; 1:2: n = 25; 1:4: n = 13). Analysis method: Student's t test (BC) and One-way ANOVA with multiple comparison test (DK). Data are expressed as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. DETAILED DESCRIPTION

[0058] At present, the cause of PD is still unclear, and there are relatively few studies on the correlation between PD and gastrointestinal dysfunction in the prior art. After extensive and in-depth research, the inventors found that Dubosiella (Dubosiella newyorkensis) bacteria can affect the secretion of serotonin / phosphatidylethanolamine, thereby affecting the diversity of intestinal flora, alleviating constipation in the elderly, and improving fine motor dysfunction in Parkinson's animal models. Regulation of the homeostasis of the intestinal microbiota is expected to provide a new target for early drug treatment of PD.

[0059] Uses of Dubosiella

[0060] The present invention relates to a new use of Dubosiella for (i) preventing and / or treating Parkinson's disease; (ii) preparing a drug for preventing and / or treating Parkinson's disease; (iii) preventing and / or treating constipation; and (iv) preparing a drug for preventing and / or treating constipation.

[0061] As used in the present invention, the term "Dubosiella" is a genus Erysipelotrichidae of the Gram-positive family with a known species (Dubosiella Newyorkensis), the full name of which is "Dubosiella Newyorkensis", which is a strict anaerobic bacterium with a strain number of DSM 103457 = ATCC TSD-64. Dubosiella bacteria as a known strain can be obtained through commercial channels and can be cultured and amplified by a culture method familiar to those skilled in the art. In one embodiment of the present invention, a suspension consisting of Dubosiella bacteria and emulsifier OP-50 is first centrifuged, a precipitate is taken, and then the precipitate is resuspended using M9 buffer and inoculated into a suitable culture plate in proportion for culture, such as an NGM nematode culture plate. The conditions for culturing the strain are well known to those skilled in the art, such as culturing at 37°C under an anaerobic environment for 18-24 hours. The "Dubosiella" used in the embodiments of the present invention was purchased from Ningbo Mingzhou Biotechnology Co., Ltd.

[0062] As used in the present invention, the term "constipation" refers to a phenomenon in which the frequency of bowel movements decreases. Patients generally have less than 3 bowel movements per week, with difficult bowel movements and dry stools.

[0063] As used in the present invention, the term "treatment and / or prevention" includes prophylactic treatment of an individual, i.e., treatment with Dubosiella bacteria is initiated before the onset of the disease in order to prevent the disease, as well as therapeutic treatment of a disease that has already occurred in an individual. In the latter case, relief of symptoms is, for example, desired, or the patient's general condition is enhanced or the patient's disease is cured. Thus, an individual may be a person who is at risk of developing an autoimmune disease or who is already a patient of the disease.

[0064] Pharmaceutical composition and use thereof

[0065] The present invention also relates to a pharmaceutical composition containing Dubosiella, which comprises Dubosiella and a pharmaceutically acceptable carrier or excipient.

[0066] As used in the present invention, the term "pharmaceutical composition" refers to a composition for the purpose of preventing and / or treating a disease, containing one or more active ingredients, and having the function of preventing and / or treating a certain disease. In addition to containing active ingredients, the pharmaceutical composition can be combined with one or more pharmaceutically acceptable carriers or excipients according to factors such as the mode of administration, the nature of the drug, and the indication. The pharmaceutical composition according to the present invention may contain a pharmaceutically acceptable carrier. For oral administration, the pharmaceutically acceptable carrier may include a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersant, a stabilizer, a suspending agent, a colorant, a flavoring agent, and the like. For topical administration, the pharmaceutically acceptable carrier may include a matrix, an excipient, a lubricant, a preservative, and the like. The pharmaceutical composition of the present invention can be formulated into various dosage forms in combination with the above-mentioned pharmaceutically acceptable carriers. For example, for oral administration, the pharmaceutical composition can be formulated into solid or liquid dosage forms, such as tablets, lozenges, capsules, elixirs, suspensions, syrups, wafers, and the like. In addition, the pharmaceutical composition can be formulated into solutions, suspensions, tablets, capsules, sustained-release products, etc. At the same time, examples of carriers, excipients, and diluents suitable for formulations may include lactose, glucose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, etc. In addition, the pharmaceutical composition may further contain fillers, anti-aggregants, lubricants, wetting agents, flavoring agents, emulsifiers, preservatives, etc. The pharmaceutical composition described herein can be formulated in a conventional manner using one or more pharmaceutically acceptable carriers, and can also be administered in a controlled-release formulation.

[0067] In some preferred embodiments of the present invention, the pharmaceutical composition is prepared as a liquid preparation or a solid preparation. As a solid preparation, it can be selected from the group consisting of tablets, lozenges, candies, chewable tablets, chewing gums, capsules, sachets, powders, granules, coated particles, coated tablets, enteric-coated tablets and capsules, and dissolving strips and films. As a liquid preparation, it can be selected from the group consisting of oral solutions, suspensions, emulsions or syrups.

[0068] Pharmaceutical compositions as described herein can be used for rectal administration in the form of suppositories. These can be prepared by mixing the agent with a suitable non-irritating excipient that is solid at room temperature but liquid at rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter, beeswax, and polyethylene glycol, among others.

[0069] The content of Dubosiella in the pharmaceutical composition of the present invention is not limited. Preferably, the content of Dubosiella per milliliter of the pharmaceutical composition is not less than 1x10 6 CFU, preferably 1x10 6 -lxl0 16 CFU / mL, for example 1x10 7 CFU / mL, 1x10 8 CFU / mL, 1x10 9 CFU / mL, 1x10 10 CFU / mL, 1x10 12 CFU / mL, 1x10 16 CFU / mL, etc.

[0070] In a preferred embodiment of the present invention, the pharmaceutical composition of the present invention can be used in combination with other intestinal probiotics, for example, with lactobacilli, bifidobacteria, Gram-positive cocci, etc., such as a group consisting of one or more of Bifidobacterium longum, Bifidobacterium breve, Bifidobacterium ovale and Bifidobacterium thermophilum; a group consisting of one or more of Lactobacillus acidophilus, Lactobacillus casei, Lactobacillus jensenii and Lactobacillus ramanni.

[0071] The present invention also relates to new uses of the above-mentioned pharmaceutical composition, (i) preventing and / or treating Parkinson's disease; (ii) preparing drugs for preventing and / or treating Parkinson's disease; (iii) preventing and / or treating constipation; (iv) preparing drugs for preventing and / or treating constipation.

[0072] Food or health products and their uses

[0073] The present invention also relates to food or health products containing Dubosiella, which comprises Dubosiella and a carrier material acceptable to health products.

[0074] As used in the present invention, the term "health care product" refers to the commonality of health care (functional) food and general food, which can regulate the functions of the human body and is suitable for consumption by specific groups of people, but is not intended to treat diseases. The health care product in the present invention can be a product in the field of medicinal food, functional food, food additive, and nutrition and food products. When it is a food product, it can be selected from beverages, yogurt vinegar, fruit juice, ice cream, bread, biscuits, cereals, health bars and paste, but is not limited thereto.

[0075] As used in the present invention, the terms "acceptable carrier material for health products" and "optional carrier material for health products" are used interchangeably and refer to excipients and other additional materials used in the production of health products. In a preferred embodiment of the present invention, the optional carrier material for health products can be independently selected from the group consisting of oatmeal, lactic acid fermented food, resistant starch, dietary fiber, carbohydrates, protein and glycosylated protein, but is not limited thereto.

[0076] The present invention also relates to the use of the above health care product composition, which is used for (i) relieving or improving constipation; (ii) preparing food or health care products for relieving or improving constipation.

[0077] Treatment

[0078] The present invention also relates to a method for preventing and / or treating Parkinson's disease, or preventing and / or treating constipation, the method comprising the steps of:

[0079] administering to the subject a therapeutically effective amount of Dubosiella; or,

[0080] A therapeutically effective amount of a pharmaceutical composition of the present invention is administered to a subject.

[0081] As used herein, the term "subject" includes animals, such as any mammal or nematode, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice, etc. In a specific embodiment, the subject is a human.

[0082] To make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples that do not specify specific conditions are usually based on conventional conditions or the conditions recommended by the manufacturer. Unless otherwise specified, percentages and parts are weight percentages and weight parts. The experimental materials and reagents used in the following examples can be obtained from commercial channels unless otherwise specified.

[0083] Unless otherwise specified, the technical and scientific terms used herein have the same meaning as commonly understood by ordinary technicians in the technical field to which the application belongs. It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments of the present application.

[0084] Unless otherwise indicated, the term "or" means and is used interchangeably with the term "and / or".

[0085] As used herein, including the appended claims, singular forms of words such as "a," "an," and "the" include their corresponding plural referents unless the context clearly dictates otherwise.

[0086] Example 1. Construction of PD mouse model

[0087] In this example, the PD mouse model was constructed by synthesizing α-Syn preformed fibrils used for injection modeling of the PD mouse model according to the following method.

[0088] (1) Preparation of α-Syn preformed fibrils (PFF)

[0089] 500 μg of α-Synuclein was prepared into a 5 mg / mL stock solution using 100 μL sterile PBS, which was placed on a magnetic stirrer and stirred at 1000 rpm at 37° C. for seven days to prepare α-Syn polymers. The α-Syn polymers were further sonicated for 30 seconds (amplitude 10% / 0.5 second pulse interval).

[0090] (2) Electron microscope

[0091] The ultrasonically treated PFF was adsorbed onto a 400-mesh carbon-coated copper grid and treated for 2 minutes, followed by rinsing with 3 drops of Tris-HCl (50 mM, pH 7.4), and further treated on a drop of 0.75% uranium formate for 30 seconds and repeated once. Finally, a Tecnai G2 spirit 120 kV cryo-transmission electron microscope was used to image the ultrasonically treated PFF to determine the length of the PFF and perform statistics.

[0092] (3) Stereotaxic brain injection

[0093] Anesthesia: The mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (0.06 mL / 10 g body weight) and fixed on a stereotaxic apparatus. The top of the head was disinfected with iodine, the hair on the head surface was trimmed, and then disinfected again with iodine.

[0094] Stereotaxic brain injection: a midsagittal incision was made, the skin on both sides of the incision was clamped with an artery clamp, and a cotton swab was dipped in 30% H2O2 to wipe the bregma. According to the mouse brain stereotaxic atlas, the position was determined (injection position of bilateral striatum: AP+0.2mm; ML±2.0mm; DV 2.6mm) and a micro-hole dental drill was used to drill holes, and the needle was slowly inserted using a self-made micro-injector with a glass electrode (α-Syn PFF: 5μg (2μL, 0.1μL / min); solvent PBS: (2μL, 0.1μL / min). After the injection was completed, the needle was stopped for 10 minutes and the glass electrode was slowly rotated out.

[0095] Suturing and feeding: After the mouse's head is sutured, it is disinfected again with iodine, and the mouse is placed in a 37°C constant temperature incubator. After it wakes up, it is returned to the cage and fed freely. Three days after the operation, sugar water is given or wet food is added to the cage for continuous feeding.

[0096] Note: 0.3 mL of normal saline was injected subcutaneously before the operation to prevent dehydration during the operation. Erythromycin eye ointment was applied to the eyes to protect the eyes. A thermometer was used to measure the rectal temperature and record the physiological status of the mice before, during and after the operation. A 37°C constant temperature pad was used to keep warm throughout the operation.

[0097] Example 2. Dubosiella treatment method

[0098] In this embodiment, Ningbo Mingzhou Bio was commissioned to culture and amplify the Dubosiella flora. The obtained Dubosiella and OP50 suspensions were first centrifuged, and after weighing the precipitate, Dubosiella and OP50 were resuspended in M9 buffer to 100 mg / mL, and then planted on NGM nematode culture plates in proportion (the configuration ratio was pure OP50; Dubosiella: OP50 = 1:1; Dubosiella: OP50 = 1:2; Dubosiella: OP50 = 1:4;). After that, the paved NGM nematode culture plates were placed in a UV crosslinker, and the parameters were set to UV999 for 5 minutes for subsequent experiments.

[0099] Example 3. Nematode rearing

[0100] For the two nematode strains, N2 and NL5901, used in the recovery experiment, after taking out the thawed part of the cryotube from the -80 degrees Celsius refrigerator, use an alcohol-scalded iron spoon to take out three small spoons of nematode freezing solution with ice crystals and place it near the edge of the nematode growth medium (NGM) plate covered with Escherichia coli (Escherichia coli: OP50). After the ice crystals melted, rotate the culture plate to allow the liquid to flow through the Escherichia coli layer. Then, seal the nematode culture plate with a sealing film and culture it on a 20°C NGM plate under standard conditions. The recovery status of the nematodes was observed the next day.

[0101] After the nematodes are successfully revived, the nematodes are treated with Bleach for synchronous culture. The specific method is: Bleach solution (5mMNaOH: bleach: ddH2O=1:3:6) is added to the NGM plate containing OP50 away from the OP50 position, and then about 20 adult nematodes containing eggs are selected and placed in the Bleach solution. After the nematodes are lysed, the status of the nematodes is observed the next day.

[0102] Example 4. Lifespan experiment

[0103] Pentofluorouracil (0.04 mg / mL) was added to the NGM plates of the nematode lifespan experiment to inhibit nematode egg-laying. Synchronized N2 and NL5901 nematodes were selected and placed on different NGM nematode culture plates and recorded as day 0. They were cultured at 20 degrees Celsius, and the number of lost, dead, and surviving nematodes was recorded every day. The remaining feeding bacteria on the culture plates were observed, and the plates were transferred in time when the amount of bacteria was less. The records were continuously kept until the last nematode died. Finally, the data were sorted and the lifespan curve was drawn using Prism 9.0.

[0104] Example 5. Experiment on intraperitoneal egg storage

[0105] L4 wild-type NL5901 nematodes were selected and fed with Dubosiella for 48 hours. The nematodes were lysed with Bleach solution, and the number of eggs stored in the abdomen of the nematodes was counted. The experiment was repeated three times independently.

[0106] Example 6. Swimming test

[0107] Wild-type NL5901 nematodes at the L4 stage were selected and fed with Dubosiella for 48 hours. The nematodes were then transferred to M9 buffer. The number of body swings within 30 seconds was counted, and a change in the direction of the middle part of the nematode's body was considered a swing. The experiment was repeated three times independently.

[0108] Example 8. Construction of PFF mouse model

[0109] In this example, a chronic PD mouse model induced by PFF was established with reference to existing experimental methods. First, the basic characteristics of the PFF used for experimental injection were detected. After treatment, electron microscopy was used to observe and photograph the PFFs and statistically analyze the length of the PFFs, which was mainly distributed in the range of 30-70 nm, with an average length of 58.52 nm ± 1.30 nm ( Figure 1A -C), 60 days and 270 days after the injection of PFF into the striatum, it can be observed that PFF gradually spread to other brain regions with the increase of time ( Figure 1D ). 270 days after injection, pSer129-α-Syn expression, i.e. the formation of Lewy-body, can be detected by immunofluorescence imaging in the striatum, cortex, hippocampus, substantia nigra and other brain regions ( Figure 1E ). In summary, after PFF injection into the striatum, α-Syn pathology can gradually spread to nearby brain regions and induce pSer129-α-Syn expression in the mouse brain, indicating the feasibility of PFF to construct PD model.

[0110] Example 9. Construction of a PD mouse model with PD-related motor dysfunction and constipation phenotypes

[0111] In this example, an experimental scheme was designed to test the modeling effect of PFF. A chronic PD mouse model was selected in which PFF was injected into the brain stereotaxically to simulate the pathological changes and behavioral changes of PD. In the PD mouse model cultured to nine months old, the development of various mice was not significantly affected ( Figure 2A ). The classic rotating rod and climbing pole test were further applied to detect the movement and balance ability of mice. The results showed that the head turning time and total time of PD mice in the climbing pole behavior test were significantly increased ( Figure 2B -C). The time that PD mice spent on the rotarod was significantly reduced ( Figure 2D ), the results of brain pathological immunofluorescence staining showed that the number of DA neurons in the substantia nigra of the WT-PFF group was significantly lower than that of the control group ( Figure 2E -F), accompanied by a significant decrease in the content of the neurotransmitter dopamine in the striatum ( Figure 2G ), the above data show that PFF model mice have obvious behavioral and pathological phenotypes of PD mice and can be used for further research.

[0112] Example 10. The most obvious change in the intestinal flora of PD mice is Dubosiella

[0113] In this example, stool samples of the PD group and the healthy control group were sequenced by 16s DNA to analyze the differential flora between the two groups.

[0114] The results showed that compared with the WT-Vehicle group, the WT-PFF group had significantly lower levels of Dubosiella, Faecalibaculum, Coriobacteriaceae-UCG-002, and unclassified-f-Eggerthellaceae, while the Candidatus-Saccharimonas, Lactococcus, norank-f-Ruminococcaceae, Lachnospiraceae-NK4A136-group, and Bacillus groups increased significantly, with the most significant change in Dubosiella ( Figure 3 ), we further designed relevant experiments to explore whether Dubosiella is a new probiotic involved in the pathogenesis of PD, hoping to find new therapeutic strategies to intervene in the progression of PD through the brain-gut axis at the gastrointestinal level.

[0115] Example 11. Neurotransmitter-related metabolic disorders exist in the intestines of PD mice

[0116] In this example, cluster heat maps and VIP histograms are used to show the changes in WT-Vehicle / WT-PFF neurotransmitters. Figure 4As shown, the results showed that the contents of Serotonin, 5-Hydroxy-L-tryptophan, L-Glutamate, L-Glutamicacid, L-Tyrosine, etc. were significantly increased in the PD mouse model. Serotonin is a key regulator of gastrointestinal motility and secretion, and about 90% of Serotonin in the human body is secreted by enterochromaffin cells of the intestinal epithelium. The Serotonin receptors expressed in gastrointestinal tissues are diverse and widely distributed, and are involved in the regulation of gastrointestinal function.

[0117] At the same time, the contents of phosphatidylethanolamine PE (15:0 / 20:2 (11Z, 14Z)), PE (15:0 / 20:4 (8Z, 11Z, 14Z, 17Z)), 1-palmitoyl-2-stearoyl-sn-glycerol DG (16:0 / 18:4 (6Z, 9Z, 12Z, 15Z) / 0:0) were significantly reduced in the PD mouse model. Among them, PE (15:0 / 20:4 (8Z, 11Z, 14Z, 17Z)) is a glycerophospholipid (hereinafter referred to as PE). Studies have shown that the PE content of SNc in sporadic PD patients is significantly lower than that in the control group, and L-dopa treatment can correct it back to normal levels, indicating that PE plays a very important role in the pathogenesis of PD. This study first found that in the PFF-induced chronic PD mouse model, the PE level in the intestine was significantly reduced, accompanied by an increase in the content of neurotransmitters such as serotonin. The results showed that there was a correlation between intestinal flora disorder and neurotransmitter metabolism disorder in the periphery of the PD mouse model.

[0118] Example 12: Dubosiella is the main intestinal flora, and the diversity of intestinal flora is intervened by regulating transmitters such as Serotonin and PE

[0119] In this example, Spearman correlation analysis was used to evaluate the correlation between bacterial flora and neurotransmitters. Figure 5A As shown in Figure 2, Dubosiella, Coriobacteriaceae-UCG-002 can significantly negatively regulate Serotonin and significantly positively regulate PE-15. Figure 5B As shown in the correlation analysis, Serotonin was significantly positively correlated with the intestinal flora diversity index Ace and Shannon, and significantly negatively correlated with MDS1; PE was significantly negatively correlated with Ace and Shannon, and significantly positively correlated with MDS1 ( Figure 5C ), the above results indicate that Dubosiella affects the diversity of intestinal flora by regulating transmitters such as Serotonin and PE.

[0120] The above revealed that Dubosiella plays a key role in mediating peripheral intestinal flora and metabolic disorders, and it is significantly correlated with Serotonin and PE that regulate gastrointestinal function. So, can Dubosiella be used as a probiotic to improve the constipation phenotype or behavioral disorders of PD mice through replenishment or other forms?

[0121] Example 13. Dubosiella can significantly improve PD motor behavior disorders and constipation phenotype in aged mice

[0122] In this example, a preliminary study was conducted on mice and nematodes. The experimental design is as follows: Fig. 6A On the one hand, the inventors' research indicated that Dubosiella significantly increased the number of fecal particles discharged within 24 hours and 2 hours, and improved the constipation phenotype of 19-month-old WT mice ( Figure 6B -C).

[0123] This example also evaluated the fine motor regulation of PD mice. The results showed that Dubosiella could significantly improve the nesting behavior of 19-month-old hA53T mice. The weight of non-nesting materials and nesting scores of mice in the Dubosiella group were significantly increased ( Fig.6D -E). The above data indicate that Dubosiella can improve the constipation phenotype of aged mice and regulate fine motor functions in PD.

[0124] The effects of Dubosiella on the lifespan and motor function of PD nematodes (NL5901 nematode strain) and WT nematodes (N2 nematode strain) were further evaluated. It was found that feeding nematodes with a UV-inactivated mixture of Dubosiella and OP50 in different ratios of 1:1 / 1:2 / 1:4 could extend the average lifespan of nematodes to varying degrees ( Figure 6G -H). The above data show that Dubosiella may play a role in delaying aging as a probiotic, so what is the specific mechanism? Based on the previous research, Spearman analysis showed that Dubosiella can significantly negatively regulate the content of Serotonin, and studies have shown that Bacillus licheniformis isolated from traditional Korean food resources enhances the longevity of C.elegans by regulating Serotonin signals, so further exploration of Dubosiella's regulatory effect on Serotonin was conducted.

[0125] The egg-laying process of nematodes is specifically regulated by serotonergic neurons - hermaphrodite-specific motor neurons (HSN) to internalize egg-laying muscles (ELMs) to lay eggs. Egg-laying behavior has become an important phenotypic analysis method for neural signal transduction mechanisms. HSN is necessary for normal egg-laying, and activation of HSN will trigger egg-laying behavior. Therefore, this behavioral paradigm was used to study the egg-laying behavior of nematodes to explore the effect of Dubosiella on Serotonin in nematodes. The results of the study are as follows Fig.6I As shown, it was found that when the feeding concentration ratio of Dubosiella:OP50=1:1 / 1:2, the nematode egg-laying was significantly inhibited, and the number of eggs in the abdomen of the nematode increased significantly, providing direct evidence for the negative regulation of Serotonin by Dubosiella. At the same time, the previous article found that Dubosiella can extend the lifespan of nematodes to varying degrees, indicating that in this study, the regulatory effect of Dubosiella on lifespan is related to the regulation of Serotonin by Dubosiella. Further, we evaluated the movement of the nematodes by real-time recording of the nematode swimming video and evaluating the number of swings of the nematodes within 30 seconds. The study showed that feeding nematodes with a UV-inactivated mixture of Dubosiella and OP50 at a ratio of 1:2 can significantly improve the movement ability of NL5901 and N2 strains of nematodes ( Figure 6J -K). In summary, combined with the experimental data of mice, it is clear that Dubosiella can improve the motor dysfunction of PD mice and nematodes. At the same time, the direct regulatory effect of Dubosiella on Serotonin was confirmed at the nematode level.

[0126] Those skilled in the art will appreciate that the above-mentioned embodiments are specific examples for implementing the present invention, and in actual applications, various changes may be made thereto in form and detail without departing from the spirit and scope of the present invention.

Claims

1. Use of Dubosiella, characterized in that: Used for: (i) prevention and / or treatment of Parkinson's disease; (ii) preparing a medicament for preventing and / or treating Parkinson's disease; (iii) prevention and / or treatment of constipation; and / or (iv) preparing a medicament for preventing and / or treating constipation.

2. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises Dubosiella and a pharmaceutically acceptable carrier or excipient.

3. The use of the pharmaceutical composition according to claim 2, characterized in that: Used for (i) preventing and / or treating Parkinson's disease; (ii) preparing drugs for preventing and / or treating Parkinson's disease; (iii) preventing and / or treating constipation; and / or (iv) preparing drugs for preventing and / or treating constipation.

4. The use according to claim 3, characterized in that Each milliliter of the pharmaceutical composition contains no less than 1x10 Dubosiella 6 CFU.

5. The use according to claim 3, characterized in that: The pharmaceutical composition is a solid preparation, and the solid preparation is selected from at least one of tablets, lozenges, candies, chewable tablets, chewing gums, capsules, powders, granules, coated microparticles, coated tablets, enteric-coated tablets and capsules.

6. The use according to claim 3, characterized in that: The pharmaceutical composition is a liquid preparation, and the liquid preparation is selected from at least one of an oral solution, a suspension, an emulsion and a syrup.

7. A health care product composition, characterized in that: The health care product is used for (i) relieving and / or improving constipation; (ii) relieving and / or improving Parkinson's disease; (iii) preparing food or health care products for relieving and / or improving constipation; (iv) preparing health care products for relieving and / or improving Parkinson's disease, and the food or health care product composition comprises Dubosiella; and a carrier or excipient acceptable to the health care product.

8. The use of the health care product composition according to claim 4, characterized in that: Used for (i) relieving and / or improving constipation; (ii) relieving and / or improving Parkinson's disease; (iii) preparing food or health products for relieving and / or improving constipation; (iv) preparing health products for relieving and / or improving Parkinson's disease.

9. The use according to claim 3, characterized in that: The health care product is selected from at least one of beverages, yogurt vinegar, fruit juice, ice cream, bread, biscuits, cereals, health bars, paste, seasoning, milk, cheese and fermented milk.

10. A method for preventing and / or treating Parkinson's disease, characterized in that: The method comprises the steps of: administering to the subject a therapeutically effective amount of Dubosiella; or, Administering a therapeutically effective amount of the pharmaceutical composition of claim 2 to a subject.

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