CN119235924A - Use of Dubosiella and pharmaceutical compositions containing same - Google Patents

Abstract

The application discloses Dubosiella and application of a pharmaceutical composition containing the Dubosiella. The application provides a new application of Dubosiella, dubosiella can regulate and control gastrointestinal functions to relieve constipation phenotype of PD mice, and researches show that the risk of PD of constipation patients is obviously increased, so Dubosiella is hopeful to relieve constipation by regulating intestinal flora diversity and further relieve disease risk and disease symptoms of PD. The application clearly supplements Dubosiella at the nematode level, can obviously relieve the dyskinesia of the PD nematodes, and suggests that Dubosiella is expected to become a potential probiotic for synergistically regulating and controlling the peripheral and central disease symptoms of the PD in the future.

Description

Dubosiella and use of pharmaceutical compositions containing the same

Technical Field

The invention relates to the field of biological medicine and food health care, in particular to Dubosiella and application of a pharmaceutical composition containing the same.

Background

Parkinson's Disease (PD) is the second most common neurodegenerative disease in the clinic. Typical clinical dyskinesias of PD include resting tremor, myotonia, etc., non-motor clinical manifestations include hypotension, sleep disorders, depression, and gastrointestinal dysfunction, and non-dyskinesias generally occur early in dyskinesias, so that 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 essentially characterized by abnormal aggregation of alpha-synuclein (alpha-Synuclein, alpha-Syn), the pathological range of which involves all areas of the brain intestinal axis, including the central, autonomic and enteric nervous systems. The highly bi-directional communication between the brain and the gut is transmitted through immunological, neuroendocrine, etc. processes, which are significantly affected by the gut microbiota. Intestinal microbiota and its related metabolites interact with the central nervous system through a range of biochemical and functional inputs, affecting the progression of the disease. The inventor finds in the study that intestinal flora imbalance exists in PD patients, gastrointestinal dysfunction is one of the most common and disabled non-motor symptoms of PD patients, constipation is the most common and painful precursor symptom of PD, and the incidence rate is as high as 70-80%. This suggests that constipation may be linked to the development of PD. However, there is little research in this area in the prior art, however, there is little research in the correlation of constipation with the presence of PD, and the corresponding mechanism is not clear. Therefore, the research on the relationship between gastrointestinal dysfunction and the development progress of PD diseases, the definition of the corresponding mechanism, is hopeful to provide a new selection target for early drug treatment of PD.

Disclosure of Invention

The invention aims to provide a purpose of Dubosiella.

It is a further object of the present invention to provide a use of the pharmaceutical composition.

It is another object of the present invention to provide a method for preventing and/or treating PD.

To solve the above technical problem, according to a first aspect of the present invention, there is provided use of Dubosiella for:

(i) Preventing and/or treating PD;

(ii) Preparing a medicament for preventing and/or treating PD;

(iii) Preventing and/or treating constipation, and/or

(Iv) Preparing the medicine for preventing and/or treating constipation.

In a second aspect of the invention, a pharmaceutical composition is provided comprising Dubosiella, and a pharmaceutically acceptable carrier or excipient.

In a third aspect of the invention, there is provided the use of a pharmaceutical composition for (i) preventing and/or treating PD, (ii) preparing a medicament for preventing and/or treating PD, (iii) preventing and/or treating constipation, and/or (iv) preparing a medicament for preventing and/or treating constipation, the pharmaceutical composition comprising Dubosiella and a pharmaceutically acceptable carrier or excipient.

In some preferred embodiments, dubosiella is not less than lxl CFU, ⁶ CFU, more preferably not less than lxl CFU, ⁸ CFU per milliliter of the pharmaceutical composition.

In some preferred embodiments, the pharmaceutical composition is a solid formulation selected from at least one of a tablet, a troche, a candy, a chewable tablet, a chewing gum, a capsule, a powder, a granule, a coated microparticle, a coated tablet, an enteric coated tablet, and a capsule.

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

According to a fourth aspect of the invention, a health-care product composition is provided, and 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 by the health-care products.

In a fifth aspect of the invention, use of a nutraceutical composition is provided for (i) alleviating and/or ameliorating constipation, (ii) alleviating and/or ameliorating PD, (iii) preparing a food or nutraceutical for alleviating and/or ameliorating constipation, and (iv) preparing a nutraceutical for alleviating and/or ameliorating PD.

In some preferred embodiments, the health product is selected from at least one of beverages, yogurt vinegar, fruit juices, ice cream, bread, biscuits, cereals, health bars, pastes, flavors, milk, cheese, and fermented milks.

In a sixth aspect of the present invention, there is provided a method of preventing and/or treating PD, the method comprising the steps of:

administering to the subject a therapeutically effective amount of Dubosiella, or

Administering to the subject a therapeutically effective amount of a pharmaceutical composition according to the second aspect of the invention.

In a seventh aspect of the present invention, there is provided a method for preventing and/or treating constipation, the method comprising the steps of:

administering to the subject a therapeutically effective amount of Dubosiella, or

Administering to the subject a therapeutically effective amount of a pharmaceutical composition according to the second aspect of the invention.

The invention has at least the following advantages over the prior art:

(1) The invention provides a new application of Dubosiella, dubosiella can regulate gastrointestinal tract and intestinal flora disturbance to further relieve constipation phenotype of PD mice, and researches show that the risk of PD of constipation patients is obviously increased, so Dubosiella is expected to relieve constipation by regulating intestinal tract and intestinal flora diversity, and simultaneously, the disease risk and disease symptoms of PD are further relieved.

(2) The invention clearly supplements Dubosiella at the level of the PD nematode model, can obviously relieve the dyskinesia of the PD nematode, and suggests that Dubosiella is expected to become a potential probiotic for synergistically regulating and controlling the peripheral and central disease symptoms of the PD in the future.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

One or more embodiments are illustrated by way of example and not limitation in the figures of the accompanying drawings.

FIG. 1A is a schematic diagram of an electron microscope at magnification of left (68000X)/right (98000X) for preparing a PFF in accordance with an embodiment of the present invention. Scale bar = 200nm.

Figure 1B is a graph of PFF length distribution at various length stages after sonication in accordance with an embodiment of the present invention. The average length of the PFF was 58.52nm±1.30nm (n=559).

Figure 1C is an α -syn stage PFF length distribution in accordance with an embodiment of the present invention.

Figure 1D is a schematic representation of the spread of PFF in various brain regions 60 days and 270 days after injection into the striatum in accordance with an embodiment of the present invention (red dots represent PFF fluorescence signals).

FIG. 1E is a graph showing the distribution of pSer 129-alpha-Syn in various brain regions of mice 270 days after PFF injection into the striatum in accordance with an embodiment of the present invention. Scale bar = 50 μm.

Fig. 2A is a graph of weight statistics for each group of mice for the ninth month in accordance with an embodiment of the present invention.

FIG. 2B is a statistical chart of the results of the turning time of each set of pole climbing experiments according to the embodiment of the invention.

FIG. 2C is a statistical chart of total time results of various sets of pole climbing experiments according to an embodiment of the invention.

Fig. 2D is a statistical plot of the results of the individual groups of mice in the rotarod experiments according to the examples of the present invention.

FIG. 2E is a schematic representation of immunofluorescence of the individual groups of mouse SNc brain region TH ⁺ neurons, scale bar,200 μm, according to an example of the present invention.

Fig. 2F is a statistical plot of unbiased stereoscopic count data for TH ⁺ neurons in each group SNc, according to an embodiment of the invention.

FIG. 2G is a statistical plot of the relative content of DA neurotransmitters detected by striatal HPLC-MS in accordance with an embodiment of the present invention. Data are expressed as mean ± s.e.m., p <0.01, p <0.001, and p <0.0001.

Fig. 3 is a graph of species analysis and diversity evaluation of intestinal flora differences between PD group and control group in accordance with an embodiment of the present invention.

FIG. 4 is a cluster map and VIP histogram showing changes in neurotransmitters between the WT-Vehicle and WT-PFF groups in accordance with an embodiment of the invention.

FIG. 5A is a Sepeaman correlation heat map in accordance with an embodiment of the invention.

FIG. 5B is a graph showing the correlation of Serotonin with the intestinal flora diversity index Ace, shannon, MDS1 according to one embodiment of the present invention.

FIG. 5C is a graph showing the correlation of PE with intestinal flora diversity index Ace, shannon, MDS according to one embodiment of the present invention.

Fig. 6A is a schematic diagram of a mouse behavioural detection procedure according to an embodiment of the present invention.

FIG. 6B is the number of fecal pellets per 24h unit time of WT aged mice in accordance with an embodiment of the present invention. Analytical method Student's t test, p <0.05, data expressed as mean ± s.e.m. (WT: n=10; WT-Dubosiella: n=9).

FIG. 6C is the number of fecal pellets per 2h unit time in WT aged mice according to an embodiment of the present invention. Analytical method Student's t test, p <0.05, data expressed as mean ± s.e.m. (WT: n=10; WT-Dubosiella: n=9).

Figure 6D is a weight statistic of non-nesting material for nesting experiments in accordance with embodiments of the present invention. (WT: n=10, WT-Dubosiella: n=9, ha53t: n=6, ha53t-Dubosiella: n=7).

Figure 6E is a statistical plot of the results of the nesting score of the nesting test in accordance with embodiments of the present invention. (WT: n=10, WT-Dubosiella: n=9, ha53t: n=6, ha53t-Dubosiella: n=7).

Fig. 6F is a statistical plot of colon length results from mice in accordance with an embodiment of the invention. (WT: n=7, WT-Dubosiella: n=5, ha53t: n=6, ha53t-Dubosiella: n=6).

FIG. 6G is a graph of survival of NL5901 strain (PD nematode) in accordance with an embodiment of the invention.

FIG. 6H is a graph of N2 line nematode (WT) survival in accordance with an embodiment of the invention.

FIG. 6I is the number of eggs stored in the abdomen of strain NL5901 according to an embodiment of the invention. (OP 50 group: n=70; 1:1 group: n=31; 1:2 group: n=67; 1:4 group: n=38).

FIG. 6J is a statistical plot of the number of swings within 30s of swimming of the NL5901 strain according to an embodiment of the invention. (OP 50: n=30; 1:2: n=30; 1:4: n=30).

FIG. 6K is a statistical plot of the number of swings within 30s of swimming of the N2 line according to an embodiment of the present invention. (OP 50: n=31; 1:2: n=25; 1:4: n=13). Analytical methods-Student's t test (B-C) and One-way ANOVA with multiple comparison test (D-K). Data are expressed as mean ± s.e.m., p <0.05, p <0.01, p <0.001, and p <0.0001.

Detailed Description

The cause of PD is currently unknown, and relatively few studies have been conducted in the prior art on the correlation between PD and gastrointestinal dysfunction. Through extensive and intensive studies, the inventors of the present invention have found that Dubosiella (Dubosiella newyorkensis) bacteria are capable of affecting the secretion of serotonin/phosphatidylethanolamine and thus the diversity of intestinal flora, alleviating senile constipation, and improving the fine motor dysfunction of an animal model for Parkinson. Modulation of homeostasis against intestinal microbiota is expected to provide a new target of choice for early drug treatment of PD.

Use of Dubosiella

The invention relates to novel application of Dubosiella in (i) preventing and/or treating parkinsonism, (ii) preparing a medicament for preventing and/or treating parkinsonism, (iii) preventing and/or treating constipation, and (iv) preparing a medicament for preventing and/or treating constipation.

As used herein, the term "Dubosiella" is a known species of genus Erysipelotrichidae of the gram-positive family (Dubosiella Newyorkensis), all known as "Dubosiella Newyorkensis", is a severe anaerobic bacterium having strain number DSM 103457 =atcc TSD-64.Dubosiella As a known strain, it is commercially available and can be amplified by culturing in a manner known to those skilled in the art. In one embodiment of the invention, a suspension of Dubosiella bacteria and emulsifier OP-50 is first centrifuged to obtain a pellet, and the pellet is then resuspended in M9 buffer and inoculated in a suitable culture plate, such as an NGM nematode culture plate, in proportion. The conditions under which the species are cultivated are well known to those skilled in the art, for example, in an anaerobic environment at 37℃for 18-24 hours. "Dubosiella" used in the examples of the present invention was purchased from Ningbo boat biotechnology Co.

As used herein, the term "constipation" refers to a phenomenon in which the number of bowel movements is reduced. Patients generally have less than 3 times per week, with difficult defecation and dry feces.

As used herein, the term "treatment and/or prophylaxis" includes prophylactic treatment of an individual, i.e., treatment with Dubosiella bacteria is initiated prior to the occurrence of a disease in order to prevent the disease, as well as therapeutic treatment of a disease that has occurred in an individual. In the latter case, relief of symptoms is for example desired, or the overall condition of the patient is enhanced or the disease of the patient is cured. Thus, an individual may be a person at risk of developing an autoimmune disease or who is already a patient with the disease.

Pharmaceutical composition and use thereof

The invention also relates to pharmaceutical compositions comprising Dubosiella, including Dubosiella, and a pharmaceutically acceptable carrier or excipient.

As used herein, the term "pharmaceutical composition" refers to a composition containing one or more active ingredients for the purpose of preventing and/or treating a disease, which has the function of preventing and/or treating a disease. In addition to containing the active ingredient, the pharmaceutical composition may be combined with one or more pharmaceutically acceptable carriers or excipients depending on the mode of administration, pharmaceutical properties, indication, etc. The pharmaceutical composition according to the present invention may contain a pharmaceutically acceptable carrier. For oral administration, pharmaceutically acceptable carriers may include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants, flavoring agents and the like. For topical administration, pharmaceutically acceptable carriers may include matrices, excipients, lubricants, preservatives, and the like. The pharmaceutical composition of the invention can be combined with the pharmaceutically acceptable carrier to prepare various dosage forms. For example, for oral administration, the pharmaceutical compositions may be formulated in solid or liquid dosage forms, such as tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. In addition, the pharmaceutical composition may be formulated as solutions, suspensions, tablets, capsules, sustained release preparations, and the like. Meanwhile, examples of carriers, excipients and diluents suitable for the formulation may include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil and the like. In addition, the pharmaceutical composition may further contain fillers, anti-coagulants, lubricants, wetting agents, flavoring agents, emulsifying agents, preservatives, and the like. The pharmaceutical compositions described herein may be formulated in conventional manner using one or more pharmaceutically acceptable carriers and may also be administered in controlled release formulations.

In some preferred embodiments of the invention, the pharmaceutical composition is prepared as a liquid formulation or as a solid formulation. The solid preparation may be selected from the group consisting of tablets, troches, candies, chewable tablets, chewing gums, capsules, sachets, powders, granules, coated particles, coated tablets, enteric coated tablets and capsules, and dissolving strips and films. The liquid preparation may be selected from the group consisting of oral solutions, suspensions, emulsions or syrups.

The pharmaceutical compositions as described herein may be used for rectal administration in the form of suppositories. These can be prepared by mixing the agent with a suitable non-irritating excipient which is solid at room temperature but liquid at the rectal temperature and will therefore melt in the rectum to release the drug. Such materials include cocoa butter, beeswax, polyethylene glycols and the like.

The content of Dubosiella in the pharmaceutical composition of the present invention is not limited. Preferably, dubosiella is not less than lxl, ⁶ CFU, preferably lxl, ⁶-lxl0¹⁶ CFU/mL, e.g. lxl0⁷CFU/mL、lxl0⁸CFU/mL、lxl0⁹CFU/mL、lxl0¹⁰CFU/mL、lxl0¹²CFU/mL、lxl0¹⁶CFU/mL, etc., per mL of pharmaceutical composition.

In a preferred embodiment of the invention, the pharmaceutical composition of the invention may be used in combination with other intestinal probiotics, for example with Lactobacillus, bifidobacterium, gram-positive cocci, etc., such as one or more of Bifidobacterium longum, bifidobacterium breve, bifidobacterium ovale and Bifidobacterium thermophilum, lactobacillus acidophilus, lactobacillus casei, lactobacillus jensenii and Lactobacillus raman.

The invention also relates to a new application of the pharmaceutical composition, (i) preventing and/or treating parkinsonism, (ii) preparing a medicament for preventing and/or treating parkinsonism, (iii) preventing and/or treating constipation, and (iv) preparing a medicament for preventing and/or treating constipation.

Food or health care product and application thereof

The invention also relates to foods or health products containing Dubosiella, including Dubosiella and carrier materials acceptable for the health products.

As used herein, the term "health product" refers to the commonality of health (functional) foods in general, which can regulate the functions of the human body, and is suitable for specific people to eat, but not for the purpose of treating diseases. The health products in the invention can be medical foods, functional foods, food additives, and products in the fields of nutrition and food products. In the case of a food product, it may be selected from the group consisting of beverages, yogurt vinegar, fruit juices, ice desert strangs, breads, biscuits, cereals, health bars and pastes, but is not limited thereto.

As used herein, the terms "nutraceutical acceptable carrier material" and "nutraceutical optional carrier material" are used interchangeably and refer to excipients and other additional materials used in the manufacture of a nutraceutical. In a preferred embodiment of the present invention, the optional carrier material of the health product may be independently selected from the group consisting of oat gruel, lactic acid fermented food, resistant starch, dietary fiber, carbohydrates, proteins and glycosylated proteins, but is not limited thereto.

The invention also relates to application of the health-care product composition in (i) relieving or improving constipation and (ii) preparing food or health-care products for relieving or improving constipation.

Therapeutic method

The invention also relates to a method for preventing and/or treating parkinson's disease, or constipation, said method comprising the steps of:

administering to the subject a therapeutically effective amount of Dubosiella, or

Administering to a subject a therapeutically effective amount of a pharmaceutical composition of the invention.

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, and the like. In a particular embodiment, the subject is a human.

The present invention will be further described with reference to specific embodiments in order to make the objects, technical solutions and advantages of the embodiments of the present invention more apparent. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental methods, in which specific conditions are not noted in the following examples, are generally conducted under conventional conditions or under conditions recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated. The experimental materials and reagents used in the following examples were obtained from commercial sources unless otherwise specified.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs, it is to be noted that the terms used herein are used merely to describe specific embodiments and are not intended to limit exemplary embodiments of the application.

The term "or" means and is used interchangeably with the term "and/or" unless otherwise indicated.

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

Example 1 construction of PD mouse model

In this example, a PD mouse model was constructed by synthesizing alpha-Syn preformed fibrils for injection molding of the PD mouse model according to the following method.

(1) Alpha-Syn preformed fibrils (PFF) preparation

500 Μg of α -Synuclein was formulated as a 5mg/mL stock solution using 100 μl of sterile PBS and placed on a magnetic stirrer with stirring at 1000rpm for seven days at constant temperature of 37 ℃. The alpha-Syn polymer was further sonicated for 30 seconds (amplitude 10%/0.5 second pulse gap).

(2) Electronic mirror

After adsorbing the sonicated PFF onto a 400 mesh carbon coated copper grid for 2 minutes, 3 drops of Tris-HCl (50 mM, pH 7.4) were added dropwise for rinsing, further treated on 0.75% uranium formate drops for 30 seconds and repeated once, and finally the sonicated PFF was imaged with a Tecnai G2 spiral 120kV freeze transmission electron microscope to determine the length of the PFF and counted.

(3) Brain stereo injection

Anesthesia the mice were anesthetized with 1% pentobarbital sodium (0.06 mL/10g body weight) and then fixed on a brain stereotactic apparatus, the head and top were sterilized with iodophor, and the hair on the head surface was cut clean and re-sterilized with iodophor.

Brain stereotactic injection, which is to make a median sagittal incision, clamp the skin on both sides of the incision with an arterial clamp, dip 30% H2O2 with a cotton swab for wiping, and expose bregma. According to the brain stereogram of the mice, the position (bilateral striatum injection position: AP+0.2mm; ML.+ -. 2.0mm; DV 2.6 mm) is determined, and a microporous dental drill is used for punching, a micro-injector self-made with a glass electrode is used for slowly inserting the needle (alpha-Syn PFF:5 mug (2 mug, 0.1 mug/min) and solvent PBS (2 mug, 0.1 mug/min), and the glass electrode is slowly screwed out after stopping the needle for 10min after the injection is completed.

Suturing and feeding, namely suturing the head of the mouse, then sterilizing again by using iodophor, placing the mouse into a 37 ℃ constant temperature incubator, and placing the mouse into a cage after the mouse wakes up for free ingestion. Sugar water is added three days after operation or wet food is added into the cages for continuous feeding.

Note that 0.3mL physiological saline is injected subcutaneously before operation to prevent dehydration during operation, erythromycin eye ointment is smeared on eyes to protect the eyes, a thermometer is used for measuring the anus temperature after operation and recording and detecting the physiological state of a mouse, and a constant temperature cushion at 37 ℃ is used for keeping warm during operation.

EXAMPLE 2 Dubosiella treatment method

In this example, the Ningbo boat organism was commissioned to culture and amplify Dubosiella colonies. For the Dubosiella and OP50 suspensions obtained, centrifugation was performed first, the pellet was weighed, resuspended Dubosiella and OP50 using M9 buffer to a formulation of 100mg/mL, and then planted in proportion on NGM nematode plates (configuration ratio was pure OP50; dubosiella: op50=1:1; dubosiella: op50=1:2; dubosiella: op50=1:4;). The laid NGM nematode plates were then placed in an ultraviolet cross-linker and treated for 5min with the set parameters UV999 for subsequent experiments.

EXAMPLE 3 nematode feeding

After the thawing part of the freezing tube is taken out from a refrigerator at the temperature of minus 80 ℃ and the two nematode strains of N2 and NL5901 used in the resuscitation experiment, three small spoons of nematode freezing solution with ice crystals are taken out by using an iron ladle burnt by alcohol, the three small spoons of nematode freezing solution are placed at the position of a Nematode Growth Medium (NGM) plate paved with escherichia coli (ESCHERICHIA COLI: OP 50) close to the edge, the culture plate is rotated after the ice crystals are thawed, the solution flows through an escherichia coli layer, then the nematode culture plate is sealed by using a sealing film, and is cultured on the NGM plate at the temperature of 20 ℃ under the standard condition, and the resuscitation state of nematodes is observed next day.

After the nematodes are successfully recovered, carrying out Bleach treatment on the nematodes for synchronous culture, dripping Bleach liquid (5 mMNaOH: bleaching agent: ddH ₂ O=1:3:6) on an NGM plate containing OP50 at a position far away from the OP50, then selecting about 20 eggs-containing nematode adults, placing the nematode adults in Bleach liquid until the nematodes are cracked, and observing the nematode state the next day.

Example 4 Life test

The NGM plate of the nematode life experiment is added with pentafluoroeturacil (0.04 mg/mL) for inhibiting the oviposition of nematodes, contemporaneous N2 and NL5901 nematodes are selected and placed on different NGM nematode culture plates, and are recorded as day 0, cultured at 20 ℃, the numbers of the lost, dead and alive nematodes are recorded every day, the residual condition of feeding bacteria on the culture plates is observed, dishes are timely rotated when the bacterial load is small, the last nematode is continuously recorded, and finally, the data are sorted, and a life curve graph is drawn by using Prism 9.0.

EXAMPLE 5 Abdominal egg storage experiment

After the L4-phase wild NL5901 nematode is selected to feed for Dubosiella hours, bleach liquid is used for cracking the nematode, the number of eggs stored in the nematode belly is counted, and the experiment is independently repeated three times.

Example 6 swimming experiments

After Dubosiella h of feeding to L4 wild type NL5901 nematodes, the nematodes were transferred to M9 buffer. Counting the times of body swinging within 30 seconds, wherein the change of the distortion direction of the middle part of the nematode body is one swinging. Experiments were independently repeated three times.

Example 8 construction of PFF mouse model

In this example, a PFF-induced chronic PD mouse model was established with reference to existing experimental methods. First, the basic characteristics of PFF used in experimental injection were examined, and after treatment, the PFF was observed and photographed by electron microscopy and counted to have a length mainly ranging from 30 to 70nm and an average length of 58.52 nm.+ -. 1.30nm (FIGS. 1A to C), and the PFF was observed to gradually spread to other brain regions with time increase 60 days and 270 days after injection into the striatum (FIG. 1D). Immunofluorescence imaging was performed on brain areas such as striatum, cortex, hippocampus, substantia nigra, etc. 270 days after injection to detect pSer 129-alpha-Syn expression, i.e., lewy-body, formation (FIG. 1E). Taken together, it is demonstrated that alpha-Syn pathology gradually spreads toward nearby brain regions after striatal injection of PFF and induces expression of pSer 129-alpha-Syn in the mouse brain, indicating the feasibility of PFF modeling in constructing PD models.

Example 9 construction of PD mice models with PD-related motor dysfunction and Constipation phenotypes

In this example, an experimental scheme was designed to test the molding effect of the PFF. A chronic PD mouse model of PFF brain stereotactic injection simulating pathological and behavioral changes in PD was selected. In the PD mice model cultivated to september, the development of each mouse was not significantly affected (fig. 2A). Further apply classical commentaries on classics stick, climbing pole test experiment detects mouse motion and balancing ability. The results showed a significant increase in turn time and total time in rod climbing behaviours for PD mice (fig. 2B-C). The residence time of PD mice in the rotating rod is significantly reduced (figure 2D), and the result of the cerebral pathology immunofluorescence staining shows that the DA energy neuron number of the substantia nigra brain region of the WT-PFF group is significantly reduced compared with that of the control group (figures 2E-F), and the data show that the PFF model-building mice have obvious behavioural and pathological phenotypes of the PD mice and can be used for the next study along with the significant reduction of neurotransmitter dopamine content in striatum bodies (figure 2G).

EXAMPLE 10 the most pronounced change in intestinal flora of PD mice at Dubosiella

In this example, stool samples from PD and healthy control groups were sequenced by 16s DNA and differential flora between the two groups was analyzed.

The results showed that the WT-PFF group showed a significant decrease in the flora at seed level Dubosiella, faecalibaculum, coriobacteriaceae-UCG-002, unclassified-f-EGGERTHELLACEAE, and a significant increase in the flora at Candidatus-Saccharimonas,Lactococcus,norank-f-Ruminococcaceae,Lachnospiraceae-NK4A136-group,Bacillus, where the Dubosiella variation was most pronounced (FIG. 3), and further we designed the relevant experiments to investigate whether Dubosiella is a novel probiotic involved in the PD pathogenesis, and hopefully could search at the gastrointestinal level for a new therapeutic strategy for pre-PD disease progression through the brain intestinal shaft.

EXAMPLE 11 neurotransmitter-related metabolic disorders in the intestinal tract of PD mice

The use of a cluster map and VIP histogram in this example to show the change in WT-Vehicle/WT-PFF neurotransmitters is shown in fig. 4, and the results indicate that Serotonin,5-Hydroxy-L-tryptophan, L-Glutamate, L-Glutamic acid, L-Tyrosine, etc. levels are significantly elevated in the PD mouse model. Wherein Serotonin is a key regulator of gastrointestinal motility and secretion, and about 90% of Serotonin in humans is secreted by enteroendocrine cells of the intestinal epithelium, enteropheochromophile cells, and Serotonin receptors expressed in gastrointestinal tissues are diverse and widely distributed and are involved in the regulation of gastrointestinal function.

Meanwhile, the content of phosphatidylethanolamine PE (15:0/20:2 (11Z, 14Z)), PE (15:0/20:4 (8Z, 11Z,14Z, 17Z)), 1-palmitoyl-2-stearoyl-sn-glycero DG (16:0/18:4 (6Z, 9Z,12Z, 15Z)/0:0) and the like are remarkably reduced in a PD mouse model, wherein PE (15:0/20:4 (8Z, 11Z,14Z, 17Z)) is glycerophospholipid (hereinafter referred to as PE), and the existing research shows that the PE content of SNc of sporadic PD patients is obviously lower than that of a control group, and the levodopa treatment can be corrected back to normal level, so that PE plays a very important role in the pathogenesis of PD. The present study for the first time found that in PFF-induced PD chronic mouse models, PE levels in the gut were significantly reduced, accompanied by increased levels of neurotransmitters such as serotonin. The results indicate that there are conditions associated with disturbances of intestinal flora and neurotransmitter metabolism at the periphery of the PD mouse model.

EXAMPLE 12 intestinal flora based on Dubosiella intervention in intestinal flora diversity by modulating Serotonin and PE-like transmitters

In this example, the correlation of the bacterial flora with neurotransmitters was evaluated using a spearman correlation analysis. The results are shown in FIG. 5A as Dubosiella, coriobacteriaceae-UCG-002 can significantly down regulate Serotonin and significantly forward regulate PE-15. Most interesting is that Serotonin is significantly positively correlated with intestinal flora diversity indexes Ace and Shannon, significantly negatively correlated with MDS1, and that PE is significantly negatively correlated with Ace and Shannon, and significantly positively correlated with MDS1, as shown in FIG. 5B, which shows that Dubosiella affects intestinal flora diversity by modulating transmitters such as Serotonin and PE.

It was revealed above that Dubosiella plays a key role in mediating peripheral intestinal flora and metabolic disorders, which are significantly related to Serotonin, PE, which regulate gastrointestinal function, then Dubosiella could be used as a probiotic to improve constipation phenotype or behavioral disorders in PD mice by anaplerosis etc?

Example 13 Dubosiella significantly improved the PD kinematic disorders and constipation phenotype of aged rats

In this example, preliminary studies were made at the mouse and nematode level, and the experimental design is shown in FIG. 6A. In one aspect, the inventors' study indicated that Dubosiella significantly increased the number of fecal particles excreted over 24h and 2h, improving the constipation phenotype of 19 month old WT aged mice (fig. 6B-C).

This example also evaluates the fine-tuning of the movement of PD mice, and the results demonstrate that Dubosiella can significantly improve nesting behavior in 19 month old hA53T mice, with significant increases in the non-nesting material weight and nesting score in Dubosiella mice (fig. 6D-E). The above data indicate Dubosiella can improve constipation phenotype and regulate fine movement of PD in aged rats.

The effect of Dubosiella on nematode longevity and PD nematode locomotor function was further assessed using PD nematodes (NL 5901 nematode line) and WT nematodes (N2 nematode line). Feeding nematodes with different ratios of UV-inactivating mixture Dubosiella and OP50 at 1:1/1:2/1:4 was found to extend the mean life of the nematodes to varying degrees (FIGS. 6G-H). The above data illustrates that Dubosiella may act as a probiotic to delay aging, and what is then the specific mechanism is based on the previous study, spearman analysis indicated that Dubosiella could significantly negatively regulate Serotonin content, and that bacillus licheniformis isolated from korean traditional food sources enhanced longevity of c.elegans by modulating Serotonin signals, thus further exploring the regulatory effect of Dubosiella on Serotonin.

The oviposition process of nematodes is laid by serotonergic neurons-hermaphrodite specific motor neurons (hermaphrodite specific motor neurons, HSN) specifically modulating internalizing oviposition muscles (egg-laying muscles, ELMs), and oviposition behavior has become an important phenotypic analysis method for neural signal transduction mechanisms. HSN is necessary for normal spawning, and activation of HSN triggers spawning. Thus, the oviposition behavior of nematodes was studied by means of this behavioral paradigm to investigate the effect Dubosiella on the nematode Serotonin. As shown in fig. 6I, the study results show that the oviposition of the nematodes is significantly inhibited under the condition that the feeding concentration ratio is Dubosiella:op50=1:1/1:2, the number of eggs in the abdomen of the nematodes is significantly increased, and direct evidence is provided for Dubosiella negative regulation Serotonin. At the same time, dubosiella was found to extend nematode life to varying degrees, indicating that durum's effect on life regulation was associated with Dubosiella regulation Serotonin in this study. Further, the number of the swinging times of the nematodes in 30 seconds is recorded and evaluated in real time through the swimming video of the nematodes, and the research shows that the ultraviolet inactivating mixture Dubosiella and the OP50 for feeding the nematodes in the ratio of 1:2 can remarkably improve the exercise capacity of the nematodes in NL5901 and N2 strains (fig. 6J-K). Taken together with the experimental data results of mice, it is clear that Dubosiella can improve motor dysfunction in PD mice and nematodes. At the same time, the direct regulatory effect of Dubosiella on Serotonin was clarified at the nematode level.

It will be understood by those of ordinary skill in the art that the foregoing embodiments are specific examples of carrying out the invention and that various changes in form and details may be made therein without departing from the spirit and scope of the invention.

Claims (10)

1. Use of dubosiella, characterized by:

   (i) Preventing and/or treating parkinson's disease;

   (ii) Preparing a medicament for preventing and/or treating parkinsonism;

   (iii) Preventing and/or treating constipation, and/or

   (Iv) Preparing the medicine for preventing and/or treating constipation.
2. 2. A pharmaceutical composition is characterized by comprising Dubosiella and a pharmaceutically acceptable carrier or excipient.
3. 3. The use of the pharmaceutical composition according to claim 2, for (i) preventing and/or treating parkinson's disease, (ii) preparing a medicament for preventing and/or treating parkinson's disease, (iii) preventing and/or treating constipation, and/or (iv) preparing a medicament for preventing and/or treating constipation.
4. 4. The use according to claim 3, wherein Dubosiella is not less than lxl to ⁶ CFU per ml of the pharmaceutical composition.
5. 5. The use according to claim 3, wherein the pharmaceutical composition is a solid formulation selected from at least one of tablets, troches, candies, chewable tablets, chewing gums, capsules, powders, granules, coated particles, coated tablets, enteric coated tablets and capsules.
6. 6. The use according to claim 3, wherein the pharmaceutical composition is a liquid formulation selected from at least one of an oral solution, a suspension, an emulsion and a syrup.
7. 7. A health-care product composition is 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 a food or health-care product for relieving and/or improving constipation, (iv) preparing a health-care product for relieving and/or improving Parkinson's disease, the food or health-care product composition comprises Dubosiella, and a carrier or excipient acceptable by the health-care product.
8. 8. The use of the health-care product composition according to claim 4, wherein the health-care product composition is used for (i) relieving and/or improving constipation, (ii) relieving and/or improving Parkinson's disease, (iii) preparing a food or health-care product for relieving and/or improving constipation, and (iv) preparing a health-care product for relieving and/or improving Parkinson's disease.
9. 9. The use according to claim 3, wherein the health product is selected from at least one of beverages, yogurt vinegar, fruit juices, ice cream, bread, biscuits, cereals, health bars, pastes, seasonings, milk, cheese and fermented milks.
10. 10. A method for preventing and/or treating parkinson's disease, said method comprising the steps of:

    administering to the subject a therapeutically effective amount of Dubosiella, or

    Administering to a subject a therapeutically effective amount of the pharmaceutical composition of claim 2.