Application research of dunaliella or metabolite of dunaliella in preparation of medicine for preventing or treating Parkinson's disease

Abstract

The invention relates to application research of Dunaliella sp. Or metabolites thereof in preparation of drugs for prevention or treatment of Parkinson's disease, Dunaliella sp. Can produce L-DOPA and analogues thereof through metabolism, and the Dunaliella sp. Is a promising drug for treatment of Parkinson's disease. In addition, the main metabolite indoleacetic acid (IAA) of the dunaliella can significantly improve the dyskinesia of PD mice, relieve the loss of dopaminergic neurons in the brain and reduce neuroinflammation, thereby delaying the PD process. The intestinal symbiotic bacterium Dunaliella sp. And the metabolite thereof provided by the invention have a good effect of treating or preventing Parkinson's disease, and the Dunaliella sp. Metabolites to generate L-DOPA and analogues thereof, so that the Dunaliella sp. Is a potential PD treatment medicine and is suitable for clinical popularization.

Description

Technical Field

[0001] The present disclosure relates to the field of biopharmaceutical technology, and particularly to an application study of a duodenal bacterium preserved in the German Collection of Microorganisms with a preservation number of DSM 103457 or its metabolite in the preparation of a drug for preventing or treating Parkinson's disease. Background Art

[0002] Parkinson's Disease (PD) is a common neurodegenerative disease in the middle-aged and elderly in China. It is mainly characterized by the pathological changes of progressive degeneration of dopaminergic neurons in the substantia nigra and the formation of Lewy bodies, the biochemical changes of decreased dopamine neurotransmitter in the striatum area and the imbalance between dopamine and acetylcholine neurotransmitters, and the clinical manifestations of motor symptoms such as tremor, muscle rigidity, bradykinesia, and postural balance disorder, as well as non-motor symptoms such as hyposmia, constipation, sleep behavior disorders, and depression. Parkinson's disease is the disease with the fastest growth rate in terms of prevalence, disability rate, and mortality among neurological diseases. The average onset age of this disease is between 50 and 60 years old, and the overall prevalence rate of people over 65 years old in China is 1700 / 100,000. With the increase in age, the prevalence rate of Parkinson's disease will gradually increase, the condition will show progressive aggravation, and Parkinson's disease cannot be cured completely, with a certain disability rate, which affects the daily work and learning ability of patients. This is not only a serious health problem, but also a huge burden on patients and social economy. Therefore, the research on the pathogenesis and intervention of Parkinson's disease has become a frontier hot spot in scientific and technological innovation. Finding and developing more effective drugs for treating Parkinson's disease has become one of the urgent tasks for mankind.

[0003] In clinical treatment, as a neurodegenerative disease, there is currently no effective cure for Parkinson's disease. The current commonly used treatment methods mainly include comprehensive interventions of drugs, surgery, and rehabilitation training. Among them, drug treatment is still the core treatment method. Drug treatment is mainly based on dopamine replacement therapy (levodopa (L-DOPA) and its analogs), which can quickly relieve motor symptoms, but long-term use may lead to fluctuations in efficacy (such as the "on-off phenomenon") and dyskinesia. Surgical treatment mainly based on deep brain stimulation (DBS) has a relatively high risk (infection, bleeding, etc.), requires strict screening of indications and is costly; "Expert Consensus on the Management of Gait Disorders in Parkinson's Disease in China" recommends physical therapy and rehabilitation medicine (whole-body vibration training, visual cue gait training, combined with traditional Chinese medicine acupuncture and massage, etc.) as a supplement to drugs and surgery, but the efficacy of this depends on the patient's compliance and it is difficult to replace drugs or surgery.

[0004] In many Parkinson's disease patients, gastrointestinal symptoms often precede typical motor symptoms by several years or even decades, such as constipation, suggesting that there may be a certain connection between the gut microbiota and Parkinson's disease. Some scholars retrospectively analyzed patients who had undergone gastric vagus nerve trunk resection and found that the risk of Parkinson's disease in patients was significantly reduced 20 years after the surgery compared to the control group that did not undergo the surgery. Therefore, it is believed that the occurrence of Parkinson's disease may be related to the gastrointestinal tract and spread to the brain through the vagus nerve. In recent years, the view that the gut is the "second brain" of humans has gradually been recognized by more and more scholars. The gut can interact with the brain through the nervous, immune, and endocrine systems to form the "gut-brain" axis. Gut microbiota directly or indirectly participate in the communication network of the "gut-brain" axis through themselves and their metabolites, thereby affecting the course of Parkinson's disease. Entering the 21st century, probiotics have received attention due to their benefits to host health. Probiotics exert their effects by maintaining the balance of the gut microbiota, enhancing the host's immune response, and inhibiting the growth of harmful microorganisms, and are considered a promising non-antibiotic treatment method. However, the effects of probiotic products on the market are often limited by the choice of strains and survival rates, which means that more effective microbial strains need to be found to combat Parkinson's disease.

[0005] The invention patent with the authorization number CN111849816B discloses that the strain Dubosiella newyorkensis NYUBLA4 of the genus Dubosiella can extend the lifespan of Caenorhabditis elegans without affecting the normal vital sign of Caenorhabditis elegans, the swallowing frequency, and is safe and non-toxic to the nematodes. The patent application of CN117018038A discloses the application of Dubosiella newyorkensis or a probiotic preparation containing Dubosiella newyorkensis in the preparation of products for preventing, alleviating, or improving Alzheimer's disease.

[0006] However, there has been no exploration of Dubosiella in the prevention and treatment of PD, and it is expected to open up a new way to treat such diseases. Summary of the Invention

[0007] Aiming at the application defects of existing diseases in clinical and medical treatment, the present invention provides the application of a strain of Dubosiella newyorkensis and its metabolites in the preparation of drugs for preventing / treating Parkinson's disease. Among them, Dubosiella can metabolize to produce L-DOPA and its analogs, which are promising drugs for the treatment of Parkinson's disease. In addition, the main metabolite of Dubosiella, indole-3-acetic acid (IAA), can significantly improve the motor disorders of PD mice, relieve the loss of dopamine neurons in the brain, and reduce neuroinflammation, thereby delaying the progression of PD.

[0008] To achieve the above object, the present invention proposes the following technical solutions:

[0009] The present invention provides the use of Dubosiella newyorkensis and / or its metabolites in the preparation of a medicament for preventing and / or treating Parkinson's disease.

[0010] In one embodiment of the present invention, the probiotic is used to improve dyskinesia.

[0011] In one embodiment of the present invention, the probiotic is used to improve neuroinflammation.

[0012] In one embodiment of the present invention, the probiotic is used to improve blood-brain barrier damage.

[0013] In one embodiment of the present invention, the probiotic is used to improve gastrointestinal symptoms such as constipation.

[0014] For the above-mentioned use, the medicament contains a pharmaceutically effective dose of Dubosiella newyorkensis and / or its metabolites and a pharmaceutically acceptable carrier.

[0015] Specifically, the pharmaceutically effective dose of Dubosiella newyorkensis is 10 8 ~10 10 CFU. The present invention preferably uses a pharmaceutically effective dose of Dubosiella newyorkensis of 10 9 CFU.

[0016] The pharmaceutically acceptable carrier is milk powder, lactose, cyclodextrin, maltose, glucose, glycerol, sodium glutamate, vitamin C, mannose, galactose, mannitol or methylcellulose.

[0017] The method of Dubosiella newyorkensis intervening in MPTP-induced PD mice is as follows: Each mouse is orally administered 1×10 9 CFU of Dubosiella newyorkensis, and the same operation is performed every other day for a total of two supplements, which can effectively relieve the PD-like symptoms of mice induced by 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine hydrochloride (MPTP).

[0018] The present invention also provides the use of the intestinal symbiotic bacterial metabolite IAA of the above-mentioned Dubosiella newyorkensis in the preparation of a medicament for treating or preventing PD.

[0019] Furthermore, Dubosiella newyorkensis can metabolize to produce L-DOPA and its analogs;

[0020] Furthermore, Dubosiella newyorkensis can metabolize to produce IAA (indoleacetic acid); Oral supplementation of IAA to mice for one week has the same function as Dubosiella newyorkensis in relieving MPTP-induced PD-like symptoms.

[0021] Compared with the prior art, the present invention has the following beneficial technical effects:

[0022] The intestinal symbiotic bacterium Bacteroides dorei and its metabolites provided in the present invention have a good effect on treating or preventing Parkinson's disease, and Bacteroides dorei metabolizes to produce L-DOPA and its analogs, which are potential therapeutic drugs for PD and are suitable for clinical promotion. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a diagram showing the effect of Bacteroides dorei on the motor dysfunction of MPTP-induced PD mice in Example 2;

[0024] Figure 2 It is a diagram showing the effect of Bacteroides dorei on dopaminergic neurons of MPTP-induced PD mice in Example 2;

[0025] Figure 3 It is a diagram showing the effect of Bacteroides dorei on the blood-brain barrier of MPTP-induced PD mice in Example 2;

[0026] Figure 4 It is a diagram showing the effect of Bacteroides dorei on neuroinflammation of MPTP-induced PD mice in Example 2;

[0027] Figure 5 It is a diagram showing the effect of Bacteroides dorei on MPTP-induced PD mice under the state of intestinal bacteria clearance in Example 2;

[0028] Figure 6 It is a diagram showing the effect of Bacteroides dorei on the changes of serum metabolites of MPTP-induced PD mice in Example 3;

[0029] Figure 7 It is a diagram showing the effect of Bacteroides dorei on the level of L-DOPA in the serum of MPTP-induced PD mice in Example 3;

[0030] Figure 8 It is a diagram showing the effect of Bacteroides dorei on the changes of fecal metabolites of MPTP-induced PD mice in Example 3;

[0031] Figure 9 It is a diagram showing the effect of the production level of dopa analogs in the culture supernatant of Bacteroides dorei in Example 3;

[0032] Figure 10 It is a diagram showing the histopathological effect of IAA on the brain injury of MPTP-induced PD mice in Example 4;

[0033] Figure 11 It is a diagram showing the effect of IAA on neuroinflammation of MPTP-induced PD mice in Example 4;

[0034] Figure 12It is a graph showing the effect of IAA on the behavior of MPTP-induced PD mice in Example 4. Specific implementation method

[0035] The reagents, equipment, applications, etc. mentioned in this example can be provided by the market or laboratory.

[0036] The Dubosiella newyorkensi used in the present invention is preserved at the German collection of microorganisms, with the preservation number DSM 103457. The preferred pharmaceutically effective dose of Dubosiella in the present invention is 1×10 9 CFU.

[0037] In this implementation method, the Dubosiella or its metabolites are only described for the application of this application, and analogs of Dubosiella or its metabolites with the same properties are also within the protection scope of this application.

[0038] Example 1

[0039] Preparation of Dubosiella agent

[0040] The frozen Dubosiella strain is thawed at 37°C, inoculated into 200 mL of sterilized cooked meat liquid medium, and 2 spoons of cooked meat beef granules, 0.05 mg of cysteine, and 0.8 g of glucose are added. It is cultured under strict anaerobic conditions at 37°C for 48 h. After filtration, the bacterial liquid is centrifuged at 3000 g and 4°C for 20 min, the supernatant is discarded, the bacterial precipitate is washed with sterile physiological saline, and the bacterial precipitate is resuspended with 30% (v / v) glycerol to obtain an active agent, which can be stored in a -80°C refrigerator.

[0041] Before use, the active agent is diluted with sterile physiological saline, and the bacterial density is measured at OD 600 nm. The concentration of the active agent is 1×10 10 CFU / mL.

[0042] Example 2

[0043] Experimental study on using Dubosiella to prevent MPTP-induced Parkinson's disease.

[0044] An acute PD mouse model is induced by intraperitoneal injection of MPTP. MPTP is dissolved in sterile physiological saline and injected once a day, with a dosage of 25 mg / kg, and continuously injected for 7 days.

[0045] Control group (normal control group): Oral supplementation of sterile physiological saline, 2 days / time, for a total of 2 times. From the fifth day, sterile physiological saline is intraperitoneally injected daily for 7 consecutive days.

[0046] MPTP group (model establishment group): Oral administration of sterile normal saline, twice a day for 2 days. From the fifth day, the model was established by intraperitoneal injection of MPTP for 7 consecutive days.

[0047] HK-Dub group (inactivated bacteria group): Inactivate the active bacteria agent of Dubu bacillus by pasteurization. Oral administration of 0.1 mL of inactivated bacteria, twice a day for 2 days. From the fifth day, the model was established by intraperitoneal injection of MPTP for 7 consecutive days.

[0048] Dub group: Supplement 1×10 9 CFU / time, twice a day for 2 days. From the fifth day, the model was established by intraperitoneal injection of MPTP for 7 consecutive days.

[0049] Abx+MPTP group (model establishment group after antibiotic clearance of intestinal flora): Mice were gavaged daily with vancomycin 2 g, ampicillin 4 g, neomycin sulfate 4 g, and metronidazole 1 g dissolved in 12 mL of sterile water respectively, and 0.1 mL was orally gavaged respectively for 5 consecutive days. After 5 days, oral administration of sterile normal saline, twice a day for 2 days. After oral administration of sterile normal saline twice, the model was established by intraperitoneal injection of MPTP for 7 consecutive days.

[0050] Abx+MPTP+Dub group (group supplemented with active bacteria of Dubu bacillus after antibiotic clearance of flora): After 5 days of antibiotic gavage, supplement 1×10 9 CFU / time, twice a day for 2 days. After the second colonization of Dubu bacillus, the model was established by intraperitoneal injection of MPTP for 7 consecutive days.

[0051] Seven days after intraperitoneal injection of MPTP, the behavior of mice was measured, and each group was sacrificed and sampled after behavioral detection.

[0052] Main indicators: Behavioral level detection, dopaminergic neuron level, blood-brain barrier level.

[0053] Secondary indicators: Neuroinflammatory level, protective effect of Dubu bacillus on PD mice after antibiotic clearance of intestinal flora.

[0054] Statistical analysis method: Experimental data were analyzed using GraphPad Prism software (v8.0), and the results were expressed as mean ± standard error of the mean (SEM). The significance of differences between groups was determined by unpaired two-tailed Student's t-test, and p<0.05 was considered statistically significant.

[0055] Results:

[0056] Behavioral level detection: The grip strength test was used to evaluate the muscle strength level of mice, and the pole climbing test and rotarod test were used to evaluate the motor coordination ability of mice. The results showed that compared with the MPTP group, the treatment with live Bacteroides dorei significantly improved the symptoms of motor dysfunction in mice. After the loss of Bacteroides dorei activity, there was no improvement in the motor impairment of PD mice( Figure 1 ).

[0057] Dopaminergic neuron level: Immunohistochemistry was used to analyze the level of dopaminergic neurons in the substantia nigra region of PD mice induced by MPTP with live Bacteroides dorei. The results showed that compared with the MPTP group, the number of dopaminergic neurons in the substantia nigra region of mice in the Dub group was closer to that of the normal control group, and the neuron morphology was good without obvious shrinkage. After the loss of Bacteroides dorei activity, there was no improvement in the loss of dopaminergic neurons in PD mice( Figure 2 ).

[0058] Blood-brain barrier level: Western blot was used to analyze the blood-brain barrier level of PD mice induced by MPTP with live Bacteroides dorei. The results showed that compared with the MPTP group, the representative proteins of the blood-brain barrier (tight junction proteins ZO1 and Occludin) in the Dub group of mice increased significantly. After the inactivation of Bacteroides dorei, the blood-brain barrier damage in mice was aggravated( Figure 3 ).

[0059] Neuroinflammation level: Immunofluorescence staining was used to analyze the neuroinflammation level of PD mice induced by MPTP with live Bacteroides dorei. The results showed that compared with the MPTP group, the activation levels of astrocytes and microglia in the substantia nigra region of mice in the Dub group decreased significantly, indicating a decrease in the neuroinflammation level. After the inactivation of Bacteroides dorei, the neuroinflammation level in mice was aggravated( Figure 4 ).

[0060] Protective effect of Bacteroides dorei on PD mice after antibiotic clearance of gut microbiota: Western blot was used to analyze the protective effect of live Bacteroides dorei on PD mice induced by MPTP in the state of antibiotic clearance of gut microbiota. The results of the Western blot experiment showed that the neuroprotective effect of live Bacteroides dorei on PD mice was not affected by the absence of gut microbiota( Figure 5 ).

[0061] In summary, Bacteroides dorei can effectively prevent MPTP-induced Parkinson's disease.

[0062] Example 3

[0063] Bacteroides dorei increases the levels of L-DOPA and IAA in PD mice.

[0064] The experimental grouping referred to Example 2.

[0065] Main indicators: Serum untargeted metabolomics.

[0066] Secondary indicators: Fecal untargeted metabolomics and bacterial culture supernatant untargeted metabolomics.

[0067] Statistical analysis method: The experimental data was analyzed using GraphPad Prism software (v8.0), and the results were expressed as mean ± standard error of the mean (SEM). The significance of differences between groups was determined by unpaired two-tailed Student's t-test, and p < 0.05 was considered statistically significant.

[0068] Results:

[0069] (1) Serum untargeted metabolomics: Through untargeted metabolomics analysis, it was found that the levels of tryptophan, indoleacetic acid (IAA), indole-3-pyruvic acid, and indole-3-acetonitrile in the serum of mice in the dub group were significantly increased, suggesting that this bacterium may play a neuroprotective role through the tryptophan metabolic pathway ( Figure 6 ). In addition, the level of L-DOPA in the serum of mice in the dub group was significantly higher than that in the MPTP group ( Figure 7 ).

[0070] (2) Fecal untargeted metabolomics: Through untargeted metabolomics analysis, it was found that the content of Carbidopa in the feces of mice in the dub group was significantly increased ( Figure 8 ).

[0071] (3) Bacterial culture supernatant untargeted metabolomics: Through untargeted metabolomics analysis of the bacterial culture supernatant, it was found that Dubosia can secrete dopamine derivatives ( Figure 9 ).

[0072] Example 4

[0073] Experimental study on using IAA to prevent MPTP-induced Parkinson's disease.

[0074] Control group (normal control group): Oral supplementation with sterile saline, once a day for 7 times. Starting from the 7th day, sterile saline was injected daily for 7 consecutive days.

[0075] MPTP group (model group): Oral supplementation with sterile saline, once a day for 7 times. Starting from the 7th day, a model was constructed using the intraperitoneal injection method of MPTP, and MPTP was intraperitoneally injected for 7 consecutive days.

[0076] IAA group (IAA treatment group): Oral supplementation with IAA, once a day for 7 times. Starting from the 7th day, a model was constructed using the intraperitoneal injection method of MPTP, and MPTP was intraperitoneally injected for 7 consecutive days.

[0077] Seven days after intraperitoneal injection of MPTP, the behavior of the mice was measured, and each group was sacrificed and sampled after the behavioral test.

[0078] Main indicators: pathological sections of the substantia nigra region of mice

[0079] Secondary indicators: immunofluorescence staining of GFAP and IBA1 in the midbrain and behavioral testing of mice

[0080] Statistical analysis method: The experimental data were analyzed using GraphPad Prism software (v8.0), and the results were expressed as mean ± standard error of the mean (SEM). The significance of differences between groups was determined by unpaired two-tailed Student's t-test, and p < 0.05 was considered statistically significant.

[0081] Results:

[0082] Pathological sections of the substantia nigra region of mice: Immunohistochemistry combined with Nissl staining was used to analyze the level of pathological damage induced by IAA in MPTP-induced PD mice. The results showed that compared with the MPTP group, the number of dopaminergic neurons in the IAA group of mice was significantly increased, and the neuronal structure was close to that of the normal group ( Figure 10 ).

[0083] Immunofluorescence staining of GFAP and IBA1 in the midbrain: Immunofluorescence staining combined with Nissl staining was used to analyze the level of neuroinflammation induced by IAA in MPTP-induced PD mice. The results showed that compared with the MPTP group, the activation levels of astrocytes and microglia in the IAA group of mice were significantly reduced, indicating a decrease in the level of neuroinflammation ( Figure 11 ).

[0084] Behavioral testing: The pole climbing test was used to analyze the behavioral level of IAA in MPTP-induced PD mice. The results showed that compared with the MPTP group, the motor level of IAA mice increased ( Figure 12 ).

[0085] In summary, IAA can effectively prevent MPTP-induced Parkinson's disease.

[0086] The embodiments of the present disclosure have been described above. The above description is exemplary and not exhaustive, and is not limited to the disclosed embodiments. Many modifications and variations are obvious to those of ordinary skill in the art without departing from the scope and spirit of the described embodiments. The selection of terms used herein is intended to best explain the principles of the embodiments, practical applications, or improvements to technologies in the market, or to enable other ordinary skill in the art in this technical field to understand the disclosed embodiments.

Claims

1. Use of the bacterium of the genus **Dubosiella** with the deposit number DSM 103457 or its metabolite in the preparation of a medicament for preventing or treating Parkinson's disease.

2. The use according to claim 1, for improving dyskinesia.

3. The use according to claim 1, for improving neuroinflammation.

4. The use according to claim 1, for improving blood-brain barrier damage.

5. The use according to claim 1, for improving gastrointestinal symptoms.

6. The application according to claim 1, characterized in that The metabolite is the intestinal symbiotic bacterium metabolite IAA of the bacterium of the genus **Dubosiella**.

7. The application according to claim 1, characterized in that, The metabolite is the intestinal symbiotic bacterium metabolite L-DOPA of the bacterium of the genus **Dubosiella**.

8. The application according to any one of claims 1-7, characterized in that, The drug contains a pharmaceutically effective dose of 8 ~10 10 CFU of Ducibacterium or its metabolites, and a pharmaceutically acceptable carrier; wherein, the pharmaceutically effective dose is 10 9. The application according to claim 8, wherein The pharmaceutically effective dose is 10 9 CFU.

10. The application according to claim 8, characterized in that The carrier is a mixture of one or more of the following carriers: milk powder, lactose, cyclodextrin, maltose, glucose, glycerol, sodium glutamate, vitamin C, mannose, galactose, mannitol or methylcellulose.

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