Application research of parabacteroides dielsii in preparation of medicine for treating Parkinson's disease

Abstract

The invention provides application of parabacteroides dieldrii in preparation of a medicine for treating Parkinson's disease. The parabacteroides dieldrii can play a neuroprotective role through multiple mechanisms of regulating intestinal flora, reducing neuroinflammation, improving a blood-brain barrier function and the like. Animal experiments show that the active parabacteroides dielsii can significantly improve PD mouse dyskinesia, protect dopaminergic neurons, repair intestinal and central barriers, and remodel a metabolite spectrum related to nerve regulation. The intestinal symbiotic bacterium parabacteroides dieldrii provided by the invention has a good effect of treating the Parkinson's disease, and has a wide application prospect in the field of micro-ecological treatment of the Parkinson's disease.

Description

TECHNICAL FIELD

[0001] The present disclosure relates to the field of biopharmaceutical technology, in particular to the application of Parabacteroides distasonii preserved in the BeNaCulture Collection (BNCC) with the accession number BNCC354946 in the preparation of a drug for treating Parkinson's disease. BACKGROUND

[0002] Parkinson's Disease (PD) is the second most common neurodegenerative disease after Alzheimer's disease. The main clinical manifestations include progressive movement disorders such as tremor, bradykinesia and muscle rigidity, as well as various non-motor symptoms such as constipation, hyposmia and sleep disorders. The core pathological feature is the selective and progressive loss of dopaminergic neurons in the substantia nigra pars compacta of the midbrain, which leads to a significant decrease in dopamine content in the striatum, and further triggers typical motor dysfunction. Developing a Parkinson's disease treatment strategy with clear efficacy, novel mechanism of action and high safety has become an important direction in the field of medical research.

[0003] Currently, the treatment of PD mainly focuses on symptom control with drugs. The most commonly used is dopamine replacement therapy, such as levodopa and its derivatives. Drug therapy can solve the problem of dopamine deficiency and has significant initial effect, but often cannot alleviate all signs and symptoms, and patients usually develop complications over time. For patients who are ineffective for oral medications, device and surgical therapies such as deep brain stimulation may be effective, but these methods have safety and therapeutic limitations. Gene therapy for patients with Parkinson's disease involves permanent genetic changes, which raises special safety and delivery considerations, and the clinical efficacy and safety need to be observed for a long time before they can be determined.

[0004] In recent years, more and more studies have revealed that there is a bidirectional regulatory relationship between the gut and the brain, that is, the "gut-brain axis", Parkinson's disease is no longer simply regarded as a central nervous system disease, and the gut as an important target in the early stage of the disease has attracted widespread attention. A large number of clinical and basic researches have confirmed that PD patients have gastrointestinal dysfunction, such as constipation, intestinal peristalsis slowing down, etc. before the appearance of motor symptoms. The imbalance of intestinal flora not only affects the host immune status, but also affects the central nervous system through metabolites, neurotransmitters and inflammatory factors, and participates in the pathogenesis of PD. Therefore, targeting the intestinal microecological intervention of PD course has become a research strategy that has been paid attention to in recent years. Fecal bacteria transplantation, probiotics, prebiotics and antibiotics are commonly used ways to regulate intestinal flora. At present, several genera of bacteria have been reported to have potential application in metabolic diseases, inflammatory bowel disease and neurodegenerative disease. However, the therapeutic effect of the existing microecological intervention strategy in Parkinson's disease is still uncertain, and its stability, strain dependence and individual differences limit its wide application and clinical transformation. Therefore, exploring new functional symbiotic strains has become an important direction of probiotic treatment of PD research.

[0005] Parabacteroides distasonis (P. distasonis) is a symbiotic bacterium widely present in the intestines of humans and animals, which can play a protective role in various disease models by regulating intestinal flora balance, enhancing intestinal barrier function, inhibiting inflammatory response and oxidative stress, etc. P. distasonis significantly improved the neurological impairment and reduced vascular injury and inflammatory response in the model of acute ischemic stroke combined with hyperuricemia by regulating the "gut flora-intestine-brain axis". The patent application CN113750121A discloses the application of P. distasonis in the preparation of Alzheimer's disease drug preparations, indicating its intervention potential in central nervous system diseases.

[0006] However, P. distasonis has not been involved in the exploration of treating PD, which is expected to open up a new way to treat such diseases. SUMMARY

[0007] In view of the application defects of the existing diseases in clinical and medical treatment, the application provides a use of P. distasonis in the preparation of a drug for treating Parkinson's disease. The active P. distasonis can significantly improve the motor dysfunction of PD mice, protect dopaminergic neurons, reduce neuroinflammation, repair the intestinal and central barriers, and reshape the metabolite profile related to neural regulation.

[0008] To achieve the above-mentioned purpose, the application provides the following technical solutions.

[0009] This invention provides the application of *Pseudomonas diffusa* in the preparation of drugs for treating Parkinson's disease, characterized in that the standard strain of *Pseudomonas diffusa* is numbered BNCC354946.

[0010] In one embodiment of the present invention, the probiotics are used to improve movement disorders.

[0011] In one embodiment of the present invention, the probiotics are used to protect dopaminergic neurons and reduce neuroinflammation levels.

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

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

[0014] In the above-described applications, the drug contains a pharmaceutically effective dose of *Pseudomonas difficile* and a pharmaceutically acceptable carrier.

[0015] Specifically, the pharmaceutically effective dose of *Pseudomonas dilatatus* is 10. 8 ~10 9 CFU.

[0016] The pharmaceutically acceptable carriers are milk powder, lactose, cyclodextrin, maltose, glucose, glycerol, monosodium glutamate, vitamin C, mannose, galactose, mannitol, or methylcellulose.

[0017] The method of intervention of *Peribrobacterium difficile* in MPTP-induced PD mice was as follows: The model was established by intraperitoneal injection of MPTP, with daily intraperitoneal injections of MPTP and probenecid for 4 consecutive weeks. Then, 10 mg of MPTP was administered orally daily. 8 ~10 9 0.1 mL of CFU-containing *Pseudomonas difficile* active bacterial agent was administered over two weeks.

[0018] The beneficial effects of this invention compared to the prior art are as follows:

[0019] This invention utilizes *Pseudomonas dell's disease* to treat Parkinson's disease and examines the therapeutic effect of *Pseudomonas dell's disease*. The results demonstrate that administration of *Pseudomonas dell's disease* can significantly improve motor dysfunction in PD mice, protect dopaminergic neurons, reduce neuroinflammation, repair the intestinal and central nervous system barriers, and reshape the metabolite profile related to neuroregulation. Attached Figure Description

[0020] Figure 1 The diagram shows the effect of *Pseudomonas dilatatus* on motor dysfunction in PD mice in Example 2; A, a schematic diagram of the open field test; B, statistical results of the open field test; C, results of the grip test; D, results of the pole climbing test.

[0021] Figure 2 This is a diagram showing the effect of *Pseudomonas dilatatus* on immunohistochemical staining of tyrosine hydroxylase (TH) in dopaminergic neurons of the substantia nigra of PD mice in Example 2; A, a schematic diagram of TH immunohistochemical staining in the substantia nigra of mice; B, statistical results of TH immunohistochemical staining in the substantia nigra of mice.

[0022] Figure 3 This is a Western Blot diagram showing the effect of *Pseudomonas dilatatus* on the striatal dopaminergic neurons (TH) in PD mice, as described in Example 2; A, Western Blot bands; B, Western Blot statistical results.

[0023] Figure 4 This is a diagram showing the effect of *Pseudomonas diffusa* on the blood-brain barrier in PD mice in Example 2; A, Western Blot bands of tight junction proteins Occludin and Claudin1; B, Occludin protein expression results; C, Claudin1 protein expression results.

[0024] Figure 5 The diagram shows the effect of *Pseudomonas dilatatus* on neuroinflammation in PD mice in Example 2; A, a schematic diagram of immunofluorescence staining of GFAP and Iba1 in the substantia nigra of mice; B, statistical results of immunofluorescence staining of GFAP in the substantia nigra of mice; C, statistical results of immunohistochemical staining of Iba1 in the substantia nigra of mice.

[0025] Figure 6 This is a diagram showing the effect of *Pseudomonas dilatatus* on colon length in PD mice in Example 2; A, Schematic diagram of mouse colon; B, Statistical results of mouse colon length.

[0026] Figure 7 This is a graph showing the effect of *Pseudomonas dilatatus* on fecal excretion in PD mice in Example 2.

[0027] Figure 8 The diagram shows the effect of *Pseudomonas dilatatus* on the histopathology of PD mouse colon tissue in Example 2; A, schematic diagram of HE and AB-PAS staining of mouse colon tissue; B, histological scoring results of HE staining of mouse colon tissue; C, statistical results of AB-PAS staining of mouse colon tissue.

[0028] Figure 9 This is a diagram showing the effect of *Pseudomonas difficile* on serum metabolomics in PD mice in Example 2. Detailed Implementation Plan

[0029] The reagents, equipment, or applications mentioned in this embodiment can be provided commercially or in a laboratory.

[0030] The *Pseudomonas dignitaria* used in this invention is deposited at the BeNa Culture Collection (BNCC), accession number BNCC354946. The pharmaceutically effective dose of *Pseudomonas dignitaria* described in this invention is 10... 8 ~10 9 CFU.

[0031] In this embodiment, *Pseudomonas dignitaria* is merely an application description of this application, while analogues of *Pseudomonas dignitaria* with similar properties are also within the scope of protection of this application.

[0032] Example 1: Preparation of *Pseudomonas difficile* inoculum

[0033] The frozen *Pseudomonas diffusa* strain was thawed at 37°C and inoculated into 200 mL of sterile minced meat carbohydrate broth, along with 2 tablespoons of beef granules, 1 mg of heme chloride, and 10 mg of vitamin K1. The culture was incubated under strictly anaerobic conditions at 37°C for 48 h. After filtration, the bacterial suspension was centrifuged at 3000 g at 4°C for 20 min, the supernatant was discarded, and the bacterial precipitate was washed with sterile physiological saline. The precipitate was then resuspended in 30% (v / v) glycerol to obtain the active bacterial agent, which can be stored at -80°C.

[0034] Before use, dilute the live bacteria agent with sterile physiological saline and determine the bacterial density at OD600. The concentration of the live bacteria agent is 10. 8 ~10 9 CFU / mL.

[0035] Example 2: Experimental study on the treatment of MPTP-induced Parkinson's disease using *Pseudomonas dilatatus*.

[0036] A chronic PD mouse model was induced by intraperitoneal injection of MPTP. MPTP was dissolved in sterile saline at a dose of 20 mg / kg, and probenecid was dissolved in dimethyl sulfoxide at a dose of 250 mg / kg. The injections were administered once daily for 4 consecutive weeks.

[0037] Control group (normal control group): Daily intraperitoneal injection of sterile saline for 4 consecutive weeks. Then, daily oral supplementation of 0.1 mL sterile saline for 2 weeks.

[0038] MPTP group (model group): The model was established by intraperitoneal injection of MPTP. MPTP and probenecid were injected intraperitoneally daily for 4 consecutive weeks. Then, 0.1 mL of sterile saline was administered orally daily for 2 weeks.

[0039] The *P. distasonis* group (live bacteria group): The model was established using the MPTP intraperitoneal injection method. MPTP and probenecid were injected intraperitoneally daily for 4 consecutive weeks. Then, 0.1 mL of *P. distasonis* live bacteria were supplemented orally daily for 2 weeks.

[0040] HK-P. distasonis group (inactivated bacteria group): The model was established by intraperitoneal injection of MPTP. MPTP and probenecid were injected intraperitoneally daily for 4 consecutive weeks. Then, the active bacteria of P. distasonis were inactivated by pasteurization, and 0.1 mL of inactivated bacteria was supplemented orally daily for 2 weeks.

[0041] Seven days later, behavioral experiments were conducted on the mice, which were then euthanized for sample collection.

[0042] Key indicators: behavioral level detection, dopaminergic neuron level, and blood-brain barrier level.

[0043] Secondary indicators: neuroinflammation level, colon length, stool volume, colon histopathology, and blood metabolomics.

[0044] Statistical analysis methods: Experimental data were analyzed using GraphPad Prism software (v9.0), and results are expressed as mean ± standard error (SEM). Significance of differences between groups was determined using one-way ANOVA with multiple comparisons; p < 0.05 was considered statistically significant.

[0045] result:

[0046] Behavioral level assessment: using the open field test ( Figure 1 A and 1B), gripping test ( Figure 1 C) and pole climbing experiment ( Figure 1 D) Quantify spontaneous activity, muscle tone, and motor coordination in mice to assess the ameliorative effect of *P. distasonis* on the behavioral performance of Parkinson's disease mice. Compared with the MPTP group, mice in the *P. distasonis* group showed significantly improved motor abilities, including significantly increased total distance traveled and movement speed in the open field test, enhanced forelimb grip strength, and shortened pole-climbing time; however, loss of *P. distasonis* activity had no ameliorative effect on motor impairment in PD mice.

[0047] Dopaminergic neuron levels: Figure 2 A shows the TH immunohistochemical staining results in the substantia nigra of the midbrain. Figure 2 B represents the corresponding quantitative analysis of optical density; Figure 3 A represents the Western blot detection of TH protein in the striatum. Figure 3B represents the quantitative result of relative grayscale values. The protective effect of *Pseudomonas dilatatus* on dopaminergic neurons was evaluated by detecting TH proteins in the substantia nigra and striatum. Live bacteria intervention significantly protected TH-positive dopaminergic neurons in the substantia nigra and striatum; however, no similar neuroprotective effect was observed with inactivated bacteria treatment.

[0048] Blood-brain barrier level: Figure 4 A shows the Western blot results of the tight junction proteins Occludin and Claudin-1 in the midbrain region. Figure 4 B and 4C represent the corresponding quantitative analyses. The protective effect of *P. distasonis* on the blood-brain barrier structure was assessed by detecting the expression levels of tight junction proteins in the midbrain region. Compared with the MPTP group, the expression levels of Occludin and Claudin-1 in mice in the *P. distasonis* group were significantly increased, suggesting an improvement in the blood-brain barrier; while inactivated bacterial treatment did not produce a similar effect.

[0049] Neuroinflammatory level: Figure 5 Image A shows immunofluorescence staining of GFAP and Iba1 in the substantia nigra region of the midbrain. Figure 5 B and 5C were used for quantitative analysis of optical density. Immunofluorescence staining was used to detect the expression levels of GFAP and Iba1 in the substantia nigra region to assess the regulatory effect of *P. distasonis* on astrocytes and microglia. Compared with the MPTP group, the expression of GFAP and Iba1 in the substantia nigra of mice was decreased, and the central inflammatory response was reduced; however, inactivated bacterial treatment did not show a significant anti-inflammatory effect.

[0050] Gastrointestinal function and structure: Figure 6 A is a picture of the colon. Figure 6 B represents the quantitative results of colon length. The effect of *P. distasonis* on gastrointestinal function was assessed by measuring colon length. Compared with the MPTP group, the colon length of mice in the *P. distasonis* group was significantly increased, suggesting that it has a role in alleviating gastrointestinal dysfunction, while inactivated bacteria treatment did not show significant improvement. Figure 7 The statistical results of the number of fecal particles expelled within 15 minutes show that, compared with the MPTP group, P. distasonis treatment significantly increased the number of stools, indicating that it helps improve intestinal motility. Inactivated bacteria intervention had no similar effect. Figure 8 Image A shows colon stained with HE and AB–PAS. Figure 8 B and 8C represent histological scores and quantitative analyses of AB–PAS-positive areas, respectively. Results showed that, compared to the MPTP group, P. distasonis treatment improved intestinal mucosal structure, including intestinal epithelial integrity and the recovery of goblet cell count, while inactivated bacteria treatment had no significant effect.

[0051] Metabolomics: Non-targeted metabolomics analysis revealed that the serum levels of several key metabolites were significantly upregulated in P. distasonis mice, including inosine, formononetin, N-acetyl-L-tyrosine, and 3′,4′-Di-O-methylquercetin. Figure 9 The aforementioned metabolites involve purine metabolism, flavonoid metabolism, and amino acid derivative metabolism, and possess antioxidant, anti-inflammatory, or neuromodulatory potential, suggesting that *Pseudomonas dignitaries* may participate in gut-brain signaling regulation and neuroprotective processes by modulating the host-microbiota metabolic network.

[0052] The various embodiments of this disclosure have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or technical improvements to the embodiments in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. Application of *Pseudomonas difficile* with accession number BNCC354946 in the preparation of drugs for treating Parkinson's disease.

2. The application of claim 1, comprising: Used to improve movement disorders.

3. The application of claim 1, comprising: Used to protect dopaminergic neurons.

4. The application of claim 1, comprising: It is used to reduce the level of neuroinflammation.

5. The application of claim 1, comprising: It is used to improve blood-brain barrier damage.

6. The application of claim 1, comprising: Used to improve gastrointestinal symptoms.

7. The application of claim 1, comprising: It is used to remodel purine metabolism, flavonoid metabolism and / or amino acid derivative metabolism related to neural regulation.

8. The application according to any one of claims 1-7, characterized in that, The drug contains a pharmaceutically effective dose of *Pseudomonas difficile* and a pharmaceutically acceptable carrier.

9. The application according to claim 8, characterized in that, The pharmaceutically effective dose is 10. 8 ~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, monosodium glutamate, vitamin C, mannose, galactose, mannitol or methylcellulose.

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