Probiotic composition for improving tourette syndrome and use thereof

Abstract

The application discloses a probiotic composition for improving Tourette syndrome and application thereof, and belongs to the technical field of biological medicine. In view of the problems of dependence and side effects of existing TS treatment drugs, the application provides a probiotic composition, wherein the probiotic composition comprises Bifidobacterium breve M-16V and Bifidobacterium animalis lactis Bb-12. The composition can be used for preparing a medicine for preventing and / or treating Tourette syndrome, so as to reduce the levels of glutamic acid, dopamine and / or gamma-aminobutyric acid in the striatum of a Tourette syndrome patient, alleviate inflammatory reaction and repair intestinal barrier damage.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology. More specifically, this invention relates to a probiotic composition for improving Tourette syndrome and its application. Background Technology

[0002] Tourette syndrome (TS) is a chronic neurodevelopmental disorder characterized by motor and vocal tics. Currently, drug treatment for TS primarily relies on alpha-2 adrenergic agonists and antipsychotics. However, these medications have varying degrees of dependence and side effects, and their tolerability and safety with long-term use require improvement. Therefore, exploring new intervention strategies is of clinical significance.

[0003] Recent studies suggest that the gut microbiota may influence central nervous system function through immune, neural, and endocrine pathways, playing a role in the pathogenesis of TS. Some literature reports abnormal gut microbiota composition in children with TS, and that fecal microbiota transplantation from healthy children can alleviate symptoms in TS patients. However, fecal microbiota transplantation, as a holistic microbiota transfer method, has a complex composition, its colonization is greatly influenced by the recipient's gut microenvironment, and its effectiveness varies significantly between individuals. Furthermore, it carries potential safety risks and is therefore difficult to use directly as a routine intervention.

[0004] Intervention strategies based on single probiotic strains or simple combinations of strains have been explored, but the effects of different strains on TS-related behaviors, neurotransmitters, and inflammatory markers vary considerably. Furthermore, some studies have employed mixed interventions with multiple probiotic strains, hoping to enhance efficacy through synergistic effects. However, multi-strain combinations may lead to decreased colonization efficiency of some strains, or even weakened expected effects, due to competition for intestinal adhesion sites and interactions of metabolites among strains. Screening for probiotic strains with clear effects on TS from a vast pool of options, and determining reasonable strain combinations, their ratios, and dosages, remain key technical challenges in current research. Summary of the Invention

[0005] One object of the present invention is to provide a probiotic composition for improving Tourette syndrome and its application therein, so as to at least solve the above-mentioned problems.

[0006] To achieve the objectives and other advantages of the present invention, a probiotic composition for improving Tourette syndrome is provided, comprising Bifidobacterium breve M-16V and Bifidobacterium animalis subsp. lactis Bb-12.

[0007] Preferably, the ratio of viable counts of the *Bifidobacterium breve* M-16V to that of *Bifidobacterium lactis* Bb-12 is 1:1.

[0008] Preferably, the total dosage of the *Bifidobacterium breve* M-16V and the *Bifidobacterium animalis* subsp. *lactamase* Bb-12 is 10. 10 CFU / kg / day.

[0009] The present invention also provides the use of the above-mentioned probiotic composition for improving Tourette syndrome in the preparation of a medicament for the prevention and / or treatment of Tourette syndrome.

[0010] Preferably, the drug is used for: a) Reduce the levels of glutamate, dopamine, and / or γ-aminobutyric acid in the striatum of patients with Tourette syndrome; b) Reduce serum levels of tumor necrosis factor-α and / or interleukin-6 in patients with Tourette syndrome; and / or c) Repair colonic mucosal damage and improve intestinal barrier function in patients with Tourette syndrome.

[0011] The present invention also provides a probiotic preparation for treating Tourette syndrome, comprising effective amounts of Bifidobacterium breve M-16V and Bifidobacterium animalis subsp. lactis Bb-12, and a pharmaceutically acceptable carrier.

[0012] Preferably, the ratio of viable counts of the *Bifidobacterium breve* M-16V to that of *Bifidobacterium animalis* subsp. *lactobacter* Bb-12 is 1:1, and the dosage of the probiotic preparation is 10. 10 CFU / kg / day.

[0013] The present invention has at least the following beneficial effects: The probiotic composition provided by this invention consists of only two strains: Bifidobacterium breve M-16V and Bifidobacterium animalis subsp. lactis Bb-12, with a live bacteria ratio of 1:1 and a dosage of 10. 10Under the condition of CFU / kg / day, continuous intervention for 4 weeks significantly reduced the stereotyped behavior scores of TS model rats, with effects comparable to the positive control drug haloperidol, indicating that the combination of these two strains can effectively improve tic symptoms. Mechanistically, this probiotic composition can simultaneously reduce the abnormal elevation of three neurotransmitters—glutamate (Glu), dopamine (DA), and γ-aminobutyric acid (GABA)—in the striatum, and decrease serum levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6), thereby regulating central neurotransmitter balance and inhibiting peripheral inflammatory responses. Furthermore, histopathological analysis of colon tissue showed that this probiotic composition can repair damaged colonic mucosa in model rats, increase the number of goblet cells, restore intestinal villi and crypt structure, and improve intestinal barrier function. In summary, the probiotic composition provided by this invention has a simple strain composition, a well-defined ratio and dosage, and can alleviate the core symptoms and related complications of Tourette syndrome through multiple pathways, including neurotransmitters, inflammation, and the intestinal barrier, providing a safe and effective new adjunctive intervention for clinical practice.

[0014] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Attached Figure Description

[0015] Figure 1 HE staining results of colon tissue from rats in each group; Figure 2 Immunohistochemical analysis results of colon tissue from rats in each group. Detailed Implementation

[0016] The present invention will now be described in further detail with reference to the embodiments and accompanying drawings, so that those skilled in the art can implement it based on the description.

[0017] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0018] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0019] I. Experimental Methods 1. Candidate probiotics The candidate probiotics, including Bifidobacterium breve M-16V, Bifidobacterium animalis subsp. lactis Bb-12, Lactobacillus rhamnosus GG, and Lactobacillus salivarius, were all obtained through direct purchase.

[0020] 2. Optimization of the probiotic combined intervention program Optimal combination screening: Thirty-five male Wistar rats (4 weeks old, 100-150g, SPF grade) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All rats were housed in an SPF-grade animal room at a temperature of 22±2℃ and humidity of 35±5%, with a 12-hour light-dark cycle. All rats had free access to food and water. After one week of acclimatization, they were divided into a normal control group (Control), a model group (TS), a haloperidol positive control group (Hal), and different bacterial strain combination treatment groups: M-16V:Bb-12 group (Treat-1), LGG:Bb-12 group (Treat-2), M-16V:Bb-12:LGG group (Treat-3), and M-16V:Bb-12:LGG:Lactobacillus salivarius group (Treat-4). The normal group and the TS model group were given saline (10 mL / kg / d) daily, the positive control group received Hal (1 mg / kg / d) daily, and the Treat-1 group received a combination of M-16V and Bb-12 (10 mL / kg / d of each probiotic) daily. 10 CFU / kg / d); Treat-2 group was given LGG and Bb-12 combination daily (10 mmol / kg of each probiotic). 10 CFU / kg / day); Treat-3 group was given a combination of M-16V, LGG and Bb-12 daily (10 mg of each probiotic). 10 CFU / kg / day); Treat-4 group was given a combination of M-16V, LGG, Bb-12 and Lactobacillus salivarius daily (10 mg of each probiotic). 10 (CFU / kg / day), the probiotics used are spray-dried powder. Once daily for 4 consecutive weeks.

[0021] Optimal formulation selection: Forty-eight male C57 mice (3-4 weeks old, 20±3 g, SPF grade) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All mice were housed in an SPF-grade animal facility at a temperature of 22±2℃ and humidity of 35±5%, with a 12-hour light-dark cycle. All mice had free access to food and water. After 7 days of acclimatization, modeling and adaptive feeding were initiated for one week, followed by division into a normal group (Control), a model group (TS), and a haloperidol positive control group (Hal). Different probiotic intervention groups with varying ratios of 1:5, 5:1, 1:1, 1:10, and 10:1 were also established for the Treat-1 group.

[0022] Optimal Dosage Screening: Thirty-six male Wistar rats (4 weeks old, 100-150g, SPF grade) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. All rats were housed in an SPF-grade animal facility at a temperature of 22±2℃ and humidity of 35±5%, with a 12-hour light-dark cycle. All rats had free access to food and water. After one week of acclimatization, they were divided into a control group, a model group (TS), a haloperidol positive control group (Hal), and a Treat-1 group, and were screened at a 1:1 ratio with a lower dose of 10... 9 CFU / kg, 10 10 CFU / kg, High 10 11 CFU / kg dosage group.

[0023] TS model construction: The model group, positive drug group, and treatment group were injected intraperitoneally with IDPN (IDPN was dissolved in physiological saline and diluted to a concentration of 15 mg / mL, and the dosage was 150 mg / (kg·d)). The normal group was injected intraperitoneally with an equal volume of physiological saline. All were administered once a day for 7 consecutive days. The mice and rats in the model group showed different degrees of stereotyped behaviors and motor behaviors, and the scores were ≥ 2 points, indicating that the model was successfully established.

[0024] Drug treatment: Administered medications starting on the second day after modeling. The positive control group was given haloperidol suspension, and the treatment group was given probiotic suspension of the corresponding concentration, administered by gavage. The normal group and the model group were given an equal volume of physiological saline by gavage, once daily for 4 consecutive weeks.

[0025] 3. Construction of behavioral evaluation methods and scoring system for the TS model group After modeling, behavioral scoring was used to observe stereotyped behaviors in each group of rats / mice to confirm successful modeling. Behavioral scoring: Each rat / mouse was observed in a double-blind manner, scoring according to the following criteria (0 points: no stereotyped behavior; 1 point: rotational behavior; 2 points: excessive up-and-down head and neck movements; 3 points: excessive up-and-down head and neck movements plus rotational behavior; 4 points: lateral head movement combined with excessive up-and-down head and neck movements). The score reflected the severity of the tic disorder in the rat / mouse. Behavioral observation was only performed in the model group, the haloperidol positive drug group, and the treatment group.

[0026] 4. Effects of combined probiotic intervention on colonic tissue and intestinal barrier in TS rats Colon HE staining analysis: Colonic HE staining was performed on rats in each group. Rat colonic tissue was collected, fixed in 4% paraformaldehyde, and cut into 5-micrometer thick sections. The sections were stained with H&E and observed under an optical microscope. Immunohistochemistry (IHC) was used to detect the expression of Claudin-1 and Occludin in rat colonic tissue. Colonic sections were incubated overnight at 4 °C with rabbit anti-substance P polyclonal antibody (1:100), followed by incubation at 25 °C with horseradish peroxidase (HRP)-labeled goat anti-rabbit IgG for 50 min. Contrast staining with hematoxylin was performed at 25 °C for 2 min, and images were captured under an optical microscope.

[0027] II. Experimental Results 1. Results of the optimal combination screening experiment The weight changes and behavioral scores of rats in each group were recorded. Control group was the normal control group, TS group was the model group, Hal group was the haloperidol positive control group, Treatment-1 was M-16V:Bb-12, Treatment-2 was LGG:Bb-12, Treatment-3 was M-16V:Bb-12:LGG, and Treatment-4 was M-16V:Bb-12:LGG:Lactobacillus salivarius. The weight changes are shown in Table 1, and the behavioral scores are shown in Table 2. In the tables, * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001 compared to the model group, and the same applies below. Table 1. Changes in body weight of rats in each group Table 2. Behavioral scores of rats in each group Table 1 shows that after 2 weeks of administration, all groups showed weight gain, with no significant difference in weight gain between groups. After 4 weeks, all groups showed significant weight gain, with treatment groups 1 and 3 showing the most significant weight gain, and the model group showing the least, suggesting that combined probiotic intervention can improve developmental delay in TS rats. Table 2 shows that at week 0 after modeling, all groups had a score ≥2, indicating successful modeling. At week 2, the scores of the Hal group and all treatment groups decreased, showing initial efficacy of the intervention. At week 4, the score of treatment group 1 decreased significantly, while the improvement effects of treatment groups 3 and 4 were not better than those of treatment group 1, suggesting that the combined use of multiple probiotics did not produce a synergistic effect.

[0028] In summary, the improvement effect of the Treat-1 group was significantly better than that of the Treat-2, Treat-3 and Treat-4 groups. Although the Treat-3 and Treat-4 groups contained Treat-1 components, they did not show synergistic effects. Therefore, the Control group, TS model group, Hal group and Treat-1 group were selected for subsequent analysis.

[0029] The inflammatory factors in the serum and the neurotransmitters in the striatum of rats in the Control group, TS model group, Hal group and Treat-1 group were detected by ELISA. The results are shown in Table 3.

[0030] Table 3. Detection results of inflammatory factors in serum and neurotransmitters in striatum of rats in each group. As shown in Table 3, the levels of Glu, DA, and GABA in TS rats increased significantly, while they decreased significantly after intervention with positive drugs and in the treatment group; the levels of inflammatory factors in TS rats increased, while they decreased after probiotic intervention.

[0031] Furthermore, pathological analysis was performed on the colon tissues of rats in the Control group, TS model group, Hal group, and Treat-1 group. The results are shown in […]. Figure 1 .Depend on Figure 1 It was found that no obvious histopathological changes were observed in the colon of the normal group (Control); the mucosa of the model group was severely damaged, with obvious inflammatory cell infiltration, reduced goblet cells, significantly shortened intestinal villi, and reduced intestinal crypt depth (TS); compared with the model group, the Hal group and Treat-1 group had intact intestinal epithelial mucosal layer structure, normal epithelial cell morphology and structure, tight arrangement, increased number of intestinal glands in the local lamina propria, reduced connective tissue hyperplasia, reduced focal lymphocyte infiltration, and significantly increased abundance of mucin / mucin-producing cells (blue), which promoted the recovery of the mucosal layer during the recovery of tic symptoms.

[0032] Immunohistochemical analysis of colon tissue from TS rats yielded the following results: Figure 2 As shown, Figure 2 (a) is a diagram showing the results of hematoxylin staining. Figure 2 (b) shows the results of Claudin-1 and Occludin protein content. Compared with the Control group, the levels of Claudin-1 and Occludin proteins in the intestinal mucosa of TS rats decreased, indicating that the intestinal barrier function of TS rats was impaired and the intestinal permeability was increased, making it easier for harmful substances to enter the body through the intestine. The Hal group and Treat-1 group significantly reversed this damage, indicating that M-16V:Bb-12 has a repairing effect on the intestinal barrier.

[0033] 2. Results of the optimal ratio screening experiment Based on the results of the optimal combination screening experiment, the composite strains of the Treat-1 group were selected for further analysis.

[0034] Screening for the optimal ratio: Changes in body weight and behavioral scores of mice in each group were recorded. Control group was the normal control group, TS group was the model group, Hal group was the haloperidol positive control group, and the M-16V:Bb-12 ratios in the treatment groups were 1:5, 5:1, 1:1, 1:10, and 10:1, respectively. The results are shown in Tables 4 and 5.

[0035] Table 4. Weight changes in mice of each group Table 5. Behavioral scores of mice in each group Table 4 shows that after 2 weeks of administration, the body weight of all groups increased, with no significant difference in weight gain between groups. At 4 weeks, compared with the model group, the normal and treatment groups (1:1 ratio) showed faster body weight gain, suggesting that a 1:1 ratio of M-16V:Bb-12 can improve developmental delay in mice. Table 5 shows that at week 0 after modeling, all groups scored ≥2 points, indicating successful modeling. At week 2, the scores of all treatment groups decreased, showing initial efficacy of the intervention. At week 4, the score of the 1:1 ratio of M-16V:Bb-12 significantly decreased, indicating that 1:1 is the optimal ratio for improving mouse development.

[0036] 3. Results of the optimal dose screening experiment Based on the results of the optimal ratio screening experiment, a 1:1 ratio of M-16V:Bb-12 in Treat-1 group was selected for further analysis.

[0037] Record the changes in body weight and behavioral scores of rats in each group. Control group was the normal control group, TS group was the model group, and Hal group was the haloperidol positive control group. 9 For the low-dose group, 10 10 For the medium-dose group, 10 11 This is the high-dose group. The results are shown in Tables 6 and 7.

[0038] Table 6. Results of weight changes in rats in each group Table 7. Behavioral scores of rats in each group Table 6 shows that after 2 weeks of administration, the body weight of rats in all groups increased, with no significant difference in weight gain between groups. After 4 weeks, the body weight of all groups increased significantly, with the medium-dose group showing the most significant increase and the model group showing the least, suggesting that probiotic intervention can improve developmental delay in TS rats. Table 7 shows that at 0 weeks after modeling, all groups scored ≥2 points, indicating successful modeling. At 2 weeks, the scores of the Hal group and all treatment groups decreased, showing initial efficacy of the intervention. At 4 weeks of administration, the scores of the Hal group and the medium-dose group decreased significantly, indicating that the medium dose of compound probiotics can improve stereotyped behaviors in rats with Tourette syndrome to some extent. There was no statistically significant difference in scores between the high-dose compound probiotic group and the model group (P>0.05), suggesting that there is a physiological threshold for the intestinal microecological capacities, and excessive probiotic supplementation is unlikely to adhere and capacitate in the intestine, and will only be excreted by the body's metabolism, resulting in ineffective supplementation.

[0039] The number of devices and processing scale described herein are for the purpose of simplifying the description of the invention. Applications, modifications, and variations of the probiotic composition for improving Tourette syndrome and its uses according to the present invention will be readily apparent to those skilled in the art.

[0040] Although embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details and illustrations shown and described herein.

Claims

1. A probiotic composition for improving Tourette syndrome, characterized in that, It contains Bifidobacterium breve M-16V and Bifidobacterium animalis subsp. lactis Bb-12.

2. The probiotic composition for improving Tourette syndrome as described in claim 1, characterized in that, The ratio of viable bacteria of the *Bifidobacterium breve* M-16V to that of *Bifidobacterium lactis* subsp. Bb-12 is 1:

1.

3. The probiotic composition for improving Tourette syndrome as described in claim 2, characterized in that, The total dosage of the *Bifidobacterium breve* M-16V and the *Bifidobacterium lactis* subsp. Bb-12 was 10. 10 CFU / kg / day.

4. The use of the probiotic composition for improving Tourette syndrome as described in any one of claims 1-3 in the preparation of a medicament for the prevention and / or treatment of Tourette syndrome.

5. The application as described in claim 4, characterized in that, The drug is used for: a) Reduce the levels of glutamate, dopamine, and / or γ-aminobutyric acid in the striatum of patients with Tourette syndrome; b) Reduce serum levels of tumor necrosis factor-α and / or interleukin-6 in patients with Tourette syndrome; and / or c) Repair colonic mucosal damage and improve intestinal barrier function in patients with Tourette syndrome.

6. A probiotic preparation for treating Tourette syndrome, characterized in that, It contains effective amounts of Bifidobacterium breve M-16V and Bifidobacterium animalis subsp. lactis Bb-12, as well as a pharmaceutically acceptable carrier.

7. The probiotic preparation according to claim 6, characterized in that, The ratio of viable counts of *Bifidobacterium breve* M-16V to *Bifidobacterium lactis* subsp. Bb-12 is 1:1, and the dosage of the probiotic preparation is 10. 10 CFU / kg / day.

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