Application of Bifidobacterium pseudolongum in the preparation of medicine for treating attention deficit hyperactivity disorder

Abstract

The present invention discloses an application of Bifidobacterium pseudolongum in a drug for treating attention deficit hyperactivity disorder. The Bifidobacterium pseudolongum is used to treat attention deficit hyperactivity disorder, and it regulates endocrine dysfunction of the brain-gut axis by balancing the intestinal microecology, thereby greatly alleviating the abnormal psychological and behavioral states associated with ADHD. The Bifidobacterium pseudolongum significantly upregulates the expression levels of ACTH and CORT hormones in serum, Nr3c1 and Nr3c2 mRNA genes in brain tissue, and GR, MR, and CRF proteins in brain tissue, thereby being able to alleviate ADHD symptoms by regulating HPA axis function. The present invention provides a strong theoretical basis for seeking the therapeutic potential of probiotics for attention deficit hyperactivity disorder, and has high social significance and potential market value.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and specifically relates to an application of Bifidobacterium pseudolongum in preparing a medicine for treating attention deficit hyperactivity disorder. Background Art

[0002] Attention-Deficit / Hyperactivity Disorder (ADHD) is one of the most common neurodevelopmental disorders in children. Its core symptoms are attention deficit, hyperactivity / impulsivity, and impairment in social and academic functions. The prevalence of ADHD in children worldwide is about 7.2%, and the prevalence of ADHD in children in my country is 6.26%. Because its symptoms mostly appear during school age, ADHD has been considered a behavioral problem limited to children and adolescents for decades. However, by the early 1980s, many scholars had conducted follow-up studies on ADHD, and the results showed that 10%-60% of ADHD patients did not have their symptoms, especially social functions, alleviated after adolescence. Only the clinical manifestations of ADHD changed, and more comorbidities and social problems appeared, which had a significant adverse effect on the patient's academic, family and social life. Therefore, ADHD has attracted more and more attention.

[0003] There is evidence that gut immune dysfunction is part of the pathophysiology of ADHD. The incidence of atopic diseases such as allergic rhinitis, asthma, allergic dermatitis, and allergic conjunctivitis has been increased in hospitalized patients with ADHD. The incidence of both constipation and fecal incontinence has been increased in children with ADHD. These findings suggest that gut dysbiosis may be involved in gastrointestinal dysfunction in patients with ADHD and may play a role in brain function and behavior in these cases.

[0004] The intestinal flora of children with ADHD is significantly different from that of normal children in terms of diversity. In addition, the discovery of the "microbe-gut-brain axis" confirms that the intestinal flora is closely related to the nervous system. Intestinal microorganisms can affect the central nervous system by affecting a variety of neurotransmitters, immune, endocrine and metabolic pathways, thereby leading to behavioral and cognitive changes. Rebuilding the homeostasis of the intestinal flora is a new means to prevent and alleviate diseases related to mental and behavioral abnormalities.

[0005] For more than 70 years, the treatment of ADHD has mainly used monoamine psychostimulants, mainly methylphenidate (MPH) and amphetamine (AMPH), plus the catecholamine non-stimulant atomoxetine (ATX). However, in clinical practice, about 20%-30% of patients have poor or even ineffective treatment with MPH and cannot achieve symptom control. Some patients cannot tolerate the side effects of MPH (sleep disorders, decreased appetite, headaches, convulsions, etc.) and reduce the dosage or even stop taking the medicine, but cannot achieve clinical control. In addition, due to the limitations of the long-term efficacy of central nervous system stimulants and the potential risk of abuse, some families refuse treatment with central nervous system stimulants. Therefore, it is very necessary and urgent to find products with few side effects, economical prices and suitable for preventing the occurrence of ADHD in sensitive populations. Summary of the invention

[0006] The present invention aims to solve one of the technical problems in the related art at least to a certain extent. To this end, the main purpose of the present invention is to provide a use of Bifidobacterium pseudolongum in the preparation of a medicament for treating attention deficit hyperactivity disorder.

[0007] The objective of the present invention is achieved through the following technical solutions:

[0008] The invention discloses an application of the pseudo-Bifidobacterium longum in a drug for treating attention deficit hyperactivity disorder. The pseudo-Bifidobacterium longum is used for treating attention deficit hyperactivity disorder. The pseudo-Bifidobacterium longum is used in a therapeutically effective amount to achieve the treatment of attention deficit hyperactivity disorder. The accession number of the pseudo-Bifidobacterium longum is Bio-74494.

[0009] Furthermore, the attention deficit hyperactivity disorder includes but is not limited to attention deficit hyperactivity disorder caused by abnormal development of brain function.

[0010] In certain specific embodiments, the effective dose is 200 μL, and the concentration of the pseudolongum Bifidobacterium bacterial solution is 2.8×10 8 CFU / kg.

[0011] In certain specific embodiments, the Bifidobacterium pseudolongum is prepared into a pharmaceutical preparation, and the pharmaceutical preparation includes any one of a liquid preparation, a solid preparation, a semisolid preparation and a gaseous preparation.

[0012] Furthermore, the dosage form of the pharmaceutical preparation includes any dosage form that is pharmaceutically acceptable.

[0013] Furthermore, the pharmaceutical preparation also includes any one or more pharmaceutical excipients that are pharmaceutically acceptable.

[0014] In certain specific embodiments, the administration method of the Bifidobacterium pseudolongum is intragastric administration.

[0015] Compared with the prior art, the present invention has at least the following advantages:

[0016] 1) The present application provides a new use of Bifidobacterium pseudolongum for preparing a drug for treating attention deficit hyperactivity disorder, which greatly alleviates the abnormal psychological and behavioral states associated with ADHD by balancing the intestinal microecology and regulating the endocrine dysfunction of the brain-gut axis. The present invention verifies that Bifidobacterium pseudolongum can improve the symptoms related to attention deficit hyperactivity disorder by regulating the ACTH and CORT hormones in serum, the Nr3c1 and Nr3c2 mRNA expression levels in brain tissue, and the GR, MR, and CRF expression levels in brain tissue.

[0017] 2) The pseudo-longum Bifidobacterium in the present application, after two weeks of oral administration of pseudo-longum Bifidobacterium bacterial solution to SHR rats, showed significant improvements in hyperactivity, spontaneous activity levels and impulsive behaviors; the expression of HPA axis-related hormones, genes and proteins were significantly upregulated. Based on the above experimental results, it is speculated that pseudo-longum Bifidobacterium may alleviate ADHD symptoms by regulating HPA axis function. The present invention provides a strong theoretical basis for seeking the therapeutic potential of probiotics for attention deficit hyperactivity disorder, and has high social significance and potential market value. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the specific embodiments of the present invention, the drawings required for use in the specific embodiments or the description of the prior art will be briefly introduced below.

[0019] Figure 1 is the growth curve of Bifidobacterium pseudolongum described in the present invention;

[0020] Figure 2 is the concentration of the pseudo-longum Bifidobacterium bacterial solution of the present invention. (A) is the concentration of the pseudo-longum Bifidobacterium bacterial solution diluted 10 -6 Colony growth after 24 hours of culture on a Bifidobacterium culture plate; (B) is a 10 dilution of the pseudolongitudinal Bifidobacterium bacterial solution. -7 Colony growth after 24 hours of culture on a Bifidobacterium culture plate; (C) is a 10 dilution of the pseudolongitudinal Bifidobacterium bacterial solution. -8 Colony growth after 24 h of culture on a Bifidobacterium culture plate;

[0021] Figure 3 This is a standard curve of the concentration of the pseudo-Bifidobacterium longum bacterial solution of the present invention;

[0022] Figure 4 The effect of the pseudo-Bifidobacterium longum of the present invention on the hyperactive behavior and spontaneous activity level of attention deficit hyperactivity disorder rats (SHR);

[0023] Figure 5The effect of the Bifidobacterium pseudolongum described in the present invention on the impulsive behavior of attention deficit hyperactivity disorder rats (SHR);

[0024] Figure 6 The effect of the Bifidobacterium pseudolongum described in the present invention on hormones related to the gut-brain axis neuroendocrine system in the brain tissue of attention deficit hyperactivity disorder rats (SHR);

[0025] Figure 7 The effect of the Bifidobacterium pseudolongum described in the present invention on the mRNA expression levels of Nr3c1 and Nr3c2 in the hippocampus of attention deficit hyperactivity disorder rats (SHR);

[0026] Figure 8 The invention relates to the influence of the Bifidobacterium pseudolongum described in the present invention on the gut-brain axis neuroendocrine system-related proteins in the brain tissue of attention deficit hyperactivity disorder rats (SHR). DETAILED DESCRIPTION

[0027] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further described below in conjunction with specific embodiments and drawings. However, the following embodiments are only preferred embodiments of the present invention and are not exhaustive.

[0028] The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.

[0029] The Bifidobacterium Pseudolongum Subsp. Pseudolongum Mitsuoka provided by the present invention is deposited in the American Type Culture Collection, the depository abbreviation is ATCC, the biological deposit number is Bio-74494, and the strain number is: 25526 TM .

[0030] Example 1: Cultivation of Bifidobacterium pseudolongum

[0031] The pseudo-Bifidobacterium longum in the present application can be cultivated by the following method:

[0032] ①Activation of Bifidobacterium pseudolongum

[0033] The activation operation of Bifidobacterium pseudolongum was carried out in an anaerobic operating table. Prepare a test tube containing 15mL of thioglycollate fluid culture medium (casein (trypsin hydrolysis) 15.0g, yeast extract powder 5.0g, glucose 5.0g, sodium thioglycollate 0.5g, L-cystine 0.5g, sodium chloride 2.5g, resazurin 0.001g, agar 0.75g, pH 7.1±0.2) (placed in an anaerobic environment for 24h in advance); disinfect the surface of the ampoule tube containing the lyophilized powder of Bifidobacterium pseudolongum strain with alcohol, burn the top of the ampoule tube of Bifidobacterium pseudolongum strain with an alcohol lamp, and then quickly move it to an anaerobic incubator to cool and break it. Add 0.5 mL of thioglycollate fluid culture medium into the ampoule tube, fully dissolve the lyophilized powder, then add the suspension of Bifidobacterium pseudolongum strain into the test tube containing the thioglycollate fluid culture medium, seal it and place it in a 15×30 cm sealed anaerobic culture bag, and immediately place it in an anaerobic gas-producing bag, and culture it in a 37°C anaerobic incubator (humidity 65%) until the bacterial solution becomes turbid or a large number of bacteria grow at the bottom.

[0034] ② Determination of the growth curve of Bifidobacterium pseudolongum

[0035] like Figure 1 As shown, in order to determine the number of Bifidobacterium pseudolongum and understand its growth and metabolism, turbidimetry was used. The relationship between the suspension of Bifidobacterium pseudolongum and the OD value was measured using a spectrophotometer to clarify the growth law of Bifidobacterium pseudolongum and correspond it with the culture time to clarify the growth curve of Bifidobacterium pseudolongum, in preparation for the homogeneity of the bacterial solution in subsequent in vitro and in vivo experiments.

[0036] ② Calculation of concentration of Bifidobacterium pseudolongum bacterial solution

[0037] like Figure 2 and Figure 3 As shown, 0.1 mL of the mixture is pipetted into a test tube containing 10 mL of culture medium. This dilution is 10 -1 Repeat the above process to make a 10-fold dilution to 10 -8 Dilution, select 10 -6 , 10 -7 , 10 -8Three dilutions, draw 0.1mL bacterial liquid droplets on the bifidobacterium culture medium (tryptic casein peptone 5.0g, soy peptone 5.0g, yeast extract powder 10.0g, glucose 10.0g, L-cysteine ​​0.5g, resazurin 0.001g, potassium dihydrogen phosphate 0.04g, dipotassium hydrogen phosphate 0.04g, sodium bicarbonate 0.4g, sodium chloride 0.08g, calcium chloride 0.008g, magnesium sulfate 0.0192g, pH 7.0) plate, after the plate is coated, use anaerobic culture method (5% CO2) to place the coated culture dish in a 37℃ incubator overnight culture. The plate is grown overnight until the colonies are clear and do not want to be connected (12-24h), and the number of colonies (n) is counted. Draw a standard curve based on the counting results and calculate the bacterial concentration.

[0038] Calculation formula: N (cfu / mL) = n*d 2 / v

[0039] n: average colony count d2: dilution concentration v: volume of bacterial solution applied on the plate

[0040] ④ Subculture and preservation of Bifidobacterium pseudolongum

[0041] Prepare 10 test tubes containing thioglycollate fluid culture medium, take 500 μL of activated Bifidobacterium pseudolongum liquid and inoculate it into the test tubes, seal them and place them in a 15×30 cm sealed anaerobic culture bag, and put them into an anaerobic gas production bag. After 1 day of static culture in an anaerobic box at 37°C, it is obvious that the bacterial liquid in the liquid culture medium is turbid. After high-pressure sterilization, the bacterial cryopreservation tube and 60% sterile glycerol (volume fraction) are placed in an anaerobic incubator, and then the bacterial liquid and glycerol are transferred to the cryopreservation tube in a ratio of 1:1. Add about 1 mL of bacterial suspension to each tube to make the final concentration of glycerol reach 30%, and store at -80°C.

[0042] ⑤ Preparation of pseudo-Bifidobacterium longum bacterial solution

[0043] Pseudolongatus Bifidobacterium reaches the logarithmic phase 8-10 hours after cultivation. Use a pipette to draw 1 mL of bacterial solution from the logarithmic growth phase culture into the spectrophotometer sample pool and use a UV spectrophotometer to detect OD 600 Optical Density and determine the bacterial concentration. 600 ≈1, the bacterial concentration is about 10 8 -10 9 After centrifuging the bacterial stock solution at 6000×g for 5 minutes, the supernatant was removed and the pellet was resuspended in sterile PBS to make about 10 8 -10 9 cfu / mL bacterial suspension. The diluted bacterial solution samples were divided into sterile centrifuge tubes and sealed, and the samples were gavaged into experimental animals according to the sterile transfer requirements.

[0044] Example 2: Performance test of Bifidobacterium pseudolongum

[0045] In this embodiment, the experimental animals used are:

[0046] Sixteen three-week-old SHR male rats were selected and maintained at room temperature of 23±1°C and humidity of 50±60%, with a 12-hour light-dark cycle and free access to clean food and water for training.

[0047] Experimental methods: SHR rats were divided into Vehicle (SHR) and Pseudo-Bifidobacterium longum gavage group (Bp), with 8 rats in each group. The SHR group was gavaged with 200 μL of normal saline every day, and the Bp group was gavaged with 200 μL of the above-mentioned Pseudo-Bifidobacterium longum bacterial solution every day. The number of viable bacteria in the Pseudo-Bifidobacterium longum bacterial solution was 2.8×10 8 CFU / kg. The daily administration time was 8:00-9:00 am for 14 consecutive days. On the 15th day, the rats in each group fasted for 12 hours for behavioral experiments. On the 16th day, the rats in each group fasted for 12 hours, blood was collected from the orbits, centrifuged at 3000r / min, and the serum was reserved for later use. Brain tissue (cortex, hippocampus) was taken for Elisa kit, Western Blot method, QPCR method and other tests. The data were expressed as mean ± standard deviation (SEM), and the independent sample T test was used to evaluate the differences between the two groups. All charts were created by GraphPadPrism 8.0.

[0048] 1) Effects of Bifidobacterium pseudolongum on hyperactive behavior and spontaneous activity levels

[0049] The mice in the above groups were given medication every day at 8:00-9:00 am for 14 consecutive days. On the 15th day, the rats in each group fasted for 12 hours and then underwent behavioral experiments, specifically:

[0050] ①Open field experiment

[0051] The open field test is used to evaluate the autonomous activity, active exploration and anxiety state of rats in a new environment. The open field apparatus is a black box with a length of 50 cm, a width of 50 cm, a height of 40 cm, and an open top. There is a camera directly above the open field apparatus connected to a computer to record the activity information of the rats in the open field. Before the experiment begins, set the open field experiment system parameters on the computer, and allow the rats to adapt to the surrounding environment for 5 minutes. Then, place the rats in the open field, and record the rats' activities in the open field for 5 minutes. After the experiment on each rat, clean up the rat feces and urine in the mine field experimental device, then thoroughly clean the mine field device with a 5% acetic acid aqueous solution, and then wipe it dry with a paper towel or dry cloth. The recorded activity information includes the total activity distance, central activity distance, central activity duration and activity trajectory diagram. The results are as follows Figure 4 As shown, Figure 4The effect of Bifidobacterium pseudolongum on the hyperactive behavior and spontaneous activity level of rats with attention deficit hyperactivity disorder (SHR). (4A) After continuous oral gavage with Bifidobacterium pseudolongum for two weeks, the total activity distance of rats in the Bp group in the open field test was significantly reduced (P<0.05); (4B) After continuous oral gavage with Bifidobacterium pseudolongum for two weeks, the central area activity distance of rats in the Bp group in the open field test was significantly reduced (P<0.05); (4C) After continuous oral gavage with Bifidobacterium pseudolongum for two weeks, the central area activity time of rats in the Bp group in the open field test was significantly reduced (P<0.05) (N=8). (4D) is the activity trajectory diagram of Vehicle rats in the open field test. (4E) is the activity trajectory diagram of rats in the Bp group in the open field test.

[0052] ②Elevated plus maze

[0053] The elevated cross maze is a vertical cross consisting of two closed walls (50cm×10cm), two open walls (50cm×10cm) and a central area (10cm×10cm), 50cm above the ground. 2h before the start of the experiment, the animals were placed in a soundproof room to adapt to the environment. The room temperature and ventilation were strictly controlled. The experimenter placed the animal in the central area at the same position each time, with the animal's head facing the direction of one of the open walls. The experiment began after the experimenter left the experimental device area. The experimental time was 5min, and the main indicators for evaluating the animal's impulsive behavior were the time the animal stayed in the open arm and the percentage of the number of times it entered the open arm to the total number of times and the total time. The results are as follows Figure 5 As shown, Figure 5 The effect of Bifidobacterium pseudolongum on the impulsive behavior of rats with attention deficit hyperactivity disorder (SHR); (5A) After continuous gavage with Bifidobacterium pseudolongum for two weeks, the total activity distance of rats in the elevated plus maze test of Bp group was significantly reduced (P<0.05); (5B) After continuous gavage with Bifidobacterium pseudolongum for two weeks, the number of times the rats in the Bp group entered the open arm space was significantly reduced (P<0.05); (5C) After continuous gavage with Bifidobacterium pseudolongum for two weeks, the percentage of activity time in the open arm space of the elevated plus maze of the Bp group was significantly reduced (P<0.05) (N=8); (5D) is the activity trajectory diagram of Vehicle rats in the elevated plus maze; (5E) is the activity trajectory diagram of rats in the Bp group in the elevated plus maze.

[0054] 2) Effects of Bifidobacterium pseudolongum on hormones related to the neuroendocrine system of the gut-brain axis in brain tissue

[0055] The serum of each group of mice in this example was measured using ELISA enzyme-linked immunosorbent to measure the levels of hormones related to the gut-brain axis neuroendocrine system in the serum: adrenocorticotropic hormone (ACTH) and cortisol (CORT). Figure 6 As shown, Figure 6 Effects of Bifidobacterium pseudolongum on hormones related to the neuroendocrine system of the gut-brain axis in the brain tissue of rats with attention deficit hyperactivity disorder (SHR); 6A shows that compared with Vehicle rats, the serum adrenocorticotropic hormone (ACTH) of rats gavaged with Bifidobacterium pseudolongum bacterial solution for two weeks was significantly increased (P<0.0001). 6B shows that the serum cortisol (CORT) level of rats gavaged with Bifidobacterium pseudolongum bacterial solution for two weeks was significantly higher than that of Vehicle rats (P<0.0001) (data are expressed as mean ± standard deviation, N=8); The results of enzyme-linked immunosorbent assay experiments show that Bifidobacterium pseudolongum can improve the endocrine system dysfunction in attention deficit hyperactivity disorder and regulate the HPA axis function.

[0056] 3) Effects of Bifidobacterium pseudolongum on genes related to the neuroendocrine system of the gut-brain axis in brain tissue

[0057] For the brain tissues (cortex, hippocampus) of each group of rats in this example, the expression levels of genes related to the gut-brain axis neuroendocrine system in the brain tissues were determined by Quantitative-PCR method: glucocorticoid receptor (Nr3c1), mineralocorticoid receptor (Nr3c2). Figure 7 As shown, 7A shows the effect of Bifidobacterium pseudolongum on the expression level of Nr3c1 mRNA in the hippocampus of rats with attention deficit hyperactivity disorder (SHR). 7B shows the effect of Bifidobacterium pseudolongum on the expression level of Nr3c2 mRNA in the hippocampus of rats with attention deficit hyperactivity disorder (SHR). Data are expressed as mean ± standard deviation, N = 6; after two weeks of intragastric administration of Bifidobacterium pseudolongum bacterial solution, the expression levels of Nr3c1 and Nr3c2 mRNA in the brain tissue of the Bp group were significantly upregulated compared with the Vehicle group. These results indicate that Bifidobacterium pseudolongum may regulate the function of the neuroendocrine system of the brain-gut axis through a certain mechanism of action, thereby improving ADHD-related symptoms.

[0058] 4) Effects of Bifidobacterium pseudolongum on analysis of proteins related to the gut-brain axis neuroendocrine system in brain tissue

[0059] For the brain tissue (cortex, hippocampus) of each group of rats in this example, Western Blot was used to determine the levels of gut-brain axis neuroendocrine system-related proteins in the brain tissue: glucocorticoid receptor (GR), mineralocorticoid receptor (MR), and corticotropin-releasing factor (CRF).

[0060] The results are as follows Figure 8 As shown, Figure 8 The effect of Bifidobacterium pseudolongum on the gut-brain axis neuroendocrine system-related proteins in the brain tissue of rats with attention deficit hyperactivity disorder (SHR). The protein expression levels of neuroendocrine system-related proteins glucocorticoid receptor (GR), mineralocorticoid receptor (MR), and corticotropin-releasing factor (CRF) were determined by immunoblotting (Western Blot). As can be seen from the figure, the WB results showed that after two weeks of gavage with Bifidobacterium pseudolongum bacterial solution, the expression levels of GR, MR, and CRF in the brain tissue of the Bp group experimental group were significantly upregulated compared with the Vehicle group. These results indicate that Bifidobacterium pseudolongum may regulate the function of the brain-gut axis neuroendocrine system through a certain mechanism of action, thereby improving ADHD-related symptoms.

[0061] The present invention shows through the above experiments that Bifidobacterium pseudolongum has the ability to alleviate ADHD-related hyperactivity and impulsive behavior symptoms. This effect may be exerted by regulating the hormone levels related to neuroendocrine function in the gut-brain axis. The present invention provides a strong theoretical basis for seeking the therapeutic potential of probiotics for attention deficit hyperactivity disorder, and has high social significance and potential market value.

[0062] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein by equivalents. These modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention, and they should all be included in the scope of the claims and specification of the present invention.

Claims

1. Bifidobacterium pseudolongum ( Bifidobacterium Pseudolongum Subsp.Pseudolongum Mitsuoka ) in the preparation of a drug for treating attention deficit hyperactivity disorder, characterized in that: The pseudo-Bifidobacterium longum is used for treating attention deficit hyperactivity disorder. The pseudo-Bifidobacterium longum is used in a therapeutically effective amount to achieve the treatment of attention deficit hyperactivity disorder. The deposit number of the pseudo-Bifidobacterium longum is ATCC-25526.

2. The use according to claim 1, characterized in that: The pseudo-Bifidobacterium longum is made into a pharmaceutical preparation, which includes any one of a liquid preparation, a solid preparation, a semisolid preparation and a gas preparation.

3. The use according to claim 2, characterized in that: The dosage form of the pharmaceutical preparation includes any pharmaceutically acceptable dosage form.

4. The use according to claim 2, characterized in that: The pharmaceutical preparation further comprises any one or more pharmaceutical excipients that are pharmaceutically acceptable.

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