Application of clostridium butyricum in treating autism

Abstract

According to the application of the clostridium butyricum in treatment of the autism, in a constructed maternal immune activated mouse autism model, the clostridium butyricum can mediate the intestinal barrier function and effectively improve the autism symptom by adjusting the balance of intestinal flora, increasing the permeability of the intestinal barrier function, inhibiting inflammatory factors and the like. The clostridium butyricum has the possibility of being used as the medicine for improving the autism, and a new application field is provided for the clostridium butyricum.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms, and particularly relates to an application of Clostridium butyricum in treating autism, providing a new application of Clostridium butyricum. Background Art

[0002] Autism, also known as autism or autism disorder, is a group of complex neurodevelopmental disorders and a representative disease of pervasive developmental disorders, which affects an individual's social interaction, communication ability, and behavior patterns. The disease begins in infancy and is characterized by social interaction disorders, communication disorders, restricted interests, stereotyped and repetitive behaviors, and most children also have varying degrees of mental retardation. Autism not only causes social disorders, communication difficulties, repetitive behaviors, and increases the risk of anxiety and depression, but also brings a significant burden to the patient's family, affects education and career development, and may also be accompanied by other health problems. Autism spectrum disorders are on the rise worldwide, and innovative and effective assessment and treatment technologies are urgently needed.

[0003] There is currently no specific drug to treat its core symptoms in the clinic, and it mainly relies on early identification, behavioral intervention and educational training. Drug treatment is usually auxiliary and is used to improve accompanying symptoms, such as emotional and behavioral disorders, ADHD, anxiety, etc. At present, the drugs approved by the US FDA for the treatment of autism include risperidone for the treatment of irritability, aggression and self-harm behavior in patients with autism; aripiprazole for the improvement of irritability, aggression and stereotyped behavior in patients with autism; other drugs such as antidepressants, anti-ADHD drugs, anti-epileptic drugs, etc. are also under study for the treatment of other symptoms associated with autism. Therefore, there is an urgent need to find more appropriate prevention and treatment measures.

[0004] Clostridium butyricum, also known as Clostridium butyricum or Miyari bacteria, is a Gram-positive anaerobic bacillus belonging to the Bacillaceae family and the genus Clostridium. It is straight rod-shaped or slightly curved, and has unique biological characteristics of high temperature resistance, acid resistance and resistance to multiple antibiotics. Clostridium butyricum has the following functions. First, Clostridium butyricum can prevent the abnormal proliferation of pathogens and spoilage bacteria in the intestine, promote the proliferation and development of beneficial intestinal flora, thereby correcting intestinal flora disorders and reducing the production of enterotoxins; secondly, according to research reports, Clostridium butyricum can activate macrophages and NK cells, increase the content of IgA and IgM in serum, and enhance the body's immunity; in recent years, Clostridium butyricum preparations have also been used in combination with other probiotics, Chinese medicine extracts and antibiotics to treat diarrhea, enteritis, irritable bowel syndrome and other diseases, and the effect is significant. As a probiotic, Clostridium butyricum has broad application prospects. Studies have found that the intestinal flora affects the nervous system through itself or its metabolism. At the same time, the central nervous system also regulates the intestinal flora to adapt to environmental changes and maintain the balance of the intestinal flora microecology. More and more studies have shown the relationship between the microbial-gut-brain axis.

[0005] As disclosed in patent application 201510491144.6, a drug for preventing and treating Alzheimer's disease, its preparation method and application, Alzheimer's disease is a progressive neurodegenerative disease with an insidious onset. Clinically, it is characterized by global dementia manifestations such as memory impairment, aphasia, apraxia, agnosia, visual-spatial skill impairment, executive dysfunction, and personality and behavioral changes. This patent application prepares drugs for preventing and treating Alzheimer's disease by using Clostridium butyricum, takes intestinal flora as a therapeutic target, and creates a new model for preventing and treating Alzheimer's disease, which not only alleviates disease symptoms but also slows down nerve cell damage, and has good social and economic benefits.

[0006] Despite this, research on the ability of Clostridium butyricum to improve and treat autism is still very limited, and further research is necessary. Summary of the invention

[0007] In view of the deficiencies in the prior art, the present invention provides an application of Clostridium butyricum in treating autism, finds that Clostridium butyricum has the effect of treating autism, and provides a new application of Clostridium butyricum in treating autism.

[0008] Another object of the present invention is to provide an application of Clostridium butyricum in the treatment of autism. Clostridium butyricum can mediate the intestinal barrier function by regulating the balance of intestinal flora, increasing the permeability of the intestinal barrier function, inhibiting inflammatory factors, improving the production of mature neurons in the hippocampus, improving cell proliferation and neurogenesis in this area, thereby effectively improving the symptoms of autism.

[0009] To achieve this object, the present invention adopts the following technical solution:

[0010] Disclosed is an application of Clostridium butyricum in treating autism, and an application of the Clostridium butyricum in a product for treating autism; the product is a medicine.

[0011] The autism is a neurodevelopmental disease, and the model is a maternal immune activation mouse autism model induced by valproic acid.

[0012] Furthermore, the drug comprises one or more of the fermentation broth, fermentation broth supernatant, fermentation broth precipitate, live bacteria, dead bacteria, lyophilized powder and cell lysate of Clostridium butyricum.

[0013] Specifically, the medicine also includes pharmaceutically acceptable excipients.

[0014] The applicant's research found that Clostridium butyricum can mediate intestinal barrier function by regulating the balance of intestinal flora, increasing the permeability of intestinal barrier function, inhibiting inflammatory factors, etc., effectively improving autism symptoms.

[0015] Specifically, Clostridium butyricum can improve the behavioral manifestations of autism when used to improve autism.

[0016] Furthermore, Clostridium butyricum can regulate the species diversity of intestinal flora and improve species composition in the application of improving autism.

[0017] Furthermore, Clostridium butyricum can improve colon barrier function in the application of improving autism.

[0018] Furthermore, Clostridium butyricum can inhibit serum inflammatory response in the application of improving autism.

[0019] Furthermore, Clostridium butyricum can inhibit colonic inflammatory response in the application of improving autism.

[0020] Furthermore, Clostridium butyricum can inhibit hippocampal inflammatory response in the application of improving autism.

[0021] Furthermore, in the application of improving autism, Clostridium butyricum can improve the production of mature neurons in the hippocampus, improve cell proliferation and neurogenesis in this area.

[0022] The research results of the present invention show that Clostridium butyricum can improve the relevant symptoms of autism in mice, so it can be used in the treatment of autism, specifically in drugs for treating autism.

[0023] Specifically, Clostridium butyricum can effectively increase the diversity and richness of a single sample of intestinal species in autistic mice, and Clostridium butyricum can effectively increase the similarity of microbial composition between intestinal species samples of autistic mice. At the phylum level, the abundance of the Clostridium butyricum group increased significantly, indicating that Clostridium butyricum can change the species composition in the intestines of autistic mice, increase their beneficial bacteria and reduce their harmful bacteria; the number of neuron-positive cells that was reduced after improvement by Clostridium butyricum was improved, indicating that Clostridium butyricum can increase the number of hippocampal neurons in mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required to be used in the description of the embodiments or the prior art are briefly introduced below.

[0025] Figure 1 Schematic diagram of the effect of Clostridium butyricum implemented in the present invention on the species diversity of intestinal flora of autistic mice (α analysis).

[0026] Figure 2 Schematic diagram of the effect of Clostridium butyricum implemented in the present invention on the species diversity (β analysis) of the intestinal flora of autistic mice.

[0027] Figure 3Schematic diagram of the effect of Clostridium butyricum implemented in the present invention on the species composition of the microbiota of autistic mice (phylum and genus level analysis).

[0028] Figure 4 This is a schematic diagram of the effect of Clostridium butyricum on the proliferation of hippocampal neurons in autistic mice implemented in the present invention. DETAILED DESCRIPTION

[0029] The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be construed as limiting the scope of the present invention. If specific conditions are not specified in the examples, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0030] The present invention is implemented as the application of Clostridium butyricum in treating autism, and the application of Clostridium butyricum in a product for treating autism; the product is a medicine.

[0031] The autism is a neurodevelopmental disease, and the model is a maternal immune activation mouse autism model induced by valproic acid.

[0032] The medicine comprises one or more of the fermentation liquid of Clostridium butyricum, the supernatant of the fermentation liquid, the precipitate of the fermentation liquid, the living bacteria, the dead bacteria, the lyophilized powder and the cell lysate.

[0033] Clostridium butyricum can mediate intestinal barrier function by regulating the balance of intestinal flora, increasing the permeability of intestinal barrier function, inhibiting inflammatory factors, etc., effectively improving autism symptoms.

[0034] The specific implementation plan is as follows:

[0035] Example 1.

[0036] 1. Materials and methods.

[0037] 1. Test materials.

[0038] 1.1 Experimental animals

[0039] Thirty female SPF mice, 10 to 12 weeks old, and 15 male SPF mice, 10 to 12 weeks old, weighing 25 to 35 g, were provided by the Experimental Animal Center of Yichun University.

[0040] 1.2 Drugs and reagents.

[0041] Valproic acid (V0033000) and claudin3 antibody (O15551) were purchased from Sigma, USA; 30% Acr-Bis (ST003), ammonium persulfate (ST005), SDS-PAGE protein loading buffer (P0015L), Tween-20 (ST828), DMSO (ST038) and Tris (ST760) were all purchased from Shanghai Biotech Co., Ltd.; glycine (A502065-0500), methanol (A601617), formaldehyde (A500684), anhydrous ethanol (A500737), and xylene (A530011) were all purchased from Shanghai Biotech Co., Ltd.; claudin1 (AF6504) antibody, occludin (AF7644) antibody, ZO (AF0321) antibody, BeyoColor TM Color prestained protein (P0072) and horseradish enzyme-labeled goat anti-rabbit / mouse IgG were purchased from Shanghai Biotime Biotechnology Co., Ltd.; IL-6 antibody (12912), TNF-α antibody (11948), and IL-1β (12703) antibodies were purchased from Cell Signaling Technology, Inc., USA; HE dye kit (G1120, Solarbio) was purchased from Beijing Solarbio Technology Co., Ltd.; ECL ultrasensitive kit (P0018FS) and BCA concentration determination kit (P0012S) were purchased from Shanghai Biotime Biotechnology Co., Ltd.

[0042] 1.3 Experimental instruments.

[0043] TGL-16A high-speed low-temperature centrifuge, Hunan Pingfan Technology Co., Ltd.; E200MV microscope, Nikon, Japan; MultiskanFC microplate reader (Thermo Fisher, USA); protein electrophoresis transfer system (Bio Rad, USA); fluorescence quantitative PCR instrument (Eppendorf, Germany).

[0044] 2. Animal grouping and drug administration.

[0045] One sexually mature male and two sexually mature female mice were selected and put into cages at 8 pm on the same day. The vaginal plugs of the female mice were tested at 8 am the next day. The mice with vaginal plugs were placed in a cage and the time was recorded as E 0.5 days. At E10.5 days, the pregnant mice were intraperitoneally injected with valproic acid (VPA) (dose of 500 mg / kg), and the concentration of valproic acid VPA was prepared to be 50 mg / ml (prepared and used immediately). At this time, the pregnant mice were kept in a single cage. After giving birth to the first generation, they were weaned 3 weeks later and randomly divided into two groups. The first group was the autism model group (AC group), and the second group was the Clostridium butyricum improved autism group, also called the treatment group (AC_C group) (0.5 ml of Clostridium butyricum suspension concentration was 2×10 9 CFU / ml by intraperitoneal gavage for 28 days); eight mice in each group, a total of 16 mice. The pregnant mice were intraperitoneally injected with an equal volume of normal saline, and after giving birth to the first generation, the blank group (NC group) was weaned after 3 weeks; the pregnant mice were intraperitoneally injected with an equal volume of normal saline, and after giving birth to the first generation, the first generation was weaned after 3 weeks, and the blank group (NC group) was used ... 9 CFU / ml of Clostridium butyricum suspension was gavaged for 28 days as the blank plus drug group (NC_C group), with 8 mice in each group, a total of 16 mice. The feces were collected at the 3rd week (T1), 4th week (T2), 5th week (T3), 6th week (T4), and 7th week (T5). In the seventh week, the animal behavior tests of each group were tested, including grooming test, marble embedding test, open field test, and three-box social interaction; the intestinal flora was tested for the structure of the phylum and genus levels as well as α diversity and β diversity; WB was used to detect the expression of claudin1, claudin3, occludin, and ZO proteins in the mouse colon; WB was used to detect the expression of IL-6, TNF-α, and IL-1β related inflammatory factors in the mouse colon and hippocampus; and immunofluorescence staining was used to detect the expression of NeuN in the dentate gyrus of the hippocampus.

[0046] 2. Experimental process.

[0047] 1. Behavioral testing.

[0048] Grooming experiment: The test mice were placed individually in a familiar cage, and the number of grooming times and grooming duration within 10 minutes were measured to evaluate the stereotyped behavior of the mice.

[0049] Marble embedding test: The test mice were placed alone in a new resin cage (length, width and height: 35*28*28cm), which was first covered with sawdust, and 20 marble beads were placed on the surface of the sawdust (4 beads in each row, 4 rows in total, and the distance between each bead was 2cm). After the mice were placed in the cage for 20 minutes, the number of beads buried by the mice was tested (the buried area was greater than 2 / 3 of its total area). The number of beads buried by the mice represents the degree of anxiety and stereotyped behavior of the mice.

[0050] Open field test: This behavioral test allows mice to move freely in a 50*50*50cm3 square box to detect the overall exercise level and anxiety of the mice. A 25*25cm2 central area is specified in the center of the bottom of the box. The distance the mouse moves in the box reflects its overall exercise level; the length of time it explores the middle area reflects its anxiety level. First, put the mouse in the box and let it explore freely in the box. Observe for 10 minutes and record the total distance the animal walks, the time it stays in the central area, the distance it walks in the central area, the number of times it stands, the number of times it urinates and defecates, etc. During this period, wipe the inner wall and bottom of the box with 75% ethanol before changing the animal to prevent the remaining information from the last animal (such as the animal's defecation, urine, and smell) from affecting the next test results.

[0051] Three-box social behavior test: The length, width and height of the box used for this behavior are 60cm, 40cm and 20cm respectively. Two transparent partitions in the middle divide the box into three areas on average. The left area, the middle area and the right area. And put an empty cage with holes in each of the left and right areas. There are small holes in both partitions, allowing mice to shuttle freely between the three areas. Adaptation stage: The test mouse is placed in the box from the middle area and freely explores any object in the box. Record the sniffing time of the test mouse to the left and right cages. Social defect detection stage: Put a mouse (S1) of the same sex and age as the test mouse in the cage in the left or right area, and put the test mouse into the box from the middle area. Record the sniffing time of the test mouse to the left and right cages. Social forgetting detection stage: Keep the mouse in the cage in the left or right area still. Put another mouse (S2) of the same sex and age as the test mouse in the cage in the other side area. The test mouse is placed in the box from the middle area. The sniffing time of the test mouse in the left and right cages was recorded.

[0052] 2. Intestinal flora testing.

[0053] After the last behavioral test, 1-2 pellets of fresh rat feces were collected, placed in a sterile centrifuge tube, and stored at -80°C for microbial testing. The microbial genomic DNA in the sample was extracted, and then the 16SrRNA gene was amplified by PCR using specific primers, followed by sequencing. Finally, the obtained data was processed and analyzed using bioinformatics tools to evaluate the composition and function of the intestinal flora. Among them, α analysis was performed as Figure 1 As shown, the β analysis is Figure 2 shown.

[0054] 3.WB detection.

[0055] The heart tissue homogenate was centrifuged at low temperature and high speed, and the supernatant was aspirated. The protein concentration was determined using a BCA protein quantification kit, and 5× protein loading buffer was added. After shaking and mixing, the sample was obtained by water bath at 95°C for 5 minutes. After the cells were lysed and the protein was extracted, the sample was loaded, electrophoresed, transferred to the membrane, blocked with milk, and washed with TBST. Then, primary antibodies such as claudin1, claudin3, occludin, ZO, IL-6, TNF-α, and IL-1β were added for overnight incubation, washed with TBST 3 times, and incubated with secondary antibodies for 1 hour, and washed with TBST 3 times. After soaking in ultrasensitive ECL chemiluminescent solution for 2 minutes, the image was developed, and the grayscale value was calculated using Image J software to compare the protein level expression between the groups. SPSS20 software was used for statistical analysis of the data, and the data results were expressed as mean ± standard deviation (xˉ±s). One-way analysis of variance was used to compare multiple groups of data, and P<0.05 was considered statistically significant. The results of detecting the expression of claudin1, claudin3, occludin, and ZO proteins in mouse colon are shown in the following figure. Figure 3 shown.

[0056] 4. Immunofluorescence staining detection.

[0057] The mice were anesthetized and perfused by heart. The tissues were fixed with 4% paraformaldehyde. The brain tissues were removed and dehydrated in 30% sucrose solution, followed by frozen sections. The sections were washed with PBS, permeabilized with 0.3% Triton X-100, and blocked with 5% normal goat serum. The primary antibody was added and incubated overnight. After washing, the fluorescently labeled secondary antibody was added and incubated for 1 hour. Finally, the nuclei were stained with DAPI. After sealing, the sections were observed under a fluorescence microscope. The results of NeuN expression in the dentate gyrus of the hippocampus were as follows: Figure 4 shown.

[0058] 3. Conclusion

[0059] 1. Clostridium butyricum can increase the internal diversity of intestinal species in a single sample.

[0060] like Figure 1 This is an α analysis of the species diversity of the intestinal flora of autistic mice by Clostridium butyricum. As shown in Figure A, the bar graph values ​​of each group are close to 1, indicating that the sequencing depth covers all species in the sample, which is sufficient to reflect the diversity contained in a given sample. Figure 1Chao 1 index, ace index and shannon index were used to represent the diversity and richness of the intestinal microbial flora of each group of mice in BD. Among them, the three indexes of the autism model group (AC group) were significantly lower than those of the normal group (P<0.05). Compared with the autism model group (AC group), the indexes of the other three groups increased (P<0.05), especially the autism group (AC_C group) improved by adding Clostridium butyricum, which showed that Clostridium butyricum could effectively increase the internal diversity and richness of a single sample of intestinal species in autistic mice.

[0061] 2. Clostridium butyricum can effectively increase the similarity of microbial composition between intestinal species samples

[0062] like Figure 2 As shown, it is the PCoA and NMDS results. It can be seen that the autism model group (AC group) is far away from the other three groups, the treatment group (AC_C group) and the blank group (NC group) and the blank plus drug group (NC_C group) are close to each other, and the bacterial flora are similar, indicating that Clostridium butyricum can effectively increase the similarity of microbial composition between intestinal species samples of autistic mice.

[0063] 3. Clostridium butyricum can change the species composition in the intestines of autistic mice.

[0064] like Figure 3 As shown in A, at the phylum level, the abundance of Bacteroidota in the autism model group was lower than that in the normal group (P<0.05), while the abundance of Bacteroidetes in the treatment group was higher (P<0.05); at the genus level, compared with the control group, the abundance of unclassified Muribacaceae and Bacteroides in the autism model group was significantly decreased (P<0.05), while the abundance in the treatment group was significantly increased (P<0.05), indicating that Clostridium butyricum can change the species composition in the intestine of autistic mice, increase their beneficial bacteria and reduce their harmful bacteria.

[0065] 4. Clostridium butyricum can increase the number of mature neurons in the hippocampus.

[0066] like Figure 4 As shown, compared with the blank group, the number of neuron-positive cells in the autism model group was significantly decreased; compared with the autism model group, the number of neuron-positive cells in the treatment group was improved after improvement by Clostridium butyricum (P<0.05), and at the same time, compared with the blank group, the number of neuron-positive cells in the blank-treated group with the addition of Clostridium butyricum was also increased, which indicates that Clostridium butyricum can increase the number of hippocampal neurons in mice.

[0067] In summary, the Clostridium butyricum of the present invention can improve the relevant symptoms of autism in mice, so it can be used in the treatment of autism, specifically in drugs for treating autism.

[0068] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A use of Clostridium butyricum in treating autism, characterized in that: The application is the use of Clostridium butyricum in products for treating autism; the product is a medicine.

2. The use of Clostridium fubutyricum in treating autism according to claim 1, characterized in that: The autism is a neurodevelopmental disease, and the model is a maternal immune activation mouse autism model induced by valproic acid.

3. The use of Clostridium butyricum in treating autism according to claim 1, characterized in that: The medicine comprises one or more of the fermentation liquid of Clostridium butyricum, the supernatant of the fermentation liquid, the precipitate of the fermentation liquid, the living bacteria, the dead bacteria, the lyophilized powder and the cell lysate.

4. The use of Clostridium butyricum in treating autism according to claim 1, characterized in that: The medicine also includes pharmaceutical excipients.

5. The use of Clostridium butyricum in treating autism according to claim 3, characterized in that: The Clostridium butyricum was 2×10 9 CFU / ml of Clostridium butyricum suspension.

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