CN109276711B

CN109276711B - Application of manganese type high-stability superoxide dismutase in improving autism and product

Info

Publication number: CN109276711B
Application number: CN201811216980.3A
Authority: CN
Inventors: 孟凡国
Original assignee: Hangzhou Redox Pharmatech Co ltd
Current assignee: Hangzhou Redox Pharmatech Co ltd
Priority date: 2018-10-18
Filing date: 2018-10-18
Publication date: 2022-04-05
Anticipated expiration: 2038-10-18
Also published as: CN109276711A

Abstract

The invention provides application of manganese type high-stability superoxide dismutase with a sequence shown as SEQ ID NO. 4 in treating autism. The manganese type high-stability superoxide dismutase adopted by the invention can improve the ecological imbalance of intestinal flora and regulate the expression of intestinal shielding related protein, thereby improving the oxidative stress of intestinal tracts and brains, achieving the aim of relieving and improving the behavior of autism, and providing a new way for treating the autism.

Description

Application of manganese type high-stability superoxide dismutase in improving autism and product

Technical Field

The invention belongs to the field of medical treatment, and relates to improvement and treatment of autism symptoms.

Background

Autism Spectrum Disorders (ASD) are a pervasive developmental disorder characterized primarily by varying degrees of social, communication impairment, repetitive and stereotyped behaviors. The severity of the disease varies from individual to individual and often involves one or more additional illnesses, including intellectual disability, anxiety, and gastrointestinal symptoms. The etiology of ASD is not clear, and the pathogenesis proposed at present includes^[1-8]:1) gene mutations cause polymorphisms in certain specific genes; 2) epigenetic variations in gene expression; 3) environmental pollution; 4) pathogen infection of the mother during pregnancy; 5) gastrointestinal metabolic dysfunction; 6) immunoregulatory abnormalities; 7) an imbalance in gut-brain connectivity; 8) oxidative stress; 9) mitochondrial dysfunction; 10) neuroinflammation during development. On the other hand, the incidence of ASD also continues to rise, from 1:110 reported in 2006 to 1:68 in 2010^[9]. Currently, there is no better treatment for ASD, and such a severe situation is a significant burden for both the family and the society. ASD has attracted a wide range of attention from all societies.

Disclosure of Invention

The inventor of the invention finds that in the autism disease state, the imbalance of oxidative stress in the intestinal tract can cause damage to the intestinal barrier and the ecological imbalance of the intestinal flora and cause autism-like behavior. SOD, as an antioxidant enzyme that specifically removes superoxide anions, improves the symptoms of autism from the standpoint of the pathogenesis of autism.

Based on this, the present invention proposes to improve the symptoms of autism using manganese-type high stability superoxide dismutase (MS-SOD).

In a first aspect, the invention provides application of manganese type high-stability superoxide dismutase (MS-SOD) in preparing a product for improving symptoms of autism, wherein the sequence of the manganese type high-stability superoxide dismutase is shown as SEQ ID NO. 4.

Preferably, the product is a medicament, food or health product.

Further preferably, when the product is a medicament, the medicament is administered orally, by injection or by gavage; still more preferably, the injection is intravenous or intraperitoneal.

Preferably, the medicament is in the form of tablets, capsules, powder injections, injections or aerosols.

Preferably, the medicament further comprises one or more pharmaceutically acceptable adjuvants, such as diluents, binders, wetting agents, disintegrants, solvents, and/or buffers, and the like. The auxiliary materials are prepared into medicaments with the manganese type high-stability superoxide dismutase in a manner known in the art, and the dosage of the auxiliary materials is also known by the person skilled in the art.

Preferably, when the product is a food or health product, it further comprises one or more pharmaceutically acceptable adjuvants, such as bulking agents, taste modifiers, solvents and/or buffers, etc. The auxiliary materials and the manganese type high-stability superoxide dismutase are prepared into products in a mode known in the field, and the dosage of the auxiliary materials is also known by the person skilled in the art.

Preferably, the improvement in symptoms of autism is alleviation or treatment of autism.

In a second aspect, the present invention provides a product for ameliorating the symptoms of autism, said product comprising manganese-type high stability superoxide dismutase (MS-SOD) having the sequence shown in SEQ ID NO. 4.

Preferably, the product is a medicament, food or health product.

According to experimental results, the manganese type high-stability superoxide dismutase adopted by the invention can improve the ecological imbalance of intestinal flora and regulate the expression of intestinal barrier-related protein, thereby improving the oxidative stress of intestinal tracts and brain, achieving the purpose of relieving and improving the behavior of autism, and providing a new way for treating the autism.

Drawings

FIG. 1A shows the result of detecting superoxide anions in the intestinal tract of mice by a WST method; NADH is marked as a matched reagent in the kit;

FIG. 1B is a photograph of mouse intestinal slice ROS level detected by a fluorescent probe ethidium dihydrogen method;

FIG. 2 is a graph showing the results of measurement of relative expression levels of mRNA of the intestinal barrier-associated tight binding protein;

FIG. 3 shows the results of the measurement of the bleeding of serotonin, interleukin-6 and endotoxin molecules in the intestinal tract;

FIG. 4 shows the result of detecting ROS level in brain tissue section of mouse after intervention of MS-SOD by using fluorescent probe ethidium dihydrogen method;

FIG. 5 is a result of expression of a protein associated with oxidative stress pathway;

FIG. 6 is an observation of engraving behavior (bead burying and hair dressing);

FIG. 7A is a mouse social behavior observation;

FIG. 7B is a mouse social preference selection observation;

FIG. 8 is the results of alpha-diversity of intestinal flora in mice;

FIG. 9 shows the results of β -diversity of intestinal flora in mice.

Detailed Description

The present invention will be described below by way of specific embodiments, but the contents of the present invention are not limited thereto. The following examples are provided to demonstrate the effect of manganese-type, highly stable superoxide dismutase in ameliorating the symptoms of autism. It will be understood by those skilled in the art that, when the manganese-type high-stability superoxide dismutase has the above-mentioned effects, it is a routine technique in the art to prepare products as an active ingredient and to perform relevant tests on the products.

Unless otherwise specified, the reagents and instruments used in the following examples are all conventional in the art and are available in commercially available forms; the experimental procedures used in the following examples are also routine in the art, and those skilled in the art can unambiguously complete the experiments and obtain corresponding results depending on the purpose of the experiments.

Example 1: preparation of manganese type high stability superoxide dismutase (MS-SOD)

The target gene was obtained by amplifying the genome of Thermus HB27 (purchased from ATCC cell Bank, accession number ATCC BAA-163) as a template with the following primer sequences: a forward primer: 5'-agaattcatgccgtacccgttcaagct-3' (SEQ ID NO:1) reverse primer: 5'-ctgtcgactcaggccttcttgaagaac-3' (SEQ ID NO: 2); the amplification product was recovered with a recovery kit (purchased from Shanghai (Co.) Ltd. in Biotechnology engineering), double-digested with enzymes EcoRI and Sal I, ligated to a plasmid vector pET28a (+) (purchased from Shanghai (Co.) in Biotechnology engineering) which was double-digested with the same enzymes, the recombinant plasmid was transformed into competent Escherichia coli BL21(DE3) (purchased from Shanghai (Co.) in Biotechnology engineering) and a strain having the correct MS-SOD nucleotide sequence was selected by sequencing and cultured to obtain MS-SOD protein. Wherein, the nucleotide sequence of the MS-SOD coding gene is as follows:

atgccgtacccgttcaagcttcctgacctaggctacccctacgaggccctcgagccccacattgacgccaagaccatggagatccaccaccagaagcaccacggggcctacgtgacgaacctcaacgccgccctggagaagtacccctacctccacggggtggaggtggaggtcctcctgaggcacctcgccgcccttccccaggacatccagaccgccgtgcgcaacaacgggggcgggcacctgaaccacagcctcttctggaggctcctcacccccgggggggccaaggagcccgtgggggagctgaagaaggccattgacgagcagttcgggggcttccaggccctcaaggagaagctcacccaggcggccatgggccggttcggctcgggctgggcctggctcgtgaaggaccccttcggcaagctccacgtcctctccacccccaaccaagacaaccccgtgatggagggcttcacccccatcgtgggcattgacgtctgggagcacgcctactacctcaagtaccagaaccgccgggccgattacctccaggccatctggaacgtcctcaactgggacgtggccgaggagttcttcaagaaggcctga(SEQ ID NO:3)。

the amino acid sequence of MS-SOD is as follows:

MPYPFKLPDLGYPYEALEPHIDAKTMEIHHQKHHGAYVTNLNAALEKYPYLHGVEVEVLLRHLAALPQDIQTAVRNNGGGHLNHSLFWRLLTPGGAKEPVGELKKAIDEQFGGFQALKEKLTQAAMGRFGSGWAWLVKDPFGKLHVLSTPNQDNPVMEGFTPIVGIDVWEHAYYLKYQNRRADYLQAIWNVLNWDVAEEFFKKA(SEQ ID NO:4)。

example 2: construction of autism mouse model

(1) The source of the mice is: adult C57 female and male mice, both purchased from experimental animals center, guangdong province. After 8 weeks, 30 mice, female (20-25g) and male (25-30g) mice were acclimatized by raising them under periodic illumination (7: 00 AM-7: 00PM) at constant temperature of 25 ℃ and constant humidity of 55% for several days. Then pressing the female: male is 1:1 ratio was caged overnight. The next morning, female mice with vaginal emboli detected were scored as E1 (embryo first day, embryoic 1), and then individually housed in cages. Pregnant females were randomly grouped.

(2) Molding the autism mouse:

VPA (sodium valproate) molding is one of the accepted molding methods and is commonly used for the study and treatment of epilepsy and other neuropsychological disorders.

VPA model (sodium valproate model): on day 12 of pregnancy, the pregnant mice were injected with valproic acid. Concentration of valproic acid: 150 mg/ml; injection amount: 5mg/10g body weight.

Control group: on the 12 th day of pregnancy, 50. mu.l of 0.9% physiological saline solution was intraperitoneally injected.

Example 3: test experiments

1. Grouping MS-SOD intervention experiments:

the mice used in this experiment (newborn mice born by the pregnant mice mentioned above) were randomly divided into the following groups (10 in each group) after weaning for three weeks, and the intervention was continued for 4 weeks, and feces were collected every week. Behaviours were tested beginning at week eight (SuperMaze animal behavioural laboratory analysis software). After the test was completed, the mice were anesthetized with chloral hydrate and dissected to collect the intestinal tissue and the brain tissue of each section.

Group A: normal mice (NC group): freely drinking water;

group B: normal mice + MS-SOD intervention group (NC + MS-SOD group): drinking MS-SOD solution (30U/10g body weight);

group C: autistic mice (ASD group): freely drinking water;

group D: autistic mice + MS-SOD intervention group (ASD + MS-SOD group): drinking MS-SOD solution (30U/10g body weight);

group E: autistic mice + MS-SOD intervention low dose group (AS _ L group): drinking MS-SOD solution (20U/10g body weight);

and F group: autistic mice + MS-SOD intervention high dose group (AS _ H group): the MS-SOD solution (30U/10g body weight) was drunk.

2. Fluorescence probe method for detecting Reactive Oxygen Species (ROS) in intestinal and brain tissues

The ROS fluorescent probe, namely Dihydroethidium (DHE), can freely penetrate through a living cell membrane to enter a cell, and is oxidized by ROS in the cell to form oxidized ethidium, and the oxidized ethidium can be doped into chromosome DNA to generate red fluorescence. The nucleus is blue (blue fluorescence is used as a substrate value to indicate that dye enters), and the amount and change of the ROS content of the cell can be judged according to the generation of red fluorescence in the cell.

Labeling a probe:

1) according to different ROS content of cells, the final concentration of the dihydroethidine can be selected within the range of 1-100 mu M, and the incubation time can be selected for 30 min. Incubations were performed at 37 ℃ with exclusion of light.

2) After incubation, the tissue was washed with fresh solution.

The operating method of fluorescence photomicrography is as follows:

1) for adherent growth cells or living tissues, the cells or living tissues can be directly observed under a fluorescence microscope; for cells growing in suspension, 25-50. mu.l of the cell suspension was dropped onto a microscope slide and covered with a coverslip.

2) Under a fluorescence microscope, exciting by blue light or green light, observing and shooting a red emission image of the cell, and dyeing ROS positive cells into red in the whole nuclear area; when excited by ultraviolet light, unoxidized dihydroethidine in cytoplasm can emit blue fluorescence.

3. Detection of superoxide anion production in intestinal tract

The superoxide detection kit of Shanghai Biyuntian biotechnology limited company is adopted for detection. The principle of the kit is as follows: the superoxide can be detected on the basis that the superoxide can reduce water-soluble tetrazole-1 (WST-1) to generate soluble colored substances.

(1) Adding 200 microliters of superoxide buffer solution, 10 microliters of WST-1 solution and 2 microliters of Catalase solution into a detection hole of the ELISA plate;

(2) adding a processed sample to be detected (intestinal feces) into the detection hole;

(3) incubation at 37 ℃ for 3 min; the absorbance was measured at 450 nm.

4. Sequencing analysis of intestinal flora bacteria 16S rDNA

And extracting the total DNA of the bacteria in the excrement sample according to the instruction of the kit for extracting the total DNA of the bacteria in the excrement by the Yirui excrement magnetic bead method. The 16S rDNA V4 variable region was amplified using the extracted total DNA as a template. In this experiment, universal primers with barcode were used, wherein V4 upstream primer F-5 '-gtgccagcmgccgcgggtaa-3' (SEQ ID NO:5) and V4 downstream primer R-5 '-ccggactachvggtwtctaat-3' (SEQ ID NO: 6). The PCR product of the 16S rDNA V4 region was sequenced using Illumina HiSeq2000 sequencing technology.

The obtained original data is processed (the adaptor sequence, the low-complexity sequence and the low-quality sequence are removed) to obtain an original base sequence of the high-throughput sequencing data after being downloaded, and the data is analyzed by QIIME. The biological analysis mainly comprises the generation of an operation classification unit (OTU), principal component analysis (PCoA), flora characteristic analysis, genus abundance analysis, community structure diversity analysis, flora difference comparison analysis (an online LefSe statistical analysis tool) and the like.

5. Determination of relative expression of proteins associated with intestinal tissue and brain tissue

Comprises the measurement of the relative expression quantity of proteins such as intestinal tissues TJP-1, TJP-2, Occludin, CLDN2, CLDN3, CLDN4, CLDN7, CLDN8, CLDN12, brain tissue Nrf-2, SOD and the like.

(1)100mg of intestinal tissue was added to lml of TRIzol reagent (Invitrogen), and lmin was homogenized electrokinetically and allowed to stand at room temperature for 5 min.

(2) Centrifuge at 12000g for 5min at 4 ℃. 200 μ l of chloroform was pre-added to a 1.5ml EP tube for use. After the centrifugation was completed, the supernatant was taken and added to chloroform. After shaking vigorously and mixing for 15s, standing at room temperature for 5 min. Centrifuge at 12000g for 15min at 4 ℃. 500. mu.l of isopropanol were prepared and pre-added to a 1.5ml EP tube for use.

(3) The upper colorless aqueous phase (middle white thin layer is protein, lower red phenol-chloroform phase contains DNA) was slowly extracted and added into isopropanol, inverted several times, and left at room temperature for 10 min. Centrifugation was carried out at 12000g for 10min at 4 ℃ to remove the supernatant and precipitate the pellet RNA at the bottom of the tube.

(4) Washing the precipitate with lml 75% precooled ethanol, centrifuging for 5min at 4 ℃ and 7500g, discarding the supernatant, depositing RNA at the bottom of the tube, and drying at room temperature.

(5) The RNA was dissolved in water after treatment with appropriate amount of diethylpyrocarbonate (EDPC).

(6) The concentration was measured using a spectrophotometer to quantify the RNA.

(7) RT-PCR (20. mu.l system) was performed using TaKaRa kit, and the expression level of mRNA was calculated using β -actin as an internal reference, thereby detecting the expression level of the intestinal barrier-associated gene.

6. Behavioral experimental method

(1) Experiment of embedding beads

12 mice were used for the bead burying experiment (marble burying test). Corn cob packing materials with the thickness of about 5cm are paved in the mouse cage, and 20 glass beads (4 glass beads in each row and 5 rows in total) are uniformly paved on the surface of the flat packing materials at equal intervals. For testing, mice were placed from one corner, allowed to move freely for 30min, and mouse activity was recorded. The mouse can use four limbs or the tip of the nose to push the padding to bury the glass beads, after the test is completed, the number of the buried beads of the mouse is counted, and the anxiety level of the mouse can be detected through the number of the buried glass beads. The evaluation criteria were more than 1/2 for the beads covered by the mat to be marked as buried glass beads.

(2) Experiment of hair-plucking behavior

The hair-conditioning behavior is a repeated stereotypical behavior which is often shown by mice in an autism model. The mouse is placed in the sound insulation observation box to prevent external interference, and the mouse can freely move in the box. The mice were allowed to acclimate in the box for 10 minutes, then the grooming behavior of the mice was recorded for 10 minutes using a camera, and the grooming behavior of the mice was recorded continuously for 5 days. The evaluation criterion for hair management in this experiment is the time for any hair on the mouse to be trimmed.

(3) Three-box experiment

The three-box social test principle is based on the characteristics that mice are naturally favored to be in social groups and have exploration tendency on new objects. While social behavior reduction is another common symptom of autism, three cases of social tests were chosen to test the social behavior of mice in this experiment. The social ability of the mice was reflected by detecting when the mice approached a metal cage in the laboratory box containing another strange mouse after the mice were familiar with the environment. The experimental box is a box with the length, the width and the height of 60cm, 45cm and 25cm, and is divided into three compartments with the same size by two white boards. The adjacent rooms had 5cm hatches through which mice could freely pass through the three rooms. The residence time of the mouse in each room was photographed and recorded using a camera. Social mice were placed in the experimental box for 30min the day before testing.

Social behavior detection: two metal cages are respectively placed in a left room and a right room, wherein one metal cage is used for placing a strange mouse (strange mouse 1), and the other metal cage is empty. The strange mice should have the same sex and age as the tested mice, and are not fed together with the tested mice for health preservation and survival. And then placing the tested mouse in a middle room and starting recording, wherein the recording time is also 5min, and after the experiment is finished, recording the difference value of the waiting time of the left room and the right room as the social contact time of the tested mouse.

Social preference detection: another strange mouse (strange mouse 2) was placed in the empty metal cage, the test mouse was placed in the middle room, and the residence time of the test mouse in the rooms of the strange mouse 1 and the strange mouse 2 was also recorded.

Results of the experiment

1. Oral SOD supplementation can improve the intestinal oxidative stress imbalance of autistic mice

FIG. 1A shows the results of detecting superoxide anions in the intestinal tract of mice by the WST method, with time on the abscissa and luminescence intensity on the ordinate, showing the concentration level of superoxide anions. From the results of this examination, it was found that the level of superoxide anion was stable in the normal group and the production of superoxide anion was significantly increased in the autism group with the change of time. When the normal mice and the autism mice are fed with MS-SOD, the concentration of superoxide anions is not increased, which indicates that the intestinal superoxide anions of the autism mice can be adjusted to normal level after being fed with MS-SOD.

The fluorescence probe-ethidium hydride method is adopted to measure and treat intestinal tissue slices, and the result can be also shown (figure 1B), the red fluorescence intensity of the intestinal tissue of the autistic mouse is higher than that of the intestinal tissue of the normal group of mice, which indicates that the superoxide anion level in the intestinal tissue of the autistic mouse is higher. No significant change in intestinal tissue superoxide anion concentration was observed in either autistic or normal mice after feeding MS-SOD. The result shows that the feeding of MS-SOD can reduce the concentration of superoxide anion in the intestinal tract of mice with autism, and the increase of the concentration of superoxide anion plays a role in the occurrence process of autism, thereby showing that MS-SOD plays a role in the treatment of autism.

2. The SOD is supplemented by oral administration, and the condition of intestinal barrier damage of the mice with autism can be obviously improved

Previous studies have shown that patients with autism are often accompanied by intestinal abnormalities, which, when the intestine becomes inflamed, can cause intestinal cell swelling, large intercellular spaces, and intestinal leakage, resulting in the penetration of macromolecular substances through the intestinal wall into the body. Serious bowel problems may be associated with breakdown and ulceration of the bowel, with the macromolecular substances entering the bowel more easily. As shown in the results of fig. 2, the expression of genes of the intestinal barrier-associated tight junction proteins TJP-1, TJP-2, Occludin, CLDN2, CLDN3, CLDN4, CLDN7, CLDN8, CLDN12, etc. was impaired in autistic mice, and the expression of intestinal barrier genes was significantly reversed after MS-SOD intervention, thereby improving the symptoms of autistic mice (see fig. 2).

3. Molecular invasion of intestinal endotoxin, interleukin-6 and serotonin

The situation that the intestinal barrier of a mouse is damaged after the autism happens can be known by detecting the molecules. Experimental results show that the contents of serotonin (5-HT), interleukin-6 (IL-6), endotoxin (LPS) and the like in the mice with the autism are obviously increased, and the indexes are obviously reduced after the MS-SOD is dried, so that the intestinal barrier is repaired (see figure 3), and the autism behavior of the mice is improved.

4. Mouse brain tissue slice ROS level after MS-SOD intervention

As can be seen in FIG. 4, after MS-SOD intervention, the oxidative stress of the brain, the brain barrier and the inflammatory state of the brain of the autistic mice were improved.

The brain tissue is measured and treated by adopting a fluorescent probe-dihydropyridine method, and the result shows that the red fluorescence intensity of the brain tissue of an autism mouse (ASD) is higher than that of the brain tissue of a normal group of mice, which indicates that the superoxide anion level in the brain tissue of the autism mouse is higher. The brain tissue superoxide anion levels were significantly reduced after feeding MS-SOD in both autistic and normal mice (see FIG. 4).

Nrf-2 is an important protein of an oxidative stress pathway, and activation of Nrf-2 can start expression of a plurality of downstream protective genes, such as expression of antioxidases such as SOD (superoxide dismutase) and the like, and improve total antioxidant capacity (T-AOC) so as to protect brain tissues from being damaged by superoxide radicals. As can be seen from the experimental results, the expression of Nrf-2 was significantly increased in the mice with autism fed with MS-SOD (see FIG. 5), indicating that MS-SOD has a therapeutic effect on autism.

5. Experimental results of engraving behavior

The pearl burying behavior and the hair trimming behavior are repeated stereotypy behaviors which are often shown by mice in an autism model. Experiments show that the number of embedded beads and hair management time of mice in the autism group are obviously increased compared with those of normal mice, and the two autism-like behaviors of the autism mice are respectively reduced by 23% and 33% after the mice are fed with MS-SOD (figure 6). The MS-SOD can reduce the anxiety symptom of the autism mouse and reduce the occurrence of the autism behavior.

6. Observation of social behaviors of mice

Mice are social animals and have a curious exploration tendency for strange things. Fig. 7A shows the social time with the familiar and unfamiliar mice, and fig. 7B shows the social time with the unfamiliar mice minus the unfamiliar time with the familiar mice in fig. 7A, with a negative value indicating that the experimental mice have a shorter social time with the familiar mice than with the unfamiliar mice, unlike the normal mice. The experimental results show that the social behaviors of the autistic mice are obviously reduced compared with the normal group of mice, the social preference is also selected to be in contact with the familiar mice, and the social behaviors are recovered after feeding SOD, and the autistic mice are also in contact communication with stranger mice (see figures 7A and 7B).

7. Experimental results of intestinal flora in mice

Metagenomic sequencing is carried out on the intestinal flora of the mice, the autism mice and normal mice show obvious difference through analysis of chao index, shannon index and the like, and different doses of MS-SOD (low dose and high dose, namely E group and F group mice) can enable the flora alpha-diversity of the autism mice to show a recovery trend (see figure 8).

The mouse intestinal flora is subjected to metagenomic sequencing, and a three-dimensional PCoA picture shows that the intestinal flora of the autism mouse and the normal mouse show obvious differentiation trend, and the MS-SOD with different dosages can restore the intestinal flora of the autism mouse to a normal state (see figure 9).

Reference documents:

[1].Hevner,R.F.,Brain overgrowth in disorders of RTK–PI3K–AKT signaling:A mosaic of malformations.Seminars in Perinatology,2015.39(1):p.36-43.

[2].Balan,S.,et al.,Exon resequencing of H3K9methyltransferase complex genes,EHMT1,EHTM2and WIZ,in Japanese autism subjects.Mol Autism,2014.5(1):p.49.

[3].Volk,H.E.,et al.,Autism Spectrum Disorder.Epidemiology,2014.25(1):p.44-47.

[4].Maternal Infection during Pregnancy and Autism Spectrum Disorders.

[5].Pediatric Neurology.

[6].Decreased Levels of EGF in Plasma of Children with Autism Spectrum Disorder.

[7].Thioredoxin:A novel,independent diagnosis marker in children with autism.

[8].Dachtler,J.,et al.,Deletion ofα-neurexin II results in autism-related behaviors in mice.Translational Psychiatry,2014.4(11):p.e484.

[9].De Angelis,M.,et al.,Autism spectrum disorders and intestinal microbiota.Gut Microbes,2015.6(3):p.207-13.

sequence listing

<110> Hangzhou Rui Dai medicine science and technology Limited

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<213> Artificial Sequence (Artificial Sequence)

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ctgtcgactc aggccttctt gaagaac 27

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<213> Thermus (Thermus thermophilus)

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atgccgtacc cgttcaagct tcctgaccta ggctacccct acgaggccct cgagccccac 60

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Claims

1. The application of the manganese type high-stability superoxide dismutase in preparing the medicine for improving the symptom of the autism, wherein the sequence of the manganese type high-stability superoxide dismutase is shown as SEQ ID NO. 4.

2. Use according to claim 1, wherein the medicament is administered by the oral, injectable or intragastric route.

3. Use according to claim 2, wherein the injection is intravenous or intraperitoneal.

4. The use of claim 2, wherein the medicament is in the form of a tablet, capsule, powder for injection, injection or aerosol.

5. The use according to claim 4, wherein the medicament further comprises one or more pharmaceutically acceptable excipients which are diluents, binders, wetting agents, disintegrants, solvents and/or buffers.

6. The use according to any one of claims 1 to 5, wherein the amelioration of symptoms of autism is a relief or treatment of autism.