Use of LP17 in the preparation of an intervention drug for preventing / treating autism

Abstract

The present invention relates to the use of LP17 in the preparation of an intervention drug for preventing / treating autism. The LP17 can inhibit the expression of TREM1 and reduce central nervous system inflammation. It has been verified by experiments that the early action of LP17 in neonatal mice can inhibit the accumulation of TREM1, significantly reduce neuroimmune inflammation in the autism model BTBR mice, and has the ability of neuroprotection and correction of the behavior of adult mice, improving the defects in the three-chamber social experiment of adult mice, reducing the stereotyped behavior of adult mice, reversing the impact of autism-related behaviors in adult mice, and providing clues for the development of autism.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technologies, and particularly relates to the application of LP17 in the preparation of intervention drugs for preventing / treating autism. Background Art

[0002] Autism is classified as a developmental disorder caused by nervous system disorders, and its symptoms include social dysfunction and restricted, repetitive behaviors or interests. At present, the demand of autistic patients and their families for professional treatment, rehabilitation and education services is increasing day by day. Patients and their families hope to obtain more effective treatment methods, more professional rehabilitation guidance and more comprehensive educational support to improve the quality of life and social adaptability of patients.

[0003] BTBR mice are currently recognized as an idiopathic autism animal model, whose core symptoms are most similar to the clinical symptoms of autistic patients and can be stably inherited to offspring. BTBR mice show obvious social dysfunction such as lack of social motivation and social ability in the three-chamber social experiment. In the direct social interaction experiment, exploratory social behaviors such as sniffing and following of BTBR mice are significantly reduced. In the USV test (ultrasonic vocalization test), when BTBR mice leave the cage of their mother mice, BTBR mice emit abnormal high-frequency and high-amplitude ultrasonic waves. Researchers believe that this behavior is similar to the loud crying of autistic children when they are forced to be separated from their mothers. In contrast, when adult BTBR mice face the smell of strange mice, the number of ultrasonic waves they emit decreases, which is similar to the language communication disorder of human autistic patients. In addition, studies have also shown that the repetitive behaviors of BTBR mice increase, such as living alone, digging, grooming and burying beads, etc. The anatomical characteristics of BTBR mice also show a certain consistency with those of autistic patients. For example, in BTBR mice, organic lesions in the central nervous system are mainly concentrated in cortical, corpus callosum and hippocampal dysplasia, and these characteristics are similar to the clinical manifestations of autistic patients.

[0004] Existing studies have proved that the occurrence of autism is related to chronic inflammation in the central nervous system, and the relevant exact pathogenesis has been determined to have reliable value for autism diagnosis or prognosis. A number of evidences show that infectious or inflammatory immune activation during pregnancy leads to a higher risk of offspring presenting with autism-related neurodevelopmental symptoms. It has been found that the offspring of maternal immune activation (MIA) model mice show autistic-like behaviors and cortical damage caused by MIA, reducing the excessive generation of CTIP2+ (cortical thymocyte transforming protein 2 positive) cortical neurons in the postnatal period. This suggests that the phenotypes induced by persistent neuroinflammation after birth are the key risk factors for inducing autism-related behaviors.

[0005] Triggering receptor expressed on myeloid cells-1 (TREM1) is a receptor belonging to the immunoglobulin superfamily, which triggers the synthesis and release of pro-inflammatory cytokines and plays an important role in acute and chronic inflammation. Regulating neuroinflammation by inhibiting the accumulation of TREM1 is an important mechanism that has not been previously emphasized in autism.

[0006] From the current research status, the exact cause of autism is still unclear, and there is no specific drug. Only some antipsychotic drugs can help relieve the symptoms of autism. Currently, the treatment of autism mainly focuses on behavioral therapy. The core symptoms of some autistic patients will persist into adulthood and require lifelong support.

[0007] In summary, discovering a drug that can directly target the core symptoms of autism and effectively improve them will be of great significance for the treatment of autism and for improving the lives of adult patients suffering from autism symptoms. Summary of the Invention

[0008] In view of the deficiencies of the existing technology, the present invention provides the application of LP17 in the preparation of an intervention drug for preventing / treating autism.

[0009] The technical solution of the present invention is as follows:

[0010] The application of LP17 in the preparation of an intervention drug for preventing / treating autism.

[0011] Further, the LP17 can inhibit the expression of TREM1.

[0012] Further, the LP17 can reduce central nervous system inflammation.

[0013] Further, the drug acts on early postnatal autistic patients.

[0014] Further, the administration method of the drug is intracranial injection.

[0015] Further, the effective dose of the drug is 20 mg / kg.

[0016] Further, the drug contains pharmaceutically acceptable excipients.

[0017] The excipients include but are not limited to: diluents, buffers, suspensions, emulsions, granules, encapsulants, excipients, fillers, binders, sprays, transdermal absorbers, wetting agents, disintegrants, absorption promoters, surfactants, colorants, flavoring agents or adsorption carriers.

[0018] Compared with the existing technology, the present invention has at least the following advantages:

[0019] The present invention relates to the use of LP17 in the preparation of an intervention drug for preventing / treating autism. LP17 can inhibit the expression of TREM1 and reduce central nervous system inflammation. Through experimental verification, the early action of LP17 in neonatal mice can inhibit the accumulation of TREM1, significantly reduce neuroimmune inflammation in the autism model BTBR mice, and has the ability of neuroprotection and correction of the behavior of adult mice. It significantly reduces the neuronal apoptosis in the cortex of BTBR mice that is higher than that of wild-type mice, improves the defects in the three-chamber social experiment of adult mice, reduces the stereotyped behavior of adult mice, reverses the influence of autism-related behaviors in adult mice, and provides clues for the development of autism. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] 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.

[0021] Figure 1 It is the enrichment situation of the KEGG pathway in Test Example 1 of the present invention;

[0022] Figure 2 It is the detection of the expression of inflammatory factors by ELISA and RT-qPCR in Test Example 1 of the present invention;

[0023] Figure 3 It is the activation degree of microglia in the cortex of BTBR mice in Test Example 2 of the present invention;

[0024] Figure 4 It is the apoptosis situation of neurons in the cortex of BTBR mice in Test Example 2 of the present invention;

[0025] Figure 5 It is that the expression level of TREM1 protein in the cerebral cortex of P7 BTBR-Vehicle mice in Test Example 3 of the present invention is significantly up-regulated;

[0026] Figure 6 It is the comparative analysis result of the homology and functional similarity of human and mouse TREM1 genes and proteins in Test Example 4 of the present invention;

[0027] Figure 7 It is the bioinformatics comparative analysis of the functions of human and mouse TREM1 proteins and their interacting proteins in Test Example 4 of the present invention;

[0028] Figure 8 It is the enrichment of the core functions of human and mouse TREM1 proteins through bioinformatics analysis in Test Example 4 of the present invention;

[0029] Figure 9This is for Test Example 5 of the present invention to detect the activation state of cortical microglia and the change in the level of TREM1 protein expressed by microglia in mice of the BTBR-Vehicle, BTBR-LP17, and C57BL / 6-Vehicle groups 7 days after birth before and after medication;

[0030] Figure 10 This is for ELISA detection of the expression of total TREM1 protein in brain tissues of the three groups of mice before and after medication in Test Example 5 of the present invention;

[0031] Figure 11 This is for the expression of pro-inflammatory factors in cerebral cortex tissues of the four groups of mice before and after medication in Test Example 6 of the present invention;

[0032] Figure 12 This is for the analysis of the change in the number of apoptotic neurons in the cerebral cortex of mice before and after medication in Test Example 6 of the present invention;

[0033] Figure 13 This is for the three-chamber social experiment test analysis of the four groups of mice before and after medication in Test Example 7 of the present invention;

[0034] Figure 14 This is for the test analysis of buried glass beads and self-grooming behavior of the four groups of mice before and after medication in Test Example 7 of the present invention. Detailed implementation manners

[0035] The present invention will be further described in detail below. It is necessary to point out here that the following specific implementation manners are only used to further illustrate the present invention and cannot be understood as limiting the protection scope of the present invention. Those skilled in the art can make some non-essential improvements and adjustments to the present invention based on the above application content.

[0036] The present invention generally and / or specifically describes the materials and test methods used in the experiments. For the test methods or testing methods involved, unless otherwise specified, they are all conventional methods; for the reagents or instruments used, unless the manufacturer is indicated, they are all commercially available conventional products and are prepared or used by conventional methods.

[0037] Source of materials:

[0038] BTBR mice (autistic mice), male and female breeding populations of the BTBR T+Itpr3tf / J (BTBR) strain were purchased from The Jackson Laboratory (Bar Harbor, ME, USA) and used for the experiments of this patent after breeding;

[0039] C57BL / 6 mice (wild-type mice), purchased from the laboratory of Army Medical University;

[0040] Feeding environment: 5 - 6 mice per cage, grouped according to a 12-hour light-dark cycle, placed in a standard animal facility, with free access to water and food.

[0041] LP17 (an inhibitory peptide targeting TREM1 with the amino acid sequence LQVTDSGLYRCVIYHPP (SEQ ID NO. 3)) was synthesized by Chongqing Biaoyue Biotechnology Co., Ltd.

[0042] Example 1 Pretreatment of BTBR mice and C57BL / 6 mice

[0043] Newborn BTBR mice were randomly divided into two groups of 10 mice each. The day of birth was designated as postnatal day 0 (P0). To avoid litter effects, animals in each treatment group were randomly selected from multiple litters, and one pup was selected from each litter. As a control group, C57BL / 6 mice were treated in the same way.

[0044] Group ①: Mice in the BTBR-Vehicle (solvent control) group were injected intracerebroventricularly with 0.9% NaCl:DMSO = 2:3 on both P5 (postnatal day 5) and P6 (postnatal day 6).

[0045] Group ②: Mice in the BTBR-LP17 group were injected intracerebroventricularly with LP17 at a concentration of 20 mg / kg on both P5 and P6.

[0046] Group ③: Mice in the C57BL / 6-Vehicle group were injected intracerebroventricularly with 0.9% NaCl:DMSO = 2:3 on both P5 and P6.

[0047] Group ④: Mice in the C57BL / 6-LP17 group were injected intracerebroventricularly with LP17 at a concentration of 20 mg / kg on both P5 and P6.

[0048] The above grouped mice were subjected to various tests, and the test results are as follows.

[0049] Test Example 1 Exacerbated cortical inflammation in P7-BTBR mice (BTBR mice at 7 days after birth)

[0050] ① In this test example, cortical tissues of P7 BTBR-Vehicle and P7 C57BL / 6-Vehicle mice were collected for transcriptome sequencing. Through bioinformatics analysis, functional annotation of DEGs (differentially expressed genes) was performed based on the Kyoto Encyclopedia of Genes and Genomes (KEGG) database and Gene Ontology (GO) category database; the changes in gene mRNA expression in the cortical tissues of early postnatal BTBR mice were detected at the transcriptome level. The detection results are as Figure 1As shown, pathways with a P-value less than 0.05 in the KEGG pathway enrichment analysis were considered significant. The pathways were sorted by P-value, and the top 20 pathways were presented as a bar chart. The x-axis represented -log10(P-value), and the bars were colored according to the proportion of upregulated or downregulated differentially expressed genes; significantly enriched gene ontology terms were shown as a dot plot, with the dots continuously colored by -log10(P-value), where lighter to darker colors indicated lower to higher P-values, and the size of the dots varied according to the number of differentially expressed genes belonging to that term; as can be seen from Figure 1 it that compared with P7 C57BL / 6-Vehicle mice, the immune system processes and inflammatory responses were significantly enriched in P7 BTBR-Vehicle mice as shown by KEGG.

[0051] ② By ELISA (enzyme-linked immunosorbent assay) and RT-qPCR (reverse transcription quantitative real-time polymerase chain reaction) detection, it was found that compared with P7 C57BL / 6-Vehicle, the mRNA expression levels of IL-6 (interleukin-6) and TNF-α (tumor necrosis factor α) in the brains of P7 BTBR-Vehicle mice were significantly increased; in the ELISA experiment, the detection results confirmed that the protein levels of pro-inflammatory factors IL-6, TNF-α, and CXCR2 (CXC chemokine receptor 2) in the cortex of P7 BTBR-Vehicle mice were significantly higher than those of the control group mice (C57BL / 6-Vehicle). At the same time, the ELISA results also confirmed that the protein level of the inflammatory factor NF-κB (nuclear factor κB) was also significantly upregulated in the cortex of P7 BTBR-Vehicle mice ( Figure 2 ), further confirming that the neuroimmune inflammatory microenvironment in the brains of early postnatal BTBR mice was altered.

[0052] Test Example 2 The activation degree of microglia in the cortex of P7-BTBR-Vehicle mice was significantly increased, and the degree of neuronal apoptosis in the cortex was significantly higher than that of the control group mice

[0053] ① In this test example, immunofluorescence staining was used to observe the activation of resident immune cells, namely microglia, in the cortex of P7 BTBR-Vehicle mice; the results were as Figure 3 shown Figure 3 A, Figure 3 B The results showed that compared with P7 C57BL / 6-Vehicle mice, the number of activated microglia in the cerebral cortex region of P7 BTBR-Vehicle mice was significantly increased, Figure 3 and as can be seen from C, the cell morphology was mostly amoeba-like, with the cell body becoming round and the branches shortening.

[0054] ② The alteration of the immune-inflammatory microenvironment in the brains of early postnatal BTBR mice may affect neuronal apoptosis. Therefore, in this test example, immunofluorescence staining was used to observe the cortical neurons of P7 BTBR-Vehicle mice; the experimental results are as Figure 4 shown. As can be seen from the figure, compared with P7 C57BL / 6-Vehicle mice, the number of NEUN+TUNEL+ double-positive cells in the cerebral cortex of P7 BTBR-Vehicle mice was significantly increased (the NEUN+TUNEL+ double-labeling technique is a method for simultaneously detecting neuronal apoptosis and the presence of neurons. NEUN (Neuronal Nuclei) is a neuronal nuclear protein, and the TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) technique is used to detect cell apoptosis), suggesting that the number of apoptotic neurons in the brains of early postnatal BTBR mice was significantly increased.

[0055] Test Example 3 The expression of TREM1 in the cortex of P7 BTBR-Vehicle mice was significantly increased

[0056] ① In this test example, transcriptome sequencing was performed on the cerebral cortex of P7 BTBR-Vehicle mice, and bioinformatics analysis was carried out on the differentially expressed genes in the cortex of P7 BTBR-Vehicle mice and P7 C57BL / 6-Vehicle mice. The results are as Figure 5 shown in A. As can be seen from the figure, the upregulation of TREM1 expression is of great significance in the differentially expressed gene expression network in the cortex of BTBR and control mice.

[0057] ② In this test example, the expression level of TREM1 protein was further detected by ELISA. The detection results are as Figure 5 shown in B, confirming that the expression level of TREM1 protein in the cortex of P7 BTBR-Vehicle mice was significantly upregulated compared with that of P7 C57BL / 6-Vehicle mice.

[0058] Test Example 4 Comparative analysis of the homology and functional similarity of human and murine TREM1 genes and proteins

[0059] ① The human and murine TREM1 gene and protein sequences were obtained from the NCBI and Uniprot databases.

[0060] Human TREM1 protein sequence: MRKTRLWGLLWMLFVSELRAATKLTEEKYELKEGQTLDVKCDYTLEKFASSQKAWQIIRDGEMPKTLACTERPSKNSHPVQVGRIILEDYHDHGLLRVRMVNLQVEDSGLYQCVIYQPPKEPHMLFDRIRLVVTKGFSGTPGSNENSTQNVYKIPPTTTKALCPLYTSPRTVTQAPPKSTADVSTPDSEINLTNVTDIIRVPVFNIVILLAGGFLSKSLVFSVLFAVTLRSFVP (SEQ ID NO.1)

[0061] Mouse TREM1 protein sequence: MRKAGLWGLLCVFFVSEVKAAIVLEEERYDLVEGQTLTVKCPFNIMKYANSQKAWQRLPDGKEPLTLVVTQRPFTRPSEVHMGKFTLKHDPSEAMLQVQMTDLQVTDSGLYRCVIYHPPNDPVVLFHPVRLVVTKGSSDVFTPVIIPITRLTERPILITTKYSPSDTTTTRSLPKPTAVVSSPGLGVTIINGTDADSVSTSSVTISVICGLLSKSLVFIILFIVTKRTFG (SEQ ID NO.2)

[0062] ② Through prediction and BLAST sequence comparison (Basic Local Alignment Search Tool, a search tool based on the local sequence alignment algorithm), from Figure 6 A and 6B, it can be seen that the human TREM1 gene is located on chromosome 6p21.1, and its gene structure contains 5 exons, encoding a single-pass transmembrane protein. The mouse TREM1 gene is located on chromosome 17 (the homologous region corresponding to human chromosome 6p21.1), and also consists of 5 exons. The gene structures of the two are highly conserved, with the same number of exons and the same protein transmembrane topology. Through BLAST comparison, the amino acid sequence homology between human and mouse TREM1 is approximately 60%; in terms of key regions, the human and mouse TREM1 proteins are highly conserved in key ligand-binding sites, such as the hydrophobic core region of the β-sheet and other core functional regions (Uniprot: mouse TREM1: Q9JK2, human TREM1: Q9NP99); and the protein structures of human and mouse were predicted by SWISS-MODEL, Figure 6 C is the predicted structure of mouse TREM1 protein, Figure 6 D is the predicted structure of human TREM1 protein.

[0063] ③ Use STRING to perform bioinformatics comparison and analysis on the functions of human and murine TREM1 proteins and their interacting proteins. The analysis results are as Figure 7 shown Figure 7 A is the prediction of murine TREM1 interacting proteins. Figure 7 B is the prediction of human TREM1 interacting proteins. It can be seen from the figure that TYPOBP, TREM2, TREML2, TREML1, CLEC5A, PGLYRP1, and HMGB1 are all present in the interacting proteins of human and murine TREM1, suggesting that the core interacting molecules of human and murine TREM1 are highly similar.

[0064] ④ Through Uniprot bioinformatics analysis of protein function enrichment, Figure 8 A is the core function of human TREM1 protein. Figure 8 B is the core function of murine TREM1 protein. It can be seen from the figure that the human and murine TREM1 proteins are highly conserved in their core functions, and are almost identical in apoptotic cell clearance (regulation of apoptotic cell clearance), regulation of interleukin-10 production, neuron apoptotic process, and microglial cell activation involved in immune response, macrophage fusion, and glial cell activation, suggesting that the core functions of human and murine TREM1 are highly conserved.

[0065] Test Example 5 The expression of TREM1 in microglia in the cortex of P7 BTBR-Vehicle mice was significantly increased; while the TREM1-specific antagonistic polypeptide LP17 significantly reduced the expression of TREM1 in the cortex of BTBR mice, and at the same time significantly reduced the activation degree of microglia in the cortex of P7-BTBR-Vehicle mice.

[0066] ① Use immunofluorescence technology to stain the cortex of P7 (postnatal day 7) BTBR-Vehicle, BTBR-LP17, and C57BL / 6-Vehicle group mice. Observe the stained cells with a Zeiss (Oberkochen, Germany) Axivert microscope connected to the Zeiss AvioVision 3.0 system and take pictures to detect the activation status of microglia (detecting IBA1) and the changes in the expression level of TREM1 in the mouse cortex.

[0067] The fluorescence detection results are as follows Figure 9 shown. As can be seen from Figure 9 A, in the C57BL / 6-Vehicle group of mice, the number of abnormally activated cortical microglia is small, and the expression level of the TREM1 molecule is low; while in the BTBR-Vehicle group of mice, the number of abnormally activated cortical microglia is significantly higher than that in the C57BL / 6-Vehicle group, and the TREM1 protein is specifically highly expressed in the microglia in the cortex of P7 BTBR-Vehicle mice. ELISA experiments also confirmed that the TREM1 protein in the microglia in the cortex of P7-BTBR mice is significantly higher than that in the C57BL / 6-Vehicle group of mice. The ELISA experiment results are as follows Figure 9 shown in B.

[0068] ② Meanwhile, ELISA experiments were used to detect the total amount of TREM1 protein in the cerebral cortex of P7 BTBR-Vehicle, P7 BTBR-LP17, and P7 C57BL / 6-Vehicle mice. As can be seen from Figure 10 it, after treatment with LP17, the total expression of TREM1 protein in the brain tissue of P7 BTBR-Vehicle mice decreased significantly.

[0069] The above results confirmed that after treatment with LP17, the expression of TREM1 in the cortex of P7-BTBR mice decreased significantly, and the level of neuroimmune inflammation in the cortex decreased significantly. LP17 can effectively inhibit the TREM1 pathway in the cortex of P7-BTBR mice and reduce the level of neuroimmune inflammation.

[0070] Test Example 6 Early postnatal use of the specific polypeptide antagonist LP17 can effectively reduce the expression of pro-inflammatory factors in the cortex of P7 BTBP mice, that is, reduce the cortical inflammation level of BTBR mice; at the same time, it can effectively reduce the neuronal apoptosis in the cortex of BTBR mice, which is significantly higher than that of wild-type mice.

[0071] ① Collect the cerebral cortex tissues of four groups of mice, namely BTBR-Vehicle, BTBR-LP17, C57BL / 6-Vehicle, and C57BL / 6-LP17, on the 7th day after birth (P7), and use ELISA to detect the protein expression levels of pro-inflammatory factors in the cerebral cortex, including interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and monocyte chemoattractant protein 1 (MCP-1).

[0072] The detection results are as follows Figure 11As shown, it can be seen from the figure that, compared with C57BL / 6-Vehicle, the expression of pro-inflammatory factors in the cortex of BTBR-Vehicle mice is abnormally increased, and the protein expression levels of TNF-α, IL-6, and chemokine MCP-1 in the cortical tissues of BTBR mice are significantly elevated (p<0.05); compared with BTBR-Vehicle, early postnatal intervention with the TREM1-specific polypeptide antagonist LP17 in BTBR-LP17 can significantly reduce the protein expression levels of TNF-α, IL-6, and MCP-1.

[0073] ② Using immunofluorescence experiments, it was confirmed that early postnatal use of the specific polypeptide antagonist LP17 could significantly reduce the number of apoptotic neurons in the cerebral cortex of P7-BTBR mice. The results are as Figure 12 shown.

[0074] Therefore, combined with the results of Test Example Five, early postnatal use of the specific polypeptide antagonist LP17 can effectively reduce the expression of TREM1 protein, inhibit the excessive immune inflammation in the central nervous system of BTBR mice, and at the same time reduce the excessive apoptosis of cortical neurons in BTBR mice, effectively inhibiting the TREM1 pathway.

[0075] Test Example Seven: Early postnatal inhibition of TREM1 improved the classical autistic-like behaviors of BTBR mice in adulthood

[0076] In this test example, through classical autistic-like behavioral experiments such as the three-chamber social experiment, bead-burying experiment, and grooming experiment, it was evaluated whether the autistic-like behaviors of BTBR mice in adulthood (8 weeks after birth) were improved after early postnatal intervention with the TREM1-specific antagonist LP17.

[0077] ① Three-chamber social experiment

[0078] The social approach test was conducted in a rectangular apparatus (40 cm * 60 cm * 22 cm), which was divided into three equal parts. Mice were allowed to enter the side chambers through retractable doorways in two partition walls. This test consisted of habituation and sociality. During the adaptation process, the animal was placed in the middle chamber and allowed to freely explore all three chambers. For the social activity, a strange mouse aged 8 - 10 weeks, with a matched sex and strain, was placed in a wire mesh cage and put in one of the two side chambers, while an identical empty cage was placed on the other side. The apparatus was cleaned with 70% ethanol after each trial. Physical contact with the cage surrounding the nose, head, and forelimbs was defined as sniffing behavior. The time the mouse spent in each chamber (Sociability Chamber Time) and the number of entries into each chamber (Chamber Entries) were analyzed by Noldus Observer software (Ethovision 11.0).

[0079] ② Marble burying experiment

[0080] Each mouse was placed in a clean standard home cage (27 cm × 16.5 cm × 12.5 cm). Twenty black glass marbles (15 mm in diameter) were arranged in a symmetric 4×5 cm grid, 2 - 3 cm deep on the top. The investigator counted the number of glass marbles buried by the mouse within 30 minutes. Marbles covered by bedding >50% were defined as "buried". The experiment was tested under dim light conditions (-15 lux).

[0081] ③ Grooming experiment

[0082] Using Noldus Observer software (Ethovision 11.0). The mouse was placed alone in a clean standard mouse cage, and the camera was placed 15 cm away from the cage. Under low light conditions (40 lux), the behavior of the mouse was recorded for 20 minutes. In order to manually score the repetitive self - grooming behavior of the mouse during the last 10 minutes of the test, the average cumulative time and the proportion of error transitions were calculated in this study. The microstructure of grooming behavior was analyzed using a grooming analysis algorithm (grooming steps: paws, face, body, legs, tail / genitals).

[0083] The test results of the three - chamber social experiment for the four groups of mice are as Figure 13 shown, from Figure 13As can be seen from Figure A, whether it is C57BL / 6-Vehicle mice or C57BL / 6-LP17 mice, C57BL / 6 mice showed normal social ability in terms of the parameter of Sociability Chamber Time, and the time they stayed in the chamber where the Novel Mouse was located was longer than the time they stayed in the chamber where the Novel Object was located. However, the BTBR-Vehicle group of mice showed typical social ability defects in terms of Sociability Chamber Time. However, in the BTBR-LP17 mice injected with the TREM1-specific polypeptide antagonist LP17 in the early stage of development, this situation of social ability deficiency was improved. Figure 13 Figure B shows the number of Chamber Entries of the four groups of mice into the social chamber. As can be seen from the figure, there was no significant difference in the number of Chamber Entries of the four groups of mice. Figure 13 Figure C shows the Sniffing Time graph of the four groups of mice for the Novel Mouse and the Novel Object. As can be seen from the figure, the C57BL / 6-Vehicle mice spent more time sniffing the Novel Mouse. The situation of C57BL / 6-LP17 mice was similar. However, there was no significant difference in the duration of the BTBR-Vehicle group of mice sniffing the Novel Mouse and the Novel Object. However, after injecting the TREM1-specific polypeptide antagonist LP17 at P5 and P6 in the BTBR-LP17 group, the time of the BTBR mice sniffing the Novel Mouse increased, and the time of sniffing the Novel Mouse was longer than the time of sniffing the Novel Object, with a significant difference. Figure 13 Figure D shows the number of times the four groups of mice entered the three chambers. As can be seen from the figure, the number of times the four groups of mice entered the three chambers was the same, and no congenital chamber-side preference was found.

[0084] As can be seen from Figure 14 Figure, after injecting the TREM1-specific polypeptide antagonist LP17 in the early stage of development, the number of Marbles Buried and the repetitive Self-grooming behavior of the BTBR mice were reduced. Figure 14 In Figure A, the number of marbles buried by the two groups of C57BL / 6 mice within 30 minutes was at a normal and similar level, significantly lower than that of the BTBR-Vehicle mice. Moreover, after the BTBR mice were treated with LP17, the number of marbles buried decreased; as can be seen from Figure 14 Figure B, the time spent on self-grooming by the two groups of C57BL / 6 mice was almost the same. The time spent on self-grooming by the BTBR-Vehicle mice was significantly longer. However, this situation was significantly improved by injecting LP17 in the BTBR mice.

[0085] 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 foregoing embodiments, those of ordinary skill in the art should understand that: they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the various embodiments of the present invention, and they should all be covered within the scope of the claims and the description of the present invention.

Claims

1. Use of LP17 in the preparation of an intervention drug for preventing / treating autism.

2. The application according to claim 1, wherein The administration method of the drug is intracranial injection.

3. The application according to claim 2, wherein The drug contains pharmaceutically acceptable excipients.

Images