Use of s100a9 protein inhibitors in preventing neurodevelopmental disorders

Abstract

The application discloses application of S100A9 inhibitors in preparation of drugs for preventing neurological developmental disorder diseases of offspring. Knocking out S100A9 of maternal neutrophils or treating a pregnant mother with a S100A9 selective inhibitor can significantly prevent and improve abnormal brain of offspring induced by psychological stress of the mother, and also significantly prevent and reduce hyperactive behavior of the offspring.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to the application of S100A9 protein inhibitors in drugs for the prevention of neurodevelopmental disorders. Background Technology

[0002] Neurodevelopmental disorders (NDD) are chronic developmental brain dysfunctions caused by various genetic or acquired factors that affect brain functional areas, including cognition, motor skills, social adaptation, and behavior, during the developmental period. According to the "Guidelines for the Diagnosis and Treatment of Mental Disorders (2020 Edition)" issued by the National Health Commission, neurodevelopmental disorders (NDD) include intellectual developmental disorders, developmental speech or language disorders, autism spectrum disorder (ASD), developmental learning disorder (DLD), developmental motor coordination disorder, attention deficit hyperactivity disorder (ADHD), stereotyped movement disorder, primary tics or tic disorders, secondary neurodevelopmental syndrome, other specified neurodevelopmental disorders, and unspecified neurodevelopmental disorders. Neurodevelopmental disorders primarily involve problems with sustained attention, concentration, and task completion (the ability to complete tasks), mainly manifesting as hyperactive behaviors, including inattention, hyperactivity, and impulsivity. Attention deficit hyperactivity disorder (ADHD), a common neurodevelopmental disorder, affects more than 4% of children worldwide, with its core phenotypes including hyperactivity, impulsivity, and inattention. The etiology of NDD is extremely complex, involving genetic, environmental, immune, and metabolic factors.Among the immune factors, the related immune mechanisms include maternal immune activation. Various maternal inflammatory factors (including obesity, bronchial asthma, autoimmune diseases, infection, mental stress, etc.) can be transmitted to the fetus through inflammatory cell signaling pathways and epigenetic mechanisms, affecting fetal neurodevelopment and significantly associated with an increased risk of offspring NDD (such as ASD, ADHD, TD).

[0003] Pregnancy is a highly sensitive period to the external environment, making pregnant women susceptible to various psychological and mental stresses. Prenatal or gestational psychological stress is a significant contributing factor to developmental defects in the fetal central nervous system. Adverse external stimuli cause psychological stress, leading to activation of the maternal immune system and increasing the risk of offspring developing attention deficit hyperactivity disorder (ADHD), schizophrenia, and neurodevelopmental disorders. Epidemiological studies and animal models have confirmed that psychological stress during pregnancy increases the risk of neurodevelopmental disorders in offspring. Currently, researchers have established chronic restraint stress (CRS) models and dexamethasone (DEX) stress models in various mammals, including mice, rats, ferrets, pigs, and rhesus monkeys. These models simulate the psychological stress perceived by pregnant mothers in a sterile environment without physiological trauma, thereby activating the maternal immune system. They can then detect the state of microglia, neuronal structure, and expression of pro-inflammatory factors in the offspring's brain, and determine the offspring's disease status through behavioral changes, thus enabling research on related mental illnesses in offspring. Currently, psychostimulants (such as amphetamines and methamphetamine) have been identified as first-line treatments for patients with attention deficit hyperactivity disorder (ADHD), but their short duration of action and drug-induced side effects are frequently reported in clinical practice. Therefore, it is essential to identify new targets for disease intervention in order to develop new treatment or prevention strategies.

[0004] S100 calcium-binding protein A9 (S100A9 protein) is an immunomodulatory cytokine that is highly expressed primarily on myeloid cells such as neutrophils, and it has both intracellular and extracellular secretory functions. The heterodimer S100A8 / A9 formed by the binding of S100A9 protein to S100 calcium-binding protein A8 (S100A8 protein) has been reported to play an important role in the progression of various diseases, such as viral infections, tumors, autoimmune diseases, cardiovascular diseases, chronic pain, and neurodegenerative diseases, but its regulatory role in neural development has not been reported. Summary of the Invention

[0005] This invention is the first to propose the S100A9 protein as a drug target for preventing neurodevelopmental disorders in offspring caused by prenatal stress.

[0006] The purpose of this invention is to provide a drug for preventing neurodevelopmental disorders in offspring caused by prenatal stress, thus filling the gaps in existing technologies. The drug can inhibit the biological activity of extracellular S100A9 protein, inhibit the expression and / or secretion of extracellular S100A9 protein by decidual neutrophils or other cells, alleviate the activation of microglia in the striatum of the embryonic and adult brain and / or the loss of γ-aminobutyric acid (GABAergic) neurons caused by various prenatal psychological stress paradigms, or improve hyperactivity-related behavioral phenotypes in offspring. In this application, "neurodevelopmental disorder" and "neurodevelopmental disorder" are used interchangeably.

[0007] In this application, "ADHD" and "Attention Deficit Hyperactivity Disorder" are used interchangeably. In this application, "mental stress" and "psychological stress" are used interchangeably.

[0008] In this application, "pregnancy" and "gestation period" are used interchangeably.

[0009] Mental stress during pregnancy includes various forms of psychological stress, which activates the mother's immune system.

[0010] Stress refers to the overall phenomenon when an individual perceives that environmental stimuli are placing an excessive burden on their physiological, psychological, and social systems, and the sum of their physiological and psychological responses. The resulting responses can be adaptive or maladaptive.

[0011] Maternal influence refers to the phenomenon where the offspring's genotype is not determined by the offspring's own genotype, but is influenced by the products encoded by the nuclear genes from the mother, resulting in the offspring exhibiting the same traits as the mother.

[0012] In this application, the terms "parent" and "original" are used interchangeably.

[0013] In this application, "parent body" and "parent parent" are used interchangeably.

[0014] This application provides the following technical solutions:

[0015] 1. Use of S100A9 inhibitors in the preparation of medicaments for the prevention of neurodevelopmental disorders.

[0016] 2. According to the use described in Project 1, the S100A9 inhibitor is characterized in that it is an anti-S100A9 antibody, an inhibitor or small molecule compound that inhibits the activity of S100A9, an oligonucleotide for knocking out the S100A9 gene, or an inhibitor that inhibits the expression of the S100A9 gene.

[0017] 3. The use according to item 1 or 2, characterized in that the amino acid sequence of the S100A9 protein is as shown in SEQ ID No: 1 or 3.

[0018] 4. The use according to item 1 or 2, characterized in that the nucleotide sequence of the S100A9 gene is as shown in SEQ ID No: 2 or 4.

[0019] 5. According to any one of items 1-4, the neurodevelopmental disorder is selected from the group consisting of intellectual disability, developmental speech or language disorder, autism spectrum disorder, developmental learning disorder, developmental motor coordination disorder, attention deficit hyperactivity disorder, stereotyped movement disorder, primary tic or tic disorder, secondary neurodevelopmental syndrome, other specific neurodevelopmental disorders and unspecified neurodevelopmental disorders.

[0020] 6. The use according to item 1 or 2, characterized in that the S100A9 inhibitor is taquimod.

[0021] 7. The use according to item 1, characterized in that the drug further comprises pharmaceutically acceptable excipients.

[0022] 8. As described in Item 1, the dosage form of the drug is an injection, infusion, powder, tablet or capsule.

[0023] 9. According to the use described in Item 5, the neurodevelopmental disorder is a neurodevelopmental disorder caused by psychological stress during pregnancy.

[0024] 10. Medications for the prevention of neurodevelopmental disorders, which contain inhibitors of the S100A9 protein.

[0025] 11. The drug described in Item 10, wherein the inhibitor of the S100A9 protein is an anti-S100A9 protein antibody, an inhibitor that inhibits the S100A9 protein, an oligonucleotide for knocking out the gene encoding the S100A9 protein, or an inhibitor that inhibits the gene encoding the S100A9 protein.

[0026] 12. The drug described in Item 10, wherein the inhibitor of the S100A9 protein is taquimod.

[0027] 13. The drug described in Item 10, wherein the neurodevelopmental disorder is selected from the group consisting of intellectual disability, developmental speech or language disorder, autism spectrum disorder, developmental learning disorder, developmental motor coordination disorder, attention deficit hyperactivity disorder, stereotyped motor disorder, primary tic or tic disorder, secondary neurodevelopmental syndrome, other specific neurodevelopmental disorders, and unspecified neurodevelopmental disorders, preferably, the neurodevelopmental disorder is developmental motor coordination disorder or attention deficit hyperactivity disorder.

[0028] 14. The drug described in Item 10, wherein the neurodevelopmental disorder is a neurodevelopmental disorder caused by psychological stress during pregnancy.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] Experiments have confirmed (see) Figure 8 and Figure 9 The administration of S100A9 protein inhibitors to the mothers significantly alleviated stress-induced hyperactivity and impaired attention in offspring, indicating that S100A9 protein inhibitors have a significant preventive effect on offspring. Attached Figure Description

[0031] Figure 1 A bar chart showing the glucocorticoid levels in the serum of pregnant mice treated with CRS and in the control group.

[0032] Figure 2 Figure A shows the immunofluorescence detection of decidual tissues treated with CRS and those in control, displaying cells expressing Ly6G (indicating neutrophils, blue) and cells expressing S100A9 protein (red); Figure B is a bar chart showing the number of neutrophils expressing S100A9 protein based on Figure A; Figure C shows the immunofluorescence detection of decidual tissues treated with DEX and those in control, displaying cells expressing Ly6G protein (indicating neutrophils, blue) and cells expressing S100A9 protein (red); Figure D is a bar chart showing the number of neutrophils expressing S100A9 protein based on Figure C.

[0033] Figure 3 To detect the expression levels of S100A8 / A9 proteins in the decidua of maternal mice under different treatments using enzyme-linked immunosorbent assay (Figure A) and the expression levels of S100A8 / A9 proteins in embryonic brain tissue (Figure B).

[0034] Figure 4 Microscopic images (Figures A and B) and statistical plots (Figures C and D) of striatal microglia in the brains of different groups of offspring, showing multiple recombination assays. In Figures C and D, the x-axis indicates the genotype of the pregnant mouse and the treatment administered, while the y-axis represents the parameters of the microglia in the offspring. Ctrl represents the control group without stress treatment, CRS represents the CRS-treated group, and wild-type indicates a wild-type pregnant mouse (a wild-type female mouse mated with a male mouse that knocked out the S100a9 gene). S100a9 KO This indicates a pregnant mouse with the S100a9 gene knocked out (this pregnant mouse is a female mouse with the S100a9 gene knocked out mated with a wild-type male mouse), Zoom indicates local zoom-in, and Rendering indicates 3D rendering.

[0035] Figure 5Figure A shows the trajectory plots of the offspring in the open field experiment from different groups, and Figure B shows a comparison of their movement data. The horizontal axis indicates the genotype of the pregnant mice and the treatments applied to them. The vertical axis represents the results of the open field experiment on the offspring of the pregnant mice. Ctrl represents the control group without stress treatment, CRS represents the CRS treatment group, and wild-type indicates wild-type pregnant mice (these are wild-type female mice mated with male mice whose S100a9 gene was knocked out). KO This refers to a pregnant mouse with the S100a9 gene knocked out (this pregnant mouse is a female mouse with the S100a9 gene knocked out mated with a wild-type male mouse).

[0036] Figure 6 The trajectory diagram (Figure A) and the comparison diagram of recognition index (Figure B) of the new object recognition experiment for different groups of offspring are shown. In Example 1, the attention phenotype of adult offspring was restored under maternal S100A9 gene defect and induced psychological stress. The horizontal axis indicates the genotype of the pregnant mice and the treatment of the pregnant mice, and the vertical axis is the result of the new object recognition experiment of the offspring of the pregnant mice. Ctrl is the control group without stress treatment, CRS is the CRS treatment group, and wild-type represents wild-type pregnant mice (the pregnant mice are wild-type female mice mated with male mice with S100a9 gene knockout). S100a9 KO This refers to a pregnant mouse with the S100a9 gene knocked out (this pregnant mouse is a female mouse with the S100a9 gene knocked out mated with a wild-type male mouse).

[0037] Figure 7 Microscopic images (Figure A) and dendritic branching statistics of striatal microglia in the brains of different groups of offspring are shown in Figure B. The horizontal axis indicates the treatment of pregnant mice, and the vertical axis represents the parameters of microglia in the brains of the offspring of pregnant mice. Saline+Ctrl is the control group treated with saline and without stress, Saline+CRS is the CRS treatment group treated with saline, and Tasq+CRS is the CRS treatment group treated with taquimod. Zoom indicates local magnification, and Rendering indicates three-dimensional rendering.

[0038] Figure 8 The figures show the trajectory plots (Figure A) and the motion data comparison plots (Figure B) of the offspring in the open field experiment for different groups. The horizontal axis indicates the treatment of pregnant mice, and the vertical axis represents the results of the open field experiment for the offspring of pregnant mice. Saline+Ctrl is the control group that was given saline and did not receive stress treatment, Saline+CRS is the CRS treatment group that was given saline, and Tasq+CRS is the CRS treatment group that was given taquimod.

[0039] Figure 9The trajectory plots (Figure A) and the comparison plot of recognition index of the offspring in different groups are shown in Figure B. The horizontal axis indicates the treatment of pregnant mice, and the vertical axis is the result of the new object recognition experiment of the offspring of pregnant mice. Saline+Ctrl is the control group that was given saline and did not receive stress treatment, Saline+CRS is the CRS treatment group that was given saline, and Tasq+CRS is the CRS treatment group that was given taquimod.

[0040] Figure 10 A comparison of S100A9 protein expression levels in the plasma of different groups of pregnant women in the second trimester (mothers). Detailed Implementation

[0041] When using the S100A9 protein inhibitor of the present invention for the prevention of neurodevelopmental disorders, the S100A9 protein inhibitor can be administered to pregnant or soon-to-be-pregnant subjects. A preferred S100A9 protein inhibitor is tasquinimod. Tasquinimod is an orally active quinoline-3-carboxamide that acts as an immunomodulator. It has a high affinity for both the S100A9 protein and histone deacetylase 4 (HDAC4 protein) and is a specific inhibitor of S100A8 / S100A9. Due to its anti-angiogenic and anti-metastatic activities, it has shown good efficacy in improving overall survival in patients with castration-resistant prostate cancer.

[0042] The S100A9 protein inhibitor can be administered via any appropriate route of administration, such as oral, inhalation, parenteral (including subcutaneous, intramuscular, intravenous, or intradermal), etc. The dosage of the S100A9 protein inhibitor is determined by the clinician based on factors such as the patient's physical condition, the subject's psychological stress during and / or before pregnancy, and the subject's family history. When using taquimod as an S100A9 protein inhibitor, the dosage can be 0.1–2 mg / kg body weight daily.

[0043] The samples used to detect the levels of S100A8 / A9 protein, S100A9 protein, and the S100A9 gene can be any suitable sample from the subject. In some embodiments, the sample is blood, tissue, cells, body fluids, urine, or fecal extract. In some preferred embodiments, the sample is blood.

[0044] Methods for determining the amount or level of S100A9 protein in a sample include performing an immunoassay, which can be performed directly or indirectly. In some embodiments, such an immunoassay is selected from the group consisting of: enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), radioimmunoassay (RIA), or immunoassays based on luminescence, fluorescence, chemiluminescence, or electrochemiluminescence detection.

[0045] The technical solution of the present invention will be described in detail below with reference to several preferred embodiments and accompanying drawings. The described embodiments are only some, not all, of the embodiments of the present invention. Other embodiments obtained by those skilled in the art based on the present invention without creative effort are also within the protection scope of the present invention.

[0046] The experimental methods used in the following examples are all conventional methods, such as behavioral testing, multiplex immunohistochemistry, liquid chromatography-mass spectrometry, electrophysiology, etc., and were performed according to the techniques and conditions described in the literature in this field or according to the product instructions. All reagents and materials involved in the examples are commercially available.

[0047] Experimental animals: The wild-type mice used in the examples were 8-week-old wild-type C57BL / 6 mice, purchased from Shanghai Silex Company; S100A9 KO The mice were C57BL / 6 mice with the S100A9 gene knocked out, purchased from Shanghai Southern Model Biotechnology Co., Ltd., catalog number: NM-KO-190143. All mice were housed at the SPF (Specific Pathogen Free) Laboratory Animal Center of the University of Science and Technology of China (USTC). The mouse housing and experimental procedures were strictly carried out in accordance with the USTC Laboratory Animal Management Regulations and were approved by the USTC Ethics Committee.

[0048] Experimental reagents: Dexamethasone (DEX) was purchased from Sigma (catalog number: D1756); multiple recombination kits were purchased from Thermo (catalog numbers: B40922, B40923, B40926); mouse S100A8 / A9 detection kits were purchased from R&D (catalog number: DY8596-05); sodium citrate antigen retrieval solution was purchased from Seville (catalog number: G1202); Iba1 antibody was purchased from CST (catalog number: 17198).

[0049] Ly6G (lymphocyte antigen 6 complex, locus G) is a specific surface marker of mouse neutrophils, which can be used to identify and detect mouse neutrophils.

[0050] Anti-Ly6G antibodies can eliminate neutrophils by utilizing the phagocytic function of mononuclear phagocytes. Anti-Ly6G antibodies were purchased from BioX Cell (catalog number: BE0075-1).

[0051] The number of days of embryonic development after fertilization is represented as E+ days, and the day the vaginal plug is discovered is designated as E0.5.

[0052] Methods for inducing psychological stress in mice include chronic restraint stress (CRS) treatment and dexamethasone (DEX) treatment.

[0053] Chronic restraint stress (CRS treatment): Following the method of Fan et al. (Fan et al., 2019), the treatment group (CRS treatment group) underwent 3 hours of stress treatment daily from day E6.5 (20:00-23:00) until day E13.5. The stress treatment involved placing a single mouse in a well-ventilated 50 mL centrifuge tube (29 mm in diameter and 115 mm in height), with the tube opening blocked by a cap and intermediate tube, preventing the mouse from moving forward or backward within the device. No food or water was provided during the stress treatment period. The control group (Ctrl group) was placed in an empty cage without stress treatment or interference. During the CRS treatment period in the treatment group, the control group mice were also not provided with food or water.

[0054] Dexamethasone treatment (DEX treatment): Following the method of Hong et al. (Hong et al., 2020), specifically: the treatment group (DEX treatment group) received dexamethasone (concentration 1.25 μg / mL) added to the drinking water provided to mice daily for free intake. The solvent control group (control group) received DMSO (the same amount of DEX added as the DEX group) added to the drinking water daily for free intake by mice.

[0055] Data analysis methods: Statistical difference analysis and graphing were performed using GraphPad Prism 9.0 software. The numbers above the bars for each group indicate the p-value for the difference analysis. The Student's t-test was used for comparisons between two groups; one-way ANOVA was used for comparisons between multiple groups under one-way factors; and two-way ANOVA was used for comparisons between multiple groups under multiple factors. Data are presented as mean ± standard error (mean ± SEM). A significant difference was defined as p < 0.05; in the graph, * represents p < 0.05; ** represents p < 0.01; *** represents p < 0.001; **** represents p < 0.0001; ns represents no significant difference.

[0056] Example 1. Psychological stress during pregnancy increases the expression of S100A8 / A9 protein on neutrophils.

[0057] 1. Inducing psychological stress during pregnancy

[0058] CRS and DEX treatments were used to induce psychological stress during pregnancy.

[0059] (1) For CRS treatment: Treatment group (CRS treatment group): 8-week-old wild-type C57BL / 6 mice were used. Wild-type C57BL / 6 female mice were mated with wild-type C57BL / 6 male mice. The vaginal plug of the female mice was checked the next day. The presence of the vaginal plug was used as a sign of successful mating. The successful mating was recorded as day E0.5. Starting from day E6.5, pregnant mice were given psychological stress treatment (CRS treatment) for 3 hours at a fixed time every day (20:00-23:00). The psychological stress treatment continued until day E13.5. Control group: The same mice were used. Pregnant mice were not given CRS treatment or interference. During the time when the treatment group was given CRS treatment, the control group mice were not provided with food or water.

[0060] The levels of glucocorticoids (cortisol) in the serum of pregnant mice were detected using the ELISA assay. The results are as follows: Figure 1 The results showed that, compared with the control group, the pregnant mice in the CRS treatment group had significantly higher levels of glucocorticoids, indicating that CRS treatment could successfully induce psychological stress in pregnant mice, and the model was successfully established, obtaining wild-type pregnant mice with psychological stress during pregnancy.

[0061] (2) For DEX treatment: Treatment group (DEX treatment group): 8-week-old wild-type C57BL / 6 mice were used. Wild-type C57BL / 6 female mice were mated with wild-type C57BL / 6 male mice. The vaginal plugs of the female mice were checked the next day. The vaginal plugs were used as the mark of successful mating. The successful mating was recorded as day E0.5. From day E6.5, the pregnant mice were given DEX treatment until day E13.5. Control group: The same mice were used without interference. An equal amount of DMSO (the same amount of DEX added as the DEX group) was added to the drinking water every day for the mice to ingest freely. During the period when the treatment group was given DEX treatment, the control group mice were not provided with food or water.

[0062] 2. S100A9 protein level detection

[0063] For wild-type pregnant mice subjected to the aforementioned psychological stress (CRS treatment or DEX treatment), pregnant mice were sacrificed at E13.5, and their decidual tissue was harvested, paraffin-embedded specimens were prepared and sectioned, stained according to standard procedures, and cells expressing S100A9 were detected using immunofluorescence. Results are as follows: Figure 2 As shown, the expression of S100A9 protein on neutrophils in decidual tissue is displayed, demonstrating that under psychological stress during pregnancy (CRS treatment or DEX treatment), the number of neutrophils expressing S100A9 in maternal decidua is significantly increased.

[0064] 3. S100 A8 / A9 protein level detection

[0065] The CRS treatment group and control group are described in section (1) above; Anti-Ly6G antibody + CRS treatment group: The same mice were used. From day 5.5 to day 13.5, pregnant mice were injected intraperitoneally with anti-Ly6G antibody (100 μg per mouse) at a fixed time every other day (20:00), and then CRS treatment was performed immediately (20:00-23:00). Pregnant mice in each group were sacrificed on day 13.5 and their decidua and embryonic brain tissue were harvested. The expression level of S100A8 / A9 protein dimer on the decidua and embryonic brain tissue was quantitatively detected by ELISA. The detection instrument was a SpectraMax iD3 / iD5 multi-functional microplate reader (MolecularDevices, USA). The results are as follows. Figure 3 As shown, compared with control mice, S100A8 / A9 protein expression was significantly increased in decidua and embryonic brain tissue under maternal psychological stress (i.e., CRS treatment). However, when anti-Ly6G antibody was used to clear maternal neutrophils, S100A8 / A9 protein expression in decidua and embryonic brain tissue returned to normal physiological levels (there was no significant difference in S100A8 / A9 protein dimer expression levels between the control group and the anti-Ly6G antibody + CRS group). Since anti-Ly6G antibody can clear maternal neutrophils, combined with the above results, it is demonstrated that maternal psychological stress (CRS treatment) increases the expression of S100A8 / A9 protein on maternal and fetal neutrophils.

[0066] Example 2. Effects of knocking out the maternal S100a9 gene or inhibiting the maternal S100a9 protein and maternal psychological stress on offspring.

[0067] 1. Inducing psychological stress in pregnant mice with knockout S100a9 gene

[0068] The impact on offspring was assessed in two ways: first, by examining the microglia in the brains of offspring mice; and second, by examining the behavior of offspring mice.

[0069] The method for detecting microglia in the brains of offspring mice was as follows: For pregnant mice under different treatments, mouse embryos were removed at E18.5, the fetal brain was isolated and fixed with 4% paraformaldehyde, then embedded in paraffin or OCT, sectioned, and antigen-retrieval was performed using sodium citrate antigen retrieval solution at pH 6.0. Staining was then performed using a multi-recombinant chemiluminescence kit, and finally, images were taken using a fluorescence microscope (3D-Histech). Iba1 antibody was used to label striatal microglia in the offspring brain.

[0070] The methods for detecting the behavior of offspring mice are as follows:

[0071] For offspring or progeny (F1 generation) of pregnant mice under different treatments, male F1 mice were subjected to the following behavioral tests at 6-12 weeks of age. Those skilled in the art know that the general motor exploration ability and attentional concentration ability of test mice can be measured through the open field test and the novel object recognition test to demonstrate that they have ADHD-like behavioral symptoms, further indicating that they have neurodevelopmental disorders, particularly developmental motor coordination disorder and attention deficit hyperactivity disorder.

[0072] (1) Open field test: Mice were placed in an open field (40 cm × 40 cm) and allowed to explore freely for 5 minutes while their activities were recorded by a camera. The time mice spent in the central area (16 cm × 16 cm) of the open field was recorded using the animal movement tracking system EthoVision XT (Noldus). This test was used to assess the general motor behavior characteristics of mice. The movement distance reflected the total activity level of the mice and was obtained by counting the total movement distance of the mice in the open field within 5 minutes. The central movement time and the frequency of entering the center reflected the activity preference of the mice in the center of the open field relative to the surrounding areas and were obtained by counting the proportion of time the mice spent in the center of the open field relative to the surrounding areas within 5 minutes and the number of times the mice entered the center of the open field, respectively.

[0073] (2) New Object Recognition Experiment: This behavior and the above behavior were conducted in the same open field, but were divided into four stages and completed over four days. On the first and second days, mice were allowed to freely explore the open field for 5 minutes to fully adapt to the environment. On the third day, two rectangular blocks (3 cm × 3 cm × 6 cm) were placed in the northwest and southeast corners of the open field, and mice were placed in them to explore freely for 5 minutes. On the fourth day, the rectangular block in the northwest corner was replaced with a cylinder (3 cm in diameter × 6 cm), and mice were placed in it to explore freely for 5 minutes. This test was used to assess the mice's attention function. The recognition index reflects the mice's ability to distinguish between new and old objects. The time spent by the mice exploring the new and old objects (at a distance of <2 cm) in the open field within 5 minutes was counted and calculated using the following formula: Recognition Index = (New Object Recognition Exploration Time - Old Object Recognition Exploration Time) / (New Object Recognition Exploration Time + Old Object Recognition Exploration Time) × 100%.

[0074] 1. Effects of knocking out the S100a9 gene in pregnant mice on offspring

[0075] 1.1 Grouping and treatment of pregnant mice

[0076] To investigate the effect of maternal genotype (rather than paternal genotype) on offspring neural development (maternal influence) and to avoid the potential impact of offspring lacking the S100a9 gene, two groups of pregnant mice were constructed, with offspring retaining the S100a9 gene. In this experiment, pregnant mice were divided into wild-type and S100a9 groups based on their genotype. KO Pregnant mouse.

[0077] Wild-type pregnant mice are: 8-week-old wild-type C57BL / 6 female mice mated with 8-week-old S100a9 female mice. KO Male mice (with the S100a9 gene knocked out) were mated, and the vaginal plugs of the female mice were checked the next day. The vaginal plugs were used as a marker of successful mating, and the successful mating diary was recorded as day E0.5 to obtain wild-type pregnant mice.

[0078] S100a9 KO The pregnant mouse is: 8-week-old S100a9 KO Female mice with the S100a9 gene knocked out were mated with 8-week-old wild-type C57BL / 6 male mice. The vaginal plugs of the female mice were examined the following day, and the vaginal plugs were used as an indicator of successful mating. The successful mating diary was recorded as day E0.5, and the S100a9 gene was obtained. KO Pregnant mouse.

[0079] For the aforementioned pregnant mice, allow natural delivery to obtain offspring mice. To verify the genotypes of the offspring mice and their parents, tail tissue can be taken, genomic DNA extracted, and gene detection performed using PCR and electrophoresis. The results showed that the wild-type offspring and S100a9... KO All offspring possess the S100a9 gene.

[0080] The treatments applied to wild-type pregnant mice included a CRS treatment group and a control group: In the CRS treatment group, pregnant mice were subjected to stress treatment (CRS treatment) for 3 hours at a fixed time every day (20:00-23:00) starting from day 6.5, and the entire stress process was terminated at day 13.5; In the control group, pregnant mice were not subjected to stress (CRS treatment) or interference, and during the time when the CRS treatment was applied in the CRS treatment group, the mice in the control group were not provided with food or water.

[0081] For S100a9 KO The treatments administered to pregnant mice included a CRS treatment group and a control group: In the CRS treatment group, pregnant mice were subjected to stress treatment (CRS treatment) for 3 hours at a fixed time every day (20:00-23:00) starting from day E6.5, and the entire stress process was terminated at day E13.5; In the control group, pregnant mice were not subjected to stress (CRS treatment) or interference, and during the time when the CRS treatment group was administered, the mice in the control group were not provided with food or water.

[0082] 1.2 Effects on offspring brain tissue

[0083] It is known in the art that changes in microglial morphology signify functional activation and indicate neurodevelopmental disorders.

[0084] The above method was used to detect microglia in the brains of offspring mice, and the results are as follows: Figure 4 As shown, compared with the wild-type control group and the CRS treatment group, maternal psychological stress caused a significant shortening and reduction in the number of dendritic branches in the striatal microglia of the offspring; compared with the wild-type control group and S100a9 KO In the control group, there was no significant difference in the branch length and number of microglia, demonstrating that knocking out the maternal S100a9 gene does not affect the activity of glial cells and neurons in the offspring; compared with S100a9... KO In the control group and the CRS treatment group, there was no significant difference in the branch length and number of microglia, which proves that knocking out the maternal S100a9 gene can restore the changes in microglia caused by psychological stress and restore the morphology of microglia to the normal level.

[0085] 1.3 Impact on offspring behavior

[0086] The behavior of offspring mice was tested using the above method, and the results are as follows: Figure 5 and 6 As shown, maternal psychological stress can cause ADHD-like behavioral symptoms in offspring. Specifically, compared to the control group, offspring experiencing maternal stress exhibited significantly hyperactive and attention-impaired phenotypes (manifested as increased total distance traveled in the open field test and decreased recognition index in the new object recognition test), similar to symptoms of ADHD in humans, indicating a neurodevelopmental disorder. Knocking out the S100a9 gene in the mother significantly alleviated and restored the hyperactive and attention-impaired behaviors in offspring caused by stress.

[0087] 2. Effects of S100a9 protein inhibitor treatment on offspring in pregnant mice

[0088] 2.1 Treatment of pregnant mice

[0089] In this experiment, based on the wild-type genotype of the female mice used, they were divided into three groups: a control group treated with saline and without stress (Saline+Ctrl group), a CRS treatment group treated with saline (Saline+CRS group), and a CRS treatment group treated with taquimod (Tasq+CRS group). Specifically, in the Tasq+CRS group: 8-week-old wild-type C57BL / 6 female mice were mated with 8-week-old wild-type C57BL / 6 male mice. The vaginal plugs of the female mice were checked the next day, and the vaginal plugs were used as a marker of successful mating. The successful mating was recorded as day E0.5. Starting from day E6.5, the pregnant mice were given 8 mg / kg of the S100A9 inhibitor taquimod by gavage at a fixed time every day (20:00), and CRS treatment was immediately initiated (20:00-23:00). The CRS treatment process was terminated at day E13.5. Saline+CRS group: The difference from the Tasq+CRS group is that pregnant mice were given an equal volume of saline via gavage (equivalent to the amount of the S100A9 inhibitor taquimod administered in the Tasq+CRS group). Saline+Ctrl group: The difference from the Saline+CRS group is that pregnant mice were not subjected to stress (CRS treatment) or interference. During the period of CRS treatment in the Saline+CRS group, the pregnant mice were not provided with food or water.

[0090] 2.2 Effects on offspring brain tissue

[0091] The microglia of the progeny mice in each group were detected using the above method, and the results are as follows: Figure 7 As shown, comparing the Saline+Ctrl group and the Saline+CRS group, maternal psychological stress caused a decrease in the branch length and number of striatal microglia in the offspring; comparing the Tasq+CRS group and the Saline+Ctrl group, there was no significant difference in the branch length and number of microglia, demonstrating that inhibiting maternal S100A9 protein with an S100A9 protein inhibitor can restore the changes in microglia caused by psychological stress and restore the morphology of microglia to normal levels.

[0092] 2.3 Impact on offspring behavior

[0093] The behavior of offspring mice in each group was tested using the above method, and the results are as follows: Figure 8 and 9As shown, maternal psychological stress can induce ADHD-like behavioral symptoms in offspring. Specifically, compared to the control group, stress-induced offspring exhibited significantly hyperactive and attention-impaired motor behaviors (manifested as increased total distance traveled in the open field test and decreased recognition index in the new object recognition test), similar to symptoms of ADHD in humans, indicating a neurodevelopmental disorder. Inhibition of the maternal S100A9 protein significantly alleviated stress-induced hyperactivity and attention-impaired behavior in offspring.

[0094] Example 3. In humans, neurodevelopmental disorders in offspring are also associated with increased S100A9 protein expression in the mother during pregnancy.

[0095] Information on healthy individuals (n=27) and individuals with ADHD (n=19) was obtained from the Birth Cohort and Biobank of the Key Laboratory of Birth Population Health, Ministry of Education, Anhui Medical University. The mothers of these healthy individuals (n=27) and individuals with ADHD (n=19) were used as subjects. Peripheral blood samples were collected from these subjects during the second trimester. Plasma was separated, and the expression level of S100A9 protein in the plasma was quantitatively detected using a multiplex cytokine detection platform. The detection instruments included MAGPIX / Luminex200 / FLEXMAP 3D / INTELLIFLEX (R&D systems, USA).

[0096] The results are as follows Figure 10 As shown, the S100A9 protein expression level during pregnancy was significantly higher in mothers of children with ADHD (mean 1533 picograms / mL) compared to that in mothers of healthy individuals (mean 945 picograms / mL). This indicates that in humans, the incidence of neurodevelopmental disorders (ADHD) in offspring is associated with increased S100A9 protein expression in their mothers during pregnancy. This also suggests that the conclusions or inferences drawn from the experiments conducted on mice are equally applicable to humans.

[0097] These results demonstrate that the S100A9 protein is a key molecule in the neurodevelopmental disorders such as ADHD mediated by maternal stress in offspring. Blocking the S100A9 protein (using an S100A9 protein inhibitor) can effectively prevent abnormal brain development during the embryonic period and prevent ADHD-related behavioral symptoms in offspring (especially in adulthood).

[0098] sequence list

[0099] SEQ ID No: 1 Mouse S100A9 protein sequence:

[0100] MANKAPSQMERSITTIIDTFHQYSRKEGHPDTLSKKEFRQMVEAQLATFMKKEKRNEALINDIMEDLDTNQDNQLSFEECMMLMAKLIFACHEKLHENNPRGHGHSHGKGCGK

[0101] SEQ ID No: 2 Nucleotide sequence of mouse S100a9 gene:

[0102] ATGGCCAACAAAGCACCTTCTCAGATGGAGCGCAGCATAACCACCATCATCGACACCTTCCATCAATACTCTAGGAAGGAAGGACACCCTGACACCCTGAGCAAGAAGGAATTCAGACAAATGGTGGAAGCACAGTTGGCAACCTTTATGAAGAAAGAGAAGAGAAATGAAGCCCTCATAAATGACATCATGGAGGACCTGGACACAAACCAGGACAATCAGCTGAGCTTTGAGGAGTGTATGATGCTGATGGCAAAGTTGATCTTTGCCTGTCATGAGAAGCTGCATGAGAACAACCCACGTGGGCATGGCCACAGTCATGGCAAAGGCTGTGGGAAGTAA

[0103] SEQ ID No: 3 Human S100A9 protein sequence:

[0104] MTCKMSQLERNIETIINTFHQYSVKLGHPDTLNQGEFKELVRKDLQNFLKKENKNEKVIEHIMEDLDTNADKQLSFEEFIMLMARLTWASHEKMHEGDEGPGHHHKPGLGEGTP

[0105] SEQ ID No: 4 Nucleotide sequence of human S100A9 gene:

[0106] ATGACTTGCAAAATGTCGCAGCTGGAACGCAACATAGAGACCATCATCAACACCTTCCACCAATACTCTGTGAAGCTGGGGCACCCAGACACCCTGAACCAGGGGGAATTCAAAGAGCTGGTGCGAAAAGATCTGCAAAATTTTCTCAAGAAGGAGAATAAGAATGAAAAGG TCATAGAACACATCATGGAGGACCTGGACACAAATGCAGACAAGCAGCTGAGCTTCGAGGAGTTCATCATGCTGATGGCGAGGCTAACCTGGGCCTCCCACGAGAAGATGCACGAGGGTGACGAGGGCCCTGGCCACCACCATAAGCCAGGCCTCGGGGAGGGCACCCCCTAA

[0107] There are many methods and approaches to implement the technical solution of this invention. The above description is only a preferred embodiment of this invention. It should be understood that for those skilled in the art, several improvements and modifications can be made without departing from the principle of this invention, and these changes should also be considered within the scope of protection of this invention.

[0108] References

[0109] 1. FAN KQ, LI YY, WANG HL, et al. 2019. Stress-Induced MetabolicDisorder in Peripheral CD4+ T Cells Leads to Anxiety-like Behavior. Cell [J], 179: 864-+.

[0110] 2. HONG JY, LIM J, CARVALHO F, et al. 2020. Long-Term Programming ofCD8 T Cell Immunity by Perinatal Exposure to Glucocorticoids. Cell [J], 180:847-861.e815.

Claims

1. Use of S100A9 protein inhibitors in the preparation of drugs for the prevention of neurodevelopmental disorders.

2. The use according to claim 1, wherein the inhibitor of S100A9 protein is an anti-S100A9 protein antibody, an inhibitor that inhibits S100A9 protein, an oligonucleotide for knocking out the coding gene of S100A9 protein, or an inhibitor that inhibits the coding gene of S100A9 protein.

3. The use as described in claim 1 or 2, wherein the inhibitor of the S100A9 protein is taquimod.

4. The use according to claim 1, wherein the neurodevelopmental disorder is selected from the group consisting of intellectual disability, developmental speech or language disorder, autism spectrum disorder, developmental learning disability, developmental motor coordination disorder, attention deficit hyperactivity disorder, stereotyped motor disorder, primary tic or tic disorder, secondary neurodevelopmental syndrome, other specific neurodevelopmental disorders, and unspecified neurodevelopmental disorders, preferably, the neurodevelopmental disorder is developmental motor coordination disorder or attention deficit hyperactivity disorder.

5. The use as described in claim 4, wherein the neurodevelopmental disorder is a neurodevelopmental disorder caused by psychological stress during pregnancy.

6. Drugs for the prevention of neurodevelopmental disorders, which contain inhibitors of the S100A9 protein.

7. The medicament of claim 6, wherein the inhibitor of the S100A9 protein is an anti-S100A9 protein antibody, an inhibitor that inhibits the S100A9 protein, an oligonucleotide for knocking out the gene encoding the S100A9 protein, or an inhibitor that inhibits the gene encoding the S100A9 protein.

8. The medicament of claim 6, wherein the inhibitor of the S100A9 protein is taquimod.

9. The medicament of claim 6, wherein the neurodevelopmental disorder is selected from the group consisting of intellectual disability, developmental speech or language disorder, autism spectrum disorder, developmental learning disorder, developmental motor coordination disorder, attention deficit hyperactivity disorder, stereotyped motor disorder, primary tic or tic disorder, secondary neurodevelopmental syndrome, other specific neurodevelopmental disorders, and unspecified neurodevelopmental disorders, preferably, the neurodevelopmental disorder is developmental motor coordination disorder or attention deficit hyperactivity disorder.

10. The medicament of claim 9, wherein the neurodevelopmental disorder is a neurodevelopmental disorder caused by psychological stress during pregnancy.

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