Application of etogliflozin in preparation of medicine for treating or improving autism

Abstract

The invention belongs to the field of medicines, and particularly relates to application of etogliflozin in preparation of a medicine for treating autism. The invention discloses an application of Eutogliflozin in preparation of autism treatment drugs, and proves that Eutogliflozin can reverse the impairment of the social ability of BTBR mice and relieve repeated engraving behaviors and anxiety-like behaviors by inhibiting oxidative stress, inflammatory response and excessive activation of microglial cells of prefrontal cortex and hippocampus. The achievement provides a new target for treating autism symptoms, reveals a new pharmacological action mechanism of the etogliflozin, and provides an important theoretical basis for further development and application of the etogliflozin.

Description

Technical Field

[0001] This invention belongs to the field of medicine, and in particular relates to the use of atoggliflozin in the preparation of drugs for the treatment or improvement of autism. Background Technology

[0002] Autism is a neurodevelopmental disorder characterized by impairments in social interaction, communication, and repetitive, stereotyped behaviors. It is caused by a combination of factors, closely related to both genetic and environmental factors. However, the pathogenesis of autism remains unclear, and effective treatment and intervention strategies are still lacking.

[0003] Numerous studies have shown that oxidative stress, inflammation, and immune activation in the prefrontal cortex (PFC) and hippocampus play crucial roles in the pathogenesis of autism. Oxidative stress is an imbalance between the production of reactive oxygen species and the antioxidant system, leading to nucleic acid and protein damage and cytotoxicity. Oxidative stress promotes elevated levels of inflammatory factors in the body, such as interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), causing inflammatory responses and cell damage. Simultaneously, inflammatory factors activate microglia, leading to a series of inflammatory responses that exacerbate neuronal dysfunction. These processes affect normal brain development, resulting in behavioral disorders in individuals with autism.

[0004] The BTBR mouse model is a stable, genotype-inbred mouse strain that has been widely used as a tool in translational research on autism to evaluate potential therapeutic efficacy. BTBR animal models exhibit core autism symptoms, such as social impairment, repetitive / stereotyped behaviors, and anxiety-like behaviors. Studies have also confirmed that BTBR mice exhibit elevated levels of oxidative stress and abnormal immune responses.

[0005] Sodium-glucose cotransporter 2 (SGLT2) inhibitors are novel oral hypoglycemic agents used to treat diabetes, ischemic stroke, epilepsy, and Alzheimer's disease. SGLT2 inhibitors possess neuroprotective properties, attributed to their antioxidant and anti-inflammatory activities. They can reduce the production of pro-inflammatory cytokines. Ertugliflozin (Ert), a SGLT2 inhibitor, has not been reported to improve autism symptoms by modulating oxidative stress and neuroinflammation. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides the use of etoragliflozin in the preparation of drugs for treating or improving autism.

[0007] Ertugliflozin (Ert) is a sodium-glucose cotransporter 2 inhibitor with the molecular formula C2. 22 H 25ClO7, with a molecular weight of 436.88 and CAS number 1210344-57-2, has the chemical name: (1S,2S,3S,4R,5S)-(2,3,4-TRIS-BENZYLOXY-5-(4-CHLORO-3-(4-ETHOXY-BENZYL)-PHENYL)-68-DIOXA-BICYCLO(3.2.1)OCT-1-YL)METHAChemicalbookNOL; (1S,2S,3S,4R,5S)-5-(4-chloro-3-(4-ethoxybenzyl)phenyl)-1-(hydroxymethyl)-6,8-dioxabicyclo[3.2.1]octane-2,3,4-trio, and its structural formula is as follows:

[0008]

[0009] Etoggliflozin is an oral hypoglycemic agent belonging to the sodium-glucose cotransporter 2 (SGLT2) inhibitor class, primarily used for glycemic control in patients with type 2 diabetes. It lowers blood glucose by selectively inhibiting the SGLT2 protein in the proximal tubules of the kidney, reducing glucose reabsorption, increasing urinary glucose excretion, and thus lowering blood sugar. It may also reduce weight and lower blood pressure.

[0010] Specifically, the technical solution of the present invention is as follows:

[0011] This invention provides the use of eletoggliflozin in the preparation of drugs for the treatment of autism, including but not limited to any of the following:

[0012] (1) Application in the preparation of drugs to reverse impaired social recognition ability in autistic animals or humans. In the three-box social recognition experiment, the social recognition index increased in the eltoggliflozin treatment group, suggesting that eltoggliflozin treatment alleviates social recognition impairment in BTBR mice.

[0013] (2) Application in the preparation of drugs for treating social novelty preference deficit caused by autism. In the three-box social novelty preference test, the social novelty preference index increased in the eltoggliflozin treatment group, suggesting that eltoggliflozin treatment alleviates social novelty impairment in BTBR mice.

[0014] (3) Application in the preparation of drugs to reduce anxiety-like behaviors in autistic animals or humans. In open field and elevated cross maze experiments, the eltoggliflozin treatment group showed an increase in open arm dwell time, indicating that eltoggliflozin treatment significantly reduced anxiety-like behaviors in BTBR mice.

[0015] (4) Application in the preparation of drugs to reduce repetitive stereotyped behaviors in autistic animals or humans. In the self-grooming and bead embedding experiments, the number of beads embedded in the edoragliflozin-treated group was reduced, indicating that edoragliflozin treatment significantly improved repetitive stereotyped behaviors in BTBR mice.

[0016] (5) Application in the preparation of drugs for treating oxidative stress, inflammatory response, and microglial overactivation in the prefrontal cortex and hippocampus caused by autism. In Nissl staining experiments, eltoggliflozin effectively reduced neuronal damage and improved neuronal morphology in the prefrontal cortex and hippocampus of BTBR mice. In ELISA experiments detecting oxidative stress and inflammatory factor levels in brain tissue, eltoggliflozin alleviated neuroinflammation in the prefrontal cortex and hippocampus of BTBR mice and significantly reduced oxidative stress levels in the CA1 region of the prefrontal cortex and hippocampus of BTBR mice. In immunofluorescence experiments, eltoggliflozin treatment reduced the number of activated microglia in the prefrontal cortex and hippocampus of BTBR mice.

[0017] Autism is a relatively serious neurodevelopmental disorder. The brains of individuals with autism exhibit structural and functional abnormalities during development, which may appear in the fetal stage or early infancy. Within neurodevelopmental disorders, autism is classified as a childhood problem distinct from mental illness. Diagnostic criteria for childhood autism are found in the International Statistical Classification of Diseases and Related Health Problems (10th Revision) (ICD-10) and the Diagnostic and Statistical Manual of Mental Disorders (5th Edition) (DSM-5). Due to the variability of autism symptoms and the differences in descriptions across different diagnostic categories, autism treatment lacks uniformity, and treatment approaches are often diverse. This results in a lack of correlation and similarity between medications used to treat autism, targeting single symptoms such as social skills or repetitive and stereotyped behaviors, while simultaneously addressing multiple symptoms. Therefore, given the unique characteristics of autism, its symptoms, especially those specified in the diagnostic criteria, are themselves treatable and can be addressed individually with appropriate pharmaceutical treatments.

[0018] The animal model used in this invention to verify the efficacy of eletoggliflozin in treating autism is the BTBR mouse model, a stable genotype of inbred mice whose unique genetic lineage is the basis for its autism-like phenotype. BTBR mice exhibit gene variations or copy number variations on multiple chromosomes. For example, some genes on chromosomes 1, 4, and 10 (such as Shank3 and Nlgn3) are related to synapse formation and nerve signal transmission, and abnormalities in these genes directly affect neural development.

[0019] Significant abnormalities in the brain structure and function of BTBR mice constitute the core pathological basis for their behavioral phenotypes. The prefrontal cortex (PFC) is a key brain region regulating social behavior and emotion; BTBR mice exhibit reduced synaptic density and impaired synaptic plasticity in PFC neurons, affecting the processing of social information. A decrease in the number of pyramidal neurons and dendritic branching in the CA1 region of the hippocampus leads to impaired learning and memory function. Abnormal activation of microglia in the central nervous system significantly promotes the development of neuroinflammatory responses. Autopsy studies of autistic patients have revealed excessive microglia activation in their brain tissue, suggesting that neuroinflammation is a core pathological feature of autism. Neuroinflammation induced by microglia in BTBR mice is closely related to autism-like behaviors.

[0020] As a further optimization of the present invention, etoragliflozin aims to treat impaired social skills, reduce repetitive stereotyped behaviors, and alleviate anxiety-like behaviors in autism by inhibiting oxidative stress, inflammatory responses, and excessive activation of microglia in the prefrontal cortex and hippocampus.

[0021] As a further optimization of the present invention, the drug comprises eltoggliflozin and a pharmaceutically acceptable carrier. The drug dosage form is one or more of tablets, capsules, lozenges, injections, suspensions, suppositories, ointments, bladder instillations, oral preparations, suppositories, or sustained-release preparations. The carrier is one or more of liposomes, nanoparticles, hydrogels, or microspheres.

[0022] As a further optimization of the present invention, the dosage of etanercept is 20 mg / kg / day, and the preferred route of administration is oral.

[0023] The beneficial effects of this invention are as follows: This invention discloses the application of eltoggliflozin in the preparation of drugs for treating autism, demonstrating that eltoggliflozin reverses impaired social skills and reduces repetitive, stereotyped behaviors and anxiety-like behaviors in BTBR mice by inhibiting oxidative stress, inflammatory responses, and excessive activation of microglia in the prefrontal cortex and hippocampus. This fully demonstrates the promising pharmaceutical prospects of eltoggliflozin in the treatment of autism. This achievement will provide a new target for treating autism symptoms, reveal a novel pharmacological mechanism of action of eltoggliflozin, and provide an important theoretical basis for its further development and application. Attached Figure Description

[0024] Figure 1To improve social deficits in BTBR mice using eletoggliflozin. (A) Representative heatmap of the three-chamber social recognition experiment; (B) Time spent by mice in the three chambers; (C) Design time of mice with unfamiliar mice (S1) and novel objects (NO); (D) Social recognition index. (E) Representative heatmap of the three-chamber social novelty preference experiment; (F) Time spent by mice in the three chambers; (G) Social time of mice with unfamiliar mice (S2) and unfamiliar mice (S1); (H) Social novelty recognition index; n=10; Compared with the novel object, unfamiliar mouse 1... *** p<0.001; Compared with stranger mouse 1, stranger mouse 2, ### p<0.001; Compared with the C57 group, the BTBR group &&& p<0.001; Compared with the BTBR group, the BTBR+Ert group $$$ p<0.001; “ns” indicates no statistical difference.

[0025] Figure 2 To reduce stereotyped and anxiety-like behaviors in BTBR mice treated with eletodaggliflozin. Self-grooming time (A) and frequency (B) in the self-grooming test; (C) number of bead embeddings in the bead embedding test; (D) representative heatmaps, time spent in the central area (E), and total distance traveled (F) in the open field test; (G) representative heatmaps, time spent in the open arm (H), and total distance traveled (I) in the elevated cruciate maze test; n=10; Compared with the C57 group, the BTBR group... ** p<0.001, *** p<0.001; Compared with the BTBR group, the BTBR+Ert group ## p<0.001, ### p<0.001; “ns” indicates no statistical difference.

[0026] Figure 3 The effects of etanercept treatment on the histomorphology of the prefrontal cortex and hippocampal CA1 region. Representative images of Nissl bodies in the prefrontal cortex (A) and hippocampal CA1 region (B); number of Nissl bodies in neurons of the prefrontal cortex (C) and hippocampal CA1 region (D); the number of Nissl bodies in BTBR mice was significantly reduced, and the number of Nissl bodies in neurons of the BTBR+Ert group was higher than that in BTBR mice; n=6; compared with C57 mice. *** p<0.001; compared with BTBR mice, ### p<0.001.

[0027] Figure 4To reduce oxidative stress levels in BTBR mice treated with etoragliflozin. The levels of MDA(A) and GSH(C), and the activities of SOD(E) and CAT(G) in the prefrontal cortex; the levels of MDA(B) and GSH(D), and the activities of SOD(F) and CAT(H) in the CA1 region of the hippocampus; n=8, compared with the C57 group, BTBR group, *** p<0.001; Compared with the BTBR group, the BTBR+Ert group ### p<0.001.

[0028] Figure 5 The effect of eletoggliflozin treatment on inflammation in BTB mice. Levels of IL-1β (A), IL-6 (C), IL-17A (E), and TNF-α (G) in the prefrontal cortex; levels of IL-1 (B), IL-6 (D), IL-17A (F), and TNF-α (H) in the CA1 region of the hippocampus; n=6; BTBR group compared to C57... *** p<0.001; Compared with the BTBR group, the BTBR+Ert group ### p<0.001.

[0029] Figure 6 Etoggliflozin treatment reduced the number of activated microglia in BTBR mice. Immunofluorescence was used to detect Iba-1 positive microglia in the prefrontal cortex (A) and the CA1 region of the hippocampus (B). Red fluorescence: Iba-1; Blue fluorescence: DAPI. Number of Iba-1 positive neurons in the prefrontal cortex (C) and hippocampus (D); n=6; Compared with C57 mice, the BTBR group... *** p<0.001; Compared with the BTBR group, the BTBR+Ert group ### p<0.001. Detailed Implementation

[0030] The present application will now be described in further detail with reference to the accompanying drawings. It should be noted that the following specific embodiments are only used to further illustrate the present application and should not be construed as limiting the scope of protection of the present application. Those skilled in the art can make some non-essential improvements and adjustments to the present application based on the above application content.

[0031] Unless otherwise specified, the preparation methods used in this embodiment are conventional methods known to those skilled in the art, and the materials or reagents used are commercially available products unless otherwise specified.

[0032] 1. Materials and Methods

[0033] 1.1 Laboratory animals and drugs

[0034] 8-week-old male BTBR mice were purchased from The Jackson Laboratory in the United States (No. 002282, body weight (26 - 28) g) [BTBR mice are a stable genotype inbred strain of mice with brain development disorders, oxidative stress, and inflammatory responses, and exhibit autistic-like behaviors]. 8-week-old male C57BL / 6 mice, weighing (25 - 28) g, were provided by Jiangsu Huachuang Xinnuo Pharmaceutical Technology Co., Ltd. [Animal license: SCXK (Su) 2020 - 0009]. All animals were housed in an environment with a relative humidity of 55% - 65% and a light / dark cycle of 12 h / 12 h, ensuring free access to food and water.

[0035] Ertugliflozin (Ert) belongs to sodium-glucose cotransporter 2 inhibitors, with the molecular formula C 22 H 25 ClO7, a molecular weight of 436.88, CAS number 1210344 - 57 - 2, chemical name: (1S,2S,3S,4R,5S)-(2,3,4-TRIS-BENZYLOXY-5-(4-CHLORO-3-(4-ETH OXY-BENZYL)-PHENYL)-68-DIOXA-BICYCLO(3.2.1)OCT-1-YL)METHAChemicalbookNOL; (1S,2S,3S,4R,5S)-5-(4-chloro-3-(4-ethoxyben zyl)phenyl)-1-(hydroxymethyl)-6,8-dioxabicyclo[3.2.1]octane-2,3,4-trio, and its structural formula is as follows:

[0036]

[0037] 1.2 Animal grouping and administration method

[0038] The 8-week-old male mice were randomly divided into four groups: C57 group (C57), ertugliflozin control group (C57 + Ert), BTBR group (BTBR), and BTBR drug treatment group (BTBR + Ert). Mice in the C57 + Ert group and the BTBR + Ert group were given ertugliflozin (20 mg / kg) by gavage, and mice in the C57 group and the BTBR group were given an equal amount of normal saline by gavage, once a day for 8 consecutive weeks. 30 minutes after the last drug treatment, three-chamber social interaction, marble burying, self-grooming, open field, and elevated plus maze experiments were conducted. After the behavioral experiments, tissues from the prefrontal cortex and hippocampal CA1 region were collected from each group of mice for Nissl staining, enzyme-linked immunosorbent assay (ELISA), and immunofluorescence experiments.

[0039] 1.3 Behavioral detection

[0040] 1.3.1 Three-Box Social Experiment

[0041] The three-box social experiment assessed the social behavior of mice and their preference for novelty. A white polycarbonate box (60cm × 40cm × 22cm) was divided into three sections: left, middle, and right. A 6cm × 6cm passageway allowed mice free passage between the partitions. The test included an adaptation period, a social recognition phase, and a social novelty preference phase. In the first phase, the mice were placed in the middle chamber and allowed to freely explore all three chambers for 10 minutes. In the second phase, a strange mouse (Stranger1) was placed in one side of the box, and an empty cage (a novel object) was placed in the same location on the other side of the box; the mice were allowed to freely explore for 10 minutes. In the third phase, a strange mouse of the same species (Stranger2) was placed in the empty metal cage from the second phase, and the test mouse was allowed to freely explore for 10 minutes. VisuTrack software was used to record the time the mice spent in the three boxes and the time they spent sniffing the mice and objects, and preference indices were analyzed. Social recognition preference index = (social time with novel mouse 1 - social time with empty cage) / (social time with novel mouse 1 + social time with empty cage) × 100%; Social novelty preference index = (social time with novel mouse 2 - social time with novel mouse 1) / (social time with novel mouse 2 + social time with novel mouse 1) × 100%.

[0042] 1.3.2 Self-reflection experiment

[0043] Self-grooming was used to assess the stereotyped behaviors and stress levels of mice in unfamiliar environments. Mice were placed in transparent cages (40cm×20cm×10cm) and allowed to adapt to the environment for 10 minutes. Their activity was then recorded by a camera over 20 minutes. The time and frequency of grooming (grooming paws, nose, midface, behind ears, head, back, perineum, and tail) were recorded.

[0044] 1.3.3 Bead embedding experiment

[0045] The experiment used marble embedding to test repetitive stereotyped behaviors in mice. The cage bottom was covered with approximately 5cm of bedding material, and 20 marble beads (1.5cm in diameter) were evenly placed on the surface of the bedding material in a 3×4 pattern. The number of marble beads buried by the mice within 10 minutes was recorded; the standard was that the bedding material covered >75% of the volume of the marble beads.

[0046] 1.3.4 Open space

[0047] The open field test was used to evaluate spontaneous behavior and anxiety changes in mice. The experimental setup consisted of a square gray resin box (50cm×50cm×45cm), with the bottom divided into 16 small squares of 4×4 grids, the middle 4 squares forming the central area. Mice were placed in the center of the open field test chamber, and the time spent in the central area and the distance traveled within 30 minutes were recorded.

[0048] 1.3.5 Elevated Cross Maze

[0049] An elevated cross maze was used to assess anxiety in animals. The elevated cross maze apparatus consisted of two open arms (75cm × 58cm × 6cm) and two closed arms (75cm × 58cm × 6cm). The central region was defined as the area between the crossed arms. Rats were placed in the central region facing the open arms, and the time spent in the open arms and the distance traveled were recorded for 10 minutes.

[0050] 1.4 Nissl staining

[0051] Mice were deeply anesthetized with sodium pentobarbital (50 mg / kg). Fresh brain tissue was fixed in 4% paraformaldehyde, dehydrated in 30% sucrose at 4°C for 48 h, embedded in OCT, and sliced ​​to a thickness of 30 μm. Brain slices were rewarmed in double-distilled water for 5 min, then stained with tar violet staining solution at 56°C for 10 min. Excess stain was removed by washing with distilled water. Nissl differentiation solution was added, and sections were differentiated for 2 min. The sections were dehydrated with graded alcohols, cleared with xylene, and mounted with neutral resin. The pathological structure of the brain tissue was examined under a microscope.

[0052] 1.5 ELISA method for detecting oxidative stress and inflammatory factor levels in brain tissue

[0053] Mice were deeply anesthetized with sodium pentobarbital. The prefrontal cortex and CA1 region of the hippocampus were isolated. After centrifugation at 5000 rpm for 10 min, the MDA content, SOD and CAT activities in the supernatant were detected by ELISA according to the instructions of the malondialdehyde (MDA), glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT).

[0054] The ELISA kit was used to further detect the concentrations of interleukin-1β (IL-1β), interleukin-6 (IL-6), interleukin-17A (IL-17A), and tumor necrosis factor (TNF-α) in the supernatant of mouse homogenates in each group. All experimental procedures were performed strictly in accordance with the manufacturer's instructions.

[0055] 1.6 Immunofluorescence

[0056] Mice were deeply anesthetized with sodium pentobarbital, fixed by perfusion with 4% paraformaldehyde, and their brain tissue was dissected and preserved with 30% sucrose for 48 hours. Brain slices were placed in blocking solution containing 5% goat / donkey serum, 2.5% bovine serum albumin and 0.3% Triton-100 and blocked on a shaker at room temperature for 2 hours. Rabbit Iba1 monoclonal antibody was added and incubated overnight at 4°C. Donkey anti-rabbit IgG bound to AlexaFlour568 was then incubated, washed and mounted, and the brain slices were observed and photographed under a laser confocal microscope. Iba1-positive neurons were counted using ImageJ software.

[0057] 1.7 Statistical Analysis

[0058] Data processing and analysis were performed using SPSS 26.0 software. All results are expressed as mean ± standard error (SEM). Data were analyzed using t-tests, two-way ANOVA, and Tukey pairwise multiple comparisons. p < 0.05 was considered statistically significant.

[0059] 2. Results

[0060] 2.1 Etoggliflozin alleviates social impairment in BTBR mice

[0061] Social interaction and communication impairments are core symptoms of autism. To determine whether etagliflozin improves social impairments in BTBR mice, a three-box test was first used to assess social interaction. Figure 1 A shows representative heatmaps of the three-box social recognition experiment for the four groups of mice. The time spent by mice in the C57 group and the C57+Ert group in the Stranger 1 (S1) box was significantly longer than the time spent in the box containing the empty cage (or the novel object, NO) (p<0.001, p<0.001). Figure 1 B) The social time of both groups of mice with unfamiliar mouse 1 was longer than that with NO (p<0.001, p<0.001); Figure 1 C). There was no significant difference in the time spent in the boxes containing the unfamiliar mouse 1 and the novel object among the BTBR group mice (p>0.05); Figure 1 B), there was no statistically significant difference in social time with unfamiliar mouse 1 and novel objects (n=10, p>0.05); Figure 1 C). The time that the BTBR+Ert group mice spent in the box containing the unfamiliar mouse 1 was significantly longer than the time spent in the box containing the new object (p<0.001); Figure 1 B), the social time spent with unfamiliar mice was significantly longer than the social time spent with new objects (p<0.001); Figure 1 C). Furthermore, compared to the C57 group, the social recognition index of mice in the BTBR group was decreased (p<0.001); Figure 1 D) The social recognition index increased in the eitogliflozin treatment group (p<0.001); Figure 1D). This suggests that etanercept treatment alleviates social recognition impairment in BTBR mice.

[0062] In novel social experiments Figure 1 E represents the heatmap of the three-box social novelty preference experiment for the four groups of mice. The time spent in the Stranger 2 (S2) box by mice in the C57 group and the C57+Ert group was significantly longer than that spent in the Novelty 1 box (p<0.001, p<0.001). Figure 1 F), the social time of both groups of mice with unfamiliar mice 2 was longer than that with novel mice 1 (p<0.001, p<0.001); Figure 1 G). There was no significant difference in the time spent in the enclosure containing the unfamiliar mouse 2 and the novel mouse 1 in the BTBR group (p>0.05); Figure 1 F), there was no statistically significant difference in social time between unfamiliar mouse 2 and novel mouse 1 (p>0.05); Figure 1 G). The BTBR+Ert group mice spent significantly more time in the enclosure containing the unfamiliar mouse 2 than in the enclosure containing the novel mouse 1 (p<0.001); Figure 1 F), the social time with unfamiliar mouse 2 was significantly longer than the social time with novel mouse 1 (p<0.001); Figure 1 G). Furthermore, compared to the C57 group, the BTBR group mice showed a decreased social novelty preference index (p<0.001); Figure 1 H), the social novelty preference index increased in the etorgali net treatment group (p<0.001); Figure 1 H). This suggests that etanercept treatment alleviates social novelty disorder in BTBR mice.

[0063] 2.2 Etoggliflozin reduces repetitive stereotyped and anxiety-like behaviors in BTBR mice

[0064] The self-grooming and bead embedding assays were used to investigate whether etanercept treatment could alter repetitive, stereotyped behaviors in BTBR mice. In the self-grooming assay, for example... Figure 2 As shown in Figures AB, compared with the C57 group mice, the BTBR group mice exhibited a significant increase in the time and frequency of repeated grooming (p<0.001, p<0.001), indicating that BTBR mice exhibit significant repetitive stereotyped behavior. Compared with the BTBR group, the eratogliflozin treatment group showed a significant decrease in the time and frequency of repeated grooming (p<0.001, p<0.001). In the bead embedding experiment, compared with the C57 group, the BTBR group showed a significant increase in the number of beads embedded (p<0.001, p<0.001). Figure 2 C). Compared with the BTBR group, the number of implanted beads was reduced in the etoragliflozin treatment group (p<0.001). Figure 2 C). This indicates that etanercept treatment significantly improves repetitive stereotyped behaviors in BTBR mice.

[0065] Open field and elevated cruciate maze tests were used to examine whether etoragliflozin treatment could alter the anxiety-like behavior of BTBR mice. In the open field test, compared with the C57 group, the BTBR group mice spent significantly less time in the central area (p<0.001). Figure 2 DE). Compared with the BTBR group, mice in the etoragliflozin treatment group spent more time in the central region (p<0.001). Figure 2 DE). There was no statistically significant difference in movement distance between the BTBR+Ert group and the BTBR group (p>0.05). Figure 2 F). In the elevated cross maze, compared with the C57 control group, the BTBR group mice had a reduced time spent in the open-arm position (p<0.001). Figure 2 GH). Compared with the BTBR group, the etoragliflozin treatment group had an increased open-arm residence time (p<0.001). Figure 2 GH). There was no statistically significant difference in distance traveled between the BTBR+Ert group and the BTBR group (p>0.05). Figure 2 I). This indicates that etoragliflozin treatment significantly reduced anxiety-like behavior in BTBR mice.

[0066] 2.3 Effects of eletoggliflozin on the morphology of the prefrontal cortex and hippocampus in BTBR mice

[0067] like Figure 3 As shown in Figures AB, neurons in the prefrontal cortex and CA1 region of the hippocampus of mice in the C57 group had abundant Nissl bodies. Compared with the C57 group, the BTBR group mice showed a reduction in Nissl bodies in neurons of the tested brain regions (p<0.001, p<0.001). Figure 3 (CD). Brain slices from the BTBR+Ert group showed extensive blue granular Nissl bodies with normal morphology. Compared with BTBR mice, the number of Nissl bodies in neurons of the tested brain regions was increased (p<0.001). Figure 3 CD). This indicates that etanercept effectively reduces damage to neurons in the prefrontal cortex and hippocampus of BTBR mice and improves neuronal morphology.

[0068] 2.4 Effect of eletoggliflozin on oxidative stress levels in BTBR

[0069] like Figure 4As shown in the AH, compared with the C57 group, the BTBR group mice showed significantly increased MDA content in the prefrontal cortex and hippocampal CA1 region (p<0.001, p<0.001), and decreased GSH content, SOD, and CAT activity (all p<0.001). Compared with the BTBR group, the BTBR+Ert group showed decreased MDA content in the tested brain regions (p<0.001, p<0.001), and significantly increased GSH content, SOD, and CAT activity (all p<0.001). This indicates that eletoggliflozin can significantly reduce oxidative stress levels in the prefrontal cortex and hippocampal CA1 region of BTBR mice.

[0070] 2.5 Effects of eletoggliflozin on inflammation in BTBR mice

[0071] like Figure 5 As shown in the ablation study, the levels of inflammatory cytokines IL-1β, IL-6, IL-17A, and TNF-α in the prefrontal cortex and hippocampus of BTBR mice were significantly higher than those in C57 mice (all p < 0.001). Compared with the BTBR group, eltoggliflozin treatment reduced the levels of IL-1β, IL-6, IL-17A, and TNF-α in the tested brain regions (all p < 0.001). These results confirm that eltoggliflozin can alleviate neuroinflammation in the prefrontal cortex and hippocampus of BTBR mice.

[0072] 2.6 Etoggliflozin inhibits the number of activated microglia in BTBR mice

[0073] like Figure 6 As shown in AD, the number of activated microglia in the prefrontal cortex and CA1 region of the hippocampus of BTBR mice was significantly higher than that in C57 mice (p<0.001, p<0.001). Etoggliflozin treatment reduced the number of activated microglia in BTBR mice (p<0.001, p<0.001). These results indicate that etoggliflozin treatment can reduce the number of activated microglia in the prefrontal cortex and hippocampus of BTBR mice.

[0074] In summary, eltoggliflozin can be used as a medication to treat or improve autism. This medication comprises eltoggliflozin and a pharmaceutically acceptable carrier. The dosage form can be any of the following: tablets, capsules, lozenges, injections, suspensions, suppositories, ointments, bladder instillations, oral preparations, suppositories, or sustained-release preparations. The carrier can be any of the following: liposomes, nanoparticles, hydrogels, or microspheres.

[0075] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various improvements without departing from the concept of the present invention, and these improvements all fall within the scope of protection of the present invention.

Claims

1. Application of etanercept in the preparation of drugs for the treatment of autism.

2. The application according to claim 1, characterized in that, The molecular formula of eletoggliflozin is C 22 H 25 ClO7, with a molecular weight of 436.88 and CAS number 1210344-57-2, has the following structural formula:

3. The application according to claim 1, characterized in that, The drug contains eltoggliflozin and a pharmaceutically acceptable carrier.

4. The application according to claim 1, characterized in that, The dosage of eletoggliflozin is 20 mg / kg / day.

5. Application of eletoggliflozin in the preparation of drugs to reverse impaired social recognition abilities in autistic animals or humans.

6. The use of atorogliptin in the preparation of drugs for treating social novelty preference deficit caused by autism.

7. The use of atorogliflozin in the preparation of drugs to reduce anxiety-like behaviors in autistic animals or humans.

8. The use of atorogliflozin in the preparation of drugs to reduce repetitive and stereotyped behaviors in autistic animals or humans.

9. The use of atorogliflozin in the preparation of drugs for the treatment of oxidative stress, inflammatory response and microglial overactivation in the prefrontal cortex and hippocampus caused by autism.

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