Application of interleukin-33 in preparation of medicine for treating autism
By using interleukin-33 to prepare therapeutic drugs, the problem of lack of effective cytokine targets in the prior art was solved, and the neurological damage to autism and inflammation were improved, and social disorders and repeated stereotyped behaviors were improved.
Patent Information
- Application Number
- CN202510289538.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-03-12
- Publication Date
- 2025-07-11
AI Technical Summary
There is a lack of cytokine target drugs to improve the therapeutic effect of autism in the prior art.
Interleukin-33 (IL-33) is used to prepare drugs for treating autism, which can regulate cytokine expression, improve neurological damage, reduce inflammatory processes, inhibit glial cell proliferation, regulate lipid metabolism and reprogramming, and improve social disorders and repetitive stereotype behaviors.
IL-33 significantly improves neurological impairment caused by autism, alleviates the inflammatory process, inhibits glial cell proliferation, regulates lipid metabolism, and improves social disorders and repetitive stereotypes.
Smart Images
Figure CN120285154A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of biomedical technologies, and in particular to the application of interleukin-33 in the preparation of drugs for treating autism. Background Art
[0002] Autism Spectrum Disorder (ASD) usually occurs around the age of 2. Due to the developmental disorder of the brain, children with ASD lose necessary social skills and life skills, bringing heavy economic and mental burdens to families and society. Currently, the treatment of ASD mainly focuses on behavioral therapy, with a long rehabilitation course and a significant lack of effective drug treatment means. Therefore, it has become extremely urgent to fully study the pathological changes after ASD in order to seek better treatment means.
[0003] A large number of studies have shown that inflammatory responses play a key role in the occurrence and development of ASD. Prenatal infections and maternal inflammatory responses are risk factors for the onset of ASD in children. Animal experiments have also shown that inflammation caused by maternal infections in mice may affect fetal brain development through the cytokine IL17A, leading to offspring suffering from ASD. In addition, there is activation of glial cells in the brains of ASD patients, and the levels of cytokines in serum and cerebrospinal fluid are also abnormally increased, including IL-1β, IL-6, IL17, IL18, TNF-α, etc. Therefore, regulating the expression of cytokines to improve autism has become an effective treatment means. However, the existing drugs for treating autism targeting cytokines still have unsatisfactory treatment effects. Summary of the Invention
[0004] For this reason, the technical problem to be solved by the present invention is to overcome the problem in the prior art of lacking a cytokine target drug that can improve the treatment effect of autism.
[0005] To solve the above technical problem, the present invention provides an application of interleukin-33 in the preparation of drugs for treating autism. Through experiments, the present invention has found that IL-33 has an improving effect on the nerve function damage caused by ASD, alleviates the inflammatory process caused by ASD, and inhibits the gliosis caused by ASD; at the same time, IL-33 can regulate the lipid metabolism reprogramming caused by ASD, alleviate the neuronal degeneration caused by ASD, and improve the abnormalities of the brain nerve synapses caused by ASD, thereby improving the social impairment and repetitive stereotyped behaviors caused by ASD. The present invention provides a basis for using IL-33 to treat ASD and provides a drug option for the clinical treatment of autism.
[0006] The first object of the present invention is to provide an application of IL-33 in the preparation of drugs for treating autism.
[0007] Furthermore, the drug for treating autism includes IL-33.
[0008] The second object of the present invention is to provide an application of an IL-33 activator in the preparation of a drug for treating autism.
[0009] Furthermore, the IL-33 activator increases the expression of IL-33.
[0010] The third object of the present invention is to provide a drug for treating autism, which is designed with IL-33 as a target to increase the expression of IL-33.
[0011] Furthermore, the drug for treating autism improves social impairment and repetitive stereotyped behaviors.
[0012] Furthermore, the drug for treating autism increases the content of lipids in the cerebral cortex and hippocampus.
[0013] Furthermore, the drug for treating autism inhibits the expression of lipoxygenase arachidonate-15-lipoxygenase. ALOX15 is a key regulatory enzyme in the metabolism of polyunsaturated fatty acids, which can catalyze various fatty acids to generate various lipid mediators and participate in the pathophysiological process of inflammation. In addition, through the action of ALOX15, polyunsaturated fatty acids generate lipid peroxidation products, which are key mediators of ferroptosis. Ferroptosis is a programmed cell death driven by ferrous ion-dependent lipid peroxidation, and one of its characteristics is the accumulation of lipid peroxidation. Treatments that inhibit ferroptosis can improve autism-related behaviors in ASD animal models. Peroxisome proliferator-activated receptor (PPAR) is a ligand-activated transcription factor in the nuclear hormone receptor family and plays an important role in regulating lipid metabolism and inflammatory responses. Impaired mitochondrial fatty acid oxidation is closely associated with ASD, and the activation of PPAR can coordinately upregulate multiple mitochondrial fatty acid oxidases, playing a neuroprotective role by reducing oxidative stress and inflammatory responses. By inhibiting ALOX15 / PPAR-mediated lipid metabolism reprogramming, lipid peroxidation and neuroinflammation involved in the pathophysiology of ASD can be reduced.
[0014] Furthermore, the drug for treating autism inhibits the abnormal expression of microglia caused by autism.
[0015] Furthermore, the drug for treating autism inhibits nerve cell apoptosis and neuronal degeneration.
[0016] Furthermore, the drug for treating autism promotes the increase in the number and length of synaptic nodes and reduces the dendritic spine density.
[0017] The beneficial effects of the present invention:
[0018] The present invention first proposes the application of IL-33 in the preparation of drugs for the treatment of autism. Through experiments, the present invention discovers that IL-33 has an improving effect on the nerve function damage caused by ASD, alleviates the inflammatory process caused by ASD, and inhibits the gliosis caused by ASD; at the same time, IL-33 can regulate the lipid metabolism reprogramming caused by ASD, alleviate the neuronal degeneration caused by ASD, and improve the abnormalities of the brain nerve synapses caused by ASD, thereby improving the social disorders and repetitive stereotyped behaviors caused by ASD. The present invention provides a basis for the treatment of ASD using IL-33 and provides drug options for the clinical treatment of autism. Brief Description of the Drawings
[0019] In order to make the content of the present invention easier to be clearly understood, the following further details the present invention according to the specific embodiments of the present invention in combination with the drawings, wherein
[0020] Figure 1 are the results of behavioral tests on mice with IL-33 and the related histogram statistical results. Among them, P28-30 represents the 28th to 30th day after the birth of the mice, P45-51 represents the 45th to 50th day after the birth of the mice, P29-P31 represents the 29th to 31st day after the birth of the mice, and P50-52 represents the 50th to 52nd day after the birth of the mice;
[0021] Figure 2 are the expression levels of inflammation-related proteins (p-STAT1, STAT1, and T-bet) in the cerebral cortex of mice after the action of IL-33 and the related histogram statistical results. Among them, P43 represents the 43rd day after the birth of the mice, and P64 represents the 64th day after the birth of the mice;
[0022] Figure 3 are the expression levels of inflammation-related proteins (RUNX1, RORγt, and IL-17) in the cerebral cortex of mice after the action of IL-33 and the related histogram statistical results;
[0023] Figure 4 are the expression levels of inflammation-related proteins (TGF-β1, SMAD1 / 2 / 3, and FOXP3) in the cerebral cortex of mice after the action of IL-33 and the related histogram statistical results;
[0024] Figure 5 are the expression levels of inflammation-related proteins (NF-KB, GATA3, and JFC1) in the cerebral cortex of mice after the action of IL-33 and the related histogram statistical results;
[0025] Figure 6 are the test results of neuroglial cells in mice after ASD with IL-33 and the related histogram statistical results;
[0026] Figure 7It is the expression level of lipid metabolism-related proteins in the cerebral cortex of mice after the action of IL-33 and the statistical results of the related histograms;
[0027] Figure 8 It is the expression level of lipid metabolism-related proteins in the cerebral cortex of mice after the action of IL-33 and the statistical results of the related histograms;
[0028] Figure 9 It is the detection results of lipid content in the cerebral cortex and hippocampus of mice after the action of IL-33 and the statistical results of the related histograms;
[0029] Figure 10 It is the test results of nerve cell apoptosis in mice after ASD by IL-33;
[0030] Figure 11 It is the degree of degeneration of cerebral cortex neurons in mice after ASD after the action of IL-33 and the statistical results of the related histograms;
[0031] Figure 12 It is the degree of degeneration of neurons in the CA1 region of the hippocampus in mice after ASD after the action of IL-33 and the statistical results of the related histograms;
[0032] Figure 13 It is the degree of degeneration of neurons in the CA3 region of the hippocampus in mice after ASD after the action of IL-33 and the statistical results of the related histograms;
[0033] Figure 14 It is the degree of degeneration of neurons in the DG region of the hippocampus in mice after ASD after the action of IL-33 and the statistical results of the related histograms;
[0034] Figure 15 It is the test results of synaptic pruning and remodeling in mice after ASD by IL-33;
[0035] Figure 16 It is the statistical results of the line chart and histogram of the synaptic pruning and remodeling test in mice after ASD by IL-33. Specific implementation manners
[0036] The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and be able to implement it, but the embodiments cited do not limit the present invention.
[0037] The behavioral experiments and molecular biology detections involved in this embodiment include:
[0038] (1) Self-grooming experiment, specifically as follows:
[0039] Before the test, the mice were allowed to adapt to the room environment for 30 minutes. The mice to be tested were gently placed into the prepared breeding cage, and the cage lid was gently covered. They were allowed to move freely for 10 minutes to adapt to the cage environment. After the 10-minute adaptation stage, the time of self-grooming behavior of the mice to be tested in the next 10 minutes was recorded. After the test, the mice to be tested were gently taken out and put back into the cage, and then the cage used for the test was cleaned with 75% ethanol solution. After it dried, the test for the next mouse was carried out. Record the time when self-grooming behavior occurred, including: the repetitive actions of the mice to be tested spontaneously grooming with their mouths or front limbs and / or any part of their whole body.
[0040] (2) Social interaction test, specifically as follows:
[0041] Before the test, the mice were allowed to adapt to the room environment for 30 minutes. The cage was lined with clean bedding, and the feed and drinking water supply bottles were removed. First, the mice to be tested were put into the cage to adapt for 10 minutes, and then a strange male mouse of the same species was put in. They were allowed to interact freely for 5 min (the first batch was recorded for 10 min). After each mouse was tested, the bedding was cleaned, and the experimental device was wiped with 75% ethanol solution until dry. After laying down clean bedding again, the test for the next mouse was carried out. Record the time when the mice to be tested actively interacted with the strange mouse. The interaction behaviors included: sniffing any part of the strange mouse, following the strange mouse, supporting the front limbs on the strange mouse, straddling the strange mouse or passing under it, grooming the body of the strange mouse, crowding and / or wrestling with the strange mouse, and biting.
[0042] (3) Immunofluorescence experiment
[0043] After deeply anesthetizing the mice, the mice were perfused through the heart with PBS and 4% paraformaldehyde. The whole brain tissue was quickly separated and obtained, and then sucrose gradient dehydration was carried out. It was embedded with OCT and frozen sections were made to obtain mouse brain sections. The sections were incubated in the primary antibody diluted with BSA at 4°C overnight, and then the sections were rinsed. They were incubated with the fluorescently labeled secondary antibody at room temperature for 2 h. Subsequently, observation was carried out using a Nikon upright fluorescence microscope, and the obtained pictures were merged and processed using Image J.
[0044] (4) Western Blotting experiment:
[0045] After anesthetizing the mice, the cerebral cortex tissues were isolated. The isolated brain tissues were added to a lysis solution containing protease inhibitors and sonicated for lysis, centrifuged, homogenized, and then the supernatant was extracted. Then, the protein concentration was measured using enhanced BCA, and an equal amount of 60 μg protein sample was loaded into each well. Each protein was separated by SDS-PAGE gel electrophoresis. Then, the proteins on the separation gel were transferred onto a PVDF membrane. Subsequently, the PVDF membrane with the transferred proteins was incubated overnight at 4°C in an antibody diluted with BSA. After washing the membrane the next day, the protein bands were incubated at room temperature for 2 h in a secondary antibody linked to horseradish peroxidase. Subsequently, the ECL chemiluminescence system was applied to detect the protein bands. Finally, the protein bands were scanned and the gray values of the protein bands were analyzed using Image J.
[0046] (5) Nile Red Staining:
[0047] After deeply anesthetizing the mice, the mice were perfused through the heart with PBS and 4% paraformaldehyde, and the whole brain tissue was quickly isolated and obtained. Then, it was dehydrated with a sucrose gradient, embedded with OCT, and frozen sections were made to obtain mouse brain sections. After rewarming the frozen sections, they were fixed with 4% PFA for 10 minutes, immersed in an appropriate amount of Nile Red staining solution (Nile Red: Hoechst 33342: detection buffer = 1:1:998), stained for 5 - 10 min, and then lipid deposition or intracellular lipid vesicles were detected.
[0048] (6) TUNEL Staining (Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick End Labeling):
[0049] After deeply anesthetizing the mice, the mice were perfused through the heart with PBS and 4% paraformaldehyde, and the whole brain tissue was quickly isolated and obtained. Then, it was dehydrated with a sucrose gradient, embedded with OCT, and frozen sections were made to obtain mouse brain sections. After rewarming the frozen sections, they were fixed with 4% PFA for 10 minutes, soaked in a 0.5% Triton X-100 solution, and incubated at room temperature for 10 minutes for membrane perforation. Subsequently, these sections were added to the TUNEL reaction system (TdT enzyme: fluorescent labeling solution = 9:1), incubated at 37°C in the dark for 60 minutes, and sealed with an anti-fluorescence quencher containing DAPI. Subsequently, observation was carried out using a Nikon laser confocal microscope.
[0050] (7) Measurement of the degree of cortical neuron degeneration, specifically as follows:
[0051] After deeply anesthetizing the mice, the mice were perfused with PBS and 4% paraformaldehyde through the heart, and the whole brain tissue was rapidly isolated and obtained. Then, sucrose gradient dehydration was performed, followed by embedding with OCT and frozen sectioning to obtain mouse brain sections. The frozen sections were subjected to Nissl staining, and subsequently, observed using a Nikon laser confocal microscope.
[0052] (8) Golgi staining
[0053] After deeply anesthetizing the mice, each group of mice was quickly sacrificed by cervical dislocation, and the skull was removed as soon as possible to obtain the brain tissue. The brain tissue was immediately rinsed with distilled water to remove the surface blood, and immediately fixed with Golgi staining fixative (G1069, Servicebio, China) for 5 days. Then, the fixed brain tissue was cut into 50-μm-thick sections, dehydrated twice with absolute ethanol (20 min each time), and cleared with xylene for 30 min. Finally, brain section images were obtained using a VS120-S6-W system (Olympus, Japan), and analyzed using Olympus ver software.
[0054] Example 1
[0055] Thirty 6-week-old male BTBR mice with basically the same body weight (BTBR mice are an inbred mouse strain that not only has the core symptoms of ASD: reduced social interaction, fewer ultrasounds emitted in social situations, and severe repetitive grooming behavior; but also has brain developmental abnormalities and abnormal immunobiochemical indexes similar to those of ASD. Therefore, BTBR mice are currently an ideal model for studying ASD) were randomly divided into 2 groups, namely: BTBR group (BTBR group), IL-33 treatment group (IL-33 + BTBR group); 15 B6 mice of the same age and body weight were randomly matched as the control group (B6 group). The mice in the IL-33 + BTBR group were intraperitoneally injected with recombinant mouse IL-33 at a dose of 300 ng / mouse per day for 7 consecutive days; the mice in the B6 group and the BTBR group were injected with the same volume of normal saline every day. The breeding conditions of the three groups of mice were the same.
[0056] On the day after the last injection, behavioral-related tests were performed on each group of mice, and the test results are as Figure 1 shown. It reflects the behavioral changes of each group of mice. Comparing Figure 1 the various test results of the BTBR group and the IL-33 + BTBR group in it, it can be seen that the mice in the IL-33 + BTBR group improved the social impairment and repetitive stereotyped behavior of BTBR mice, indicating that IL-33 improved the social behavior function deficit of ASD.
[0057] Immunoblotting experiments were performed on the cerebral cortex tissues of each group of mice, and the results are shown in Figures 2 to 9。From the test results, it can be seen that the expression of inflammation-related proteins in the IL-33 + BTBR group of mice was lower than that in the BTBR group, indicating that IL-33 has an inhibitory effect on the inflammatory process caused by ASD. At the same time, the expression of lipid metabolism-related proteins ALOX15 and PPARβ in the IL-33 + BTBR group of mice was lower than that in the BTBR group, indicating that IL-33 inhibits the lipid metabolism reprogramming mediated by ALOX15 / PPARβ.
[0058] Immunofluorescence experiments were performed on brain sections of mice in each group, and the results are as Figure 6 。Among them, blue is the nuclear marker DAPI, green is the microglia marker IBA-1, and the third column is the combination of the first and second columns. From the test results, it can be seen that the expression of microglia in the IL-33 + BTBR group of mice was lower, indicating that IL-33 has an inhibitory effect on the expression of microglia caused by ASD.
[0059] Nile red staining was performed on brain sections of mice in each group, and the results are shown in Figures 7 to 9 。From the test results, it can be seen that IL-33 treatment reversed the decrease in lipid content in the cerebral cortex and hippocampus of BTBR group mice, indicating that IL-33 inhibits abnormal lipid metabolism degradation.
[0060] TUNEL staining was performed on brain sections of mice in each group, and the results are shown in Figure 10 。From the test results, it can be seen that IL-33 inhibits the neuronal apoptosis caused by ASD.
[0061] Nissl staining was performed on brain sections of mice in each group, and the results are shown in Figures 11 - 14 。From the test results, it can be seen that the degree of neuronal degeneration in the IL-33 group of mice was reduced, indicating that IL-33 has an attenuating effect on the neuronal degeneration caused by ASD.
[0062] Golgi staining was performed on brain tissues of mice in each group, and the results are shown in Figure 15 and Figure 16 。From the test results, it can be seen that the dendritic nodes and lengths of cortical neurons in the IL-33 + BTBR group of mice increased, while the dendritic spine density decreased, indicating that IL-33 can improve the structure and function of neurons by promoting synaptic pruning and remodeling.
[0063] Obviously, the above embodiments are only examples for clear illustration and are not limitations on the implementation manners. For those of ordinary skill in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to enumerate all the implementation manners here. And the obvious changes or modifications derived therefrom still fall within the protection scope of the present invention.
Claims
1. Use of interleukin-33 in the preparation of a therapeutic drug for autism.
2. The application according to claim 1, wherein The therapeutic drug for autism includes interleukin-33.
3. Use of an interleukin-33 activator in the preparation of a therapeutic drug for autism.
4. The application according to claim 3, wherein The interleukin-33 activator increases the expression of interleukin-33.
5. A drug for treating autism, characterized in that, The therapeutic drug for autism is designed targeting interleukin-33 to increase the expression of interleukin-33.
6. The autism treatment drug according to claim 5, characterized in that, The therapeutic drug for autism improves social impairment and repetitive stereotyped behaviors.
7. The autism treatment drug according to claim 5, characterized in that, The therapeutic drug for autism inhibits the expression of lipoprotein arachidonate-15-lipoxygenase.
8. The autism treatment drug according to claim 5, characterized in that, The therapeutic drug for autism inhibits microglial hyperplasia caused by autism.
9. The autism treatment drug according to claim 5, wherein The therapeutic drug for autism inhibits nerve cell apoptosis and neuronal degeneration caused by autism.
10. The autism treatment drug according to claim 5, characterized in that, The therapeutic drug for autism promotes the increase of synaptic nodes and length and reduces dendritic spine density.