Establishment method and verification method of hydrogen peroxide induced mouse autism model

Abstract

The invention discloses an establishment method and a verification method of a hydrogen peroxide induced mouse autism model, and relates to the technical field of medical biologication.The establishment method of the hydrogen peroxide induced mouse autism model comprises the following steps that a hydrogen peroxide solution is injected into the abdominal cavity of a pregnant mouse, hydrogen peroxide induction is conducted, and the mouse autism model is obtained. And obtaining a hydrogen peroxide induced offspring mouse, namely the mouse autism model. Hydrogen peroxide is used for near intraperitoneal injection of a female mouse during pregnancy, the oxidative stress state of the female mouse during pregnancy and the oxidative stress state of an offspring mouse embryo are induced through independent hydrogen peroxide, and obvious immune response of the female mouse is not caused, so that an ASD mouse model specifically induced by oxidative stress is formed; the obtained ASD offspring mouse has a remarkable ASD phenotype, but does not show obvious dyskinetic disorder.

Description

Technical Field

[0001] The invention relates to the field of medical biotechnology, and in particular to a method for establishing and verifying a hydrogen peroxide-induced autism model in mice. Background Art

[0002] Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder characterized by deficits in social interaction and communication, repetitive and restricted behavior patterns, and a narrow range of interests or activities. In order to study the pathological mechanisms of ASD and develop treatments, animal models of autism, especially mouse models, are usually constructed to simulate the symptoms and biological characteristics of human ASD.

[0003] Currently, there are two common ASD mouse models: (1) induction models represented by maternal immune activation (MIA) mice, and (2) transgenic models represented by BTBR mice and specific transgenic mice. The MIA inflammatory induction model is consistent with clinical ASD in terms of disease simulation and ASD phenotype, but it not only introduces exogenous immunogenic substances such as lipopolysaccharide (LPS) and polyinosinic acid [Poly(I:C)], but also causes extensive immune activation and release of inflammatory factors, as well as activation of various inflammatory pathways. At the molecular level, it is mixed with the interference of various complex factors such as the toxic effects of exogenous substances themselves, immune damage, oxidative stress, etc., which to some extent hinders the screening of key pathways leading to autism. However, the neurological phenotypes of BTBR mice and transgenic mice do not necessarily form typical autism disorders, and are often accompanied by more serious cognitive dysfunction, movement disorders, etc. At the molecular level, changes in specific genes may cause changes in more extensive signaling pathways closely related to the gene. These changes may interfere with or dilute the changes in the key neural pathways that cause autism disorders, which is not conducive to molecular level research.

[0004] Therefore, there is still a lack of universal animal models for studying ASD. Summary of the invention

[0005] The main purpose of the present invention is to propose a method for establishing and verifying a hydrogen peroxide-induced autism model in mice, aiming to solve the problem of lack of a universal animal model for studying ASD in the prior art.

[0006] To achieve the above object, the present invention provides a method for establishing a hydrogen peroxide-induced autism model in mice, comprising the following steps:

[0007] A hydrogen peroxide solution was injected into the abdominal cavity of pregnant mice for hydrogen peroxide induction, and the resulting hydrogen peroxide-induced offspring mice were the mouse autism model.

[0008] In one embodiment, the method for establishing a hydrogen peroxide-induced autism model in mice comprises:

[0009] The hydrogen peroxide solution was intraperitoneally injected into pregnant mice on embryonic day 12.5 to induce hydrogen peroxide, thereby obtaining hydrogen peroxide-induced offspring mice, i.e., a mouse autism model.

[0010] In one embodiment, the amount of hydrogen peroxide injected into the peritoneal cavity of the pregnant mouse is 180-220 μmol / kg.

[0011] In one embodiment, the hydrogen peroxide induction time is 6 to 72 hours.

[0012] The present invention also provides a method for verifying a mouse autism model, wherein the method for verifying a mouse autism model is used to verify the mouse autism model obtained by the aforementioned method for establishing a mouse autism model induced by hydrogen peroxide;

[0013] The verification method of the mouse autism model comprises the following steps:

[0014] Assessing motor skills, grooming stereotypes, and social interaction in mouse models of autism;

[0015] Assessing brain synaptic changes in mouse models of autism;

[0016] Assess the expression of autism spectrum disorder-associated pathogenic genes in mouse models of autism.

[0017] In one embodiment, the step of evaluating brain synaptic changes in a mouse autism model comprises:

[0018] Assessing brain synaptic histology in mouse models of autism;

[0019] Assessment of brain synaptic markers in a mouse model of autism.

[0020] In one embodiment, the step of evaluating the expression of autism spectrum disorder-related pathogenic genes in a mouse autism model comprises:

[0021] Assess the expression of autism spectrum disorder-related pathogenic genes in mouse autism models;

[0022] Evaluation of autism spectrum disorder differentially expressed gene enriched pathways in a mouse model of autism.

[0023] In one embodiment, the brain synaptic marker comprises at least one of Synaptophysin and Postsynapticdensity protein-95.

[0024] In one embodiment, the autism spectrum disorder-related pathogenic genes include at least one of Adcy5, Adora2a, Agtr2, Cep135, Cep290, Cx3cr1, Ddc, Drd1, Drd2, Ebf3, En2, Esrrb, Fhit, Gpr139, Htr7, Il16, Lmx1b, Nckap5, Nfe2l3, P2rx5, Patj, Pax5, Pcdha4, Pcdh9, Slc29a4, Slc6a3, Slc6a4, Ppp1r1b, Sh3rf2, Tph2, Ttn and Wnt2.

[0025] In one embodiment, pathways enriched for differentially expressed genes in autism spectrum disorder include neurotransmitter-receptor interactions, dopaminergic neurons, retrograde synaptic signaling, and oxidative phosphorylation.

[0026] In the technical scheme of the present invention, hydrogen peroxide is injected intraperitoneally during the pregnancy of the mother mouse, and the oxidative stress state of the pregnant mother mouse and the oxidative stress state of the offspring mouse embryo are induced by hydrogen peroxide alone, and no obvious immune response is caused in the mother mouse, which is different from the prior art; and H2O2 is decomposed into two products, H2O2 and O2, after the redox reaction, and both products are not toxic or irritating to mice, thereby forming an ASD mouse model specifically induced by oxidative stress; the ASD offspring mice obtained by the present invention have a significant ASD phenotype, but do not show obvious motor impairment, highlighting that the ASD characteristics of the mouse model of the present invention are typical; the present invention achieves the effect of embryonic oxidative stress state by only intraperitoneal injection of hydrogen peroxide during the pregnancy of the mother mouse, which is convenient to operate and low in cost; and no obvious immune inflammatory response is caused in the mother mouse, which effectively reduces the pain of the experimental mouse, and is very beneficial to the protection and implementation of experimental animal welfare and ethics. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying creative work.

[0028] Figure 1This is a graph showing the results of detecting the CRP immune index in the serum of female mice at 0 days, 1 day, 2 days and 3 days after H2O2 injection in Example 2 provided by the present invention;

[0029] Figure 2 (A) is a graph showing the CAT detection results in the embryos from the first pair of H2O2 injected pregnant mice (H2O2) and non-injected control pregnant mice (CTR) in Example 2 provided by the present invention, Figure 2 Middle (B) is a graph showing the CAT detection results in embryos from the second pair of H2O2-injected pregnant mice (H2O2) and non-injected control pregnant mice (CTR) in Example 2 provided by the present invention;

[0030] Figure 3 (A) is a diagram showing the results of an open field test of offspring mice induced by H2O2 during pregnancy in Example 3 provided by the present invention. Figure 3 (B) is a diagram showing the results of the rotarod test of offspring mice induced by H2O2 during pregnancy in Example 3 provided by the present invention;

[0031] Figure 4 (A) is a total time statistics chart of the grooming behavior of offspring mice induced by H2O2 during pregnancy in Example 4 provided by the present invention, Figure 4 (B) is a frequency statistical diagram of the grooming behavior of offspring mice induced by H2O2 during pregnancy in Example 4 provided by the present invention;

[0032] Figure 5 (A) is a schematic diagram of the three-box social experiment scheme of offspring mice induced by H2O2 during pregnancy in Example 5 provided by the present invention, Figure 5 Middle (B) is a diagram showing the results of the three-box social ability assessment of offspring mice induced by H2O2 during pregnancy in Example 5 provided by the present invention;

[0033] Figure 6 (A) is a diagram showing the results of synaptic histology detection of offspring mice induced by H2O2 during pregnancy in Example 6 provided by the present invention. Figure 6 Middle (B) is a diagram showing the statistical results of the number of dendritic spines of offspring mice induced by H2O2 during pregnancy in Example 6 provided by the present invention;

[0034] Figure 7 This is a diagram showing the results of synaptic marker detection in offspring mice induced by H2O2 during pregnancy in Example 7 provided by the present invention;

[0035] Figure 8 (A) is a volcano plot of differentially expressed genes in the brain tissue of offspring mice induced by H2O2 during pregnancy in Example 8 provided by the present invention. Figure 8 Middle (B) is a statistical chart of down-regulated genes (green) and up-regulated genes (red) that are clearly associated with the onset of autism in the brain tissue of offspring mice induced by H2O2 during pregnancy in Example 8 provided by the present invention;

[0036] Fig. 9 This is a graph showing the results of GO enrichment analysis of differentially expressed genes in the brain tissue of offspring mice induced by H2O2 during pregnancy in Example 8 provided by the present invention;

[0037] Fig.10 This is a graph showing the results of KEGG enrichment analysis of differentially expressed genes in the brain tissue of offspring mice induced by H2O2 during pregnancy in Example 8 provided by the present invention.

[0038] The realization of the purpose, functional features and advantages of the present invention will be further explained in conjunction with embodiments and with reference to the accompanying drawings. DETAILED DESCRIPTION

[0039] To make the purpose, technical scheme and advantages of the embodiment of the present invention clearer, the technical scheme in the embodiment of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiment, it is carried out according to the normal conditions or the conditions recommended by the manufacturer. If the reagents or instruments used do not specify the manufacturer, they are all conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes, taking "A and / or B" as an example, including scheme A, or scheme B, or schemes that A and B meet at the same time. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of technical solutions is contradictory or cannot be achieved, it should be considered that the combination of such technical solutions does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work belong to the scope of protection of the present invention.

[0040] Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder characterized by deficits in social interaction and communication, repetitive and restricted behavior patterns, and a narrow range of interests or activities. In order to study the pathological mechanisms of ASD and develop treatments, animal models of autism, especially mouse models, are usually constructed to simulate the symptoms and biological characteristics of human ASD.

[0041] Currently, there are two common ASD mouse models: (1) induction models represented by maternal immune activation (MIA) mice, and (2) transgenic models represented by BTBR mice and specific transgenic mice. The MIA inflammatory induction model is consistent with clinical ASD in terms of disease simulation and ASD phenotype, but it not only introduces exogenous immunogenic substances such as lipopolysaccharide (LPS) and polyinosinic acid [Poly(I:C)], but also causes extensive immune activation and release of inflammatory factors, as well as activation of various inflammatory pathways. At the molecular level, it is mixed with the interference of various complex factors such as the toxic effects of exogenous substances themselves, immune damage, oxidative stress, etc., which to some extent hinders the screening of key pathways leading to autism. However, the neurological phenotypes of BTBR mice and transgenic mice do not necessarily form typical autism disorders, and are often accompanied by more serious cognitive dysfunction, movement disorders, etc. At the molecular level, changes in specific genes may cause changes in more extensive signaling pathways closely related to the gene. These changes may interfere with or dilute the changes in the key neural pathways that cause autism disorders, which is not conducive to molecular level research.

[0042] Therefore, there is still a lack of universal animal models for studying ASD.

[0043] In view of this, the present invention provides a method for establishing a hydrogen peroxide-induced autism model in mice, comprising the following steps: injecting a hydrogen peroxide solution into the abdominal cavity of pregnant mice to induce hydrogen peroxide, thereby obtaining hydrogen peroxide-induced offspring mice, i.e., a mouse autism model.

[0044] In the technical scheme of the present invention, hydrogen peroxide is injected intraperitoneally during pregnancy of mother mice, and the oxidative stress state of pregnant mother mice and the oxidative stress state of offspring mouse embryos are induced by hydrogen peroxide alone, without causing obvious immune response in mother mice, which is different from the prior art; and H2O2 is decomposed into two products, H2O2 and O2, after redox reaction, and both products are not toxic or irritating to mice, thereby forming an ASD mouse model specifically induced by oxidative stress; the ASD offspring mice obtained by the present invention have a significant ASD phenotype, but do not show obvious motor impairment, highlighting the typicality of the ASD characteristics of the mouse model of the present invention.

[0045] Since the present invention realizes the effect of embryonic oxidative stress state by intraperitoneal injection of hydrogen peroxide only during the pregnancy of the mother mouse, the operation is convenient and the cost is low; and it does not cause obvious immune inflammatory response of the mother mouse, effectively reduces the pain of the experimental mouse, and is very beneficial to the protection and implementation of experimental animal welfare and ethics.

[0046] It should be noted that the substances used to induce oxidative stress are very extensive, and many of them are biological toxic substances. For example, heavy metals, antibiotics, anticancer drugs, immune antigens, etc., while producing their own metabolic toxicity or inflammation, are accompanied by the occurrence of oxidative stress, and their exposure is mixed and unclear. Therefore, these substances are not suitable for the construction of ASD mouse models induced by simple oxidative stress. In the medical field, hydrogen peroxide is usually only used for clinical skin wound disinfection, and is rarely used for in vivo administration.

[0047] In some embodiments of the present invention, the method for establishing the hydrogen peroxide-induced mouse autism model includes: injecting the hydrogen peroxide solution into the peritoneal cavity of pregnant mice on embryonic day 12.5 to induce hydrogen peroxide, thereby obtaining hydrogen peroxide-induced offspring mice, i.e., a mouse autism model.

[0048] In the technical solution of the present invention, preferably, the pregnant mouse is intraperitoneally injected at day 12.5 of embryonic development. Selecting pregnant mice at this stage can ensure that the development of the nervous system of the offspring mouse embryos can be better affected, and a mouse autism model can be obtained more quickly.

[0049] In some embodiments of the present invention, the amount of hydrogen peroxide injected into the peritoneal cavity of the pregnant mouse is 180-220 μmol / kg. The amount of hydrogen peroxide injected into the peritoneal cavity of the pregnant mouse can be 180 μmol / kg, 200 μmol / kg or 220 μmol / kg. The injection amount within the above range can ensure a good oxidative stress induction effect, so that the obtained mouse autism model is more stable and has a significant ASD phenotype.

[0050] It should be pointed out that the H2O2 injection time (E12.5) and injection dose (200 μmol / kg) involved in this Example 1 represent a typical special case of an implementation method, that is, a preferred scheme. However, since the entire pregnancy period is an important stage of embryonic neural development, it can be inferred that injection at other time points, or using lower or higher injection doses, can theoretically also induce the occurrence of ASD in offspring mice.

[0051] In some embodiments of the present invention, the hydrogen peroxide induction time is 6 to 72 hours. The hydrogen peroxide induction time can be 24 hours, 48 ​​hours, 72 hours or longer. The induction time within the above range can ensure that the obtained mouse autism model is relatively stable and has a significant ASD phenotype.

[0052] It should be noted that in the present invention, the injection dose of hydrogen peroxide is a relatively low safe dose among non-lethal doses (4000 μmol / kg is a lethal dose). The injection amount of hydrogen peroxide and the induction time will not cause death to pregnant mice. This is because most of the injected H2O2 will quickly decompose into O2 and H2O after entering the body, and the body can completely absorb these gases, so it will not cause direct death of pregnant mice. At a lethal dose of 4000 μmol / kg, excessive oxygen generation will cause a rapid increase in abdominal pressure, and pregnant mice will be physically oppressed to death, which is a very special feature compared to other drugs.

[0053] The present invention also provides a method for verifying a mouse autism model, which is used to verify the mouse autism model obtained by the aforementioned method for establishing a mouse autism model induced by hydrogen peroxide; the method for verifying a mouse autism model comprises the following steps: evaluating the motor ability, stereotyped grooming movements, and social interaction ability of the mouse autism model; evaluating the brain synaptic changes of the mouse autism model; and evaluating the expression of autism spectrum disorder-related pathogenic genes in the mouse autism model. The above evaluation indexes can be used to quickly determine whether the obtained offspring mice have ASD characteristics.

[0054] In some embodiments of the present invention, the step of evaluating the changes in brain synapses of the mouse autism model includes: evaluating the histology of brain synapses of the mouse autism model; evaluating the synaptic markers of the brain of the mouse autism model. By observing the histology of brain synapses and synaptic markers, it can be quickly determined whether the synapses of the mouse brain have changed.

[0055] In some embodiments of the present invention, the step of evaluating the expression of autism spectrum disorder-related pathogenic genes in the mouse autism model includes: evaluating the expression of autism spectrum disorder-related pathogenic genes in the mouse autism model; evaluating the enrichment pathways of differentially expressed genes in autism spectrum disorder in the mouse autism model. By analyzing the expression of autism spectrum disorder-related pathogenic genes and the enrichment pathways of differentially expressed genes in autism spectrum disorder, it is possible to more accurately determine whether the mouse is an ASD model, and to more quickly confirm the specific pathway type that led to the establishment of the model.

[0056] In some embodiments of the present invention, the brain synaptic markers include at least one of Synaptophysin and Postsynaptic density protein-95 (PSD95). The above markers can quickly reflect the changes in synapses.

[0057] In some embodiments of the present invention, the autism spectrum disorder-related pathogenic genes include adenylatecyclase 5 (Adcy5), adenosine A2a receptor (Adora2a), angiotensin II receptor type2 (Agtr2), centrosomal protein 135 (Cep135), centrosomal protein 290 (Cep290), C-X3-C motif chemokine receptor 1 (Cx3cr1), dopa decarboxylase (Ddc), dopamine receptor D1 (Drd1), dopamine receptor D2 (Drd2), EBF transcription factor 3 (Ebf3), engrailed homeobox 2 (En2), estrogen related receptor beta (Esrrb), fragile histidine triad diadenosine triphosphatase (Fhit), Gprotein-coupled receptor 139 (Gpr139), 5-hydroxytryptamine receptor 7 (Htr7), interleukin 16(Il16), LIM homeobox transcription factor 1beta(Lmx1b), NCK associated protein5(Nckap5), NFE2 like bZIP transcription factor 3(Nfe2l3), purinergic receptorP2X 5(P2rx5), PATJ crumbs cell polarity complex component(Patj), paired box 5(Pax5), protocadherin alpha 4(Pcdha4), protocadherin 9(Pcdh9), solute carrier family 29member 4(Slc29a4), solute carrier family 6member 3(Slc6a3), solutecarrier family 6member 4(Slc6a4), protein phosphatase1regulatoryAt least one of inhibitorsubunit 1B (Ppp1r1b), SH3 domain containing ring finger 2 (Sh3rf2), tryptophanhydroxylase 2 (Tph2), titin (Ttn) and Wnt family member 2 (Wnt2). The above-mentioned autism spectrum disorder-related pathogenic genes can better reflect the successful establishment of the mouse autism model provided by the present invention.

[0058] In some embodiments of the present invention, the pathways enriched for differentially expressed genes in autism spectrum disorders include neurotransmitter-receptor interactions, dopaminergic neurons, reverse synaptic signaling, and oxidative phosphorylation. The above pathways can indicate the formation pathways of the method for establishing a mouse autism model provided by the present invention.

[0059] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention, and are not used to limit the present invention.

[0060] Example 1 Establishment of autism model in mice

[0061] Mice were raised in an SPF (Specific Pathogen Free) environment. The start time of pregnancy was determined by cage combination and vaginal plug examination. The pregnant mice were intraperitoneally injected with H2O2 at E12.5 (i.e., day 12.5 of embryonic development). The injection volume was 200 μmol / kg, i.e., 30% H2O2 solution was diluted 1:100 to obtain a diluted H2O2 solution. 500 μL of the diluted H2O2 solution was intraperitoneally injected into a 25g pregnant mouse. The pregnant mice were then placed in a normal SPF environment for culture until the offspring mice were born and breastfed normally. When the offspring mice reached adulthood (9 weeks old), the mouse autism model was obtained.

[0062] Example 2 Evaluation of H2O2-induced oxidative stress and immune response in maternal and offspring rat embryos during pregnancy

[0063] The levels of C-reactive protein (CRP), an inflammatory sensitive indicator, in the blood of maternal mice at 0 days (6 h after injection), 1 day (24 h after injection), 2 days (48 h after injection) and 3 days (72 h after injection) after H2O2 injection were detected by enzyme-linked immunosorbent assay (ELISA). After 12 pregnant mice were injected with H2O2 on E12.5 days, 3 pregnant mice were completely anesthetized with Avertin at each time point after injection, and blood was collected by enucleation of the eyeballs. The collected blood was allowed to stand at room temperature for 30 minutes. , centrifuge at 2500×g for 10 minutes, take the transparent supernatant as serum, dilute the serum with normal saline at 1:400 for subsequent detection; prepare standard curve samples: dilute 10 ng / mL mouse-derived CRP standard (purchased from Abcam, catalog number #222511) by 50% into 7 gradients (10000 pg / mL, 5000 pg / mL, 2500 pg / mL, 1250 pg / mL, 625 pg / mL, 156 pg / mL), plus a tube of 0 concentration blank control. Add 50 μL / well of sample to a 96-well plate with CRP antibody (horseradish peroxidase [HRP]) pre-coated on the plate, then add 50 μL of CRP antibody (HRP) diluent, mix thoroughly, and incubate at room temperature for 1 hour on a horizontal shaker at 400 rpm; then wash the plate thoroughly, remove excess liquid as much as possible, and then add 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) colorimetric solution for color reaction, and calculate the urine protein content in the samples at different time points according to the standard curve. The test results are as follows: Figure 1 As shown. Figure 1 In the figure, the horizontal axis is the detection time, from left to right, it is 6 hours (0), 1 day (1), 2 days (2), and 3 days (3) after injection; the vertical axis is the detected serum CRP protein level. The results show that the dot column graph shows that the serum CRP level of pregnant mice has not changed significantly with the passage of time, indicating that intraperitoneal injection of hydrogen peroxide does not cause a significant immune response in pregnant mice.

[0064] At the same time, the hydrogen peroxide metabolic enzyme (CAT) in the offspring mouse embryos of the mother mice one day after H2O2 injection was detected by tissue fluorescence staining: two pairs of H2O2-injected pregnant mice (recorded as H2O2) and sterile water-injected control pregnant mice (recorded as CTR) were taken, the first pair was marked as Pair#1, and the second pair was marked as Pair#2. The two pairs of mice were euthanized 24 hours after the injection and the offspring mouse embryos were taken out, and the offspring mouse embryos were fixed with 4% paraformaldehyde solution overnight, then embedded, the sagittal plane of the embryos was sliced, dewaxed with xylene, and hydrated with 100%, 75%, 50%, and 20% ethanol gradients. After hydration, the slices were transferred to distilled water for immersion, and then immersed in sodium citrate antigen repair solution and microwave heated for 10 minutes for antigen repair, and then The slices were removed and immersed in 3% hydrogen peroxide solution for 25 min in a light-proof environment to inactivate the peroxidase. The treated slices were washed three times with phosphate buffered saline (PBS), and then completely covered with phosphate buffered saline containing 5% bovine serum albumin (BSA-PBS), and placed in a humidified box at 37°C for blocking; then the H2O2 reactive protein (i.e., catalase CAT) was fluorescently localized and stained, and the blocked slices were incubated at 4°C overnight (in a humidified box) using CAT antibody (purchased from Servicebio, item number GB11681), and then incubated again with fluorescent secondary antibody, incubated at room temperature for 1 h, counterstained with 4',6-diamidino-2-phenylindole (DAPI), and then observed and scanned under a fluorescence microscope. The scanning results are shown in Figure 2. Figure 2 As shown. Figure 2 In the figure, DAPI staining (blue fluorescence) indicates the cell nucleus, CAT staining (red) indicates catalase, Merge is a schematic diagram of the color superposition of the two, H2O2 is the group treated with intraperitoneal injection of hydrogen peroxide in pregnant mice, and CTR is the control group treated with intraperitoneal injection of sterile water in pregnant mice.

[0065] Depend on Figure 2 It can be seen that Figure 2 (A) shows the embryos from the first pair (Pair#1) of H2O2-injected pregnant mice (H2O2) and non-injected control pregnant mice (CTR). Figure 2 Middle (B) is another pair (Pair#2) of embryos (H2O2 and CTR). Red fluorescence indicates CAT protein staining, DAPI indicates nucleus background staining, and Merge indicates the overlap of the two signals. The staining of the two pairs of embryos showed that the distribution range and staining intensity of the red signal of CAT in the H2O2 treatment group were significantly increased compared with the control group. That is, after the mother was injected with H2O2 (1 day after injection), the offspring mouse embryos produced a significant oxidative stress response, which was specifically manifested by a significant increase in the catalase CAT that decomposes H2O2 in the embryo. In particular, it can be observed that the expression of CAT in the embryonic brain region was also significantly increased, proving that the oxidative stress state of the embryo was successfully induced.

[0066] In summary, the present invention only uses hydrogen peroxide to induce oxidative stress in pregnant mice, and it has been verified that no obvious immune response is induced. H2O2 decomposes into H2O2 and O2 after redox reaction, neither of which is toxic or stimulating to the body, thus forming an ASD mouse model specifically induced by oxidative stress.

[0067] Example 3 Evaluation of motor ability of offspring mice induced by H2O2 during pregnancy

[0068] When the offspring mice induced by H2O2 during pregnancy reached adulthood (9 weeks old), the motor ability of the offspring mice was evaluated in real time through open field test and rotarod test. At the same time, the offspring mice (9 weeks old) without H2O2 injection during pregnancy were used as the first control group (negative control), and the BTBR autism mice were used as the second control group (positive control).

[0069] Open Field Test: After fully adapting to the behavioral environment, place the mouse to be tested in the center of a 60cm×60cm open field and start filming. Allow the mouse to move freely in the area, record the mouse's movement trajectory, and stop the timing after 10 minutes. Before the next test, clean the open field with alcohol and let it dry naturally. Use the Panlab SMART motion trajectory analysis system to measure the total distance of the mouse and evaluate the mouse's athletic ability. The evaluation results are as follows: Figure 3 As shown in (A), the ordinate represents the total moving distance of the mouse in the open field, and the abscissa represents the offspring of pregnant mice without H2O2-induced oxidative stress (HC) as a negative control, the offspring of pregnant mice with H2O2-induced oxidative stress (OS) as a positive control, and the recognized autism model mouse strain (BTBR) as a positive control. Figure 3 Middle (A) shows that the total moving distance of the three groups of offspring mice in the open field did not change significantly.

[0070] Rotarod experiment: One week before the test, all mice to be tested need to be familiarized with and initially practice the rotarod in the same environment. The practice settings are as follows: the first step is accelerated from 0 to 5 rpm for 5 minutes, and then the speed is maintained at 5 rpm for 5 minutes. The last two gradients are accelerated from 5 to 40 rpm for 5 minutes, so that the mice can become familiar with the environment and coping methods of the rotarod. After they have fully adapted to the behavioral environment, the rotarod test is started. The test program is set as follows: the initial speed of the first gradient of the rotarod is set to 5 rpm, with an acceleration time of 30 seconds; followed by a second gradient acceleration of 60 rpm for 250 seconds, until the last mouse falls and the single test ends. The evaluation results are as follows: Figure 3As shown in (B), the ordinate represents the time taken by the mouse from the start of the experiment to the fall of the rotating rod, or the delayed fall time, and the abscissa represents the time taken by the mouse from the start of the experiment to the fall of the rotating rod, or the delayed fall time. Figure 3 Same as (A), Figure 3 Middle (B) shows that the delayed falling time of OS offspring mice is shorter than that of HC offspring mice, but is significantly longer than that of the positive reference BTBR autism model mice.

[0071] Depend on Figure 3 It can be seen that there was no significant decrease in the motor ability of OS offspring mice induced by H2O2 during pregnancy compared with the control-treated HC offspring mice and the autism model mice BTBR.

[0072] Example 4 Evaluation of Grooming Stereotyped Behavior of Offspring Mice Induced by H2O2 During Pregnancy

[0073] When the offspring mice induced by H2O2 during pregnancy reach adulthood (9 weeks old), the frequency and duration of the mice's grooming behavior are observed in real time within the same time period (10 minutes) to evaluate the level of stereotyped grooming in the mice, in order to reflect the core symptoms of stereotyped repetitive behaviors of autism. At the same time, the offspring adult mice (9 weeks old) that were not injected with H2O2 during pregnancy were used as the first control group (negative control), and the BTBR autism mice were used as the second control group (positive control). The evaluation results are as follows: Figure 4 shown. Figure 4 In (A), the ordinate represents the total time of mouse grooming in 10 minutes, and the abscissa represents the negative control offspring mice (HC), the offspring mice of H2O2 oxidative stress pregnant mice (OS), and the positive control BTBR autism model mice (BTBR). Figure 4 As shown in (A), the total grooming time of mice in the OS group was significantly longer than that of the HC in the control group; Figure 4 In (B), the ordinate represents the frequency of mouse grooming behavior within 10 minutes. Figure 4 As can be seen in (B), the grooming frequency of OS mice was significantly higher than that of HC offspring mice.

[0074] Depend on Figure 4 It can be seen that compared with the offspring treated with negative control, the stereotyped repetitive behaviors of the offspring mice induced by H2O2 during pregnancy were significantly increased, reflecting the typical core symptoms of ASD.

[0075] Example 5 Evaluation of the social ability of offspring mice induced by H2O2 during pregnancy in three boxes

[0076] When the offspring mice induced by gestational H2O2 reached adulthood (9 weeks old), the Three Chamber Social experiment was applied. Under the premise of fully adapting to the behavioral environment, stranger mouse 1 (same age and sex as the offspring mice induced by gestational H2O2, without induction treatment) and toys were placed in the left and right interaction boxes respectively, and the test subject mice were placed in the middle box to allow them to communicate freely. The timing started for 10 minutes, which was stage I. The Panlab SMART trajectory analysis software was used to analyze the social preference of the test subject mice. After the test subject mice took a short rest and wiped the test box, the toy was replaced by another stranger mouse 2 (same age and sex as the offspring mice induced by gestational H2O2, without induction treatment), and the timing was also 10 minutes. This was stage II, and the social preference of the test subject mice towards the new stranger mouse, that is, social novelty, was analyzed. The above two groups of results comprehensively evaluated social ability. At the same time, the offspring adult mice (9 weeks old) that did not receive H2O2 injection during pregnancy were used as the first control group (negative control), and the BTBR autism mice were used as the second control group (positive control). Figure 5 As shown, Figure 5 (A) is a schematic diagram of the three-box social experiment scheme. The left box contains stranger mouse 1 in both stage I and stage II, and the right box contains toys and stranger mouse 2 in stage I and stage II, respectively. The preference of mice to socialize on the left side in stage I is recorded as Preference (P), and the preference of mice to socialize on the right side in stage II is recorded as Novelty (N). Figure 5 The vertical axis in (B) represents the percentage of social tendency of mice, which is calculated as (social time on the left side - social time on the right side) / total social time × 100%, Figure 5 Middle (B) shows that the HC offspring mice that did not receive H2O2 injection during pregnancy have complete social function, and their P and N are significantly different; while the OS offspring mice that received H2O2 injection during pregnancy have obvious social function defects, and the same is true for the BTBR model mice used as a positive reference.

[0077] Depend on Figure 5 It can be seen that compared with the offspring treated with negative control, the social ability of the offspring mice induced by H2O2 during pregnancy was significantly weakened, reflecting the typical core symptoms of ASD.

[0078] Example 6 Histological Detection of Synapses in Offspring Mice Induced by H2O2 During Pregnancy

[0079] When the offspring mice induced by H2O2 during pregnancy reached adulthood (9 weeks old), they were euthanized and their brain tissues were quickly isolated. Golgi fixation and staining were performed on the brain tissues, and synaptic tissues were observed under an optical microscope. At the same time, the offspring adult mice (9 weeks old) that were not injected with H2O2 during pregnancy were used as the first control group (negative control), and the BTBR autism mice were used as the second control group (positive control). The evaluation results are as follows Figure 6 As shown, Figure 6 (A) is a schematic diagram of the divergent dendrites of the neuron cell body as the center observed under a Golgi staining microscope in the brain tissue of each group of mice. Magnify is a schematic diagram of the magnification of dendrites in a specific segment. Figure 6 As shown in (A), compared with the HC offspring mice treated with negative control, the density of neuronal dendritic spines in OS offspring mice induced by H2O2 during pregnancy and BTBR autism model mice was significantly decreased; Figure 6 The vertical axis in (B) indicates the number of dendritic spines within a distance of 50 μm from the neuron dendrites in the microscopic staining image. Figure 6 Middle (B) also shows that compared with the negative control HC offspring mice, the number of neuronal dendritic spines in OS offspring mice and BTBR autism model mice was significantly decreased.

[0080] Depend on Figure 6 It can be seen that compared with the offspring mice treated with the control, the observation under an optical microscope that some dendritic spines in the brain tissue of the offspring mice induced by H2O2 during pregnancy were significantly reduced, which is consistent with the typical core pathological manifestations of ASD.

[0081] Example 7 Detection of synaptic markers in offspring mice induced by H2O2 during pregnancy

[0082] The adult offspring of OS mice induced by H2O2 during pregnancy and the HC offspring treated as negative controls (9 weeks old) were anesthetized with Avertin and fixed by cardiac perfusion with 4% paraformaldehyde solution. The brain tissue was isolated and fixed overnight, and then immunofluorescence staining of synaptic markers Synaptophysin (red fluorescence) and PSD95 (green fluorescence) was performed. DAPI (blue fluorescence) was used to mark the cell nucleus and observed under a scanning electron microscope. At the same time, the adult offspring mice (9 weeks old) who were not injected with H2O2 during pregnancy were used as the control group. The evaluation results are as follows: Figure 7 shown.

[0083] Figure 7 Hippocampis refers to the hippocampus, and Amygdala refers to the amygdala. Figure 7It can be seen that compared with the offspring treated with control treatment, the distribution of synaptic markers Synaptophysin and PSD95 staining in the offspring induced by H2O2 during pregnancy was observed to be significantly changed under scanning electron microscopy, suggesting changes in synaptic connectivity and function, which is consistent with the typical core molecular pathological changes of ASD.

[0084] Example 8 Detection of molecular pathological changes in the brain of offspring mice induced by H2O2 during pregnancy

[0085] When the offspring mice induced by H2O2 during pregnancy reached adulthood (9 weeks old), they were euthanized, and their brain tissues were quickly isolated for RNA extraction and reverse transcription, and transcriptome sequencing was performed. At the same time, the offspring mice (9 weeks old) that were not injected with H2O2 during pregnancy were used as the control group, and the differentially expressed genes in the transcriptome sequencing results of the brain tissues of the two groups were analyzed and compared. The results are as follows: Figure 8 As shown, Figure 8 (A) is a volcano plot of differentially expressed genes between the two groups. The ordinate indicates the significant p value of the expression difference (in logarithmic form), and the abscissa indicates the multiple of the expression difference (in logarithmic form). Figure 8 In (A), we can see that there are significantly differentially expressed genes between the two groups. Figure 8 Middle (B) Figure 8 Among the genes with significant differential expression in (A), down-regulated genes (green) and up-regulated genes (red) that are clearly associated with the onset of autism were found; Figure 8 It can be seen that the differentially expressed up-regulated and down-regulated genes include several currently known key pathogenic genes of ASD, suggesting the comprehensive driving effect of oxidative stress on the occurrence of ASD at the molecular level.

[0086] The differentially expressed genes were subjected to GO (Gene Ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis. Fig. 9 and Fig.10 shown.

[0087] Fig. 9 Displays the biological process network diagram enriched by differentially expressed genes. The circles are the specific biological processes enriched. The size of the circles represents the number of genes contributing to the enriched items. The color depth represents the significance of the enriched items. The lines between the circles represent the inherent biological connections between these nodes. Fig. 9It can be seen that the differentially expressed genes are concentrated in molecular pathways related to synaptic transmission, including synaptic transmission, dopaminergic, trans-synaptic signaling, chemical synaptic transmission, synaptic signaling, amine transport, dopamine transport, catecholamine transport and other biological processes related to synaptic signaling transmission and synaptic transmitter transmission, which are consistent with the molecular pathogenesis of ASD.

[0088] Fig.10 The KEGG pathways enriched by differentially expressed genes. The items on the left are the names of KEGG pathways. The horizontal axis represents the enrichment of the corresponding pathway. The size of the circle represents the number of genes contributing to the enrichment of the item. The gradient color represents the significance of the enriched item, with red being the most significant and blue being the least significant. Fig.10 It can be seen that the enrichment results are mainly reflected in neurotransmitter-receptor interaction, dopaminergic neurons and other synaptic functional pathways, as well as retrograde endocannabinoid signaling and oxidative phosphorylation and other neurometabolism pathways, which are consistent with the molecular pathogenesis of ASD. In summary, the differentially expressed genes are concentrated in the functions and pathways related to synaptic connection and signal transduction.

[0089] In summary, the present invention induces a simple oxidative stress environment by hydrogen peroxide to produce ASD offspring mice, which have the following characteristics after detection: 1) obvious stereotyped grooming behavior; 2) significantly reduced social tendencies; 3) significantly abnormal synaptic distribution in brain tissue; 4) significantly different ASD-related gene expression in brain tissue; 5) significantly different synaptic-related pathway regulation in brain tissue. This shows that the modeling method provided by the present invention is relatively simple, and ASD model mice can be obtained under a simple oxidative stress environment.

[0090] The above are only preferred embodiments of the present invention, and do not limit the patent scope of the present invention. For those skilled in the art, the present invention may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the patent protection scope of the present invention.

Claims

1. A method for establishing a hydrogen peroxide-induced autism model in mice, characterized in that: The following steps are involved: A hydrogen peroxide solution was injected into the abdominal cavity of pregnant mice for hydrogen peroxide induction, and the resulting hydrogen peroxide-induced offspring mice were the autism model in mice.

2. The method for establishing a hydrogen peroxide-induced autism model in mice according to claim 1, characterized in that: The method for establishing a hydrogen peroxide-induced autism model in mice comprises: The hydrogen peroxide solution was intraperitoneally injected into pregnant mice on embryonic day 12.5 to induce hydrogen peroxide, thereby obtaining hydrogen peroxide-induced offspring mice, i.e., a mouse autism model.

3. The method for establishing a hydrogen peroxide-induced autism model in mice according to claim 1, characterized in that: The amount of hydrogen peroxide injected into the abdominal cavity of the pregnant mouse is 180-220 μmol / kg.

4. The method for establishing a hydrogen peroxide-induced autism model in mice according to claim 1, characterized in that: The hydrogen peroxide induction time is 6 to 72 hours.

5. A method for verifying a mouse autism model, characterized in that: The method for verifying the mouse autism model is used to verify the mouse autism model obtained by the method for establishing the hydrogen peroxide-induced mouse autism model as described in any one of claims 1 to 4; The verification method of the mouse autism model comprises the following steps: Assessing motor skills, stereotyped grooming, and social interaction in mouse models of autism; Assessing brain synaptic changes in mouse models of autism; Assess the expression of autism spectrum disorder-associated pathogenic genes in mouse models of autism.

6. The method for verifying a mouse autism model according to claim 5, characterized in that: The steps of evaluating brain synaptic changes in a mouse autism model include: Assessing brain synaptic histology in mouse models of autism; Assessment of brain synaptic markers in a mouse model of autism.

7. The method for verifying a mouse autism model according to claim 5, characterized in that: The step of evaluating the expression of autism spectrum disorder-related pathogenic genes in the mouse autism model comprises: Assess the expression of autism spectrum disorder-related pathogenic genes in mouse autism models; Evaluation of autism spectrum disorder differentially expressed gene enriched pathways in a mouse model of autism.

8. The method for verifying a mouse autism model according to claim 6, characterized in that: The brain synaptic marker includes at least one of Synaptophysin and Postsynaptic density protein-95.

9. The method for verifying a mouse autism model according to claim 7, characterized in that: The autism spectrum disorder-related pathogenic genes include at least one of Adcy5, Adora2a, Agtr2, Cep135, Cep290, Cx3cr1, Ddc, Drd1, Drd2, Ebf3, En2, Esrrb, Fhit, Gpr139, Htr7, Il16, Lmx1b, Nckap5, Nfe2l3, P2rx5, Patj, Pax5, Pcdha4, Pcdh9, Slc29a4, Slc6a3, Slc6a4, Ppp1r1b, Sh3rf2, Tph2, Ttn and Wnt2.

10. The method for verifying autism model in mice according to claim 7, characterized in that: Pathways enriched for differentially expressed genes in ASD included neurotransmitter-receptor interactions, dopaminergic neurons, antidromic synaptic signaling, and oxidative phosphorylation.

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