Application of IFIT1 in treatment of autism

Abstract

The invention discloses an application of FIT1 as a target in screening a medicine for treating autism, an application of an IFIT1 expression inhibitor in preparing a medicine for treating autism and an application of shRNA (short hairpin Ribonucleic Acid) for interfering an IFIT1 gene in preparing a medicine for treating autism. Research experiments show that IFIT1 protein expression of VPA-induced ASD rats is increased, and after IFIT1 is knocked down, autism-like behaviors and cerebellar behaviors are improved to a certain extent. IFIT1 may be an important gene affecting the pathogenesis of ASD and may be a target for treating autism, and inhibition of expression of IFIT1 can improve the motion coordination ability of a subject and improve the behavior of autism.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and specifically to the application of IFIT1 in the treatment of autism. Background Art

[0002] Autism spectrum disorder (ASD) is a neurodevelopmental disorder with a global incidence rate of approximately 1-2%, and the incidence rate in males is significantly higher than that in females. Its risk factors include genetic factors (such as family history and gene mutations), environmental factors (such as prenatal infections, exposure to harmful substances, premature birth, etc.), and older parental age. The behavioral manifestations of ASD mainly focus on difficulties in social interaction (such as lack of eye contact, difficulty in understanding others' emotions), communication disorders (such as delayed language development, repetitive language), repetitive stereotyped behaviors (such as fixed daily routines, excessive focus on specific objects), and sensory sensitivity (such as abnormal reactions to sounds and lights). Early diagnosis and intervention (such as behavioral therapy, speech therapy) are crucial for improving the quality of life of patients. VPA is a commonly used anti-epileptic drug in clinical practice. Prenatal administration of VPA can increase the probability of offspring developing ASD. After pregnant mid-gestation rats are exposed to VPA, their offspring will exhibit autistic-like behavioral manifestations such as social disorders, repetitive stereotyped behaviors, and interest preference disorders, and it has become a recognized classic ASD model.

[0003] The cerebellum is mainly responsible for coordinating movement, maintaining balance, and regulating muscle tone in the central nervous system. At the same time, it is also involved in non-motor functions such as cognitive function, emotional regulation, and language processing. In autism spectrum disorder (ASD), the abnormal function and structure of the cerebellum are considered to be one of its pathological mechanisms. Research shows that there may be volume changes, abnormal neuronal development, or connection dysfunction in the cerebellum of ASD patients, and these abnormalities may affect motor coordination, cognitive flexibility, as well as social and communication abilities. Abnormal connections between the cerebellum and the cerebral cortex and limbic system may also exacerbate the core symptoms of ASD, such as repetitive behaviors and difficulties in social interaction. Therefore, the role of the cerebellum in ASD is not limited to motor coordination, but may also participate in regulating the complex behavioral manifestations of ASD through its extensive neural circuits.

[0004] IFIT1 protein (Interferon-induced Protein with Tetratricopeptide Repeats 1) is a protein induced by interferon and plays an important role in the innate immune response. It mainly inhibits virus replication and translation by recognizing and binding to virus RNA lacking 2'-O methylation, thereby exhibiting broad-spectrum antiviral activity. In addition, IFIT1 participates in the interferon signaling pathway, enhances the antiviral state of cells, and regulates the inflammatory response to avoid excessive immune damage. In terms of cell function, IFIT1 can inhibit cell proliferation and induce apoptosis, and may play a role in anti-tumor. Its expression level is also related to the prognosis of certain cancers. IFIT1 also participates in the regulation of RNA metabolism, affects cellular RNA homeostasis, and plays a key role in the interaction between the host and pathogens. In summary, IFIT1 has multiple functions in antiviral, immune regulation, cell proliferation and apoptosis, cancer research, and RNA metabolism, and is an important molecule in immunology and disease research. Summary of the Invention

[0005] The object of the present invention is to provide the application of IFIT1 in the treatment of autism in view of the above problems.

[0006] In order to achieve its object, the technical solution adopted by the present invention is:

[0007] The first aspect of the present invention provides the application of IFIT1 as a target in screening drugs for the treatment of autism.

[0008] The drug inhibits the expression of IFIT1.

[0009] The second aspect of the present invention provides the application of an inhibitor of IFIT1 expression in the preparation of a drug for the treatment of autism.

[0010] The expression inhibitor is selected from nucleic acid molecules, protein molecules or small molecule compounds.

[0011] The nucleic acid molecule includes shRNA.

[0012] The third aspect of the present invention provides the application of shRNA interfering with the IFIT1 gene in the preparation of a drug for the treatment of autism.

[0013] The fourth aspect of the present invention provides the application of a vector or recombinant lentivirus containing shRNA interfering with the IFIT1 gene in the preparation of a drug for the treatment of autism.

[0014] The nucleotide sequence of the shRNA is:

[0015] Sense strand: 5’-GGCUCUGUUACAAGCAACATT-3’;

[0016] Antisense strand: 5’-UGUUGCUUGUAACAGAGCTT-3’.

[0017] The vector containing shRNA that interferes with the IFIT1 gene is a lentiviral vector.

[0018] In the application described in any one of the above, the drug improves the motor coordination ability of the subject and improves autistic-like behaviors.

[0019] The beneficial effects of the present invention are as follows: The research experiments of the present invention show that the expression of IFIT1 protein increases in VPA-induced ASD rats. After knocking down IFIT1, both autistic-like behaviors and cerebellar behaviors have been improved to a certain extent. IFIT1 may be an important gene affecting the pathogenesis of ASD and may be a target for treating autistic-like conditions. Inhibiting the expression of IFIT1 can improve the motor coordination ability of the subject and improve autistic-like behaviors. Description of the Drawings

[0020] Figure 1 Shows the expression of IFIT1 protein in the cerebellum of ASD rats: A. Representative bands of Western blot; B. Statistical analysis results of the gray values of each group of bands, (n = 5, ***P < 0.001).

[0021] Figure 2 Shows the expression of IFIT1 protein in the cerebellum of ASD rats after injection of the knockdown virus: A. Representative bands of Western blot; B. Statistical analysis results of the gray values of each group of bands, (n = 5, *P < 0.05, **P < 0.01, ***P < 0.001).

[0022] Figure 3 Shows the effect of knocking down IFIT1 in the cerebellum of ASD rats on their motor coordination ability: A. Time to pass the balance beam; B. Number of slips of the hind paws, (n = 10, *P < 0.05, ***P < 0.001, ****P < 0.0001).

[0023] Figure 4 Shows the effect of knocking down IFIT1 in the cerebellum of ASD rats on their muscle tone: A. Hanging time; B. Hanging posture score, (n = 10, **P < 0.01, ****P < 0.0001).

[0024] Figure 5 Shows the effect of knocking down IFIT1 in the cerebellum of ASD rats on their spatial exploration, motor ability and repetitive stereotyped behaviors: A. Number of rearing; B. Self-grooming time; C. Number of defecations; D. Time staying in the central square, (n = 10, *P < 0.05, **P < 0.01, ***P < 0.001).

[0025] Figure 6 Shows the effect of knocking down IFIT1 in the cerebellum of ASD rats on their social interaction ability: A. Bedding digging time; B. Time chasing unfamiliar rats; C. Attack time on unfamiliar rats; D. Sniffing and touching time on unfamiliar rats, (n = 10,

[0026] *P < 0.05, ****P < 0.0001).

[0027] Figure 7 Shows the effect of knocking down IFIT1 in the cerebellum of ASD rats on their social interaction ability and social preference: A. Social interaction ability; B. Social preference; In the figure, Chamber A: Residence time in Chamber A (the chamber where Stranger1 is located), Chamber B: Residence time in Chamber B (the middle chamber), Chamber C: Residence time in Chamber C (the chamber where the object or Stranger2 is located), Stranger1: Sniffing and touching time with unfamiliar rat 1, Stranger2: Sniffing and touching time with unfamiliar rat 2, Object: Sniffing and touching time with the object, (n = 10 - 13, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001).

[0028] In the figure, VPA - SI, VPA - SI - NC represent the experimental groups with gene knockdown using shRNA. Detailed implementation manners

[0029] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0030] The experimental methods in the following embodiments are all conventional methods unless otherwise specified.

[0031] Embodiment 1

[0032] 1. Materials and methods

[0033] 1.1 Materials

[0034] 1.1.1 Experimental animals

[0035] Sprague - Dawley (SD) rats in the reproductive age, 20 male rats and 40 female rats, were purchased from the Experimental Animal Center of Chongqing Medical University. The rats reproduced and raised their offspring under normal feeding, drinking, and living environment conditions.

[0036] 1.2 Experimental methods

[0037] 1.2.1 Establishment of VPA - induced ASD rat model

[0038] After the rats purchased from the Experimental Animal Center had adapted to the feeding environment for two weeks, female and male rats were caged together at a ratio of 1:1 at 20:00 every night. The cages were separated the next morning at 8:00, and the vaginal plugs were checked, and vaginal smears were taken to observe the presence of sperm. If a vaginal plug was seen or sperm were seen in the vaginal smear, it was recorded as pregnancy day 1. On day 12.5 of pregnancy in female rats, a solution of VPA (sodium valproate) (50 mg / ml, dissolved in sterile saline) was intraperitoneally injected at a dose of 500 mg / kg. The pregnant rats were placed in separate breeding cages after injection, and their male offspring were used as the research subjects.

[0039] 1.2.2 Behavioral tests (PND 42 - 49)

[0040] 1.2.2.1 Autism-like behavioral tests

[0041] Open field test and self-grooming test: The bottom of a transparent, roofless (100×100×40 cm) acrylic glass box was divided into 25 equal squares. The 9 squares in the center formed the central zone, and the 16 squares connected to the four walls of the box were marked as the peripheral zone. Before starting the recording, each pup had 10 minutes to adapt to the arena. Then they were placed into the central area of the box and allowed to explore freely for 10 minutes. The Smart behavioral software was used to record the time the pups spent in the central and peripheral zones, and the number of times the observed rats stood upright and climbed the wall was manually recorded. At the same time, the duration of self-grooming (reflecting repetitive stereotyped behavior) and the number of defecations (reflecting anxiety) were counted. After each test, the box was thoroughly wiped with 75% alcohol and allowed to dry before the next round of experiments.

[0042] Juvenile social play: Clean bedding was placed in the above-mentioned transparent acrylic box. Before the start of the experiment, the rats to be tested were allowed to adapt to the box for 10 minutes. Subsequently, a social rat of the same age and sex as the test rat was placed into the experimental box. During the test, the interactions between the test rat and the social rat (including sniffing, touching, licking, and following the social rat) and the time of attacking the social rat and the time of digging the bedding were recorded.

[0043] Three-chamber social test: The experimental apparatus consisted of a rectangular transparent acrylic box (120×45×40 cm), which was divided into three chambers of the same size. They were labeled as Chamber A, Central Chamber, and Chamber B from left to right. The test process included three stages:

[0044] ① Adaptation stage: Place wire cages of the same size in Chambers A and B. Put the test mice into the middle chamber and let them move freely for 10 minutes.

[0045] ② Social test stage: Put a male SD rat of the same age (stranger 1) in the wire cage in Chamber A, and an object in the cage in Chamber B. Put the test mice into the middle chamber and observe their activities in the box and the time of olfactory and tactile communication with the strange social mouse or the object.

[0046] ③ Social preference test stage: After the above stage, put another male strange rat of the same age (stranger 2) in the wire cage in Chamber B. Continue to observe the time the test mice stay in each chamber and the time of olfactory and tactile communication with the two social mice.

[0047] 1.2.2.2 Cerebellar behavioral tests (motor coordination ability and muscle tone)

[0048] Static beams: The pups need to pass through a 70 - centimeter - long wooden stick (2 - centimeter diameter), which is horizontally placed 40 centimeters above the ground. One day before the test, train the mice to be tested and let them adapt to the equipment. After the training, each mouse undergoes three tests of passing through the static beam, and record the time to pass through the static beam and the number of times the hind feet slip during the test. If the pup remains stationary on the wooden stick or falls off the wooden stick, the maximum passing time (60 s) is taken as the final result. Calculate the proportion of each mouse successfully passing through the static beam through three static beam experiments.

[0049] Traction test: The forelimbs of the pups are independently placed on a steel wire 50 centimeters above the ground (2 - mm diameter, 120 - cm length), and record the time of falling off the steel wire. At the same time, score the hanging posture of the pups: 1 point, forelimbs hanging on the wire; 2 points, trying to climb onto the wire; 3 points, forelimbs and at least one hind paw hanging on the wire; 4 points, all four limbs and the tail wrapped around the wire; 5 points, trying to escape to the horizontal end. Each mouse undergoes three experiments, with an interval of 20 minutes between each experiment, and take the average value of the three experiments for statistical analysis.

[0050] 1.2.3 shRNA - mediated knockdown of IFIT1

[0051] Entrust GenePharma to construct AAV lentivirus of shRNA. The sequences of shRNA are as follows:

[0052] Sense strand (SEQ ID NO.1): 5’ - GGCUCUGUUACAAGCAACATT - 3’;

[0053] Antisense strand (SEQ ID NO.2): 5’ - UGUUGCUUGUAACAGAGCTT - 3’.

[0054] Perform stereotaxic injection into the brain:

[0055] Grouping: The male offspring rats exposed to VPA were divided into two groups, namely the VPA+IFIT (shRNA) group and the VPA+IFIT1 (shRNA-NC) group.

[0056] Anesthesia: After weighing each rat, anesthetize the rats by intraperitoneal injection with pentobarbital at a concentration of 40 mg / kg.

[0057] Fixation: After successful anesthesia, fix the head of the neonatal rats on the stereotaxic apparatus, keeping the head plane parallel to the operating plane.

[0058] Positioning: Cut the hair at the surgical site, disinfect the head skin with iodophor, and then make a hole at a position 2.5 mm posterior and 2.5 mm to the left or right of the midline with the lambda at the back as the origin. Do not drill too deep to ensure only the skull is penetrated.

[0059] Injection: After determining the injection position, gently penetrate the skull with the needle tip of the injection needle, slowly advance the micro-injection needle to 3.0 mm below the skull plane, and then inject the following reagents at a speed of 0.25 μl / min:

[0060] VPA+IFIT (shRNA) group: Inject 2.5 μl into each side of the cerebellum, with a virus titer of 1.56×10^13.

[0061] VPA+IFIT1 (shRNA-NC) group: Inject 2.5 μl into each side of the cerebellum, with a titer of 1.68×10^13.

[0062] Suture: Leave the needle in place for 10 min after injection and then slowly withdraw the needle. After suturing the incision, place the rat on a heating pad and wait for the anesthesia to wear off. After complete recovery, return the neonatal rats to the cage of the mother rats and continue to raise them.

[0063] 1.2.4 Determination of protein concentration by BCA method

[0064] 1.2.5 Western Blot

[0065] 1.2.6 Statistical methods

[0066] Use GraphPad Prism software to perform statistical analysis on the data, and the results are expressed as mean ± standard error (mean±

[0067] SEM). The data between two groups were analyzed by independent samples t-test, and one-way ANOVA was used for multiple group comparisons, with Tukey's multiple comparisons as the post hoc test. P<0.05 indicates statistical significance.

[0068] 2. Results

[0069] 2.1 Increased expression of IFIT1 protein in the cerebellum of VPA-induced ASD rats

[0070] To observe whether there were differences in the expression of IFIT1 protein in the cerebellum between the WT group and the VPA group, we detected the expression level of IFIT1 protein in the cerebellar tissues of rats in the two groups at 35 days after birth by Western blot. The results showed that the expression level of IFIT1 in the VPA group was significantly higher than that in the WT group (P<0.05)( Figure 1 ).

[0071] 2.2 Protein changes after intracerebellar injection of IFIT1 knockdown virus in VPA rats

[0072] To verify whether there were differences in the expression of IFIT1 protein in the cerebellum of the VPA group injected with IFIT1 knockdown virus, we detected the expression level of IFIT1 protein in the cerebellar tissues of rats 6 weeks after virus injection by Western blot. The results showed that the expression level of IFIT1 in the VPA group injected with the knockdown virus was significantly lower than that in the VPA group (P<0.01)( Figure 2 ).

[0073] 2.3 Intracerebellar injection of IFIT1 knockdown virus improves the motor coordination ability of ASD rats

[0074] 2.3.1 Balance beam experiment

[0075] This experiment mainly detected the balance ability, muscle strength and motor coordination ability of rats. The experimental results ( Figure 3 ) showed that the motor coordination ability of rats in the VPA group was decreased compared with that in the normal group, while their behavior was significantly improved after intracerebellar injection of IFIT1 knockdown virus.

[0076] 2.3.2 Suspension experiment

[0077] The suspension experiment was mainly used to detect the situation of limb muscle tone. The experimental results ( Figure 4 ) showed that the suspension time of rats in the VPA group was longer and the suspension score was lower than that in the normal group, while their behavior was significantly improved after intracerebellar injection of IFIT1 knockdown virus.

[0078] 2.4 Intracerebellar injection of IFIT1 knockdown virus improves the autistic-like behavior of ASD rats

[0079] 2.4.1 Open field experiment

[0080] The open field experiment was mainly used to detect the spontaneous behavior and exploratory behavior of experimental animals in a new environment, and at the same time record their self-grooming time to reflect repetitive stereotyped behavior. The experimental results ( Figure 5) It was shown that, compared with the normal group, the VPA group exhibited poorer motor ability (P<0.001) and exploration desire (P<0.01), and at the same time showed obvious repetitive stereotyped behaviors (P<0.05). After injecting the IFIT1 knockdown virus, the motor ability of VPA rats was significantly improved, the residence time in the central grid increased significantly (P<0.01), and the self-grooming behavior was significantly improved (P<0.01).

[0081] 2.4.2 Young social experiment

[0082] The young social experiment mainly detected the social ability of rats and abnormal behaviors such as aggression in social interaction. The experimental results ( Figure 6 ) showed that, compared with the normal group, the time for the VPA group to contact and communicate with strange rats was significantly reduced, showing abnormal social interaction ability (P<0.0001). After injecting the IFIT1 knockdown virus, the social interaction time of VPA rats increased significantly (P<0.0001), and the time to chase strange rats also increased significantly (P<0.05).

[0083] 2.4.3 Three-chamber social experiment

[0084] The three-chamber social experiment is a behavioral test used to detect the social interaction ability and social preference of rats. This experiment was divided into two stages. The first stage (0-10 min) detected its social interaction ability. The experimental results showed that, compared with the normal group, the time of the VPA group in chamber A where stranger 1 was located was significantly reduced compared with the normal group (P<0.05), and the interaction time with stranger 1 was significantly reduced (P<0.001). After injecting the IFIT1 knockdown virus, the time of VPA rats in chamber A increased significantly (P<0.001), and the interaction time of VPA rats with stranger 1 increased significantly (P<0.0001)( Figure 7 A).

[0085] The second stage (10-20 min) was mainly used to detect the social preference of rats. The experimental results showed that, compared with the normal group, the time of the VPA group in chamber A where stranger 1 was located was significantly increased compared with the normal group (P<0.05), the interaction time with stranger 1 increased significantly (P<0.01), while the time in chamber C where the new stranger 2 was located was significantly reduced (P<0.001), and the communication and interaction time with the new stranger 2 was significantly reduced (P<0.001). After injecting the IFIT1 knockdown virus, the time of VPA rats in chamber A where stranger 1 was located was significantly reduced (P<0.05), and the communication and interaction time was also significantly reduced (P<0.05). At the same time, the time in chamber C where the new stranger 2 was located increased significantly (P<0.01), and the communication and interaction time with the new stranger 2 also increased significantly (P<0.001)(Figure 7 B).

[0086] 3. Analysis

[0087] We explored the expression level of IFIT1 in the cerebellum of VPA-induced ASD rats, and at the same time explored whether knocking down the IFIT1 protein would improve its autistic-like behavior and cerebellar behavior.

[0088] The results showed that the expression of IFIT1 protein increased in VPA-induced ASD rats. Subsequently, we injected the knockdown virus into the cerebellar region by stereotaxic injection, and then verified the knockdown effect by WB, indicating that the knockdown effect was good. The results of behavioral tests showed that obvious autistic-like behaviors appeared in the VPA group and the cerebellar coordination ability decreased. After knocking down IFIT1, both the autistic-like behavior and cerebellar behavior were improved to a certain extent. In summary, IFIT1 may be an important gene affecting the pathogenesis of ASD and may be a target for treating autistic-like conditions.

Claims

1. Application of IFIT1 as a target in screening drugs for the treatment of autism.

2. The use according to claim 1, characterized in that: The drug inhibits the expression of IFIT1.

3. Application of IFIT1 expression inhibitors in the preparation of drugs for the treatment of autism.

4. The use according to claim 3, characterized in that: The expression inhibitor is selected from nucleic acid molecules, protein molecules or small molecule compounds.

5. The use according to claim 4, characterized in that: The nucleic acid molecule includes shRNA.

6. Application of shRNA interfering with IFIT1 gene in the preparation of drugs for the treatment of autism.

7. Use of a vector or recombinant lentivirus containing shRNA that interferes with the IFIT1 gene in the preparation of a drug for treating autism.

8. The use according to any one of claims 5 to 7, characterized in that: The nucleotide sequence of the shRNA is: positive strand: 5'-GGCUCUGUUACAAGCAACATT-3'; Antisense strand: 5′-UGUUGCUUGUAACAGAGCTT-3′.

9. The use according to claim 7, characterized in that: The vector containing the shRNA interfering with the IFIT1 gene is a lentiviral vector.

10. The use according to any one of claims 1 to 9, characterized in that: The drug improves the subject's motor coordination ability and autism-like behavior.

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