Application of FTO inhibitor in preparation of medicine for improving cognitive impairment of schizophrenia

Abstract

The invention provides application of an FTO inhibitor in preparation of drugs for improving cognitive impairment of schizophrenia, and belongs to the technical field of targeted therapy of neurological and mental diseases. Based on a schizophrenia animal model, the invention finds that FTO protein expression in a prefrontal cortex of a key brain region related to memory recognition in schizophrenia is obviously increased; the earlier-stage research finds that the recognition and memory functions of model animals can be remarkably improved by specifically inhibiting FTO (Fluorine-doped Tin Oxide). Therefore, a brand-new treatment strategy is provided: the targeted FTO improves the schizophrenia recognition memory.

Description

Technical Field

[0001] The present invention relates to the technical field of targeted treatment of neuropsychiatric diseases, and in particular to the use of an FTO inhibitor in the preparation of a drug for improving cognitive impairment in schizophrenia. Background Art

[0002] The problem with existing technology is that there is no effective treatment for cognitive impairment in schizophrenia (especially recognition and memory functions). For example, existing antipsychotic drugs (such as olanzapine and risperidone) mainly improve positive symptoms and are not effective for core cognitive impairments such as recognition and memory.

[0003] Although studies have linked FTO to obesity-related cognitive impairment (PMID: 39984825), the etiologies and neural mechanisms of schizophrenia and obesity are distinct. Schizophrenia is primarily associated with abnormal neurodevelopment, glutamate / GABAergic circuitry disorders, and prefrontal cortex dysfunction, while obesity-related cognitive impairment is primarily linked to metabolic inflammation and insulin resistance. Therefore, existing technologies do not suggest that FTO inhibitors can improve the specific recognition memory deficits in schizophrenia.

[0004] Based on this, the present invention is proposed. Summary of the Invention

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides the use of an FTO inhibitor in the preparation of a drug for improving recognition memory dysfunction in schizophrenia.

[0006] Preferably, the cognitive disorder is recognition memory dysfunction.

[0007] Preferably, the FTO inhibitor is a small molecule compound FB23-2.

[0008] This invention is based on the discovery in animal models of schizophrenia that FTO protein expression is significantly elevated in the prefrontal cortex, a key brain region associated with recognition memory in schizophrenia. Our previous research has shown that specific inhibition of FTO significantly improves recognition memory in these animals. Therefore, we propose a novel therapeutic strategy: targeting FTO to improve recognition memory in schizophrenia. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 The expression of FTO in Example 1; Figure 2 The results of the FTO inhibitor in Example 2 inhibiting the expression of FTO protein in the prefrontal cortex of mice; Figure 3 These are the experimental results for new object recognition in Example 3. DETAILED DESCRIPTION

[0010] The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0011] Example 1 Verification of FTO expression in schizophrenia model

[0012] Establishment of MK-801-induced schizophrenia mouse model: The experimental animals were 6-9 weeks old male C57BL / 6J mice weighing approximately 18-25g. MK-801 was injected intraperitoneally at a concentration of 0.5mg / kg. Three hours later, an MK-801-induced acute schizophrenia mouse model was established. Mice injected with an equal volume of saline served as a control group.

[0013] qRT-PCR detection of Fto mRNA levels in the prefrontal cortex: (1) Treat the equipment needed for the experiment with DEPC water to remove RNase in advance for subsequent use.

[0014] (2) Pre-cool Trizol and add 1 ml to approximately 50 mg of prefrontal cortex tissue obtained from frozen sections.

[0015] (3) Use a 1 ml injection needle to repeatedly extract the Trizol solution containing the brain tissue until the tissue is completely lysed.

[0016] (4) Add 200 μl of chloroform, shake vigorously for 15 seconds, place on ice for 5 minutes, and centrifuge at 12,000 rpm for 15 minutes at 4°C.

[0017] (5) Take the upper colorless aqueous phase, add an equal volume of 200 μl of isopropanol, mix well, let stand at room temperature for 10 minutes, and centrifuge at 4°C, 12,000 rpm for 15 minutes.

[0018] (6) Remove the supernatant and add 1 ml of 75% ethanol prepared with DEPC·H2O. Gently shake and wash the precipitate, then centrifuge at 7,500 rpm for 5 min at 4°C. Repeat this step once more to fully remove the salt ions in the precipitate.

[0019] (7) After removing the supernatant, dry the precipitate and add 20 μl of DEPC·H2O preheated at 55°C. Incubate in a 55°C water bath for 5 minutes and measure the nucleic acid concentration and absorbance using Nanodrop.

[0020] (8) RNA concentration and purity determination: Take 1 μl of DEPC·H2O to calibrate the NanoDrop, then take 1 μl of the extracted RNA sample for concentration and purity determination. The remaining sample is stored in a -80℃ ultra-low temperature freezer for a long time for reverse transcription.

[0021] (9) The reverse transcription experiment was completed using the SuperScript® Ⅲ RT reverse transcription kit from Invitrogen, USA. The recommended total reaction volume of the kit is 20 μl, the starting amount of total RNA is 1 μg, and the starting amount of mRNA is 50 ng.

[0022] (10) We used real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) to perform biological verification on the differential gene expression after threshold screening of transcriptome sequencing data analysis. For each sample with a gene to be tested, the internal reference gene Gapdh also needs to be amplified while amplifying the target gene to quantify the gene to be tested. At least two replicates were made for each gene in each sample to reduce systematic error and human error. After the reaction, the Ct value of the target gene and the Gapdh gene were compared, and the 2 -△Ct The differences in gene expression between the experimental group and the control group were calculated by the method. The data were expressed as Mean ± SEM, and the p value was calculated by Student's t test. p<0.05 was considered statistically significant.

[0023] Western Blot detection of FTO protein levels in the prefrontal cortex: (1) Protein sample preparation: The mouse prefrontal cortex tissue was removed and added with RIPA lysis buffer. The sample was repeatedly aspirated with a 1 mL syringe needle and then ultrasonically lysed (5 minutes in total, with ultrasound on for 4 seconds and off for 6 seconds). After lysis, 6× loading buffer was added and the sample was placed in a 98°C metal bath for protein denaturation. After about 15 minutes, the sample was quickly placed on ice, cooled, and stored at -20°C.

[0024] (2) Glue preparation: When the separation gel is filled to about 2 / 3 of the glass plate, seal the gel immediately with isopropyl alcohol. After sealing, let it stand until the gel solidifies. After the gel solidifies, pour out the isopropyl alcohol and rinse with deionized water. Then prepare the concentrated gel and insert the comb teeth for the appropriate number of samples. After the gel is completely solidified, it can be used.

[0025] (3) Electrophoresis: Take the denatured protein sample out of the -20℃ refrigerator, denature it at 98℃ for 10 minutes, and then quickly place it on ice. After the sample is completely cooled, add it to the lane of the stacking gel. The sample loading volume for each well is 20μL. When the sample is in the stacking gel, the electrophoresis voltage is 80V. After about 30 minutes of electrophoresis, the sample is electrophoresed to the separation gel, and the voltage is adjusted to 100V to continue protein separation. The final electrophoresis time is adjusted according to the molecular weight of the protein.

[0026] (4) Transfer: After the electrophoresis is completed, transfer the membrane. Before transfer, immerse the PVDF membrane in methanol for about 30 seconds to hydrate it. After taking it out, rinse it with deionized water and then place it in the transfer solution. After the electrophoresis is completed, take out the PAGE gel and place it in the transfer folder according to the "sandwich" structure (sponge pad, filter paper, gel, PVDF membrane, filter paper, sponge pad). Connect the gel side to the negative electrode and the PVDF membrane side to the positive electrode. Do not leave bubbles or wrinkles between the sandwich structure. Place the transfer folder in the transfer tank, pour in the transfer solution, and use the transfer system for wet transfer. The transfer condition is 100V and the transfer time is 1 hour.

[0027] (5) Blocking: After the transfer is completed, remove the PVDF membrane and place it in 10 mL of blocking solution (0.01% prepared 5% skim milk) and incubate slowly at room temperature for 1 hour.

[0028] (6) Incubate with the primary antibody. Place the PVDF membrane in the primary antibody (FTO antibody) prepared in 0.1% TBST and incubate on a shaker at 4°C overnight.

[0029] (7) Wash three times with 0.05% TBST to remove any residual non-specifically bound or unbound primary antibody on the membrane, with each wash lasting 5 minutes.

[0030] (8) Incubate with secondary antibody. Place the PVDF membrane in the secondary antibody prepared with 0.1% TBST and incubate on a shaker at room temperature for 1 hour.

[0031] (9) Wash three times with 0.05% TBST to remove any residual non-specifically bound or unbound secondary antibody on the membrane, with each wash lasting 5 minutes.

[0032] (10) Chemiluminescence (ECL): Using the HRP-ECL luminescence method, mix liquid A and liquid B in a 1:1 ratio, evenly drop them on the PVDF membrane, place it on the gel imaging system for analysis and scanning, and perform marker scanning and photography.

[0033] Results The FTO expressions in the control group (Vehicle) and the model group (MK-801) are shown in the figure. Figure 1 A in the middle is the mRNA expression, Figure 1 Figure B shows the expression of FTO protein. The results showed that the mRNA level of Fto in MK-801 model mice was significantly higher than that in the control mice; the FTO protein level in MK-801 mice was significantly higher than that in the control mice.

[0034] Example 2 FB23-2 can inhibit the expression of FTO protein in the prefrontal cortex of mice

[0035] MK-801 mouse models were established according to the method in Example 1 and intraperitoneally injected with the FTO small molecule inhibitor FB23-2 (2 mg / kg / day) for 14 days (the control group received the same treatment). Prefrontal cortex tissue was extracted from the mice and subjected to western blot analysis (using the same method as in Example 1) to measure FTO protein expression levels.

[0036] The results are as follows Figure 2 As shown: When MK-801 mice were intraperitoneally injected with FB23-2, the FTO protein expression level was no longer higher than that of the control group.

[0037] Example 3: FTO inhibitors improve cognitive impairment

[0038] Methods: MK-801 model mice were intraperitoneally injected with the FTO small molecule inhibitor FB23-2 (2 mg / kg / day) for 14 days and then subjected to a novel object recognition test.

[0039] Novel Object Recognition Test: This test assesses the cognitive abilities of mice. The experiment consists of an acclimation phase and a test phase. During the acclimation phase, a test box is prepared and the test mouse is placed in the center. The mouse is allowed to explore freely for 5 minutes before being returned to its cage. Two small balls of the same color and size are placed on either side of the test box. The mouse is then placed back in the box and allowed to explore the two identical balls for 5 minutes. One hour later, the test phase begins. One of the balls is replaced with a different color and size, placed on either side of the test box. The mouse is then placed back in the box and allowed to explore the balls for 5 minutes. This behavior is videotaped for 5 minutes. If the mouse sniffs or touches the object with its forepaw, it is considered to be exploring the novel object. The time spent sniffing the novel object (TN) and the time spent exploring the old object (TF) during these 5 minutes is recorded. The novel object discrimination index is calculated as TN / (TF + TN). After each experiment, the test box and balls are cleaned and wiped with 75% ethanol. After drying, the next mouse is tested for behavioral analysis.

[0040] The results are as follows Figure 3 As shown: In the novel object recognition experiment, the time ratio of MK-801 model mice to sniff novel objects was significantly lower than that of the control group (vehicle) mice. However, when MK-801 mice were treated with FB23-2, the time ratio of sniffing novel objects was not significantly different from that of the control group mice.

[0041] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Application of FTO inhibitors in the preparation of drugs for improving cognitive impairment in schizophrenia.

2. The use according to claim 1, characterized in that The cognitive impairment is recognition memory dysfunction.

3. The use according to claim 1 or 2, characterized in that The FTO inhibitor is a small molecule compound FB23-2.

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