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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
  • A61K38/10—Peptides having 12 to 20 amino acids
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
  • A61K38/1787—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/22—Anxiolytics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/24—Antidepressants
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
  • C07K14/70571—Receptors; Cell surface antigens; Cell surface determinants for neuromediators, e.g. serotonin receptor, dopamine receptor
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
  • C07K7/04—Linear peptides containing only normal peptide links
  • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/70—Vectors or expression systems specially adapted for E. coli
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2207/00—Modified animals
  • A01K2207/35—Animals modified by environmental factors, e.g. temperature, O2
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2227/00—Animals characterised by species
  • A01K2227/10—Mammal
  • A01K2227/105—Murine
• • A—HUMAN NECESSITIES
  • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
  • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
  • A01K2267/00—Animals characterised by purpose
  • A01K2267/03—Animal model, e.g. for test or diseases
• • C—CHEMISTRY; METALLURGY
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  • C12N2800/00—Nucleic acids vectors
  • C12N2800/10—Plasmid DNA
  • C12N2800/106—Plasmid DNA for vertebrates
  • C12N2800/107—Plasmid DNA for vertebrates for mammalian

Definitions

• the present invention relates to the field of drug research and development, and specifically relates to the medical use of polypeptides.
• Posttraumatic stress disorder refers to an individual experiencing, witnessing or encountering one or more actual deaths involving themselves or others, or being threatened with death, or being seriously injured, or having their physical integrity threatened. Later, the resulting delayed onset and persistence of mental disorders in individuals.
• PTSD is triggered by a failed response to stress during trauma, so it brings about a series of changes, including hormonal, neurochemical and brain structural defects.
• cortisol dysregulation during trauma may delay the synaptic effects of norepinephrine in the peripheral and central nervous systems, affecting memory consolidation of the event.
• adrenergic activation facilitates learning at low cortisol levels.
• the physicochemical disorders of PTSD affect serotonergic and dopaminergic mechanisms.
• the inventors conducted research and found that there is a dimer between the D2 receptor in the dopamine (Dopamine (DA) receptor) and the 5-HT2CR in the 5-hydroxytryptamine (5-HT) receptor.
• DA dopamine
• 5-HT2CR 5-hydroxytryptamine
• the DA receptor is a receptor located in the organism that functions through its corresponding membrane receptor.
• DA receptors can be divided into five types: D1, D2, D3, D4 and D5.
• D2 receptor (D2R) is widely expressed in the brain.
• 5-HT receptors are a group of G protein-coupled receptors and ligand-gated ion channels that appear in the center of the central nervous system and the periphery of the peripheral nervous system.
• 5HT receptors can be divided into seven subfamilies 5-HT1, 5-HT2, 5-HT3, 5-HT4, 5-HT5, 5-HT6 and 5-HT7.
• 5-HT2 receptors are also divided into A, B and C. There are three subtypes of receptor proteins, namely, 5-HT2A, 5-HT2B and 5-HT2C.
• 5-HT2CR is widely expressed throughout the brain, including limbic-mesolimbic cortex and striatal regions, such as VTA, NAc, PFC, amygdala, hippocampus, and dorsal striatum, mainly in presynaptic localization.
• D2R for example, can interact with 5-
• the HT2CR interaction was subsequently verified by animal models to interfere with the interaction of the D2R/5-HT2CR complex through KL peptide, which can play a role in treating PTSD.
• the invention provides a polypeptide having the amino acid sequence shown in SEQ ID NO:1.
• the use of the polypeptide of the present invention in the preparation of medicaments for the treatment and/or prevention of PTSD is also provided.
• polypeptides of the invention may be 15 to 30 aa in length.
• polypeptides of the invention may be derived from D2Rs.
• the D2R from which the polypeptide is derived can be queried through biological information databases (such as Genbank, EMI, DDBJ, etc.), and the sequence of the polypeptide can be confirmed based on this.
• the polypeptide in addition to the sequence shown in SEQ ID NO: 1 (i.e., positions 226 to 240 of the amino acid sequence in CAB56463), the polypeptide may also include one or more multiple additional Amino acid residues; for example, the sequence of the polypeptide may be as shown at positions 225 to 240 in CAB56463, in which case it has 16 amino acid residues.
• the D2R is derived from primates.
• the D2R is of human origin.
• a polypeptide of the invention consists of up to 30 contiguous amino acid residues derived from human D2R and contains the sequence set forth in SEQ ID NO: 1.
• a polypeptide of the invention consists of at most 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16 contiguous amino acid residues derived from human D2R It is composed of bases and contains the sequence shown in SEQ ID NO:1.
• the human-derived D2R amino acid sequence is shown in SEQ ID NO: 2.
• treating means causing a desired or beneficial effect in a patient, which may include reducing the frequency or severity of one or more symptoms of a disease, or inhibiting or Inhibit the further development of a disease, condition or disorder.
• prevention means preventing or delaying the onset of a disease, or preventing the occurrence of clinical or subclinical symptoms thereof.
• polypeptide of the present invention is also useful for depression and anxiety disorders.
• the use of the polypeptide of the present invention in the preparation of a medicament for treating and/or preventing depression is provided.
• the use of the polypeptide of the present invention in preparing medicaments for treating and/or preventing anxiety disorders is provided.
• depression refers to a predominantly depressed state of mind that is out of proportion to one's situation and can range from sullenness to grief and even stupor.
• anxiety disorder refers to a neurosis in which anxiety is predominant.
• the polypeptide of the present invention plays an anti-PTSD role through a new mechanism in pathology (destroying the D2R/5-HT2CR complex), and has good clinical development value.
• the polypeptide of the present invention can also achieve antidepressant and antianxiety effects.
• polypeptide of the present invention disrupts the interaction of the D2R/5-HT2CR complex, but has no effect on the expression of DA or 5-HT. and has no significant impact.
• the polypeptides of the invention act by specifically disrupting the interaction of the D2R/5-HT2CR complex.
• the invention provides a nucleic acid molecule encoding a polypeptide of the invention.
• a nucleic acid molecule of the invention is the reverse complement of the sequence encoding a polypeptide of the invention.
• nucleic acid molecule of the invention is shown in SEQ ID NO: 4.
• nucleic acid molecules of the invention in the preparation of medicaments for the treatment and/or prevention of PTSD, depression and/or anxiety disorders is also provided.
• nucleic acid molecules of the present invention can be used to produce polypeptides of the present invention. According to the expression system used, those skilled in the art can appropriately adjust the sequence of the nucleic acid molecules (such as based on the codon preference of the selected expression system).
• the invention provides an expression vector comprising a nucleic acid molecule of the invention.
• the use of the expression vector of the present invention in preparing medicaments for treating and/or preventing PTSD, depression and/or anxiety disorders is also provided.
• nucleic acid molecules of the invention can be inserted into expression vectors using a variety of known methods.
• the nucleic acid molecule can be inserted into appropriate restriction endonuclease sites.
• Standard techniques for cloning, isolation, amplification and purification, as well as enzymatic reactions involving DNA ligases, DNA polymerases, restriction endonucleases, etc., as well as various separation techniques, are known to those skilled in the art. and commonly used technologies.
• the invention provides a host cell comprising a nucleic acid molecule or expression vector of the invention.
• the host cells of the present invention are also provided for use in the treatment and/or prevention of PTSD, depression and/or anxiety. Use in medicines for disease.
• a variety of expression systems can be used to produce the polypeptides of the invention.
• a mammalian expression system is taken as an example.
• Host cells can include the COS-7 cell line of monkey kidney fibroblasts and other cell lines that can express compatible vectors, such as C127, 3T3, CHO, Hela and BHK cells. Tie.
• Mammalian expression vectors should contain an origin of replication, an appropriate promoter and enhancer as well as any necessary ribosome binding sites, polyadenylation sites, splice donor and acceptor sites, transcription termination sequences, and 5' flanking non- Transcribed sequence.
• DNA sequences derived from, for example, the SV40 splicing and polyadenylation sites can be used to provide the desired non-transcribed genetic elements.
• the expression vector can be introduced into the host cell by a variety of methods familiar to those skilled in the art, including but not limited to, for example, calcium phosphate transfection, DEAE-dextran mediated transfection, or electroporation.
• the present invention provides a complex comprising a polypeptide of the present invention and a (transport) carrier for blood-brain barrier permeability connected thereto.
• the complex of the present invention is intended to make the polypeptide of the present invention more suitable for delivery to the human or animal body to treat and/or prevent PTSD, depression and/or anxiety disorders.
• the carrier for permeating the blood-brain barrier can be: HIV-1 Tat protein, insulin, cationized albumin, anti-rat transferrin receptor monoclonal antibody (OX26), human insulin receptor One or more of human mouse-derived monoclonal antibody (HIRMAb), Penetratin, the transduction domain of Tat protein, Pep-1 peptide, S4 13 -PV, Magainin 2 and Buforin 2.
• the TAT transduction domain can be transduced across the membrane into cells, and its amino acid is YGRKKRRQRRR (as shown in SEQ ID NO: 5).
• the polypeptides of the present invention can be linked to carriers used to permeate the blood-brain barrier through appropriate linking techniques.
• Exemplary connection technologies may be avidin-biotin technology, polyethylene glycol (PEG)-based space arm technology, fusion protein technology, etc.
• PEG polyethylene glycol
• fusion protein technology etc.
• the transduction domain of the HIV-1 Tat protein is used as a carrier to penetrate the blood-brain barrier
• the polypeptide of the present invention can be directly connected to the transduction domain of the Tat protein through fusion protein technology.
• a linker when using fusion protein technology, can also be used to connect the polypeptide of the present invention to a carrier for permeating the blood-brain barrier.
• exemplary linkers may be flexible linkers with glycine, such as G, GSG, GSGGSG, GSGGSGG, GSGGSGGG, GGGGSGGG, GGGGS, and SGG, etc.
• the present invention provides a method of treating or preventing PTSD, depression and/or anxiety in a subject, comprising:
• An effective amount of a polypeptide or complex of the invention is administered to the subject.
• the polypeptide of the present invention achieves the above method by specifically destroying the interaction of the D2R/5-HT2CR complex.
• an effective amount refers to an amount of active agent sufficient to induce the desired biological result.
• the result may be an alleviation of signs, symptoms or causes of disease, or any other desired change in a biological system.
• therapeutically effective amount is used herein to mean any amount of a formulation that causes a substantial improvement in a disease condition when repeatedly applied to the affected area over a period of time. This amount will vary with the condition being treated, the stage of progression of the condition, and the type and concentration of formulation used. Those skilled in the art will be able to determine appropriate amounts through routine experimentation.
• Subject refers to a vertebrate, preferably a mammal, more preferably a human. Mammals include, but are not limited to, rats, monkeys, humans, farm animals, and pets. Also included are tissues, cells and their progeny of biological entities obtained in vitro or cultured in vitro.
• a polypeptide or complex of the invention for use in the treatment or prevention of PTSD, depression and/or anxiety disorders.
• Figure 1 shows the immunoblotting results of co-immunoprecipitation of mouse hippocampal tissue
• Figure 2 shows the immunoblotting results of co-immunoprecipitation performed in the SPS mouse model
• Figure 3 shows the position of the fragments involved in Embodiment 3 on D2R
• Figure 4 shows the immunoblotting results of GST pulldown analysis on mouse hippocampal tissue using the fragments in Example 3;
• Figure 5 shows the experimental design flow chart of Example 4.
• Figure 6 shows the immunoblotting results of co-immunoprecipitation of 293T cells treated with polypeptides
• FIG 7 shows the immunoblotting results of co-immunoprecipitation of hippocampal tissue of SPS mice treated with the polypeptide (left); the effect of polypeptide treatment on the resting level of SPS mice (right);
• Figure 8 shows the effect of polypeptides on corticosterone levels in the hippocampus of SPS mice
• Figure 9 shows the effect of polypeptides on cortisol levels in the hippocampus and cortex of SPS mice
• Figure 10 shows the effect of polypeptides on DA levels in the hippocampus and cortex of SPS mice
• Figure 11 shows the effect of polypeptides on 5-HT levels in the hippocampus and cortex of SPS mice
• Figure 12 shows the results of OFT, TST, FST and SPT analysis on CRS mice treated with polypeptides in Example 6; among them, *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001;
• Figure 13 shows the results of OFT analysis on CRS mice treated with polypeptides in Example 7, where *P ⁇ 0.05, **P ⁇ 0.01;
• Figure 14 shows the results of EPM analysis on CRS mice treated with polypeptides in Example 7, where **P ⁇ 0.01.
• the conditions should be followed according to the conventional conditions or the conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not indicated, they are conventional products that can be purchased commercially.
• Balb/c mice were purchased from the Medical Experimental Animal Center of Guangdong province, China. They were male, 12-14 weeks old, and were kept at 18-22°C with 12 hours of light/12 hours of darkness. Diet and running water were freely available throughout the procedure.
• mice Male, 12-14 weeks were housed at 18-22°C, 12 hours light/12 hours dark. Diet and running water were freely available throughout the procedure.
• Results are expressed as mean + s.e.m.
• Statistical analysis was performed using Graphpad Prism 8 software.
• One-way analysis of variance (ANOVA) and post hoc comparisons were used to assess potential differences between means. Unless otherwise stated, independent samples t-tests were used to compare any given two groups throughout the study. Unless otherwise stated, the significance level for tests was set at P ⁇ 0.05.
• Co-immunoprecipitation was performed using protein samples (100-500 ⁇ g of protein) from Balb/c mouse hippocampal tissue. Precipitation was performed using mouse monoclonal antibodies against 5-HT2CR and D2R, D2R or 5-HT2CR with 25 ⁇ l of protein A/G + agarose bead slurry (SantaCruz Biotechnology, sc-2001).
• the 5-HT2CR antibody pulldowns D2R in hippocampal tissue, confirming the coupling of D2R and 5-HT2CR.
• mice Male Balb/c mice were used to establish the SPS model.
• a modified SPS protocol was used to include a series of stress exposures: restraint stress (in a 50 ml plastic tube with several ventilation holes) for 2 hours, rest for 3 minutes, forced swimming in a clear glass tank for 12 minutes ( ⁇ 24°C), After resting for 15 min (drying under a warm lamp), the predator (rat) odor was exposed for 20 min, followed by ether anesthesia (until the mouse completely lost consciousness). Afterwards, mice were transferred back to their home cages with fresh bedding. Mice in the control group were housed in groups and did not receive any stress exposure. After the model was established, all mice were raised without disturbance for one week.
• the constructed SPS mouse model was used to study the interaction between D2R and 5-HT2CR in the model according to the co-immunoprecipitation and immunoblotting methods in Example 1. The results are shown in Figure 2.
• D2R/5-HT2CR complex In order to confirm the mutual binding site of the D2R/5-HT2CR complex, first amplify the D2R full-length cDNA clone (Genbank accession number: cDNA fragment of the loop (IL3) region (D2R-IL3, K211-Q373). These fragments were subcloned into the BamH1/EcoR1 or BamH1/Xho1 sites of pGEX-4T-3 plasmid (Ubao no: VT1255). Initiate methionine residues and stop codons were incorporated where appropriate. All constructs were resequenced to confirm that splicing fusions were appropriately achieved.
• E.coli BL21 active cells (Kansi Health Life Technology Company, no: KTSM104L) were used for expression, and the GST fusion protein containing the IL3 region of D2R (GST-D2R-IL3) and the D2R-containing IL3 region were purified from the bacterial lysate.
• GST fusion protein in CT region (GST-D2R-CT). Among them, the specific positions of the coding sequences of the CT region and IL3 region on D2R are shown in Figure 3.
• Solubilized mouse hippocampal tissue extract 500 ⁇ g protein was diluted with 1 ⁇ PBS/1% Triton Beads were washed 1-8 times with 1 ⁇ PBS/1% Triton X-100. The bound proteins were eluted with SDS-PAGE protein loading buffer (YeSen, no: 20315ES05), separated by SDS-PAGE, and immunoblotted with respective antibodies. The results are shown in Figure 4, A.
• the IL3 region of D2R can pulldown 5-HT2CR.
• the IL3 region into: KVC (K211-V270) region of D2R, ES (E271-S321) region of D2R and D2R
• the PQ (P322-Q373) region the specific locations of these regions on D2R are shown in Figure 3.
• a GST fusion protein containing the KVC region of D2R (GST-D2R-KVC), a GST fusion protein containing the ES region of D2R (GST-D2R-ES), and a GST containing the PQ region of D2R were used.
• the fusion protein (GST-D2R-PQ) was subjected to pulldown analysis, and the immunoblotting results are shown in Figure 4, B.
• the KVC area of D2R can pulldown 5-HT2CR.
• D2R's KT K211-T225
• D2R's KL K226-L240
• D2R's KV K241-V255
• D2R's IV I256-V270
• a GST fusion protein containing the KT region of D2R (GST-D2R-KT), a GST fusion protein of the KL region of D2R (GST-D2R-KL), and a GST fusion protein of the KV region of D2R were used.
• GST-D2R-KV GST fusion protein containing the IV region of D2R
• Figure 4 the immunoblotting results are shown in Figure 4, C.
• D2R can bind to 5-HT2CR from mouse hippocampal tissue through the KL polypeptide (K226-L240), indicating that D2R can interact with 5-HT2CR through the K226-L240 region.
• TAT N-terminal fusion of TAT
• FIG. 5 The specific process is shown in Figure 5, in which the human 293T cell line was cultured in high-glucose modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS) added to the medium. Cells were maintained in a 37°C incubator in a 95% air, 5% carbon dioxide atmosphere. When cells were grown to 70% density, transfection was performed using a 1:1 ratio of 5-HT2CR and D2R plasmids (purchased from Addgene and Vigenebio (Cat#66411 and CH805293)).
• DMEM high-glucose modified Eagle medium
• FBS fetal bovine serum
• TAT-D2R-KL treatment significantly reduces the interaction between 5-HT2CR and D2R.
• an SPS mouse model was established according to Example 2.
• mice were tested under scenario B, with 5 or 15 CS+ and CS- sessions, respectively.
• scenario B mice were treated with TAT-D2R-KL for SPS mice for 1 and a half hours (intraperitoneal administration, 3nm TAT peptide, 100 ⁇ l/10g mouse), and then in scenario A in a pseudo-random manner (ITI: 90s) received CS+ and CS-, where CS+ was paired with US and CS- was never reinforced with foot shock.
• peptide-treated mice were tested again in scenario B.
• TAT-D2R-KL treatment reduced resting levels in SPS mice as 5-HT2CR and D2R interactions decreased (Fig. 7).
• TAT-D2R-KL significantly reduced the elevated corticosterone (Fig. 8) levels in the hippocampus of SPS mice.
• TAT-D2R-KL did not significantly change the levels of cortisol (Fig. 9) and DA (Fig. 10) in the hippocampus and cortex of SPS mice, and did not reduce the upregulated 5-HT in the hippocampus of SPS mice (Fig. 11).
• mice were horizontally immobilized for 6 hours (10:00 to 16:00) every day in an acrylic cylindrical flat-bottomed first restraint (25 ⁇ 90 mm) for 2 weeks.
• the filter has several slots to securely restrain mice depending on the size of each mouse and inhibit physical movement of the limbs without causing pain.
• mice were immediately returned to their home cages. Unrestrained mice (controls) remained in their home cages when not undergoing CRS procedures, and both control and CRS mice had no access to food and water during CRS exposure.
• C57BL/6J mice were subjected to various stressors including: restraint (4 hours), cage tilting (45 times of 12 hours each), light-dark cycle reversal (once), flash (12 hours), and dirty cage (2 times) , 14 hours each time). The program lasts 6 weeks. Control mice were not stressed.
• mice were treated with TAT and TAT-D2R-KL (both treatment methods were single intraperitoneal administration, 3 nmol/g); open field test (OFT), forced swimming test (FST), and tail suspension test were performed 1 hour after treatment. (TST) and SPT (sugar water preference test).
• the test method is as follows:
• OFT for total distance tested: Mice adapted to the experimental environment for 1 hour and were placed in a 45 ⁇ 45 ⁇ 30 cm chamber. Record a 5-minute video to observe the mouse's locomotor activity. The total distance covered by the mice was measured and analyzed and expressed in millimeters.
• mice were placed in a plexiglass cylinder (height 70 cm, diameter 30 cm) filled with water (water temperature 23 ⁇ 1°C), and the height of the water was above 30 cm. The mice were videotaped for 5 minutes and analyzed, and the duration of inactivity was recorded. Mice were defined as stationary when floating motionlessly in the water or with their noses above the water; swimming when they moved horizontally across the entire cylinder; and climbing when they moved vertically against the cylinder wall.
• TST Mice were suspended 40 cm above the floor with tape in a rectangular compartment (length 55 cm ⁇ width 20 cm ⁇ depth 11.5 cm). Record the video for a total of 5 minutes and record the duration of stillness. EthoVisionXT software was used for recording and analysis.
• SPT Sud Water Preference Test: conducted using a two-bottle free-choice paradigm. Mice were habituated to 1% sucrose solution for 3 days and randomly divided into groups. To assess their individual sucrose intake, mice were deprived of water and food for 24 hours over 3 days. The next day, each rat had free access to two bottles containing sucrose and water. After 2.5 hours, the positions of the water and sucrose-containing bottles were changed, and the total test time was 5 hours. Finally, record the volume of water and sucrose solution consumed and calculate it according to formula (I):
• TAT-D2R-KL treatment has no effect on the total exercise capacity of mice. Compared with TAT-treated mice, TAT-D2R-KL-treated mice significantly reduced the immobility time, and significantly It significantly enhanced sucrose preference and showed significant antidepressant effects.
• Example 7 Using a drug evaluation model to evaluate the anti-anxiety ability of polypeptides
• Example 6 In order to evaluate the anti-anxiety-like effect of the polypeptide of the present invention, the method in Example 6 was used to construct a CRS mouse model, and the mice were treated with TAT and TAT-D2R-KL (both treatment methods were single intraperitoneal administration, 3 nmol/g) , the open field test (OFT) and the elevated plus maze test (EPM) used to test the residence time in the central zone were performed 1 hour after treatment.
• the test method is as follows:
• OFT used to test the residence time in the central area: Divide the bottom surface into 25 grids of equal area, with 9 grids in the middle The grid area is the central area. After receiving polypeptide treatment for 1 hour, the mice were placed in the central area and recorded for 5 min. The time the mice stayed in the central area was recorded.
• EPM It consists of two open arms (25cm ⁇ 8cm) and two closed arms (25cm ⁇ 8cm). The intersection is the central area (8cm ⁇ 8cm), 40cm high from the ground.
• the mice were placed in the central area facing the open arms, and their free activities were recorded for 5 min. After each animal is tested, wipe the device with 70% alcohol. Record the time the mouse stays in the open arm, the time it stays in the closed arm, and the total movement distance of the mouse.
• TAT-D2R-KL polypeptide can significantly increase the residence time of mice in the central zone.
• the EPM results are shown in Figure 14.
• TAT-D2R-KL polypeptide significantly increased the open arm residence time of mice, indicating that the polypeptide of the present invention effectively reduced anxiety.

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