Antidepressant and anti-anxiety uses of peptides and complexes

Abstract

The present invention relates to the anti-depression and anti-anxiety uses of polypeptides, and provides the use of a polypeptide having a sequence as shown in SEQ ID NO: 1 in the preparation of a drug for treating and / or preventing depression or anxiety. The polypeptide of the present invention has a fast onset of action, good activity and few side effects, and has good clinical development value. The present invention also relates to a complex containing the polypeptide.

Description

Technical Field

[0001] The present invention relates to the field of anti-depression and anti-anxiety, and in particular to the use of polypeptides in anti-depression and anti-anxiety. Background Art

[0002] Depression is a mental disorder caused by various reasons with low mood as the main clinical symptom. It has the characteristics of high morbidity, high disability rate and high suicide rate. The pathogenesis of depression has not been fully elucidated. Most of the antidepressants commonly used in clinical practice are developed based on the "monoamine neurotransmitter hypothesis". This hypothesis believes that the occurrence of depression is related to the lack of monoamine neurotransmitters such as dopamine (DA), 5-hydroxytryptamine (5-HT), and norepinephrine (NA) in the brain; antidepressants block the reuptake of monoamine neurotransmitters by inhibiting the function of 5-HT and NA transporters, increasing their concentration in the synaptic cleft, thereby improving depressive symptoms.

[0003] According to the mechanism of action of antidepressants and the time of their development, commonly used antidepressants are clinically divided into the following categories, namely, the first generation of antidepressants: monoamine oxidase inhibitors (MAOIs) and tricyclic antidepressants; the second generation of antidepressants: selective serotonin reuptake inhibitors (SSRIs); the third generation of antidepressants: dual serotonin and norepinephrine reuptake inhibitors, norepinephrine reuptake inhibitors (SNRIs) and norepinephrine and serotonergic antidepressants, etc.

[0004] Although this type of drug developed based on the "monoamine neurotransmitter hypothesis" has clear therapeutic effects, its effectiveness rate is only 60%, and its antidepressant effect is slow to show (usually 2 to 3 weeks after taking the medicine). In addition, this type of drug has the disadvantages of large adverse reactions when used for a long time and easy relapse after stopping the drug.

[0005] In addition to the classic "monoamine neurotransmitter hypothesis" of depression, in recent years, as researchers have conducted in-depth studies on the pathogenesis of depression, some new antidepressant drug molecular targets based on non-monoamine neurotransmitters have been discovered, such as NMDA receptors, CRF1 receptors, δ receptors, κ receptors, GABAB receptors, M cholinergic receptors, IDO, CysLT1R, PDE4, PPARγ, PPARδ, NOS, etc. Some substances with antidepressant activity have been discovered around these new targets, and some of them have entered the clinical research stage.

[0006] In order to provide richer and better treatment strategies and better elucidate the pathogenesis, there is still a strong need to explore and discover more important pathogenesis and therapeutic targets in the field of depression. Summary of the invention

[0007] To achieve the above objectives, the inventors conducted a large number of studies based on the molecular changes of depression models and discovered a new mechanism of depression, thereby completing the present invention.

[0008] Specifically, 5-HT receptors are a group of G protein-coupled receptors and ligand-gated ion channels that appear in the central nervous system and 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 have three subtypes: A, B, and C, namely, 5-HT2A, 5-HT2B, and 5-HT2C. Among them, 5-HT2A receptor (5-HT2AR), as an excitatory 5-HT receptor, is widely expressed in the brain of mammals.

[0009] DA receptors are receptors located in the body that act through their corresponding membrane receptors. There are five types of DA receptors: D1, D2, D3, D4, and D5. D1 receptors (D1R) are widely expressed in the brain.

[0010] The inventors have recognized that 5-HT2AR regulates neurotransmitters in addition to its individual form, but also in the form of complexes, such as 5-HT1AR / 5-HT2AR and 5-HT2AR / oxytocin receptor (OXTR).

[0011] At present, there is no report showing that 5-HT2AR forms a protein complex with D1R in vivo, and there is no report revealing the association between the 5-HT2AR / D1R complex and the occurrence of depression. However, the inventors unexpectedly discovered that 5-HT2AR forms a protein complex with D1R through its carboxyl terminus, and that the complex has the pathogenicity of depression. Subsequently, through further research, a polypeptide was found to reduce the interaction of 5-HT2AR / D1R and thus alleviate the symptoms of depression and anxiety.

[0012] Accordingly, in a first aspect, the present invention provides a polypeptide having an amino acid sequence (SKDNSDGVNEKVSCV) as shown in SEQ ID NO: 1. In addition, the present invention also provides the use of the polypeptide of the present invention in preparing a medicament for treating and / or preventing depression or anxiety.

[0013] In some embodiments, the length of the polypeptide of the present invention may be 15 to 90aa, in particular 15 to 50aa, 15 to 30aa, 15 to 20aa. For example, 16aa, 17aa, 18aa, 19aa, 20aa, 21aa, 22aa, 23aa, 24aa, 25aa, 26aa, 27aa, 28aa, 29aa, 30aa, 31aa, 32aa, 33aa, 34aa, 35aa, 36aa, 37aa, 38aa, 39aa, 40aa, 41aa, 42aa, 43aa, 44aa, 45aa, 46aa, 47aa, 48aa or 49aa.

[0014] In some embodiments, the polypeptide of the present invention may be derived from 5-HT2AR. The 5-HT2AR from which the polypeptide is derived may be queried through a biological information library (such as Genbank, EMI, DDBJ, etc.), and the polypeptide derived from 5-HT2AR may be confirmed on this basis. For example, in the case where the polypeptide is derived from 5-HT2AR (Genbank accession number: NP_001365853), in addition to containing the sequence shown in SEQ ID NO: 1 (i.e., positions 457 to 471 of the amino acid sequence in NP_001365853), the polypeptide may also include one or more amino acid residues in the N-terminal direction.

[0015] In an exemplary embodiment, the polypeptide of the present invention consists of positions 442 to 471 of the amino acid sequence corresponding to NP_001365853.

[0016] In an exemplary embodiment, the polypeptide of the present invention consists of positions 385 to 471 of the amino acid sequence corresponding to NP_001365853.

[0017] In some embodiments, the 5-HT2AR is of primate origin.

[0018] In a preferred embodiment, the 5-HT2AR is of human origin.

[0019] As used herein, the term "treat" refers to 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 suppressing or inhibiting further development of the disease, condition or disorder.

[0020] As used herein, the term "prevention" refers to preventing or delaying the occurrence of a disease, or preventing the appearance of clinical or subclinical symptoms thereof.

[0021] The polypeptide of the present invention plays an antidepressant role through a new mechanism (5-HT2AR / D1R complex) in the pathology of depression, has the characteristics of rapid onset, good activity and small side effects, and has good clinical development value. In addition, the polypeptide of the present invention can also achieve an anti-anxiety effect.

[0022] It should be noted that, as in Examples 4 and 7 below, and Figure 5 , 6 , 9 and 10, the polypeptides of the present invention disrupt the interaction of the 5-HT2AR / D1R complex, but have no significant effect on the expression of 5-HT2AR or D1R. In other words, the polypeptides of the present invention act by specifically disrupting the interaction of the 5-HT2AR / D1R complex.

[0023] In a second aspect, the present invention provides a nucleic acid molecule encoding a polypeptide of the present invention. In addition, the present invention also provides the use of the nucleic acid molecule of the present invention in the preparation of a medicament for treating and / or preventing depression or anxiety.

[0024] The nucleic acid molecule of the present invention is used to produce the antigen peptide of the present invention. According to the expression system used, those skilled in the art can appropriately adjust the sequence of the nucleic acid molecule.

[0025] In an exemplary embodiment, the nucleic acid molecule has a sequence as shown in SEQ ID NO:2.

[0026] In a third aspect, the present invention provides an expression vector comprising the nucleic acid molecule of the present invention. In addition, the present invention also provides the use of the expression vector of the present invention in the preparation of a drug for treating and / or preventing depression or anxiety.

[0027] Nucleic acid sequences of the present invention can be inserted into expression vectors using a variety of known methods. For example, nucleic acid molecules can be inserted into appropriate restriction endonuclease sites. Standard techniques for cloning, separation, amplification and purification, and enzymatic reactions involving DNA ligase, DNA polymerase, restriction endonucleases, etc., and various separation techniques are known and commonly used by those skilled in the art.

[0028] In a fourth aspect, the present invention provides a host cell comprising the nucleic acid molecule or expression vector of the present invention. In addition, the present invention also provides the use of the host cell of the present invention in preparing a drug for treating and / or preventing depression or anxiety.

[0029] Can use multiple expression system, as the expression vector and host cell in prokaryotic expression system and eukaryotic expression system to produce polypeptide of the present invention.Next, take mammalian expression system as example and illustrate, host cell can comprise the COS-7 cell line of monkey kidney fibroblast and other cell lines that can express compatible vector, as C127, 3T3, CHO, Hela and BHK cell line.Mammalian expression vector should comprise replication origin, suitable promoter and enhancer and any necessary ribosome binding site, polyadenylation site, splicing donor and acceptor site, transcription termination sequence and 5 ' flanking non-transcribed sequence.Dna sequence dna that is derived from for example SV40 splicing and polyadenylation site can be used to provide required non-transcriptional genetic element.Can be by the multiple methods that those skilled in the art are familiar with, include but not limited to for example calcium phosphate transfection, DEAE-dextran-mediated transfection or electroporation, expression vector is introduced into host cell.

[0030] In a fifth aspect, the present invention provides a complex comprising the polypeptide of the present invention and a (transport) carrier for permeating the blood-brain barrier linked thereto.

[0031] In an exemplary embodiment, the carrier used to permeate the blood-brain barrier can be: HIV-1 Tat protein, insulin, cationized albumin, anti-rat transferrin receptor monoclonal antibody (OX26), human insulin receptor mouse monoclonal antibody (HIRMAb), Penetratin, the transduction domain of Tat protein, Pep-1 peptide, S4 13 -One or more of PV, Magainin 2 and Buforin 2. For example, the TAT transduction domain can be transduced across the membrane into the cell, and its amino acid sequence is YGRKKRRQRRR (as shown in SEQ ID NO: 3).

[0032] The polypeptide of the present invention can be connected to a carrier for permeating the blood-brain barrier by an appropriate connection technology. Exemplary connection technologies can be avidin-biotin technology, spacer arm technology based on polyethylene glycol (PEG), fusion protein technology, etc. For example, when the transduction domain of HIV-1 Tat protein is used as a carrier for permeating the blood-brain barrier, the polypeptide of the present invention can be directly connected to the transduction domain of Tat protein by fusion protein technology.

[0033] In some embodiments, when fusion protein technology is used, a linker can also be used to connect the polypeptide of the present invention to a carrier for permeating the blood-brain barrier. Exemplary linkers can be flexible linkers with glycine, such as G, GSG, GSGGSG, GSGGSGG, GSGGSGGG, GGGGSGGG, GGGGS and SGG, etc. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The immunoblot results of co-immunoprecipitation of mouse hippocampal tissue are shown;

[0035] Figure 2 Immunoblotting results of co-immunoprecipitation performed in CRS, CMS, and CSDS mouse models are shown;

[0036] Figure 3 The position of the fragment involved in Example 3 on 5-HT2AR is shown;

[0037] Figure 4 The immunoblot results of GST pulldown analysis on mouse hippocampal tissue using the fragments in Example 3 are shown;

[0038] Figure 5 shows the results of immunoblotting of co-immunoprecipitation of hippocampal tissues from mice treated with the polypeptide;

[0039] Figure 6 The results of immunoblotting of hippocampal tissues from mice treated with the polypeptide are shown;

[0040] Figure 7 The results of OFT, FST and TST analysis of the polypeptide-treated mice are shown, where *: P < 0.05; **: P < 0.01;

[0041] Figure 8 The results of FST, TST and SPT analysis of CRS, CMS and CSDS mice treated with polypeptides are shown, where *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001;

[0042] Fig. 9 The immunoblot results of co-immunoprecipitation of hippocampal tissues from CRS, CMS, and CSDS mice treated with peptides are shown;

[0043] Fig.10 The results of immunoblotting of hippocampal tissues of CRS mice treated with polypeptides are shown;

[0044] Fig.11 The results of OFT (for central zone residence time) analysis of the polypeptide-treated mice are shown, where **: P < 0.01;

[0045] Fig.12 The results of EPM analysis of the polypeptide-treated mice are shown, where *: P<0.05; **: P<0.01. DETAILED DESCRIPTION

[0046] Throughout the specification, unless otherwise specifically stated, the terms used herein should be understood as meanings commonly used in the art. Therefore, unless otherwise defined, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art to which the present invention belongs. In the event of a conflict, the present specification takes precedence.

[0047] The following will describe the embodiments of the present invention in detail in conjunction with the examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are used to illustrate the present invention, rather than to limit the present invention.

[0048] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments is not specified, they are conventional products that can be obtained commercially.

[0049] Unless otherwise specified, the experimental animals used in the examples were adult C57BL / 6J male mice (12-14 weeks old), which were housed at 18-22° C., 12 hours light / 12 hours dark, and had free access to food and tap water throughout the process.

[0050] The statistical analysis involved in the examples used one-way analysis of variance (anova) followed by post hoc comparisons to assess differences between mean values. Unless otherwise stated, independent sample t-tests were used to compare differences between any given two groups.

[0051] Example 1 Confirmation of the formation of 5-HT2AR / D1R complex in vivo

[0052] Protein samples (100-500 μg of protein) from mouse hippocampal tissue were used for co-immunoprecipitation using anti-5-HT2AR mouse antibody (Santa Cruz Biotechnology, sc-166775) and 25 μl of protein A / G + agarose beads slurry (Santa Cruz Biotechnology, sc-2001).

[0053] After precipitation, immunoblot analysis was performed: the denatured proteins were separated on 8% SDS-PAGE gel and then transferred to nitrocellulose membrane, which was blocked with TBST; then incubated with anti-D1R (SantaCruz Biotechnology, sc-33660) and anti-D2R (SantaCruz Biotechnology, sc-5303) mouse antibodies at 4°C overnight; finally, HRP-conjugated secondary antibodies were used for 1 hour and the signals were detected using the ECL Super Signal Chemiluminescence Kit, and the bands were analyzed for density using the Image Lab software. The results are shown in Figure 2. Figure 1 shown.

[0054] Depend on Figure 1 It can be seen that anti-5-HT2AR antibodies can precipitate D1R but not D2R, which indicates that 5-HT2AR can form a complex with D1R but not with D2R in vivo.

[0055] Example 2 Verification of the pathological significance of the complex

[0056] In order to explore the pathological significance of this complex, we constructed different depression models, including chronic restraint stress (CRS), chronic moderate stress (CMS) and chronic social stress (CSDS). The construction methods of each model are as follows:

[0057] Construction of CRS mouse model: Mice were fixed horizontally for 6 hours a day (10 o'clock to 16 o'clock) in an acrylic cylindrical flat-bottom head restrainer (25×90mm) for 2 weeks. The filter has several slots to firmly restrain the mouse according to the size of each mouse and inhibit the body movement of the limbs without causing pain. After being restrained, the mouse was immediately returned to its own cage. Non-restrained mice (controls) remained in their home cages when the CRS procedure was not performed, and both control mice and CRS mice had no access to food and water during CRS exposure.

[0058] Construction of the CMS mouse model: Mice were subjected to various stresses, including: restraint (4 hours), cage tilt (45 times, 12 hours each), light-dark cycle reversal (once), flash (12 hours), and dirty cage (2 times, 14 hours each). This regimen lasted for 6 weeks. Control mice were not subjected to corresponding stress.

[0059] Construction of the CSDS mouse model: Mice were exposed to different CD1 aggressive mice (target mice) for 10 min per day for 10 days. After 10 min of exposure, the experimental mice were separated by a plastic separator containing holes. In addition, the experimental mice were exposed to chronic stress for 24 h by placing the separator in the middle of the cage. A similar procedure (non-social stress) was also used for the control mice, but the same strain of mice was used instead of aggressive mice.

[0060] The constructed CRS mouse model, CMS mouse model and CSDS mouse model were used to study the interaction between D1R and 5-HT2AR in these models according to the immunoprecipitation and immunoblotting methods in Example 1. The results are as follows: Figure 2 shown.

[0061] Depend on Figure 2 It can be seen that a significant increase in the 5-HT2AR / D1R complex was found in the CRS, CMS and CSDS models, confirming the causal role of this complex in depression.

[0062] Example 3 Confirmation of the binding sites of the complex

[0063] In order to confirm the mutual binding sites of the 5-HT2AR / D1R complex, the 5-HT2AR full-length cDNA clone (Genbank accession number: NM_001378924) was first amplified to obtain cDNA fragments of the CT region (K385-V471) of the 5-HT2AR and the third intracellular loop (IL3) region (F255-V324) of the 5-HT2AR. These fragments were subcloned into the BamH1 / EcoR1 or BamH1 / Xho1 site of the pGEX-4T-3 plasmid (Ubc no: VT1255). The initiator methionine residue and the stop codon were incorporated as appropriate. All constructs were resequenced to confirm that the splicing fusion was properly achieved. E. coli BL21 active cells (Kangti Health Life Technology Co., Ltd., No.: KTSM104L) were used for expression, and the GST fusion protein containing the third intracellular loop (IL3) of 5-HT2AR (GST-5HT2AR-IL3) and the GST fusion protein containing the CT region of 5-HT2AR (GST-5HT2AR-CT) were purified from the bacterial lysate. The specific positions of the coding sequences of the IL3 region and the CT region on 5-HT2AR are as follows: Figure 3 shown.

[0064] 500 μg of mouse hippocampal tissue extract was diluted with 1× PBS / 1% Triton X-100 and then incubated with 20 μl protein GST resin saturated with GST protein or 15 μg GST fusion protein at 4°C overnight. The beads were washed 1-8 times with 1× PBS / 1% TritonX-100. The bound proteins were eluted with 2× loading buffer, separated by SDS-PAGE, and immunoblotted with the respective antibodies. The results are shown in Figure 4 As shown in A.

[0065] Depend on Figure 4 As shown in Figure A, the CT region of 5-HT2AR can pull down D1R.

[0066] Next, in order to further explore the correct interaction sequence / site between 5-HT2AR and D1R, we divided the CT region into: the KV (K385-V411) region of 5-HT2AR, the NN (N412-N441) region of 5-HT2AR, and the DV (D442-V471) region of 5-HT2AR. The specific locations of these regions on 5-HT2AR are as follows Figure 3 shown.

[0067] According to the method of this example, pulldown analysis was performed using a GST fusion protein containing the KV region of 5-HT2AR (GST-5HT2AR-KV), a GST fusion protein containing the NN region of 5-HT2AR (GST-5HT2AR-NN), and a GST fusion protein containing the DV region of 5-HT2AR (GST-5HT2AR-DV). The results of immunoblotting are shown in FIG. Figure 4 As shown in B.

[0068] Depend on Figure 4 As shown in B, the DV region of 5-HT2AR can pull down D1R.

[0069] Furthermore, we divided the DV region into the DA (D442-A456) region of 5-HT2AR and the SV (D442-V471) region of 5-HT2AR. The specific locations of these regions on 5-HT2AR are as follows Figure 3 shown.

[0070] According to the method of this example, pulldown analysis was performed using a GST fusion protein containing the DA region of 5-HT2AR (GST-5HT2AR-DA) and a GST fusion protein containing the SV region of 5-HT2AR (GST-5HT2AR-SV). The results of immunoblotting are shown in FIG. Figure 4 As shown in C.

[0071] Depend on Figure 4 As shown in Figure C, the 5HT2AR-SV peptide (S457-V471) can bind to D1R from mouse hippocampal tissue, indicating that 5-HT2AR can interact with D1R through the carboxyl tail.

[0072] Example 4 Confirmation of the Destructive Effect of Peptides on Complexes in Vivo

[0073] In order to verify the effect of the 5-HT2AR carboxyl-terminal polypeptide on the 5-HT2AR / D1R complex in vivo, the C-terminus of the SV region (S457-V471) of 5-HT2AR was fused to the N-terminus of the transduction domain of the HIV-1 Tat protein (as shown in SEQ ID NO: 3, hereinafter referred to as TAT) to obtain a fusion protein that can permeate the blood-brain barrier, named TAT-5HT2AR-SV. Similarly, the DV region (D442-V471) of 5-HT2AR was fused to TAT to obtain TAT-5HT2AR-DV; the CT region (K385-V471) of 5-HT2AR was fused to TAT to obtain TAT-5HT2AR-CT.

[0074] Mice were treated with TAT-5HT2AR-SV, TAT-5HT2AR-DV, and TAT-5HT2AR-CT, and mice treated with TAT alone were used as controls (all treated by single intraperitoneal administration, 3 nmol / g). After 1 hour of treatment, the mice were analyzed according to the immunoprecipitation method in Example 1. The results are shown in FIG. Figure 5 shown.

[0075] Depend on Figure 5 It can be seen that 5HT2AR-SV successfully destroyed the interaction of 5-HT2AR / D1R in mouse hippocampal tissue.

[0076] In addition, hippocampal tissues from TAT-5HT2AR-SV and TAT-treated mice were directly subjected to immunoblotting analysis. Figure 6 It can be seen that there was no significant change in the expression of 5-HT2AR and D1R after treatment.

[0077] On the other hand, TAT-5HT2AR-DV and TAT-5HT2AR-CT also successfully disrupted the 5-HT2AR / D1R interaction and did not cause significant changes in the expression of both 5-HT2AR and D1R.

[0078] Example 5 Evaluation of the antidepressant-like effect of polypeptides

[0079] In order to evaluate the antidepressant-like effect of the polypeptide of the present invention, mice treated with TAT-5HT2AR-SV, TAT-5HT2AR-DV, and TAT-5HT2AR-CT (all treated by single intraperitoneal administration, 3 nmol / g) were subjected to open field test (OFT), forced swimming test (FST) and tail suspension test (TST) 1 hour after treatment, and mice treated with Tat alone were used as controls. The test method is as follows:

[0080] OFT: Mice were acclimated to the experimental environment for 1 h and placed in a 45 × 45 × 30 cm chamber. A 5-min video was recorded to observe the locomotor activity of the mice. The total distance covered by the mice was measured and analyzed and expressed in millimeters.

[0081] FST: The mice were placed in a plexiglass cylinder (70 cm in height and 30 cm in diameter) filled with water (water temperature 23 ± 1 °C), with the water height above 30 cm. The mice were videotaped for 5 minutes and analyzed, and the duration of immobility was recorded. Mice were defined as immobility when they floated motionlessly in the water or raised their nose above the water surface; horizontal movement across the cylinder was defined as swimming; and vertical movement against the cylinder wall was defined as climbing.

[0082] TST: Mice were suspended with tape 40 cm above the floor in a rectangular compartment (length 55 cm × width 20 cm × depth 11.5 cm). Video was recorded for 5 minutes and the duration of immobility was recorded. EthoVisionXT software was used for recording and analysis.

[0083] Some of the results are shown in Figure 7 In the figure, it can be seen from the OFT results on the left that the total movement distance of mice treated with TAT-5HT2AR-SV was not significantly different from that of the TAT-treated group, indicating that the polypeptide had no effect on the motor ability of mice; in addition, it can be seen from the FST results in the middle and the TST results on the right that the immobility time of the TAT-5HT2AR-SV-treated group was significantly reduced compared with the control group (Tat-treated only). The TAT-5HT2AR-DV-treated group and the TAT-5HT2AR-CT-treated group obtained similar results to the TAT-5HT2AR-SV-treated group. The above confirms the antidepressant effect of the present invention.

[0084] Example 6: Using a drug evaluation model to evaluate the antidepressant ability of a polypeptide

[0085] To further evaluate the ability of the peptide as an antidepressant, CRS, CMS and CSDS mouse models were constructed according to the method described in Example 2; 1 hour after construction, 10 μM TAT-5HT2AR-SV, TAT-5HT2AR-DV, TAT-5HT2AR-CT or TAT were used for treatment (all treatments were single intraperitoneal administration, 3 nmol / g); then FST and TST were performed on CRS, and FST, TST and sucrose preference test (SPT) were performed on CMS and CSDS mice, respectively.

[0086] The FST and TST operations are performed as described in Example 6; the SPT operation is as follows:

[0087] The SPT was 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 h for 3 days. On the second day, each mouse had free access to two bottles containing sucrose and water. The positions of the water and sucrose-containing bottles were changed after 2.5 h, and the total test time was 5 h. Finally, the volumes of water and sucrose solutions consumed were recorded and calculated as follows (I):

[0088]

[0089] Depend on Figure 8The results of the analysis showed that for all three drug evaluation models, TAT-5HT2AR-SV treated mice significantly reduced the stress-induced increase in immobility time (A to C, FST and TST) compared with TAT treated mice, and significantly enhanced sucrose preference (A to C, SPT). The TAT-5HT2AR-DV treated group and the TAT-5HT2AR-CT treated group obtained similar results to the TAT-5HT2AR-SV treated group.

[0090] Example 7 Verifying the effectiveness of peptides in disrupting complex interactions in animal models

[0091] According to the immunoprecipitation method in Example 1, the CRS, CMS and CSDS mice (TAT-5HT2AR-SV, TAT-5HT2AR-DV, TAT-5HT2AR-CT and TAT) treated in Example 6 were analyzed, and the immunoblot results were as follows: Fig. 9 shown.

[0092] As expected, 5-HT2AR / D1R coupling levels were significantly increased in all three models; TAT-5HT2AR-SV treatment significantly reduced the interaction between 5-HT2AR and D1R in the hippocampus of stressed mice compared with TAT treatment ( Fig. 9 (A to C).

[0093] Furthermore, the hippocampal tissues of the treated CRS mice were directly subjected to immunoblotting analysis to examine the expression levels of D1R and 5-HT2AR. Fig.10 Interestingly, the levels of D1R and 5-HT2AR did not change significantly between the TAT-5HT2A-SV and TAT-treated groups, indicating that the polypeptides of the present invention are specific for the coupling of D1R and 5-HT2AR.

[0094] In addition, CRS, CMS, and CSDS mice treated with TAT-5HT2AR-DV or TAT-5HT2AR-CT were tested, respectively. The results showed that the TAT-5HT2AR-DV treatment group and the TAT-5HT2AR-CT treatment group significantly reduced the interaction between 5-HT2AR and D1R in the hippocampal tissue of stressed mice, but did not cause significant changes in the levels of D1R and 5-HT2AR.

[0095] Example 8 Evaluation of the anxiolytic-like effect of polypeptides

[0096] In order to evaluate the antianxiety-like effect of the polypeptide of the present invention, mice treated with TAT-5HT2AR-SV, TAT-5HT2AR-DV, and TAT-5HT2AR-CT (all treated by single intraperitoneal administration, 3nmol / g) were subjected to an open field test (OFT) and an elevated plus maze test (EPM) for testing the central zone residence time 1 hour after treatment, and mice treated with only TAT were used as controls. The test method is as follows:

[0097] OFT (used to test the time spent in the central area): the open bottom surface was divided into 25 grids of equal area, and the middle 9 grid areas were the central area. One hour after the mice were treated with the peptide, they were placed in the central area and recorded for 5 minutes, and the time the mice spent in the central area was recorded.

[0098] EPM: It consists of two open arms (25cm×8cm) and two closed arms (25cm×8cm), with the intersection as the central area (8cm×8cm), 40cm above the ground. One hour after receiving peptide treatment, the mice were placed in the central area facing the open arms, and their free activities were recorded for 5 minutes. After each animal was tested, the device was wiped with 70% alcohol. The time the mice stayed in the open arms, the time they stayed in the closed arms, and the total distance the mice moved were recorded.

[0099] Some OFT results are shown in Fig.11 In the experiment, TAT-5HT2A-SR peptide significantly increased the time mice stayed in the central area; similarly, TAT-5HT2AR-DV peptide and TAT-5HT2AR-CT also significantly increased the time mice stayed in the central area (P<0.01), indicating that they effectively reduced anxiety.

[0100] Some EPM results are shown in Fig.12 In the experiment, TAT-5HT2A-SR polypeptide significantly increased the open arm residence time of mice (A), reduced the closed arm residence time (B), without affecting the total motor ability (C); similarly, TAT-5HT2AR-DV polypeptide and TAT-5HT2AR-CT also significantly increased the open arm residence time of mice (P<0.01), reduced the closed arm residence time (P<0.05), without affecting the total motor ability. This shows that the polypeptide of the present invention effectively reduces anxiety. Sequence Listing <110> Shenzhen Chenyang Biotechnology Co., Ltd. <120> Antidepressant and anti-anxiety uses of peptides and complexes <130> 2112018-I-CP-CYSW-SC <160> 3 <170> PatentIn version 3.5 <210> 1 <211> 15 <212> PRT <213> Homo sapiens <400> 1 Ser Lys Asp Asn Ser Asp Gly Val Asn Glu Lys Val Ser Cys Val 1 5 10 15 <210> 2 <211> 45 <212> DNA <213> Homo sapiens <400> 2 tctaaagaca atagcgacgg agtgaatgaa aaggtgagct gtgtg 45 <210> 3 <211> 11 <212> PRT <213> Synthetic <400> 3 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg 1 5 10

Claims

1. Use of a polypeptide in the preparation of a drug for treating depression or anxiety, wherein: The amino acid sequence of the polypeptide is shown in SEQ ID NO:

1. The polypeptide is derived from a 5-HT2A receptor, and the 5-HT2A receptor is derived from humans.

2. Use of a complex in the preparation of a drug for treating depression or anxiety, the complex comprising the polypeptide according to claim 1 and a vector connected thereto for permeating the blood-brain barrier, wherein the vector is a transduction domain of a Tat protein having a sequence as shown in SEQ ID NO: 3.

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