Igfbp-2 derived polypeptides and their use in the preparation of antidepressants

Abstract

The application discloses an IGFBP-2 derived polypeptide and application thereof in preparation of an antidepressant, and relates to the technical field of biological medicines. The amino acid sequence of the polypeptide is Pro-Lys-Lys-Leu-Arg-Pro, the N terminal of which is acetylated and the C terminal of which is amidated, and the amino acid sequence is shown as SEQ ID NO:1. The polypeptide has a small molecular weight, can easily penetrate the blood-brain barrier, and has good stability after terminal modification. Animal experiments prove that the polypeptide can significantly improve the depressive behavior of Shank3 gene knockout mice, including improving the spatial cognitive ability, enhancing the social interaction and curiosity, and relieving the anxiety behavior. Cell experiments prove that the polypeptide can significantly improve the viability of hippocampal neuron cells from Shank3 gene knockout mice, and up-regulate the expression level of synaptic plasticity related proteins GluA1 and SYN1. The polypeptide can be prepared by a chemical synthesis method, has low cost, and can be used for preparing a medicine for preventing or treating depression (especially depression related to Shank3 gene mutation).

Description

Technical Field

[0001] This application relates to the field of biomedical technology, specifically to a polypeptide derived from insulin-like growth factor binding protein 2 (IGFBP-2) and its use in the preparation of drugs for the prevention and / or treatment of depression. Background Technology

[0002] Major depressive disorder (MDD) is widely considered one of the most pressing mental health problems. Globally, the number of cases has increased by nearly 50% over the past 30 years, affecting more than 264 million people of all ages. Relapse throughout the life course after the initial depressive episode is particularly problematic, estimated to occur in rates as high as 75-90%. Because many current therapies are ineffective for many patients, and some cause serious side effects, new strategies are needed to overcome depression.

[0003] Shank proteins are scaffold proteins located in the postsynaptic density of glutamatergic synapses, regulating synapse formation, development, and plasticity. The Shank family of proteins is encoded by three genes: Shank1, Shank2, and Shank3. Studies have found a strong association between mutations in Shank family genes and neurodevelopmental disorders, including autism spectrum disorder, intellectual disability, schizophrenia, depression, and Alzheimer's disease. Major depressive disorder (MDD) is the most prevalent mood disorder globally and a leading cause of disability. Currently, the primary treatment for depression is a selective serotonin reuptake inhibitor (SSRI). However, approximately half of patients with depression do not respond to SSRI treatment. Recently, the FDA approved intranasal ketamine for the treatment of depression, but various side effects have been reported in patients with depression, and more than one-third of them do not respond to ketamine. Therefore, investigating the mechanisms of MDD is crucial for developing new and effective treatments.

[0004] Insulin-like growth factor binding protein-2 (IGFBP-2) is a pleiotropic polypeptide that can act as an autocrine and / or paracrine growth factor. IGFBP-2 is the most abundant IGFBP in cerebrospinal fluid (CSF), and its expression is highest in the developing brain. IGFBP-2 is expressed in the hippocampus, cortex, olfactory lobe, cerebellum, and amygdala, and its signaling influences the brain's development from primary to advanced stages. Currently, research on IGFBP-2 mainly focuses on gliomas. Previous studies have shown that IGFBP-2-derived peptides promote neuroplasticity and alleviate autism spectrum disorders, but their application in the treatment of depression has not yet been reported.

[0005] The therapeutic efficacy of peptides is greatly limited by many factors, with ineffective drug transport across the blood-brain barrier being the biggest challenge. The efficiency of drug transport across the BBB largely depends on properties such as molecular size, hydrophilicity, and degree of dissociation. Large protein molecules (such as IGFBP-2, approximately 36 kDa) are difficult to penetrate the blood-brain barrier and suffer from poor stability and high production costs. Therefore, developing small molecule peptides that can overcome these limitations and possess antidepressant activity has significant clinical implications and application value. Summary of the Invention

[0006] To address or partially address the problems existing in related technologies, this invention aims to provide a novel small molecule polypeptide that can be used to prevent and / or treat depression, overcoming the problems of low response rate and significant side effects of existing antidepressants, as well as the difficulty of large molecule proteins to penetrate the blood-brain barrier, poor stability, and high production costs.

[0007] This application provides an IGFBP-2 derived polypeptide, the amino acid sequence of which is Pro-Lys-Lys-Leu-Arg-Pro, and the N-terminus of the polypeptide is acetylated and the C-terminus is amidated; the amino acid sequence of the polypeptide is shown in SEQ ID NO:1.

[0008] Furthermore, the polypeptide is derived from the heparin binding site 1 (HBD1) region in the intermediate linker domain of insulin-like growth factor binding protein 2 (IGFBP-2).

[0009] This application also provides a pharmaceutical composition comprising a therapeutically effective amount of the aforementioned polypeptide, and a pharmaceutically acceptable carrier or excipient.

[0010] Furthermore, the dosage form of the pharmaceutical composition is an injection, a lyophilized powder injection, or a nasal spray.

[0011] This application also provides the use of the above-described polypeptide or the pharmaceutical composition in the preparation of a medicament for the prevention and / or treatment of depression.

[0012] Furthermore, the aforementioned application of depression includes depression or depressive-like behavior associated with Shank3 gene mutations.

[0013] This application also provides the use of the above-described polypeptide or the pharmaceutical composition in the preparation of a medicament for improving at least one of the following symptoms: decreased spatial cognition, impaired social interaction, lack of curiosity, and anxiety behavior.

[0014] This application also provides the use of the above-described polypeptide or the pharmaceutical composition in the preparation of a medicament for promoting neuronal cell viability, upregulating the expression level of the glutamate receptor subunit GluA1 and / or upregulating the expression level of synaptophysin SYN1.

[0015] Furthermore, in the above-described application, the neuronal cells are hippocampal neurons.

[0016] Compared with the prior art, the present invention has the following beneficial effects: 1. The polypeptide provided by this invention has a small molecular weight (about 700 Daltons), which makes it easy to penetrate the blood-brain barrier and overcomes the limitations of IGFBP-2 as a large molecule protein in therapeutic applications.

[0017] 2. The polypeptide provided by this invention has better in vivo stability after N-terminal acetylation and C-terminal amidation modification.

[0018] 3. Experiments have shown that this peptide can significantly improve depressive-like behaviors in Shank3- / - mice, including improving spatial cognitive ability, enhancing social interaction and curiosity, and alleviating anxiety behaviors.

[0019] 4. Through cell experiments, this invention demonstrates that the polypeptide can significantly improve the viability of HT22 hippocampal neurons derived from Shank3- / - mice and upregulate the expression levels of synaptic plasticity-related proteins GluA1 and SYN1, suggesting that its antidepressant effect may be related to promoting neuronal survival and synaptic plasticity.

[0020] 5. The polypeptides provided by this invention can be prepared by chemical synthesis, which is inexpensive and easy to mass-produce. Attached Figure Description

[0021] Exemplary embodiments of this application will be described in more detail with reference to the accompanying drawings.

[0022] Figure 1 This is a schematic diagram of the 1Porsolt experiment results; in the figure, compared with the NC group, *p<0.5; Figure 2 This is a schematic diagram of the Y-maze experiment results; in the diagram, A represents the spontaneous alternation rate; B represents the total number of arms that enter; and C represents the resting time. Compared with the NC group, *p<0.5; compared with the shank3- / - group, #p<0.5, ##p<0.01; Figure 3These are the results of the three-box social novelty test; in the figure, A represents the time the mouse spent visiting the left, middle, and right sides in the preference experiment; B represents the distance the mouse traveled in the left, middle, and right sides in the preference experiment; C represents the time the mouse spent visiting the left, middle, and right sides in the novelty experiment; and D represents the distance the mouse traveled in the left, middle, and right sides in the novelty experiment. Compared with the NC group, *p<0.5; compared with the shank3- / - group, #p<0.5; Figure 4 This is a schematic diagram of the results of the water maze experiment; in the diagram, A represents the latency period in the positioning and navigation experiment; C represents the latency period in the space exploration experiment; compared with the NC group, *p<0.5; compared with the shank3- / - group, #p<0.5; Figure 5 This is a schematic diagram of the open field experiment results; in the diagram, A represents the distance traveled; B represents the distance traveled in the central region; C represents the number of times the participant entered the central platform; D represents the time spent traveling on the central platform; and E represents the average speed of the movement. Compared with the NC group, *p<0.5; compared with the shank3- / - group, #p<0.5; Figure 6 This is a schematic diagram showing the changes in HT22 in the Shank3- / - group mice; in the figure, A represents cell viability detected by CCK-8; B and C represent the expression of GluA1 and SNY1 detected by RT-qPCR, respectively. Figure 7 The results show the effect of PKKLRP on HT22 cells in the shank3- / - group; in the figure, A represents cell viability detected by CCK-8 assay; B and C represent the expression of GluA1 and SNY1 detected by RT-qPCR, respectively. Figure 8 This is a high-performance liquid chromatography (HPLC) detection report of the final product obtained in Example 1; Figure 9 The mass spectrum is shown for the final product prepared in Example 1. Detailed Implementation

[0023] Embodiments of this application will now be described in more detail with reference to the accompanying drawings. While embodiments of this application are shown in the drawings, it should be understood that this application may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to make this application more thorough and complete, and to fully convey the scope of this application to those skilled in the art.

[0024] Example 1: Synthesis and Modification of Peptides This embodiment provides a method for synthesizing the polypeptide of the present invention.

[0025] The amino acid sequence of the polypeptide is: Pro-Lys-Lys-Leu-Arg-Pro (abbreviated as PKKLRP).

[0026] The peptide was synthesized using a solid-phase synthesis method (Fmoc / tBu strategy). After synthesis, the N-terminus of the peptide was acetylated (Ac-) and the C-terminus was amidated (-CONH2) to obtain the final product: Ac-PKKLRP-CONH2. The product was purified by high-performance liquid chromatography (HPLC) with a purity ≥95%. Mass spectrometry (MS) confirmed that the molecular weight was consistent with the theoretical value.

[0027] Figure 8 The image shows the high-performance liquid chromatography (HPLC) detection report of the final product prepared in Example 1; Figure 9 The image shows the mass spectrum of the final product prepared in Example 1.

[0028] Example 2 To verify that the IGFBP-2 derivative peptide PKKLRP prepared in Example 1 can alleviate depression in mice, this study provides a preliminary foundation for subsequent drug development and the investigation of the mechanism by which PKKLRP alleviates Shank3- / - depression.

[0029] 1. Experimental research was conducted from the following two aspects: 1.1. Animal-level investigation of the effect of IGFBP-2 derivative peptide PKKLRP on Shank3.

[0030] A six-amino acid peptide, PKKLRP, was isolated from IGFBP-2. After treatment with different concentrations (50 ng / kg, 100 ng / kg, and 200 ng / kg), mice underwent Y-maze, three-box social interaction, water maze, and open field tests to assess whether PKKLRP could improve depressive or anxious behaviors.

[0031] 1.2. Investigating the effect of IGFBP-2 derivative peptide PKKLRP on Shank3- / - mouse HT22 cells at the cellular level.

[0032] HT22 cells were isolated from normal mice (NC) and Shank3 double knockout mice (shank3- / -), and then treated with 1 μM PKKLRP to detect cell viability. Simultaneously, the expression of GluA1 and SYN1 was detected by RT-qPCR.

[0033] 2. The specific research route and plan are as follows: 2.1 Technical Approach animal cell 2.2 Research Plan 2.2.1 Screening for the concentration of IGFBP-2-derived peptide PKKLRP at the animal level.

[0034] Animal: Mouse Groups: shank3- / - group, IGFBP-2 (1000 ng / kg) and PKKLRP group (20 ng / kg and 200 ng / kg) Detection: a. Porsolt test to screen the concentration of PKKLRP.

[0035] 2.2.2 Animal-level study revealed the effect of IGFBP-2-derived peptide PKKLRP on depression in mice.

[0036] Animal: Mouse Groups: NC group, shank3- / - group, and PKKLRP group (50 ng / kg, 100 ng / kg, and 200 ng / kg) Detection: a. Water maze test to assess the spatial ability of mice b. The three-box social experiment was used to test the mice's social interaction and curiosity.

[0037] c. Y-maze test to detect the spatial learning and memory abilities of mice.

[0038] d. Open field test is a method for detecting the exploratory behavior, autonomous behavior, and stress level of mice in an open environment.

[0039] 2.2.3. Cellular level reveals changes in HT22 in Shank3- / - mice.

[0040] Cell: HT22 Grouping: NC group and shank3- / - group Detection: a. CCK-8 assay for cell viability.

[0041] b. RT-qPCR detection of GluA1 and SYN1 expression.

[0042] 2.2.4. Cellular level analysis of the effect of IGFBP-2-derived peptide PKKLRP on HT22 in depressed mice.

[0043] Cell: HT22 Groups: NC group, shank3- / - group, and PKKLRP (1μM) Detection: a. CCK-8 assay for cell viability.

[0044] b. RT-qPCR detection of GluA1 and SYN1 expression.

[0045] 3. Specific experimental methods 3.1 Open Field Experiment The open field test can detect the potential effects of AAV injection on spontaneous movement, changes in motor ability, and changes in anxiety levels in mice. The experimental procedure was based on previous literature

[76] . The open field test chamber was made of gray acrylic material and had a size of 40 cm × 40 cm × 30 cm. When analyzing the behavioral state of mice, the bottom of the chamber was evenly divided into 16 small rectangles (4 × 4). The four small rectangles in the middle were defined as the central area, and the remaining small rectangles were defined as the surrounding area. The ambient light was adjusted to 50 lx. Each mouse was placed in the central area and allowed to explore freely in the chamber for 30 minutes. A high-definition camera on the top of the test chamber recorded the mouse's activities during this period. The activity trajectory, total distance traveled, and time spent in the central area were recorded using Ethovision 11.5 software.

[0046] 3.2 Y-maze Experiment The Y-maze was used to assess working memory in the mouse hippocampus. The Y-shaped device, made of gray resin, had three arms, designated A, B, and C, and measured 40 cm × 9 cm × 16 cm. Mice were placed in the central area of ​​the Y-shaped enclosure and allowed to explore freely for 8 minutes. A mouse was considered to have fully entered an arm when its center point was completely within that arm. Each instance of exploring the three arms sequentially without repetition was recorded as a correct spontaneous alternation (SAP). The number of SAPs and the total number of times mice entered each arm were recorded using an Ethovision 11.5 microscope, and the alternation rate (AR) was calculated using a formula.

[0047] AR = (Total number of times entered each arm / Total number of times entered each arm - 2) 3.3 Three-Box Social Experiment The three-box social test box is a rectangular device (60 cm × 40 cm × 22 cm) made of transparent polyurethane. The experimental subjects are placed alone in the middle box and can freely explore the two boxes separated by the movable door. Refer to previous literature

[79] . The experiment is divided into three stages: adaptation stage, social stage and social recognition stage. The time interval between each stage is 120 min. First stage: adaptation stage, the experimental subjects are placed in the middle box and allowed to freely explore the three boxes. After 10 min, they are taken out to end the adaptation. Second stage: social stage, mice of the same species, sex and age (S1) and unfamiliar objects (O) are placed in the mouse cages of the two boxes respectively, ensuring that the distance between the mouse cage and the movable door of the corresponding box is consistent. The experimental subjects are placed in the middle box and allowed to freely explore the three boxes. After 10 min, they are taken out. Third stage: social recognition stage, the unfamiliar object in the second stage is replaced with unfamiliar mice of the same species, sex and age (S2). The experimental subjects are placed in the middle box and allowed to freely explore the three boxes. After 10 min, they are taken out. The time spent by mice exploring each enclosure at each stage was analyzed using the Nordas software Ethovision 11.5, as well as the sniffing time when the distance between the nose and the cage was < 2 cm. The preference coefficient was calculated based on the enclosure time and sniffing time according to the formula. 3.4 Water Maze Experiment The water maze test can assess the hippocampal-dependent spatial learning and memory abilities of rodents. Considering it is an invasive stimulus test, it is planned to be conducted last in the behavioral experiments. The water maze test pool is a circular pool with a diameter of 120 cm and a height of 50 cm, filled with water mixed with milk, and geometric markers to aid the mice's spatial orientation are affixed around the pool. The room containing the pool is kept quiet and dimly lit, and a high-definition camera is placed directly above the pool to record the mice's movements within it. The pool is divided into four quadrants of equal area: the target quadrant (T), the contralateral quadrant (O), the left quadrant (L), and the right quadrant (R). The water maze test consists of forward and reverse water maze tests, lasting 11 days. After each test, the mice are wiped dry, placed on a heated blanket to dry, and then returned to their original cages. The forward water maze test consists of three phases to assess the mice's hippocampal spatial learning abilities. Phase 1: Adaptation phase (1 day). To rule out differences in motor skills and vision among the mice, on Day 1, a circular platform with a diameter of 12 cm was placed 1-2 cm above the water surface and centered in the quadrant. A flag was placed on the platform to guide the mice. The platform was placed sequentially in each quadrant, with the mice facing the wall and entering from the opposite quadrant. When the exploration time exceeded 60 seconds, researchers used tools to guide the mice to the platform and allow them to adapt for 30 seconds before removing them from the pool. Normal mice swam normally and could clearly see the guide. Second stage: Training phase (5 days). The circular platform was placed 1-2 cm below the water surface, ensuring the mice could not see it. The platform was then fixed in the T quadrant, and the mice were randomly placed in other quadrants from their starting position. Training was conducted 4 times a day (60 seconds / experiment) for 5 consecutive days. The time it took for the mice to find the platform within 60 seconds was recorded. Third stage: Exploration and detection phase (1 day). On Day 7, the platform was removed, and each mouse was tested for 60 seconds, analyzing the time spent in each quadrant. The reverse water maze consisted of two phases to assess the plasticity of mouse memory. Phase 1: Training (3 days). During the forward water maze training, the platform was placed in the contralateral quadrant (O), 1-2 cm below the horizontal plane. Mice were randomly placed in other quadrants as their starting position. Training was conducted 4 times per day (60 seconds / experiment) for 3 consecutive days. The time it took for the mice to find the platform within the specified time was recorded. Phase 2: Testing (1 day). On day 11, the platform was removed, and each mouse underwent a 1-minute test. The time spent in each quadrant was recorded. Ethovision 11.5 software was used to record and analyze the escape latency, time spent in the target quadrant and the contralateral quadrant, and the distance traveled.

[0048] 3.5. HT22 cells were isolated and extracted from mice. Neuronal cells (HT22) were isolated from normal mice and Shank3- / - mice. The specific procedure is as follows: Under aseptic conditions, atrial tissue from 1-3 day old SD rats is taken out, and the tissue block is washed twice with PBS to obtain a size of about 1 mm3. 4 mL of enzyme digestion solution (0.1% and 0.1% type I collagenase) is added to the tissue block, suspended for 10 seconds, and digested at 37°C for 10 minutes. Afterwards, the tissue is pipetted to form a single-cell suspension, allowed to settle naturally, and the supernatant is collected. Digestion is terminated with medium containing 10% FBS and then placed at 4°C. 3-4 mL of enzyme digestion solution is added to the remaining tissue, suspended for 10 seconds, and digested at 37°C for 10 minutes. The supernatant is collected and digestion is terminated using the same method, and the tissue is placed at 4°C. This step is repeated 2-3 times until the tissue is completely digested. The cell digestion solution is filtered through a 200-mesh stainless steel sieve, centrifuged at 1200 rpm for 10 minutes, the supernatant is discarded, and the precipitated cells are suspended in DMEM / F12 medium containing 10% FBS. The cells are then seeded into 25 cm2 culture flasks and placed at 37°C with 5% CO2. Incubate in an incubator; after 1 hour of differential adhesion, aspirate the culture medium and inoculate it into 6-well plates as needed for further incubation.

[0049] 3.6 CCK-8 Experiment Logarithmic growth phase cells were collected and seeded into 96-well plates at a density of 1 × 10⁴ cells. Three replicates were created for each group: a control group (culture medium, CCK8, DMSO, cell-free), a normal group, and an OGD / R group. After treatment, 20 μl of CCK8 (5 mg / ml) was added to each well of each group, and the plates were incubated at 37°C in a tri-gas incubator for 2 h. Then, 150 μl of dimethyl sulfoxide was added, and the plates were shaken for 10 minutes in the dark. The absorbance was measured at 560 nm using a microplate reader. Cell viability (%) = absorbance of each group × 100 / control group.

[0050] 3.7 RT-qPCR detection of SYN1 and GluA1 expression Total RNA was extracted from cells using TRI reagents (Molecular Research Center) according to the manufacturer's instructions. The isolated RNA was reverse transcribed using the ImProm-II reverse transcription system (Promega) as recommended by the manufacturer. RNA levels were detected by qPCR using the StepOnePlus real-time system (Applied Biosystems) and FastStart UniversalSYBR Green Master (Roche) according to the manufacturer's instructions. Specific primer pairs were used to detect and quantify RNA levels of SYN1 and GluA1, with GAPDH as an internal control.

[0051] 4. Research Results 4.1 Effects of IGFBP-2 and its derivative peptides on the floating time of Shank3- / - mice In the Porsolt assay, the floating time of mice treated with IGFBP-2 was significantly reduced compared to the shank3- / - group. A six-amino acid peptide, PKKLRP, was isolated from IGFBP-2. PKKLRP at concentrations of 20 ng / kg and 200 ng / kg was found to be more effective than IGFBP-2. Figure 1 Therefore, we subsequently used different concentrations of PKKLRP peptide for further experiments.

[0052] 4.2 Y-maze Experiment To investigate the effects of different concentrations of PKKLRP peptide (50 ng / kg, 100 ng / kg, and 200 ng / kg) on ​​spatial cognitive ability in shank3- / - mice, we conducted a Y-maze test. The results showed that, compared with the NC group, the spontaneous alternation rate of mice in the shank3- / - group was significantly reduced (…). Figure 2 A), there was no significant difference in the total number of arm insertions among the groups of mice ( Figure 2 B). Furthermore, the resting time of mice in the shank3- / - group was significantly increased, while treatment with different concentrations of PKKLRP peptide significantly reduced the resting time of the mice. Figure 2 C), among which 100 ng / kg of PKKLRP peptide showed the best effect. In conclusion, different concentrations of PKKLRP peptide can improve the spatial cognitive ability of Shank3- / - mice.

[0053] 4.3 Three-Box Social Experiment In the three-box social experiment, the social preference test and the social novelty test were conducted. In the social preference test, mice were placed in the middle, with one mouse on the left and nothing on the right. In the preference test... Figure 3 In novel experiments (AB), knockout of the shank gene significantly increased visit time and movement distance in the left ventricle. After shank gene knockout, mice were treated with three different concentrations of the peptide PKKLRP (50 ng / kg, 100 ng / kg, and 200 ng / kg). Different concentrations significantly increased visit time and movement distance in the left ventricle and significantly decreased visit time and movement distance in the right ventricle. Figure 3In the CD test, mice were placed in the middle, with one mouse on the left and an unfamiliar mouse on the right. Knocking out the shank gene increased the mice's visit time and movement distance in the left ventricle, and also increased the visit time and movement distance in the right ventricle. However, there was no significant difference in visit time and movement distance in the middle ventricle among the groups. Then, different doses of the virus were used for treatment. Low concentrations of the peptide significantly reduced the mice's visit time and movement distance in the left ventricle, and increased the mice's visit time and movement distance in the right ventricle. In conclusion, treatment with different concentrations of the peptide PKKLRP increased impaired social interaction and curiosity in mice, indicating that depressive behavior in Shank3- / - mice was alleviated.

[0054] 4.4 Water Maze Experiment In the navigation experiment, knocking out the shank gene significantly shortened the time required for mice to successfully locate the platform. Treatment with different concentrations of the peptide PKKLRP (50 ng / kg, 100 ng / kg and 200 ng / kg) further shortened the time required for mice to successfully locate the platform. Figure 4 A). In space exploration experiments, treatment with peptides at doses of 100 ng / kg and 200 ng / kg significantly reduced the latency in mice ( Figure 4 B).

[0055] 4.5 Open Field Experiment In the open field test, after knocking out the shank gene, the distance mice moved on the central platform ( Figure 5 B) Time of visit ( Figure 5 D) and the number of visits to the central platform ( Figure 5 C) Significantly reduced, total travel distance ( Figure 5 A) and average speed ( Figure 5 E) No significant difference. Treatment with different concentrations of peptide PKKLRP (50 ng / kg, 100 ng / kg and 200 ng / kg) resulted in different distances of movement in mice on the central platform. Figure 5 B), the time spent visiting the central area ( Figure 5 D) and frequency ( Figure 5 C) All significantly increased. In conclusion, different concentrations of the peptide PKKLRP can alleviate anxiety behavior in mice.

[0056] 4.6 Changes in HT22 in Shank3- / - mice HT22 cells were extracted from mice. Cell viability analysis revealed that the viability of HT22 cells extracted from the shank3- / - group was significantly lower than that from the NC group. Figure 6 A). Meanwhile, the expression of GluA1 and SNY1 was significantly reduced in HT22 cells of the shank3- / - group ( Figure 6 B).

[0057] 4.7 Effect of PKKLRP on HT22 in shank3- / - group.

[0058] HT22 cells were extracted from mice. Treatment of HT22 cells with 1 μM PKKLRP resulted in enhanced cell viability. Figure 7 A). Meanwhile, the expression of GluA1 and SNY1 was significantly increased in PKKLRP-treated HT22 cells ( Figure 7 B).

[0059] in conclusion This study successfully explored the potential value of PKKLRP, a peptide derived from IGFBP-2, in treating depression. The effects of PKKLRP on anxiety and depressive behaviors in Shank3- / - mice were investigated, revealing a significant improvement. Therefore, different concentrations of PKKLRP (50 ng / kg, 100 ng / kg, and 200 ng / kg) were used. The results showed that treatment with different concentrations of PKKLRP improved spatial exploration ability, memory, and social skills in mice, while reducing depressive and anxious behaviors.

[0060] Example 3: Preparation of the pharmaceutical composition The PKKLRP polypeptide (Ac-PKKLRP-CONH2) prepared in Example 1 was used as the active ingredient and mixed with a pharmaceutically acceptable carrier to prepare an injection.

[0061] Prescription: PKKLRP peptide 1 mg, mannitol 50 mg, water for injection to 10 mL.

[0062] Preparation method: Dissolve PKKLRP peptide and mannitol in an appropriate amount of water for injection, filter aseptically, fill into vials, and freeze-dry to obtain lyophilized powder for injection. Dissolve in water for injection before use.

[0063] The various embodiments of this application have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or improvement of the technology in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. An IGFBP-2 derived polypeptide, characterized in that, The amino acid sequence of the polypeptide is Pro-Lys-Lys-Leu-Arg-Pro, and the N-terminus of the polypeptide is acetylated and the C-terminus is amidated; the amino acid sequence of the polypeptide is shown in SEQ ID NO:

1.

2. The polypeptide according to claim 1, characterized in that, The polypeptide is derived from the heparin binding site 1 (HBD1) region in the intermediate linker domain of insulin-like growth factor binding protein 2 (IGFBP-2).

3. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises a therapeutically effective amount of the polypeptide of claim 1 or 2, and a pharmaceutically acceptable carrier or excipient.

4. The pharmaceutical composition according to claim 3, characterized in that, The dosage form of the pharmaceutical composition is an injection, a lyophilized powder injection, or a nasal spray.

5. The use of the polypeptide of claim 1 or 2 or the pharmaceutical composition of claim 3 or 4 in the preparation of a medicament for the prevention and / or treatment of depression.

6. The application according to claim 5, characterized in that, The depression mentioned includes depression or depression-like behavior associated with mutations in the Shank3 gene.

7. The use of the polypeptide of claim 1 or 2 or the pharmaceutical composition of claim 3 or 4 in the preparation of a medicament for improving at least one of the following symptoms: decreased spatial cognition, impaired social interaction, lack of curiosity, and anxious behavior.

8. The use of the polypeptide of claim 1 or 2 or the pharmaceutical composition of claim 3 or 4 in the preparation of a medicament for promoting neuronal cell viability, upregulating the expression level of glutamate receptor subunit GluA1 and / or upregulating the expression level of synaptophysin SYN1.

9. The application according to claim 8, characterized in that, The neurons are hippocampal neurons.

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