Polypeptide grn-e and use thereof in preparing medicine or preparation for perimenopausal emotional disorder

Abstract

The application discloses a polypeptide GRN-E and application thereof in preparation of a medicine or preparation for perimenopausal emotional disorder. It is found for the first time that a GRN-E fragment (a C-terminal domain of PGRN, an amino acid sequence is SEQ ID NO. 1 in a sequence listing) can pass through a blood-brain barrier and has a unique effect in relieving perimenopausal anxiety and depression. As a key functional fragment, the GRN-E has a more concentrated effect on a central nervous system and can effectively avoid off-target effects of a full-length protein. The polypeptide GRN-E of the application can be applied to preparation of a medicine or preparation for perimenopausal emotional disorder as an innovative treatment target and a novel biological preparation and has a high clinical conversion value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to the polypeptide GRN-E and its application in the preparation of drugs or formulations for perimenopausal mood disorders. Background Technology

[0002] Perimenopause women experience a significantly increased risk of mood disorders such as anxiety and depression due to the continued fluctuation and decline in estrogen levels. Epidemiological studies show that women without a history of depression have approximately twice the risk of developing depression after entering perimenopause compared to premenopausal women. Current clinical interventions primarily rely on hormone replacement therapy (HRT) and antidepressants, such as selective serotonin reuptake inhibitors (SSRIs) and norepinephrine-serotonin reuptake inhibitors (SNRIs). While HRT is effective in alleviating some perimenopausal mood symptoms, its long-term use carries significant safety risks, including an increased risk of breast cancer, thrombosis, and cardiovascular events. SSRIs and SNRIs have limited efficacy in these patients, and menopausal-related mood depression is often difficult to significantly improve. Furthermore, these medications frequently cause adverse reactions such as emotional apathy and weight gain, affecting patient adherence. Overall, existing treatments suffer from insufficient safety, weak targeting, and significant individual differences, and safe and effective molecularly targeted drugs are still lacking.

[0003] Recent studies have shown that declining estrogen levels not only affect neurotransmitter metabolism but also disrupt neurotrophic factor signaling and the balance of neuroinflammation. Animal experiments and clinical studies have both found that estrogen deficiency can lead to reduced synaptic plasticity in mood-regulating brain regions such as the hippocampus and prefrontal cortex, impaired neuronal lysosomal-autophagy function, mitochondrial metabolic disorders, and decreased microvascular endothelial barrier function. These changes disrupt neuronal energy homeostasis, cause synaptic structural degeneration, and abnormal neural circuit activity, thereby triggering mood disorders. Therefore, there is an urgent need to develop novel drugs or formulations based on small secretory protein fragments to achieve safe, effective, and targeted treatment of perimenopausal mood disorders. Summary of the Invention

[0004] The technical problem to be solved by this invention is how to develop or prepare drugs to alleviate or assist in the relief of perimenopausal mood disorders and / or how to develop or prepare drugs to treat or assist in the treatment of perimenopausal mood disorders and / or how to develop and / or prepare drugs that can cross the blood-brain barrier to alleviate or assist in the relief, treatment or assist in the treatment of perimenopausal mood disorders.

[0005] To address the aforementioned technical problems, the present invention first provides the use of polypeptides or pharmaceutical salts thereof in the development and / or preparation of remedies for treating or preventing perimenopausal mood disorders; the polypeptide may be any of the following:

[0006] A1) The amino acid sequence is the polypeptide of SEQ ID NO.1 in the sequence listing;

[0007] A2) The amino acid sequence is the polypeptide of SEQ ID NO.2 in the sequence listing;

[0008] A3) A polypeptide derived from A1) or A2) or having the same function as the polypeptide shown in A1) or A2) by substitution and / or deletion and / or addition of amino acid residues;

[0009] A4) is a fusion polypeptide obtained by attaching a protein tag to the N-terminus and / or C-terminus of A1), A2) or A3).

[0010] In one specific embodiment of the present invention, the label is a Flag label.

[0011] In the above applications, the symptoms of perimenopausal mood disorders may include at least one of the following:

[0012] 1) Depression

[0013] 2) Anxiety.

[0014] To address the aforementioned technical problems, the present invention also provides the use of the aforementioned polypeptide or its pharmaceutical salt in the development and / or preparation of medicaments for treating and / or alleviating and / or preventing diseases caused by ovarian dysfunction or loss.

[0015] In the above applications, the symptoms of the disease may include at least one of the following:

[0016] 1) Depression

[0017] 2) Anxiety.

[0018] To address the aforementioned technical problems, the present invention also provides the use of the polypeptide or its pharmaceutical salt described above in the development and / or preparation of medicaments that promote autophagy and / or synapse formation in primary neurons.

[0019] To address the aforementioned technical problems, the present invention also provides a medicine, which may be a medicine for treating or alleviating or adjuvantly treating or adjuvantly alleviating perimenopausal mood disorders or a medicine for treating or alleviating or adjuvantly treating or adjuvantly alleviating diseases caused by ovarian dysfunction or loss, wherein the medicine contains the polypeptide or its pharmaceutical salt described above.

[0020] The polypeptides or their pharmaceutical salts described above are also within the scope of protection of this invention.

[0021] To address the aforementioned technical problems, the present invention also provides biomaterials related to the polypeptides described above, wherein the biomaterials may be any of the following:

[0022] B1) The gene encoding the polypeptide mentioned above;

[0023] B2) has the expression box of B1);

[0024] B3) has a recombinant vector similar to B1;

[0025] B4) has a recombinant vector of B2);

[0026] B5) contains recombinant cells with B1);

[0027] B6) contains recombinant cells with B2);

[0028] B7) recombinant cells with B3);

[0029] B8) contains recombinant cells with B4).

[0030] To address the aforementioned technical problems, the present invention also provides a method for preparing a polypeptide, comprising the following steps: expressing the gene encoding the polypeptide shown in SEQ ID NO.1 in cells using a baculovirus expression system, and purifying it by enzyme digestion to obtain the polypeptide shown in SEQ ID NO.2.

[0031] The gene encoding the polypeptide mentioned above may be any of the following:

[0032] (c1) DNA molecules with coding regions as shown in SEQ ID NO.3, numbers 1189-1476;

[0033] (c2) DNA molecules with coding regions as shown in positions 1164-1476 of SEQ ID NO.3;

[0034] (c3) The coding region is like the DNA molecule in SEQ ID NO.3.

[0035] The method described above may include the following steps:

[0036] (1) The recombinant plasmid was introduced into Escherichia coli DH10Bac to obtain recombinant Escherichia coli; the recombinant plasmid was obtained by inserting a DNA molecule containing the gene into a baculovirus transfer vector;

[0037] (2) After culturing the recombinant Escherichia coli obtained in step (1), the plasmid is extracted, which is the recombinant bacmid;

[0038] (3) Transfect Sf9 cells with the recombinant bacmid obtained in step (2) and culture them. Collect the culture supernatant, which is the P1 generation virus solution.

[0039] (4) Infect Sf9 cells with the P1 generation virus solution and culture them, and collect the culture supernatant, which is the P2 generation virus solution;

[0040] (5) Infect Sf9 cells with the P2 generation virus solution and culture them, and collect the culture supernatant, which is the P3 generation virus solution;

[0041] (6) Infect Sf9 cells with the P3 generation virus solution, collect the culture supernatant, and purify it to obtain the recombinant protein.

[0042] In one specific embodiment of the present invention, the baculovirus transfer vector is the pFastBac Dual vector.

[0043] Specifically, the recombinant plasmid is a recombinant plasmid obtained by replacing the small fragment between the BamHI and HindIII restriction enzyme recognition sequences in the pFastBacDual vector with the double-stranded DNA molecule shown in SEQ ID NO.3.

[0044] The method described above utilizes a baculovirus expression system to express the coding gene of the recombinant protein in cells. The baculovirus-insect cell expression system (BICS) has advantages such as high protein expression levels, complete post-expression translational modification, and short culture cycles. This system can efficiently express target proteins in insect cells (such as Sf9 or High Five cells) and exhibits good protein folding ability and secretion mechanism, making it suitable for the preparation of vaccine and diagnostic antigens. Therefore, developing a method for large-scale, soluble protein expression based on a baculovirus expression system, and combining it with the construction of a simple, highly specific, and sensitive ELISA detection system, has become a key path to overcome the current technological bottlenecks.

[0045] The recombinant cells mentioned above are recombinant animal cells, recombinant plant cells, or recombinant microorganisms.

[0046] In some embodiments, the animal cell line may be non-propagating material. In some embodiments, the animal cells may be isolated mammalian cells. In some embodiments, the animal cells may be mammalian cells, avian cells, amphibian cells, fish cells, or insect cells. Mammal cells include, but are not limited to: Chinese hamster ovary cells (CHO cells), Chinese hamster ovary cell subline (CHO-K1 cells), African green monkey kidney cells (Vero cells), SV40-transformed African green monkey kidney cells (COS cells), young hamster kidney cells (BHK cells), mouse breast cancer cells (C127 cells), human embryonic kidney cells (HEK293 cells), human HeLa cells, fibroblasts, bone marrow cell lines, T cells, or NK cells, etc. Avian cells include, but are not limited to: chicken cells, duck cells, or goose cells, etc. Amphibian cells include, but are not limited to: African clawed frog cells or giant salamander cells, etc. Fish cells include, but are not limited to: grass carp cells, carp cells, rainbow trout cells, or catfish cells, etc. Insect cells include, but are not limited to: Sf21 cells, Sf9 cells, or High Five cells (Hi5 cells), etc. In some embodiments, the mammalian cells do not include animal germ cells, animal fertilized eggs, and animal embryonic stem cells. In some embodiments, the mammalian cells may be somatic cells or cell lines.

[0047] The term "microorganism" typically includes bacteria, viruses, fungi, actinomycetes, rickettsiae, mycoplasma, chlamydia, spirochetes, algae, etc. The bacteria may originate from the genus *Corynebacterium*, such as *Corynebacterium glutamicum*, *Corynebacterium pekinensis*, and *Corynebacterium obliterans*. The bacteria may originate from the genus *Brevibacterium*, such as *Brevibacterium lactis*, *Brevibacterium flavum*, and *Brevibacterium ammoniacum*. The bacteria may originate from the genus *Escherichia*, such as *Escherichia coli*. The bacteria may originate from the genus *Erwinia*. The bacteria may originate from the genus *Agrobacterium*, such as *Agrobacterium tumefaciens*. The bacteria may originate from the genus *Flavobacterium*. The bacteria may originate from the genus *Alcaligenes*. The bacteria may originate from the genus *Pseudomonas*. The bacteria may originate from the genus *Bacillus*, such as *Bacillus*. For example, the *Escherichia coli* may be *Escherichia coli* DH10Bac. The virus may include rotavirus, baculovirus, retrovirus (such as lentivirus), adenovirus, adeno-associated virus, poxvirus, papillomavirus, influenza virus, papillomavirus (such as SV40), and herpesvirus (such as herpes simplex virus). The fungus may be from the genus *Saccharomyces*, such as *Saccharomyces cerevisiae*, *Candida*, *Methanolaxyl*, *Pichia pastoris*, etc. The fungus may be from the genus *Fusarium*. The fungus may be from the genus *Rhizoctonia*. The fungus may be from the genus *Verticillium*. The fungus may be from the genus *Penicillium*. The fungus may be from the genus *Aspergillus*. The fungus may be from the genus *Cephalosporium*. The actinomycete may be from the genus *Streptomyces*, such as *Streptomyces*. The algae may originate from the phylum Cyanophyta, such as cyanobacteria. The algae may originate from the genus *Fucus*. The algae may originate from the genus *Achnanthes*. The algae may originate from the genus *Amphiprora*. The algae may originate from the genus *Amphora*. The algae may originate from the genus *Ankistrodesmus*. The algae may originate from the genus *Asteromonas*. The algae may originate from the genus *Boekelovia*.

[0048] For example, the recombinant cell is a recombinant cell obtained by introducing the coding gene of the polypeptide into *E. coli* DH10Bac. For example, the recombinant cell is a recombinant cell obtained by introducing the recombinant vector into *E. coli* DH10Bac. For example, the recombinant cell is a recombinant cell obtained by introducing the coding gene of the recombinant protein into Sf9 cells. For example, the recombinant cell is a recombinant cell obtained by introducing the recombinant vector into Sf9 cells.

[0049] In one specific embodiment of the present invention, the ovarian dysfunction or loss described above is caused by the removal of the ovaries.

[0050] In the above applications, the term "prevention" generally refers to methods implemented to prevent or delay the occurrence of a disease, condition, or symptom in a subject.

[0051] The term "treatment" generally refers to a method implemented to achieve a beneficial or desired clinical outcome. Beneficial or desired clinical outcomes include, but are not limited to, reduction of symptoms, lessening of disease severity, reduction of disease extent, stabilization of disease (i.e., cessation of disease progression), delay or slowing of disease progression, improvement or relief of disease status (whether partial or complete remission), whether detectable or undetectable. Furthermore, treatment can also refer to an extension of survival compared to the expected survival of a subject without treatment.

[0052] In the above applications, the drug can be used in animals with ovaries, such as mammals.

[0053] The mammals mentioned above can be mice or humans.

[0054] The behavioral experiments described above may include sucrose preference tests, open field tests, elevated cross maze tests, elevated zero maze tests, and / or forced swimming and tail suspension tests. The mammal may be a mouse.

[0055] The applications or methods described above are not for disease diagnosis. They are not intended to directly obtain disease diagnoses or health status results from living humans or animals.

[0056] The above applications or methods are for non-disease treatment purposes. They are not intended to restore or restore health or reduce suffering in living human or animal bodies.

[0057] This invention is the first to discover that the GRN-E fragment (the C-terminal domain of PGRN) can cross the blood-brain barrier and plays a unique role in alleviating perimenopausal anxiety and depression. Mechanistically, GRN-E can maintain lysosomal and metabolic balance by regulating lysosomal protease activity, autophagic flux, and the AMPK signaling pathway, thereby improving perimenopausal mood disorders. Full-length PGRN, as a pleiotropic growth factor, not only acts on the central nervous system but is also associated with various peripheral pathological processes such as tumor proliferation, obesity, and insulin resistance; long-term exogenous supplementation may cause potential side effects. GRN-E, as a key functional fragment, has a more concentrated effect on the central nervous system, effectively avoiding the off-target effects of the full-length protein. In summary, GRN-E, as an innovative therapeutic target and novel biological agent, demonstrates unique mechanistic advantages and broad application prospects in the intervention of perimenopausal mood disorders, and deserves further research and development.

[0058] This invention mainly includes the following contents:

[0059] The establishment and behavioral evaluation of the GRN-E intervention mouse model: A perimenopausal mouse model was first established using bilateral ovariectomy (OVX). After the model stabilized, intervention was performed by intraperitoneal injection of 5 mg / kg / 3 days of GRN-E (PBS was used as a control). Subsequently, behavioral tests (open field test, elevated zero maze test, tail suspension test, and forced swimming test) were used to assess anxiety and depression-like behaviors in the mice, verifying the potential role of GRN-E in alleviating perimenopausal mood disorders.

[0060] GRN-E Crossing the Blood-Brain Barrier: Brain tissue sections from mice that received intraperitoneal injection of GRN-E (5 mg / kg / 3 days) were analyzed. Immunofluorescence staining was used to detect the expression and distribution of GRN-E in the hippocampus and to observe whether it could cross the blood-brain barrier.

[0061] Molecular-level mechanism analysis involved treating cultured primary neurons with either GRN-E or PGRN, while the control group received PBS intervention. Western blotting was performed on day 16 to observe changes in key molecular markers after GRN-E intervention, including molecules related to energy metabolism pathways (AMPK and its phosphorylation level), lysosomal functional markers (LC3), and the phosphorylation level of the lysosomal hydrolase Cathepsin D (CTSD). These assays systematically evaluated the regulatory mechanisms of GRN-E on energy metabolism and the autophagy-lysosomal pathway.

[0062] The GRN-E fragment (derived from the C-terminal domain of the precursor protein) provided by this invention has the following beneficial effects in the preparation of drugs or formulations for alleviating or treating perimenopausal mood disorders:

[0063] 1. It possesses good blood-brain barrier permeability and central nervous system function characteristics.

[0064] The GRN-E described in this invention has a small molecular weight and stable structure, allowing it to effectively cross the blood-brain barrier and distribute in the hippocampus, a brain region that influences mood regulation, after exogenous administration, thereby exerting a regulatory effect at the central level. Compared to hormone replacement therapy or traditional antidepressants, this invention can directly act on brain targets, avoiding systemic side effects caused by peripheral hormone intervention.

[0065] 2. It can improve the neurological imbalance associated with decreased estrogen levels.

[0066] GRN-E can regulate lysosomal autophagy activity, energy metabolism, and synaptic homeostasis at the cellular level, thereby improving neural network plasticity and functional stability under estrogen deficiency. Animal experiments have shown that GRN-E treatment can reduce anxiety and depression-like behaviors and restore neuromorphic and metabolic indicators.

[0067] 3. It features high safety, simple preparation, and wide application.

[0068] GRN-E is a small, naturally derived secretory polypeptide that is readily obtained through recombinant expression or chemical synthesis, exhibiting good water solubility and biocompatibility. This molecule can be used as an active ingredient in various drug delivery routes (such as injection, nasal administration, and oral delivery systems), offering flexible formulation options that facilitate commercialization and industrialization.

[0069] 4. It overcomes the limitations of existing treatment methods.

[0070] Existing hormone replacement and antidepressant treatments suffer from slow onset of action, significant side effects, and difficulty in crossing the blood-brain barrier. This invention utilizes the central accessibility and multi-target regulatory characteristics of GRN-E to provide a novel, non-hormonal, highly targeted, and safe treatment strategy for perimenopausal mood disorders.

[0071] In summary, this invention is the first to propose a technical solution in the field of prevention and treatment of perimenopausal mood disorders, which utilizes the small secretory protein fragment GRN-E as the active pharmaceutical ingredient. It has the advantages of unique mechanism of action, clear therapeutic target, feasible preparation process and high potential for clinical translation, and can effectively make up for the shortcomings of existing technologies. Attached Figure Description

[0072] Figure 1The results show the behavioral tests of the perimenopausal model mice. (A) Open field test: the trajectory diagrams of mice in the sham-operated group and the OVX group, as well as the percentage of time and number of times mice entered the central area within 10 minutes; (B) Elevated zero maze test: the time and number of times mice in the sham-operated group and the OVX group entered the open arm; (C) Elevated cross maze test: the trajectory diagrams of mice in the sham-operated group and the OVX group, as well as the percentage of time and number of times mice entered the open arm; (D) Tail suspension test: the immobility time of mice in the sham-operated group and the OVX group; (E) Forced swimming test: the immobility time of mice in the sham-operated group and the OVX group. Black represents the sham-operated group, n=8; pink represents the OVX group, n=8. Independent samples t-test: p < 0.05 was considered statistically significant compared to the sham-operated group.

[0073] Figure 2 This is an analysis of behavioral test results in GRN-E-interventional perimenopausal model mice. (A) In the open field test, the trajectory diagrams of mice in the control group and the GRN-E group, and the percentage of time the mice spent entering the central area within 10 minutes. (B) In the elevated zero maze test, the trajectory diagrams of mice in the control group and the GRN-E group, and the percentage of time the mice spent entering the open arm. (C) In the tail suspension test, the percentage of immobile time of mice in the control group and the GRN-E group. (D) In ​​the forced swimming test, the percentage of immobile time of mice in the control group and the GRN-E group. Black represents the control group (PBS), n=6; green represents the GRN-E intervention group, n=6. Independent samples t-test, p < 0.05 was considered statistically significant compared to the control group.

[0074] Figure 3 The distribution of GRN-E in the hippocampus after systemic injection of Flag-GRN-E. (A) Flowchart of the Flag-GRN-E intervention experiment in OVX mice. (B) Immunostaining of Flag tags to confirm the distribution of GRN-E in hippocampal neurons after systemic injection.

[0075] Figure 4 PGRN and GRN-E can promote autophagy and synapse formation in primary neurons. (A) Technical roadmap for intervention of GRN-E and PGRN in primary hippocampal neurons. (BE) Western blotting detection of LC3, AMPK, and pCTSD expression levels and statistical results (n=6). (FG) Representative map and statistical results of sparse labeling (n=10). (HJ) Representative map and statistical results of synaptic markers PSD95 and Synapsin I (SYN1) expression (n=15). (KL) Representative map and statistical results of c-FOS immunofluorescence (n=12). Independent samples t-test, p < 0.05 was considered statistically significant compared with the control group. Detailed Implementation

[0076] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0077] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0078] The following examples use statistical software to process the data. The experimental results are expressed as mean ± standard deviation. One-way ANOVA test was used. P < 0.05 (*) indicates a significant difference, and P < 0.01 (**) indicates a highly significant difference.

[0079] The antibody sources in this embodiment of the invention are as follows, wherein the amino acid sequence of the mouse PGRN protein is NP_032201.3,09-JAN-2024.

[0080] In this embodiment of the invention, the pFastBac Dual vector and Sf9 insect cells were obtained from the research group of Liang Ling at Peking University School of Medicine. The relevant literature is: Yu S, Ding JH, Wang JL, Wang W, Zuo P, Yang A, Dai Z, Yin Y, Sun JP, Liang L. Structural insights into cholesterol sensing by the LYCHOS-mTORC1 pathway. Nat Commun. 2025 Jul 23;16(1):6792. doi: 10.1038 / s41467-025-61966-w. This information is publicly available from the applicant and is used solely for replicating this invention; it may not be used for any other purpose.

[0081] Table 1. Names and sources of antibodies involved in the embodiments of the present invention

[0082]

[0083] The experimental methods in this embodiment of the invention are as follows:

[0084] 1. Ovarian removal surgery:

[0085] Female C57BL / 6J mice aged 8-10 weeks were randomly divided into an ovariectomy group (OVX) and a sham operation group (Sham). Both surgeries were performed under sterile conditions using isoflurane inhalation anesthesia (5% for induction, 1-2% for maintenance). Mice were placed in a prone position, and skin preparation and disinfection were performed based on the location of the ovary on the body surface. An incision of approximately 0.5-1.0 cm was made on the back corresponding to the ovary. Subcutaneous tissue was bluntly dissected, and the peritoneum was incised to enter the abdominal cavity. After gently pulling on the fat pad below the kidneys, the ovary and fallopian tubes, which were encased in fat, were exposed. Blood vessels and ovarian tissue were ligated proximal to the fallopian tube using 5-0 sutures, followed by ovarian removal. After confirming hemostasis, the peritoneum was sutured layer by layer, and the skin incision was closed with 5-0 sutures to obtain the ovariectomy mouse (OVX). The procedure for the Sham group was the same as for the OVX group, but after exposing the ovary and fallopian tubes, ligation and removal were not performed; only simple pulling was used to confirm the ovary's location before the peritoneum and skin were sutured layer by layer.

[0086] 2. Behavioral tests:

[0087] All animal behavioral tests were recorded using an infrared camera (with supplemental infrared lighting) and analyzed using SMART software. One hour before each test, the mice were placed in the testing room to acclimatize, and the room was kept quiet during the test. The equipment was thoroughly cleaned and dried with 75% ethanol before each test.

[0088] 1) Open field test: Mice were placed in a square open field with sides of 50 cm (internal dimensions 50×50×50 cm), and their activity was recorded for 10 minutes. The overall movement and anxiety-like behaviors of the mice were assessed by analyzing the time they spent in the central area.

[0089] 2) Elevated Zero Maze Experiment: The zero maze is a circular platform (outer diameter: 60 cm, width: 5 cm) 50 cm above the ground, consisting of two open areas and two closed areas. Mice are placed in a closed area and allowed to explore for 10 minutes. The time spent in the open areas is used as an indicator of anxiety-like behavior.

[0090] 3) Tail Suspension Test: The mouse's tail was secured to a horizontal bar with adhesive tape, keeping it suspended at a height of approximately 20 cm. The time spent at rest was recorded over a 5-minute test. Prolonged resting time was interpreted as depressive-like behavior.

[0091] 4) Forced swimming test: Mice were placed in a transparent cylindrical water tank (height: 40 cm, diameter: 20 cm) filled with water (temperature: 23~25 degrees Celsius, water depth: 20 cm). The experiment lasted for 6.5 minutes. The first 1.5 minutes were used as an adaptation period. The immobility time of the mice in the last 5 minutes was recorded to assess their level of depressive-like behavior.

[0092] 3. Immunofluorescence staining

[0093] Mice were deeply anesthetized, and after cardiac perfusion, they were rinsed with ice-cold PBS and then fixed with 4% paraformaldehyde (PFA). Brain tissue was collected and fixed overnight in 4% PFA at 4°C, followed by gradient dehydration with 20%–30% sucrose solution. After OCT embedding, 30 μm thick coronal sections were cut using a cryostat (CM1950, Leica).

[0094] The sections were first incubated in blocking buffer (3% BSA, 0.3% Triton X-100 in PBS), followed by overnight incubation at 4°C with five primary antibodies (corresponding to Mouse anti-NeuN, Rabbit anti-Flag, Mouse anti-PSD95, Rabbit anti-Synaptophysin, and Rabbit anti-cfos in Table 1). After washing with PBS, the sections were incubated at room temperature with two Alexa Fluor-labeled secondary antibodies (corresponding to Donkey anti-Rabbit in Table 1). 594, Donkey anti-mouse, Cells were incubated at 647°C (Invitrogen) for 1 h. Nuclei were then counterstained with DAPI and mounted with anti-quenching mounting medium (S2110, Solarbio) to preserve fluorescence signal. Finally, imaging and analysis were performed using a confocal microscope (Stellaris 8, Leica).

[0095] 4. Western blotting

[0096] Mouse brain tissue proteins were extracted using RIPA lysis buffer containing protease and phosphatase inhibitors (C0001, C0002, C0003, TargetMol). The lysates were clarified by centrifugation at 12,000 × g for 15 min (4 °C). Protein concentration was determined using a BCA protein quantification kit (Thermo Fisher). Equal amounts of protein (20–30 µg) were separated by SDS-PAGE or Native PAGE and transferred to nitrocellulose membranes (10600043, Cytiva). The membranes were blocked for 1 h at room temperature in TBST solution containing 5% skim milk powder, and then incubated overnight at 4 °C with five primary antibodies (corresponding to Rabbit anti-LC3B, Mouse anti-β-actin, Rabbit anti-Phospho-AMPKα, Rabbit anti-CTSD, and Rabbit anti-AMPKα in Table 1). After washing with TBST, the membrane was incubated with two HRP-conjugated secondary antibodies (corresponding to Donkey anti-Rabbit, HRPConjugated, Donkey anti-Mouse, and Jackson in Table 1) and detected by colorimetric analysis using enhanced chemiluminescence reagents (ECL, WBKLS0500, Millipore). Protein band signal intensity was quantitatively analyzed using ImageJ software.

[0097] 5. Sparse labeling and imaging of neurons

[0098] In primary cultured hippocampal neurons, sparse labeling was achieved by infecting cells with a lentiviral vector expressing EGFP (LV-hysn-EGFP) at DIV3 (fold of infection 0.01). Neurons were fixed and imaged at DIV16–18. All labeled neurons were imaged using a confocal microscope (Stellaris 8, Leica) equipped with a 40× oil immersion objective (magnification 3). Z-shaped images were acquired using a 0.2 μm step size, and maximum intensity projection maps were generated. Dendritic spine density was quantified in a double-blind manner using ImageJ based on clearly distinguishable secondary dendritic segments.

[0099] Example 1. GRN-E (the C-terminal domain of PGRN) can cross the blood-brain barrier to alleviate perimenopausal anxiety and depression.

[0100] 1. GRN-E intervention significantly restored depression- and anxiety-like behaviors in perimenopausal model mice.

[0101] 1.1 Preparation of perimenopausal mouse model

[0102] Ovariectomized mice (OVX) were obtained by ovariectomy of female C57BL / 6J mice:

[0103] Experimental grouping: Sixteen female C57BL / 6J mice aged 8-10 weeks, weighing 19±1g, were randomly divided into two groups: sham operation group and ovariectomy group (OVX), with 8 mice in each group.

[0104] 1.2 Ovariectomized mice (OVX) exhibited mood disorders

[0105] Behavioral tests were performed on ovariectomized mice 4 weeks after the procedure. The results showed that, compared with the sham group, ovariectomized mice (OVX) exhibited significantly more depressive and anxiety-like behaviors.

[0106] In the open field experiment, compared with the sham group, ovariectomized mice (OVX) showed a significantly lower total distance traveled into the open field within 30 minutes and a significantly lower number of times they entered the center of the open field (central region) in the first 10 minutes. Figure 1 (A)

[0107] In the elevated zero maze and elevated cross maze experiments, compared with the sham group, ovariectomized mice (OVX) had significantly reduced time to enter the open arm and significantly fewer entries into the open arm. Figure 1 (Chinese BC)

[0108] In forced swimming and tail suspension tests, the immobility time of ovariectomized mice (OVX) was significantly increased compared to the sham group. Figure 1 middle DE).

[0109] The behavioral results above indicate that, compared with the sham group, ovariectomized mice (OVX) exhibited significant mood disorders such as depression and anxiety-like behaviors. Therefore, this invention demonstrates that the ovariectomized mouse model was successfully established by removing the ovaries of mice; the ovariectomized mouse (OVX) is the perimenopausal model mouse.

[0110] 1.3 Preparation of GRN-E

[0111] Recombinant mouse GRN-E protein was expressed in insect cells (Spodoptera frugiperda, Sf9) using a Bac-to-Bac baculovirus system. The expression vector encoded a secretory fusion protein containing an N-terminal His-MBP-TEV-Flag tag (pFastBac-GP64-His-MBP-TEV-Flag-GRN-E). The constructed recombinant bacmid DNA was transfected into Sf9 cells to obtain high-titer baculovirus. For protein expression, Sf9 cells were cultured at 2 × 10⁻⁶ cells per cell line. 6 Infected at a density of cells / mL and cultured in serum-free SIM SF Expression Medium (SinoBiological, MSF-AF) at 27°C for 60–72 hours. The culture supernatant was collected by centrifugation and filtered through a 0.22 μm filter membrane. Specifically:

[0112] 1.3.1 Construction of recombinant plasmids

[0113] The recombinant plasmid pFastBac-GRN-E was obtained by replacing the small fragment between the BamHI and HindIII restriction sites in the pFastBac Dual vector with the double-stranded DNA molecule shown in SEQ ID NO.3, which was fully synthesized by Beijing Xianghong Biotechnology. Sequencing confirmed that the recombinant plasmid pFastBac-GRN-E contains the DNA shown in SEQ ID NO.3 in the sequence listing.

[0114] SEQ ID NO.3 (5'-3'):

[0115]

[0116] 1.3.2 Construction of recombinant baculovirus expression vector

[0117] 1) Add 1 ng of recombinant plasmid pFastBac-GRN-E to 100 μL of DH10Bac (Beijing Bomei, BC112-01) competent cells, mix well, and incubate on ice for 30 min. Then, place the centrifuge tube in a 42℃ metal bath for heat shock treatment for 45 s, immediately incubate on ice for 2 min, add 900 μL of liquid LB medium, and culture at 37℃ and 220 rpm for 4 h with shaking.

[0118] 2) After completing step 1), take 100 μL of bacterial suspension and spread it onto a solid LB agar plate containing 50 μg / ml kanamycin, 7 μg / ml gentamicin, 10 μg / ml tetracycline, 100 μg / ml X-gal and 40 μg / ml IPTG. Incubate at 37°C in the dark for 24 h. At this time, white single colonies can be observed.

[0119] 3) After completing step 2), pick a uniform white colony from the plate and inoculate it into 1 mL of liquid LB medium containing 50 μg / ml kanamycin, 7 μg / ml gentamicin and 10 μg / ml tetracycline. Incubate at 37°C and 220 rpm for 12 h with shaking.

[0120] 4) After completing step 3), take a sample of bacterial culture and streak it onto a solid LB medium plate containing 50 μg / ml kanamycin, 7 μg / ml gentamicin, 10 μg / ml tetracycline, 100 μg / ml X-gal and 40 μg / ml IPTG. Incubate at 37°C upside down for 24 h.

[0121] 5) After completing step 4), pick a uniform white colony from the plate and inoculate it into 1 mL of liquid LB medium containing 50 μg / ml kanamycin, 7 μg / ml gentamicin and 10 μg / ml tetracycline. Incubate at 37°C and 220 rpm for 12 h with shaking.

[0122] 6) After completing step 5), sample the bacterial culture and perform Sanger sequencing using primers F1 and R1. The sequencing results were completely compared with the designed plasmid pattern, indicating that a recombinant Bacmid containing the DNA shown in SEQ ID NO.3 was obtained;

[0123] F1: 5'-ATACCGTCCCACCATCGGG-3';

[0124] R1: 5'-ACGGCCACAACTCCTCATA-3'.

[0125] 1.3.3 Preparation of recombinant baculovirus

[0126] 1) Take 2 mL of the bacterial culture containing recombinant Bacmid obtained in step 1.3.2, inoculate it into 200 mL of liquid LB medium containing 50 μg / ml kanamycin, 7 μg / ml gentamicin and 10 μg / ml tetracycline, and culture at 37℃ and 220 rpm for 14 h with shaking. Then, use the endotoxin-free small-volume extraction kit (Tiangen, DP106) to extract plasmid according to the instructions to obtain recombinant Bacmid.

[0127] 2) Transfect Sf9 cells with the recombinant Bacmid obtained in step 1) (transfection was performed using...) II. Reagent (Thermo Fisher Scientific, 10362100), then incubate at 27°C for 72 hours (cytopathic effects can be observed), and harvest the supernatant, which is the P1 generation virus solution;

[0128] Sf9 insect cells were transfected with 3 μg of recombinant Bacmid to obtain 2 mL of P1 generation virus solution.

[0129] 1.3.4 Passage amplification of recombinant baculovirus

[0130] 1) Inoculate the P1 generation virus solution obtained in step 1.3.3 into the cell suspension (virus solution to cell suspension volume ratio is 1:10), then culture at 27℃ and 110 rpm with shaking until more than 80% of the cells show cytopathic effects. Harvest the supernatant, which is the P2 generation virus solution. Cell suspension preparation method: Collect Sf9 cells in logarithmic growth phase and resuspend them in serum-free SIM SF Expression Medium (SinoBiological, MSF-AF) to achieve a cell concentration of 2.0 × 10⁻⁶ cells / mL. 6 cells / mL;

[0131] 2) Inoculate the P2 generation virus solution obtained in step 1) into the cell suspension (the volume ratio of virus solution to cell suspension is 1:10), and then culture at 27°C and 110 rpm with shaking until more than 80% of the cells show cytopathic effects. Harvest the supernatant, which is the P3 generation virus solution. The preparation method of the cell suspension is the same as that in step 1).

[0132] Approximately 200 mL of P3 generation virus solution was obtained by passage and amplification of 2 mL of P1 generation virus solution.

[0133] 1.3.5 Large-scale expression and purification of proteins

[0134] 1) Inoculate the P3 virus solution obtained in step 1.3.4 into the cell suspension (virus solution to cell suspension volume ratio is 1:100), incubate at 27℃ with shaking at 110 rpm for 60 h, then centrifuge at 1500 rpm for 15 min and collect the supernatant. Cell suspension preparation method: Take Sf9 cells and resuspend them in serum-free SIM SF Expression Medium to achieve a cell concentration of 2.0 × 10⁶ cells / mL. 6 cells / mL. SIM SF Expression Medium. Approximately 2 L of supernatant was prepared, collected by centrifugation, and then filtered through a 0.22 μm filter membrane;

[0135] 2) The supernatant obtained in step 1) was used to purify the His-tagged target protein using a Ni-NTA affinity chromatography column (Smart-Lifesciences, SA003100). The eluted protein fraction was concentrated using a centrifugal ultrafiltration tube (10 kDa molecular weight cutoff, Amicon Ultra, UFC9010) and incubated overnight at 4°C with TEV protease (Shanghai Sangon Biotech, C500302) to remove the His-MBP tag. The tag-removed protein was further concentrated using a centrifugal ultrafiltration tube with a 3 kDa molecular weight cutoff (Amicon Ultra, UFC9003) and purified by gel filtration on an AKTA Pure system (Cytiva) using a Superdex 75 Increase 10 / 300 GL column (Cytiva). The mobile phase was pre-equilibrated TBS, yielding a solution containing the target protein, also known as the Flag-GRN-E solution. The target protein is the protein shown in SEQ ID NO.2, and it is named Flag-GRN-E protein. Flag-GRN-E protein is a GRN-E protein with a Flag tag. The amino acid sequence of the GRN-E protein is SEQ ID NO.1 in the sequence listing. The coding sequence of Flag-GRN-E protein is the DNA molecule shown in positions 1189-1476 of SEQ ID NO.3; the coding sequence of GRN-E protein is the DNA molecule shown in positions 1164-1476 of SEQ ID NO.3.

[0136] SEQ ID NO.1:

[0137] EKDVDFIQPPVLLTLGPKVGNVECGEGHFCHDNQTCCKDSAGVWACCPYLKGVCCRDGRHCCPGGFHCSARGTKCLRKKIPRWDMFLRDPVPRPLL.

[0138] SEQ ID NO.2:

[0139] GDYKDDDDKEKDVDFIQPPVLLTLGPKVGNVECGEGHFCHDNQTCCKDSAGVWACCPYLKGVCCRDGRHCCPGGFHCSARGTKCLRKKIPRWDMFLRDPVPRPLL.

[0140] The purity of the final protein was verified by SDS-PAGE and Coomassie Brilliant Blue staining. The protein used for in vivo administration was prepared without endotoxin; the final endotoxin level was confirmed to be less than 0.1 EU / μg protein by colorimetric LAL assay (Beyotime, C0276S).

[0141] 1.4 Behavioral Test Analysis of GRN-E-Intervention in Perimenopause Model Mice

[0142] The Flag-tagged GRN-E (Flag-GRN-E, SEQ ID NO.2 in the sequence listing) was dissolved in PBS to prepare a 0.5 mg / mL GRN-E solution. Twelve perimenopausal model mice were randomly divided into two groups for GRN-E intervention analysis: a control group (OVX+PBS) and a GRN-E intervention group (OVX+GRN-E), with six mice in each group.

[0143] GRN-E intervention group: Each perimenopausal model mouse was administered GRN-E solution via intraperitoneal injection. The volume of GRN-E administered to each mouse was 200-250 uL, and the dosage was 5 mg / kg / 3 days.

[0144] Control group (OVX+PBS): Each perimenopausal model mouse was administered the control group via intraperitoneal injection of the solvent (PBS solution), with each mouse receiving 200-250 uL.

[0145] Three weeks after intraperitoneal injection of GRN-E, behavioral tests were performed on mice in the GRN-E intervention group and the control group. Results showed that compared with the control group (… Figure 2 Compared to PBS (represented by PBS): In the open field experiment, the time spent in the central region by mice in the GRN-E intervention group was significantly longer ( Figure 2 (A); In the elevated zero-maze experiment, the time it took for mice in the GRN-E group to enter the open arm was significantly increased ( Figure 2 In the tail suspension and forced swimming experiments, the immobility time of mice in the GRN-E group was significantly reduced (B). Figure 2 (CD) Behavioral results suggest that GRN-E intervention significantly alleviated and improved mood disorders such as depression and anxiety-like behaviors in perimenopausal model mice.

[0146] 2. GRN-E can cross the blood-brain barrier and act on hippocampal neurons.

[0147] Brain tissue sections were taken from mice that received intraperitoneal injection of GRN-E (5 mg / kg / 3 d). Immunofluorescence staining was used to detect the expression and distribution of GRN-E in the hippocampus and to observe whether it could cross the blood-brain barrier.

[0148] In step 1.3, after 3 weeks of intervention with flag-tagged GRN-E in OVX model mice, brain tissue was harvested for immunofluorescence detection. The results showed that significant flag signals appeared in the hippocampus of the GRN-E intervention group mice. Figure 3 GRN-E in B is represented by B, and it clearly co-localizes with the neuronal marker NeuN. Figure 3 (Middle B). This result indicates that exogenous GRN-E can cross the blood-brain barrier and specifically act on hippocampal neurons.

[0149] 3. PGRN and GRN-E can promote autophagy and synapse formation in primary neurons.

[0150] Cultured primary neurons were treated with either GRN-E or PGRN (R&D, 2420-PG), while the control group received PBS intervention. Western blotting was performed on day 16 to observe changes in key molecular markers after GRN-E intervention, including molecules related to energy metabolism pathways (AMPK and its phosphorylation level), lysosomal functional markers (LC3), and the phosphorylation level of the lysosomal hydrolase Cathepsin D (CTSD). These assays systematically evaluated the regulatory mechanism of GRN-E on energy metabolism and the autophagy-lysosomal pathway.

[0151] 3.1 Primary cell culture

[0152] To further verify the mechanism of action of PGRN and GRN-E, this invention treated primary hippocampal neurons with recombinant proteins from day 8 to day 16 of primary culture. Figure 4 (A)

[0153] Primary hippocampal neurons were isolated from C57BL / 6J mouse embryos at day 16.5 (E16.5). The hippocampal bodies were dissected in ice-cold Dulbecco's Modified Eagle Medium (DMEM, CM15020, Macgene) and digested with protease (LS003119, Worthington) and DNase I (SL20761, coolaber) at 37°C for 30 minutes. After gentle homogenization and centrifugation, the dispersed cells were suspended in DMEM containing 10% fetal bovine serum (FBS, A5669701, Gibco) and seeded onto poly-D-lysine (PDL, P1149, Sigma) coated culture plates or coverslips. After 4-6 hours of cell attachment, the medium was replaced with phenol red-free neural basal medium (12348017, Gibco), and supplemented with 2% B27 (17504044, Gibco), 1% GlutaMAX (35050061, Gibco), and 0.5% penicillin-streptomycin (15140122, Gibco). Neurons were maintained for growth in a humidified 5% CO2 incubator at 37°C, with half of the medium replaced every 3 days. From day 8 to day 16, three treatment groups were administered: control group treated with PBS, positive control group treated with PGRN (100 ng / ml), and intervention group treated with GRNE (100 ng / ml). Western blotting was then performed on day 16 to observe changes in key molecular markers after GRN-E intervention. Neurons aged 16-18 days were used for dendritic spine imaging and immunofluorescence (c-FOS) detection.

[0154] The results showed that both treatments significantly increased the LC3-II / LC3-I ratio, accompanied by a slight decrease in p62 levels, suggesting enhanced autophagic flux. Figure 4 (BD). Meanwhile, both PGRN and GRN-E interventions upregulate AMPK phosphorylation levels ( Figure 4 (BE). At the structural level, these two interventions significantly increased dendritic spine density and enhanced the expression of synaptic markers PSD95 and Synapsin I (SYN1), further supporting the direct role of PGRN and GRN-E in promoting synaptic plasticity. Figure 4 (Middle HI). Furthermore, as a classic marker of neuronal excitation, c-Fos was observed in neurons chronically exposed to full-length PGRN or its fragmented GRN-E, and this invention showed that c-Fos... + The number of cells increased significantly ( Figure 4(KL). The above results suggest that PGRN may enhance the activity of CTSD enzymes through its C-terminal domain, promoting the production of cleavage fragment GRN-E. GRN-E can then independently promote autophagy and synapse formation in primary neurons, thus exerting an antidepressant effect.

[0155] GRN-E (the C-terminal domain of PGRN) can cross the blood-brain barrier to alleviate perimenopausal anxiety and depression. Mechanistically, GRN-E can maintain lysosomal and metabolic balance by regulating lysosomal protease activity, autophagic flux, and the AMPK signaling pathway, thereby improving synaptic function and neuronal activity and ultimately alleviating perimenopausal mood disorders. This invention discovers GRN-E as an innovative therapeutic target and novel biological agent, exhibiting unique mechanistic advantages and broad application prospects in the intervention of perimenopausal mood disorders, and possessing high clinical translational value.

[0156] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. The application of polypeptides in the preparation of drugs for treating perimenopausal mood disorders; characterized in that: The polypeptide is any one of the following: A1) The amino acid sequence is the polypeptide of SEQ ID NO.1 in the sequence listing; A2) The amino acid sequence is the polypeptide of SEQ ID NO.2 in the sequence listing; The symptoms of perimenopausal mood disorders include at least one of the following: 1) Depression 2) Anxiety.

2. A medicine, wherein the medicine is a medicine for treating, alleviating, or adjuvantly treating or adjuvantly treating perimenopausal mood disorders or a medicine for treating, alleviating, or adjuvantly treating or adjuvantly treating diseases caused by ovarian dysfunction or loss, characterized in that: The drug contains the polypeptide as described in claim 1.

3. A polypeptide, which is the polypeptide described in claim 1.

4. A biomaterial related to the polypeptide described in claim 1, characterized in that: The biomaterial is any one of the following: B1) The gene encoding the polypeptide of claim 1; B2) has the expression box of B1); B3) has a recombinant vector similar to B1; B4) has a recombinant vector of B2); B5) contains recombinant cells with B1); B6) contains recombinant cells with B2); B7) recombinant cells with B3); B8) contains recombinant cells with B4).

5. A method for preparing a polypeptide, comprising the following steps: expressing the gene encoding the polypeptide shown in SEQ ID NO.2 in cells using a baculovirus expression system to obtain the polypeptide shown in SEQ ID NO.

2.

6. The method according to claim 5, characterized in that: The method includes the following steps: (1) The recombinant plasmid was introduced into Escherichia coli DH10Bac to obtain recombinant Escherichia coli; the recombinant plasmid was obtained by inserting a DNA molecule containing the gene into a baculovirus transfer vector; (2) After culturing the recombinant Escherichia coli obtained in step (1), the plasmid is extracted, which is the recombinant bacmid; (3) Transfect Sf9 cells with the recombinant bacmid obtained in step (2) and culture them. Collect the culture supernatant, which is the P1 generation virus solution. (4) Infect Sf9 cells with the P1 generation virus solution and culture them, and collect the culture supernatant, which is the P2 generation virus solution; (5) Infect Sf9 cells with the P2 generation virus solution and culture them, and collect the culture supernatant, which is the P3 generation virus solution; (6) Infect Sf9 cells with the P3 generation virus solution, collect the culture supernatant, and purify it to obtain the polypeptide.

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