A polypeptide and its use in preventing and treating hair loss

Abstract

The present application relates to the technical field of active peptide, and discloses a kind of polypeptide and its application in preventing and treating alopecia;The amino acid sequence of the polypeptide is Phe-Trp-Ala-Phe-Phe-His, obtained by alkaline protease hydrolysis Lechriurus, it is verified by experiment, the polypeptide has the promoting effect to frizzled class receptor 7 protein (frizzled class receptor 7), can promote the growth and development of HDPCs cell, improve its migration activity, and can activate Wnt / β-catenin signal pathway, and up-regulate the expression of β-catenin, C-myc, CyclinD1 and LEF1 and other downstream key proteins, while promoting the expression of Wnt10b, also can alleviate the decrease of protein expression amount caused by inhibitor DKK1 and FZ7-21, therefore the polypeptide can promote hair follicle cell proliferation by Wnt / β-catenin signal pathway, regulate hair growth cycle, improve local microcirculation and other various ways to play hair growth effect, can be used for preparing hair growth drug, also can be used for preparing the cosmetics that help preventing and treating alopecia.

Description

Technical Field

[0001] This invention relates to the field of active peptide technology, specifically to a polypeptide and its application in the prevention and treatment of hair loss. Background Technology

[0002] Hair loss is a disease that causes hair to fall out naturally from the head or other parts of the body. It is a common skin disease in clinical practice. Although hair loss does not directly endanger physical health, it seriously affects the mental health and quality of life of patients. For example, hair loss problems may damage personal image, leading to psychological problems such as anxiety and low self-esteem, and may also pose potential obstacles to social and career development. With increasing social pressure and changes in lifestyle, hair loss has become an increasingly common skin health problem worldwide. Therefore, the development of safe and effective hair growth products has important social demand and market prospects.

[0003] The Philippine medicinal leech (also known as the Manila leech or Hirudo medicinalis) belongs to the phylum Annelida, class Hirudinea, and family Hirudinidae. It is a typical tropical-subtropical organism, mainly distributed in Southeast Asia, including the Philippines, Thailand, Vietnam, Malaysia, Indonesia, and Myanmar. In China, it is mainly distributed in southern provinces such as Guangxi, Yunnan, and Guangdong. As a traditional Chinese medicine, it has important medicinal value. Its medicinal properties are characterized by a bitter and salty taste, neutral nature, and slight toxicity. It mainly acts on the liver meridian and has the effects of "breaking blood stasis, promoting menstruation, and eliminating stagnation." In modern medical research, increasing evidence shows that bioactive peptides in natural plant and animal extracts can have a positive impact on human health through multiple mechanisms. The Philippine medicinal leech contains a variety of bioactive components, among which polypeptides have received widespread attention due to their good cell affinity and bioregulatory functions. Modern pharmacological studies have confirmed that Philippine medicinal leech polypeptide extracts can promote cell proliferation.

[0004] The Wnt / β-catenin signaling pathway regulates biological processes such as cell proliferation, differentiation, and migration. Current research has reported that the Wnt / β-catenin signaling pathway is closely related to the occurrence and development of seborrheic alopecia. Among them, β-catenin, C-myc, Wnt10b, CyclinD1, and LEF1 play key regulatory roles in the Wnt / β-catenin signaling pathway. Changes in the expression levels of these factors can affect the activation of hair follicle stem cells and the proliferation and differentiation of hair follicle epithelial cells, thereby participating in biological processes such as hair follicle development, hair growth cycle regulation, and hair follicle regeneration. When the activity of this signaling pathway decreases, the activation of hair follicle stem cells is inhibited, and hair follicles gradually become miniaturized, which leads to hair entering the regression or resting phase, aggravating the occurrence and development of hair loss.

[0005] Based on the above background, a polypeptide derived from Hirudo medicinalis is being developed. This polypeptide can promote hair growth through multiple pathways, such as promoting hair follicle cell proliferation, regulating the hair growth cycle, and improving local microcirculation, via the Wnt / β-catenin signaling pathway. It can not only provide a new type of natural raw material with stable preparation process and significant hair growth effect, expanding the research and development ideas of hair growth products, but also open up a new industrial path for the comprehensive and high-value utilization of Hirudo medicinalis resources, which has important scientific research significance and market value. Summary of the Invention

[0006] To address the problems existing in related technologies, the primary objective of this invention is to provide a polypeptide whose amino acid sequence is shown in SEQ ID NO: 1, specifically Phe-Trp-Ala-Phe-Phe-His (FWAFFH).

[0007] The second objective of this invention is to provide a method for preparing the aforementioned polypeptide, wherein the polypeptide is obtained by hydrolyzing leeches with an alkaline protease, and the specific steps are as follows: (1) Tris-HCl buffer was added to the freeze-dried leech powder and ultrasonic extraction was performed.

[0008] (2) Collect the filtrate after separation using an ultrafiltration membrane.

[0009] (3) The filtrate was freeze-dried, reconstituted, and purified by column chromatography using high performance liquid chromatography.

[0010] Preferably, the pH of the Tris-HCl buffer solution in step (1) of the present invention is 8.

[0011] Preferably, the conditions for ultrasonic extraction in step (1) of the present invention are: ultrasonic extraction at 25°C for 3 hours.

[0012] Preferably, the ultrafiltration membrane in step (2) of the present invention has a molecular weight cutoff of 3 kDa and 10 kDa.

[0013] Preferably, the column temperature for high performance liquid chromatography purification in step (3) of the present invention is 30°C; phase A is water containing 0.1% formic acid, phase B is water containing 0.1% formic acid and 80% acetonitrile; and the flow rate is 0.6 mL / min.

[0014] The third objective of this invention is to propose the following two applications of the aforementioned polypeptide: (1) Application of the polypeptide in the preparation of anti-hair loss drugs.

[0015] (2) Application of the polypeptide in the preparation of anti-hair loss daily chemical products.

[0016] The beneficial effects of this invention are: (1) The peptide FWAFFH of the present invention is non-toxic to HDPCs, can significantly promote the migration of HDPCs cells, and can improve the migration of HDPCs cells inhibited by the inhibitors FZ7-21 and DKK-1, indicating that the peptide FWAFFH helps cells homing in hair follicle morphogenesis, cycle reconstruction and regeneration therapy.

[0017] (2) The peptide FWAFFH described in this invention has a significant agonistic effect on key proteins such as β-catenin, C-myc, Wnt10b, CyclinD1 and LEF1 in the Wnt / β-catenin signaling pathway, indicating that the peptide FWAFFH can promote hair follicle cell proliferation, regulate hair growth cycle and improve local microcirculation through the Wnt / β-catenin signaling pathway. Attached Figure Description

[0018] Figure 1 This is a diagram showing the molecular docking of the FWAFFH polypeptide with Frizzled class receptor 7 (PDB: 5T44) in Example 3 of this invention.

[0019] Figure 2 This refers to the cytotoxicity test results in Example 5 of the present invention, wherein... Figure 2 (a) shows the effect of FZ7-21 on the viability of HDPCs. Figure 2 (b) shows the effect of FWAFFH peptide on HDPCs. Figure 2 (c) shows the effect of DKK-1 on HDPCs.

[0020] Figure 3 This describes the effect of FWAFFH polypeptide and inhibitors FZ7-21 and DKK-1 on the migration ability of HDPCs in Example 6 of this invention.

[0021] Figure 4 This is the effect of the FWAFFH polypeptide and its inhibitors FZ7-21 and DKK-1 on the expression of key proteins in the Wnt / β-catenin signaling pathway, as shown in Example 7 of this invention.

[0022] Figure 5 This is a quantitative diagram illustrating the effects of the FWAFFH peptide and its inhibitors FZ7-21 and DKK-1 on the expression of key proteins in the Wnt / β-catenin signaling pathway, as shown in Example 7 of this invention. Figure 5 (a) Effects of FWAFFH peptide and its inhibitors FZ7-21 and DKK-1 on β-catenin protein expression. Figure 5 (b) Effects of FWAFFH peptide and its inhibitors FZ7-21 and DKK-1 on C-myc protein expression. Figure 5(c) shows the effects of FWAFFH peptide and its inhibitors FZ7-21 and DKK-1 on Cyclin D1 protein expression. Figure 5 (d) shows the effects of FWAFFH peptide and its inhibitors FZ7-21 and DKK-1 on LEF1 protein expression. Figure 5 (e) shows the effects of FWAFFH peptide and its inhibitors FZ7-21 and DKK-1 on Wnt10b protein expression. Detailed Implementation

[0023] In vitro experiments The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, but the scope of protection of the present invention is not limited thereto; unless otherwise specified, all reagents used in the present invention are commercially available analytical grade reagents, and all raw materials used can be purchased through conventional commercial channels.

[0024] The material information used in the embodiments of this invention is as follows: Freeze-dried leech powder: 300 mesh, Huazhi Pharmaceutical Co., Ltd., product batch number: HZFYZ-20240315.

[0025] Example 1 This embodiment describes the preparation of a phenothiurin-derived polypeptide, and the specific steps are as follows: (1) Add 100 mL of Tris-HCl buffer to 10 g of lyophilized leech powder and extract by sonication at 25 °C for 3 h.

[0026] (2) The extract was added to a centrifuge tube containing a 10 kDa ultrafiltration membrane and centrifuged at 8000 rpm for 20 min to obtain concentrate I (>10 kDa) and permeate I (<10 kDa); permeate I was added to a centrifuge tube containing a 3 kDa ultrafiltration membrane and centrifuged at 8000 rpm for 20 min to obtain concentrate II (3-10 kDa) and permeate II (<3 kDa); permeate II was placed in a rotary evaporator for concentration and concentrated under reduced pressure at low temperature (≤40℃) until the volume was significantly reduced or the required concentration was reached to obtain concentrate III (<3 kDa).

[0027] (3) The three concentrates were freeze-dried and named N3 (<3kDa), N6 (3-10kDa), and N9 (>10kDa), and their anticoagulant activity was determined: the freeze-dried N3 (<3kDa), N6 (3-10kDa), and N9 (>10kDa) samples were dissolved in Tris-HCl buffer to prepare a solution with a concentration of 0.5mg / mL; their anticoagulant activity was determined using the coagulation time method: the sample was added to fresh plasma and mixed well, and the volume of the sample and fresh plasma was... The ratio was 1:1. In addition, 5 U / mL, 10 U / mL, 20 U / mL and 40 U / mL hirudin were added to fresh plasma and mixed well. The plasma without hirudin was set as a blank control. The coagulation time of plasma was observed at 37℃. A standard curve was plotted with the coagulation time of hirudin. The anticoagulant activity of the samples was calculated, and the anticoagulant activity values ​​of N3, N6 and N9 were obtained, which were 300 ATU / mL, 550 ATU / mL and 800 ATU / mL, respectively. N6 (3-10 kDa) was further separated.

[0028] (4) Rinse the dextran gel G-15 with distilled water to fully hydrate it, then load it into the gel column, flush the column for 30 min, and after rinsing it clean, connect the column to the constant flow pump and the automatic collector, set the collector time to 10 min, prepare a 5 mg / mL N6 (3-10 kDa) sample solution for loading, connect the high-level tank when the sample is close to the gel, and then start collecting the sample. After the sample has passed through the column, rinse the column with distilled water, detect the collected sample in a spectrophotometer, measure the absorbance value at a wavelength of 220 nm, obtain the peptide mass spectrometry information, combine the fractions with similar absorbance values ​​and in the same peak region, freeze-dry them separately and use them for further separation.

[0029] (5) Analyze the peptide mass spectrometry information of the components obtained in step (4) using liquid chromatography-mass spectrometry (HPLC-MS).

[0030] The chromatographic separation conditions are as follows: ①Chromatographic column system: Pre-column: 150μm id×50mm, packing material is Reprosil-Pur 120 C18-AQ, 3μm.

[0031] Analytical column: 150μm id×170mm, packing material is Reprosil-Pur 120 C18-AQ, 1.9μm.

[0032] ② Column temperature: 30℃.

[0033] ③Mobile phase: Phase A is water containing 0.1% formic acid, and Phase B is water containing 0.1% formic acid and 80% acetonitrile.

[0034] ④ The elution gradient and flow rate are shown in Table 1: Table 1 Elution gradient and flow rate of mobile phase in liquid chromatography Mass spectrometry conditions are: ① Primary mass spectrometry parameters: Resolution: 70000.

[0035] Automatic gain control target value (AGC target): 3e6.

[0036] Maximum injection time: 100ms.

[0037] Scan range: 100-1500 mass-to-charge ratio.

[0038] ② Secondary mass spectrometry parameters: Resolution: 17500.

[0039] Automatic gain control target value (AGC target): 1e5.

[0040] Maximum injection time: 50ms.

[0041] TopN: 20.

[0042] Normalized collision energy (NCE / stepped NCE): 28.

[0043] Ionization mode: Positive ion mode (ESI+).

[0044] Spray voltage: 3.2kV.

[0045] Capillary temperature: 350℃.

[0046] Dryer temperature: 350℃.

[0047] Mass spectrometry data were analyzed using PeaksStudio 8.0 software. Target proteins were retrieved from the UniProtKB_Taxonomy_Poecilobdellamanillensis database, and de novo sequencing was used to analyze the identified peptide fragments. Peptide fragments meeting the threshold "-10lgP>15" were screened, and then PeptideRanker (http: / / distilldeep.ucd.ie / PeptideRanker / ) was used for bioactivity prediction scoring, with a threshold of PeptideRanker prediction score >0.95. Through this process, 16 peptides with high-confidence identification results and potential biological activity were screened. After further screening and activity evaluation, one key active peptide was finally determined for subsequent functional studies. Its amino acid sequence is FWAFFH, as shown in Table 2, and it will be used for subsequent research. Other candidate peptides are not specifically disclosed in this application.

[0048] Table 2 shows peptides with potential biological activity after screening for "-10lgP>15". The target functions of the peptides shown in Table 1 were evaluated using the computer bioinformatics tool BIOPEP-UWM (https: / / biochemia.uwm.edu.pl / ). The focus was on analyzing whether they have activities related to growth and development, such as promoting cell proliferation and regulating the cell cycle. The key amino acid sites of the target peptides were also evaluated. The results are shown in Table 3. A total of 1 protein that can act on developmental regulatory peptides, promote cell proliferation, and regulate the cell cycle and proliferation was screened.

[0049] Table 3 Results of targeted bioactivity analysis based on the BIOPEP-UWM database Example 2 Predict the absorption, distribution, metabolism, excretion, and toxicity properties of the peptide obtained in Example 1 (ADMET). First, the amino acid sequences of the peptides shown in Table 2 were converted into the Simplified Molecular Input Line Canonical System (SMILES). AdmetSAR (https: / / lmmd.ecust.edu.cn / admetsar2) was used to predict the ADME properties of the peptides. Peptides with easy absorption, good metabolic performance, and low (or non-toxic) toxicity were further analyzed through molecular docking. The screening results are shown in Table 4. Peptide FWAFFH has good solubility, long average retention time in the human body, good metabolic performance, and low acute oral toxicity.

[0050] Table 4 shows the ADME characteristic prediction of the screened peptides using SMILES codes. Example 3 The peptide FWAFFH was molecularly docked with Frizzled class receptor 7, one of the important receptors in the Wnt / β-catenin signaling pathway. The molecular docking method is as follows: (1) Ligand: The 3D structure of the polypeptide was constructed using Chem Draw 20.0 and the energy was minimized. The structure was then saved in “mol2” format.

[0051] (2) Receptor: The structure of Frizzled class receptor 7 was obtained from the RCSB protein database (http: / / www.rcsb.org), PDBID 5T44; the receptor protein (Frizzled class receptor 7) was dehydrated, impurity removed and hydrogenated using PyMol 3.0.4 and AutoDockTools 1.5.7 and then exported as a file as the receptor.

[0052] (3) Molecular docking execution: Molecular docking was performed using AutoDock Vina. All results were visualized and analyzed using PyMol, PLIP, and Discovery Studio. The optimal binding conformation of the peptide with Frizzled class receptor 7 was predicted based on the binding energy score. The interaction between the peptide and the active site of Frizzled class receptor 7 was constructed, and the results are as follows: Figure 1 As shown in Table 5; Figure 1In the 3D diagram, blue lines represent hydrogen bonds, and gray dashed lines represent hydrophobic interactions. The minimum binding energy between the peptide and Frizzled class receptor 7 is -9.4 kcal / mol, and the negative binding energy indicates that the interaction between the peptide and Frizzled class receptor 7 is spontaneous. The diagram clearly shows that its interaction region consists of 12 amino acid residues: THR-28, GLU-52, GLN-55, TYR-57, GLN-111, ASP-48, LEU-51, PHE-56, PRO-58, LEU-59, LYS-61, and PHE-110. The peptide interacts with the Frizzled class receptor... The 6 amino acids (ASP-48, LEU-51, PHE-56, PRO-58, LEU-59, LYS-61) at the 7 active sites form 8 hydrophobic interactions, 7 hydrogen bonds with 5 amino acids (THR-28, GLU-52, GLN-55, TYR-57, GLN-111), 2 π-Stacking interactions with PHE-110, and 1 π-Cation interaction with LYS-61. The π-π stacking and π-cation interactions... Figure 1 The 3D diagram does not show the residues and bond lengths in line form; see Table 5 for details. It can be seen that hydrogen bonds and hydrophobic interactions are the main forces driving the catalytic binding of the peptide to Frizzled class receptor 7. Only hydrogen bond interactions smaller than 5 Å are shown in the 3D diagram.

[0053] Table 5. Interaction between FWAFFH and Frizzled class receptor 7 Example 4 Artificial synthesis of FWAFFH The resin used in this embodiment is 2-Chlorotrityl Chloride resin with a degree of substitution of 1.1 mmol / g. A solid-phase synthesis method was employed, following the synthetic sequence from the C-terminus to the N-terminus (i.e., histidine-phenylalanine-phenylalanine-alanine-tryptophan-phenylalanine). The specific steps are as follows: (1) Initiation and coupling: After swelling the resin in N,N-dimethylformamide (DMF), the carboxyl group of the first amino acid at the C-terminus (i.e. histidine, whose α-amino group is protected by the Fmoc group) is covalently linked to the resin until the target sequence is completed.

[0054] (2) Deprotection: Remove the Fmoc protecting group of the newly attached amino acid with a decapping solution (N,N-dimethylformamide containing piperidine) to expose the free α-amino group.

[0055] (3) Activation and coupling of the next amino acid: Repeat the following steps in the sequence phenylalanine-phenylalanine-alanine-tryptophan-phenylalanine: Activation: The next Fmoc protected amino acid was pre-activated with activators O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU, CAS: 94790-37-1) and N,N-diisopropylethylamine (DIEA, CAS: 7087-68-5) in piperidine-containing N,N-dimethylformamide.

[0056] Coupling: An activating solution is added to the resin, causing the carboxyl group of the new amino acid to undergo a condensation reaction with the exposed amino group on the resin to form a peptide bond.

[0057] Detection and capping: The coupling was verified to be complete using the ninhydrin detection method. For unreacted amino groups, the coupling was capped with an acetic anhydride / pyridine mixture to prevent the generation of byproducts with deleted sequences. The acetic anhydride / pyridine mixture was prepared in the laboratory, with acetic anhydride and pyridine mixed at a volume ratio of 1:1.

[0058] (4) Final cleavage and purification: After synthesizing the target sequence, the complete peptide chain was cleaved from the resin using a strong acid cleavage buffer (the volume ratio of trifluoroacetic acid, ethylenedithiol, triisopropylsilane and distilled water in the strong acid cleavage buffer was 95:2:2:1), and all side chain protecting groups were removed at the same time; the filtrate containing the product was poured into cold diethyl ether to precipitate the crude peptide, and the crude product was obtained after centrifugation and washing, and the crude product was purified to 98% by high performance liquid chromatography.

[0059] (5) Preparation of peptide solution: The prepared peptide was prepared into an FWAFFH active peptide solution with a concentration of 8 mg / mL using sterile water containing 1% DMSO (dimethyl sulfoxide).

[0060] Example 5 The viability assays of HDPCs cells using FWAFFH, Frizzled class receptor 7 inhibitor (FZ7-21), and Wnt / β-catenin signaling pathway inhibitor (DKK-1) were performed using the following steps: (1) Observe the growth status of HDPCs under a microscope and select cells in the logarithmic growth phase for the experiment.

[0061] (2) After removing cell waste fluid, rinse with 2 mL of PBS buffer (phosphate buffer) to remove cell metabolic waste and dead cells. Then digest the cells with 1 mL of trypsin and gently blow the bottom of the culture dish with 2 mL of DMEM high sugar medium to detach the cells and disperse them evenly to obtain a cell suspension.

[0062] (3) Take 20 μL of cell suspension, dilute it 20 times with PBS buffer, and count the cells. Calculate the total number of cells. Based on the requirement of 5 × 10⁶ cells per well in a 96-well plate. 3 Calculate the amount of cells and prepare the required cell suspension. Add 90 µL of cell suspension evenly to each well.

[0063] (4) After culturing for 24 hours, the supernatant was discarded and replaced with basal medium. Then, 400, 200, and 100 μg / mL of FWAFFH active peptide (603) solution, 5, 20, 10, 5, 2.5, and 1 μM of FZ7-21, and 1, 0.5, and 0.25 μg / mL of DKK-1 were added to the wells respectively. The wells with the added active peptide solution and inhibitor were set as experimental groups; the wells containing only cells were used as control groups; and the wells without cells, active peptide solution, and inhibitor were set as apoptosis groups. Three replicate wells were set up for each group.

[0064] (5) After culturing for 24 hours, add 25 μL of TCA fixative (trichloroacetic acid solution) to each well and fix for 1 hour and 20 minutes. Wash the culture plate with running water and air dry.

[0065] (6) Add 100 μL of SRB staining solution (sulfonylrhodamine B) to each well of the fixed dry culture plate and stain for 30 min. Wash the plate with a plate washer and air dry.

[0066] (7) Add 100 μL of Tris-HCl (tris(hydroxymethyl)aminomethane) to each well of the stained culture plate to dissolve the cells, shake on a shaker for 10 min, and measure the absorbance at 570 nm using a UV spectrophotometer.

[0067] (8) Calculate cell viability using the following formula: Cell viability (%) = [(OD)] （实验组） -OD （对照组） ) / OD （对照组） ×100% (5.1) The calculation results are as follows Figure 2 As shown, FWAFFH and its inhibitors at different concentrations have no toxic effects on cells.

[0068] Example 6 The effect of FWAFFH on the migration activity of HDPCs was investigated using the following steps: (1) Before starting cell plating, you need to draw even horizontal lines on the back of the 6-well plate with a marker. The lines that pass through the wells should be spaced about 0.5-1cm apart to ensure that the lines are clear and equidistant, which will facilitate subsequent observation.

[0069] (2) Observe the cell growth status under a microscope, and select cells in the logarithmic growth phase and in good growth condition for the experiment; after removing cell waste, rinse with 2 mL of PBS buffer to remove cell metabolic waste and dead cells, then digest the cells with 1 mL of trypsin, and gently blow the bottom of the culture dish with 2 mL of culture medium to detach the cells and disperse them evenly. Take 20 μL of cell suspension, dilute it 20 times with PBS, and count the cells to calculate the total number of cells. According to the requirement of 5 × 10⁶ cells per well of a 6-well plate. 3 Calculate the number of cells and prepare the required cell suspension.

[0070] (3) Then, the cells were evenly seeded into the 6-well culture plate at a rate of 1.8 mL per well to ensure that the cells could completely cover the well plate the next day.

[0071] (4) After 24 hours, using a ruler perpendicular to the horizontal line behind the cell, the same pipette tip was used to make cell scratches in each well, so that the scratches and the lines on the culture plate formed a "#" shape. Then, the cells were washed with PBS to remove the floating cells generated during the scratching. Serum-free culture medium containing 400 μg / mL FWAFFH, 0.5 μg / mL DKK1 + 400 μg / mL FWAFFH, and 2.5 μM FZ7-21 + 400 μg / mL was added to the well as the experimental groups. At the same time, control groups with only culture medium added, and control groups with only DKK1 and only FZ7-21 added were also set up. The concentrations of the inhibitors DKK1 and FZ7-21 were obtained from Example 5 and had no effect on cell viability.

[0072] (5) Subsequently, the 6-well plate was placed under an inverted fluorescence microscope for photographic recording, and images were acquired at 0h and 24h after scratching.

[0073] (6) Finally, the cell migration in the photographs was analyzed using ImageJ software, and the results are as follows: Figure 3 As shown, FWAFFH improved the migration ability of HDPCs cells. The inhibitors DKK1 and FZ7-21 inhibited the migration ability of HDPCs cells. However, after adding FWAFFH to the inhibitors DKK1 and FZ7-21, the inhibited migration ability of HDPCs cells was improved, indicating that FWAFFH has a promoting effect and can promote the growth and development of HDPCs cells and improve their migration activity.

[0074] Example 7 The effects of FWAFFH on the expression levels of key proteins in the Wnt / β-catenin signaling pathway, including Beta-catenin, C-myc, WNT10B, GAPDH, Cyclin-D1, LEF1, and β-ACTIN, were investigated using the following steps: (1) Observe the growth status of cells under a microscope, and select cells in the logarithmic growth phase and in good growth condition for the experiment; after removing cell waste, rinse with 2 mL of PBS buffer to remove cell metabolic waste and dead cells, then digest the cells with 1 mL of trypsin, and gently blow the bottom of the culture dish with 2 mL of culture medium to detach the cells and disperse them evenly. Take 20 μL of cell suspension, dilute it 20 times with PBS, and count the total number of cells. According to the requirement of 5 × 10⁶ cells per well of a 6-well plate, the total number of cells is 1000. 3 Calculate the number of cells and prepare the required cell suspension.

[0075] (2) Then, the cells were evenly seeded into the 6-well culture plate at a rate of 1.8 mL per well to ensure that the cells could completely cover the well plate the next day.

[0076] (3) After culturing for 24 hours, the waste culture medium was removed and replaced with serum-free basal culture medium. The experimental groups were prepared by adding 400 μg / mL FWAFFH, 0.5 μg / mL DKK1 + 400 μg / mL FWAFFH, and 2.5 μM FZ7-21 + 400 μg / mL serum-free culture medium to the wells. At the same time, a control group was set up by adding only culture medium.

[0077] (4) Continue incubation for 24 hours. After the drug incubation is completed, aspirate the cell supernatant, rinse 1-2 times with pre-cooled 1×PBS buffer, add 200 μL of protein lysis buffer (the volume ratio of protein lysis buffer: β-mercaptoethanol: PMSF is 100:1:1, and it should be prepared fresh for use), and incubate on ice for 10 min for lysis. After lysis is completed, use a pipette to blow the cells from the bottom of the dish into a 1.5 mL EP tube.

[0078] (5) The sample was broken up in a cell ultrasonic disruptor with a power of 30W for 4 minutes. The sample was repeatedly operated until it was blown into a watery state. Then the sample was placed in a metal bath at a temperature of 98℃ for 10 minutes to denature the protein. Finally, it was centrifuged at 4℃ and 14200rpm for 10 minutes. The supernatant was aliquoted as needed and stored in a -80℃ refrigerator for later use.

[0079] (6) Preparation of separating and stacking gels: First, after cleaning two glass plates and ensuring there is no residual gel, assemble them and add pure water to check for leaks; according to the molecular weight of the target protein, select the appropriate concentration of separating gel for preparation; after preparing 10% separating gel, add TEMED and mix thoroughly, then immediately pour it between the glass plates to 1 cm below the comb, then add isopropanol to press the gel to the same horizontal line. After the separating gel solidifies for about 60 minutes, pour out the isopropanol and use absorbent paper to absorb the residual liquid in the plate; next, add 5% stacking gel and slowly insert the comb along the gap (to avoid the generation of air bubbles). After the gel solidifies, it can be temporarily stored in a -4℃ refrigerator.

[0080] (7) Electrophoresis: After the gel has completely solidified, take out the sample prepared in step (5), put it in a 98℃ metal bath and boil for 3 minutes. After shaking and mixing, centrifuge, clamp the solidified gel in the electrophoresis tank, pour in the electrophoresis solution, and after ensuring no leakage, vertically pull up the comb and add the prepared sample to the electrophoresis well. Add electrophoresis solution to the outer tank to cover the electrodes. Use the segmented electrophoresis method. First, use a voltage of 60V to press the protein sample into a straight line, and then use a constant voltage of 130V to separate the protein in the separating gel. When the sample indicator is close to the bottom of the glass plate by about 0.5cm, the electrophoresis can be stopped.

[0081] (8) Transfer: Before transfer, the 0.45 μm polyvinylidene fluoride (PVDF) membrane is activated by soaking in methanol for about 5 min. At the same time, the sponge filter paper is soaked in pre-cooled 1× transfer solution to ensure that it is fully wetted. Then, the gel after electrophoresis is removed from the electrophoresis tank and the protein separated in the SDS-PAGE gel is transferred to the 0.45 μm PVDF membrane by the "sandwich" wet transfer method at a constant current of 250 mA. During the transfer process, it is necessary to avoid the formation of air bubbles between the PVDF membrane, the gel and the sponge pad. Since heat is generated during the transfer process, ice boxes should be placed around the transfer device to help dissipate heat. The transfer time should be adjusted appropriately according to the molecular weight of the target protein to ensure that the protein is completely transferred to the membrane.

[0082] (9) After electroporation, remove the PVDF membrane with protein bands and marker labels, wash away the residual electroporation solution with 1×TBST solution, then immerse the membrane in 5% skim milk powder (dissolved in 1×TBST) and block it on a decolorizing shaker at room temperature for 2 hours; after blocking, wash the PVDF membrane with 1×TBST on a horizontal decolorizing shaker for 5 minutes, repeat 3 times, cut according to the size of the target protein, and then incubate overnight at 4°C with primary antibody. The primary antibody used includes: Beta-catenin rabbit polyclonal antibody (Proteintech), diluted 1:5000.

[0083] β-actin mouse monoclonal antibody (Proteintech), diluted at a ratio of 1:10000.

[0084] GAPDH mouse monoclonal antibody (Proteintech), diluted at a ratio of 1:10000.

[0085] Cyclin D1 Rabbit polyclonal antibody (Proteintech), diluted 1:5000.

[0086] Lef-1 rabbit monoclonal antibody (ABclonal, diluted 1:5000).

[0087] Wnt10b rabbit antibody (Signalway Antibody), diluted at a ratio of 1:5000.

[0088] After 12 hours, the membrane was removed and placed on a room temperature decolorizing shaker. It was washed with 1×TBST for 10 minutes, repeated 3 times. Secondary antibody was then incubated at room temperature for 2 hours. The secondary antibody used was: Horseradish peroxidase-conjugated goat anti-rabbit IgG (Proteintech), diluted 1:7000.

[0089] Horseradish peroxidase-conjugated goat anti-mouse IgG (Proteintech), diluted 1:7000.

[0090] Continue washing with a decolorizing shaker for 10 minutes, repeating 3 times; the primary and secondary antibodies used in the experiment were prepared with 2% skim milk powder (1×TBST diluted), while the phosphorylated proteins were prepared with 2% BSA (1×TBST dissolved).

[0091] (10) After washing the membrane, perform chemiluminescence detection. Use the MeilunBio Fibre Ultrasensitive ECL Chemiluminescence Detection Kit (MeilunBio, Meilun Biotechnology, China, Catalog No.: MA0186-1). According to the instructions, mix MeilunBio Fibre Ultrasensitive ECL Solution A (luminescent substrate) and MeilunBio Fibre Ultrasensitive ECL Solution B (oxidant) in a 1:1 volume ratio. Add the mixture evenly to the surface of the PVDF membrane to completely cover the membrane. Incubate at room temperature for 1-2 min and then perform chemiluminescence signal detection and imaging.

[0092] (11) Immunoblotting results are as follows Figure 4 As shown, after the addition of peptide FWAFFH, the expression levels of key proteins in the Wnt / β-catenin signaling pathway, namely β-catenin, C-myc, Wnt10b, Cyclin D1, and LEF1, increased, and peptide FWAFFH could alleviate the inhibitory effects of inhibitors FZ7-21 and DKK-1 on these key proteins. Quantitative analysis of the expression levels of these key proteins yielded the following results: Figure 5 As shown, compared to the control group, the peptide FWAFFH significantly promoted the expression of β-catenin, C-myc, Wnt10b, CyclinD1, and LEF1; indicating that the peptide FWAFFH can activate the Wnt / β-catenin signaling pathway and upregulate the expression of downstream key proteins such as β-catenin, C-myc, CyclinD1, and LEF1, while also promoting the expression of Wnt10b and alleviating the decrease in protein expression caused by the inhibitors DKK1 and FZ7-21.

[0093] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A polypeptide, characterized in that, Its amino acid sequence is: Phe-Trp-Ala-Phe-Phe-His.

2. The use of the polypeptide of claim 1 in the preparation of a drug for preventing and treating hair loss.

3. The use of the polypeptide of claim 1 in the preparation of daily chemical products for preventing and treating hair loss.

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