Polypeptide and application thereof in preparation of medicine for promoting wound healing
By preparing the peptide VTHFPDSRRPIS, the problem of delayed or abnormal wound healing was solved, and HaCaT cell proliferation and migration were significantly promoted, EGFR expression was significantly upregulated, and wound healing and skin barrier repair were promoted.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- YUNNAN MINZU UNIV
- Filing Date
- 2026-03-27
- Publication Date
- 2026-05-19
AI Technical Summary
In existing technologies, delayed or abnormal wound healing can lead to increased pain and infection risk, affecting quality of life and potentially causing psychological problems. Furthermore, existing methods for promoting wound healing have limited effectiveness.
A polypeptide VTHFPDSRRPIS was prepared by hydrolyzing lyophilized leech powder with alkaline protease to isolate and identify polypeptides with wound-healing activity. The specific steps included dissolution, enzymatic hydrolysis, ultrafiltration and freeze-drying. It can be applied to the preparation of wound-healing drugs and skin barrier cosmetics.
The peptide VTHFPDSRRPIS significantly promoted HaCaT cell proliferation and migration at different concentrations, significantly upregulated EGFR phosphorylation expression levels, and promoted wound healing and skin barrier repair.
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Figure CN122060033A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of polypeptide technology, specifically to a polypeptide and its application in the preparation of drugs that promote wound healing. Background Technology
[0002] Wound healing is a complex biological process that refers to the process by which the skin or other tissues restore their structure and function after being damaged through a series of orderly cellular and molecular events. It is the most common tissue repair phenomenon in clinical practice. Although most acute wounds heal smoothly, delayed or abnormal healing not only affects physical appearance but may also significantly reduce the patient's quality of life. For example, chronic wounds may cause pain and an increased risk of infection, which in turn affects daily activities and may even trigger psychological problems such as anxiety and depression, causing long-term impacts on social and mental health.
[0003] Wound healing is a complex and highly ordered biological process involving the synergistic regulation and dynamic interactions among various cells, cytokines, and the extracellular matrix. Keratinocytes (HaCaT), as the main constituent cells of the epidermis, play a crucial role in wound repair. Their proliferation and migration activities directly determine the efficiency of re-epithelialization and are the core link in wound closure and tissue regeneration. The stronger the cell proliferation and migration capacity, the stronger the wound repair capacity. Epidermal growth factor receptor (EGFR), as a key molecule regulating wound healing, can effectively promote wound closure and tissue repair and reconstruction by mediating key biological processes such as re-epithelialization, angiogenesis, inflammatory response, and collagen deposition. The higher the expression level of EGFR, the stronger the wound healing capacity.
[0004] Hirudo medicinalis ( Poecilobdella manillensis The Mani medicinal leech, also known as the golden-edged (copper-edged) leech, belongs to the phylum Annelida, class Hirudinea, family Hirudinidae, genus Hirudinea. It is a higher invertebrate that is hermaphroditic. It is relatively large, with a body length of 107-113 mm and a width of 8-12.5 mm. It feeds on the blood of mammals and mainly lives in paddy fields, ditches, and ponds. The individuals are relatively large, and when alive, their backs are olive green or yellowish-brown with a broken or continuous bluish-gray longitudinal stripe on their backs.
[0005] Previous studies have reported that Hirudo medicinalis possesses biological activities such as anticoagulation, antithrombosis, antiplatelet aggregation, and angiogenesis. Summary of the Invention
[0006] To address the shortcomings of existing technologies, one objective of this invention is to provide a polypeptide with the following amino acid sequence: Val-Thr-His-Phe-Pro-Asp-Ser-Arg-Arg-Pro-Ile-Ser (VTHFPDSRRPIS).
[0007] A second objective of this invention is to provide a method for preparing the aforementioned polypeptide, wherein the polypeptide is prepared from lyophilized leech powder hydrolyzed by alkaline protease, and the specific steps are as follows: (1) Dissolve the freeze-dried leech powder in ultrapure water, add alkaline protease for enzymatic hydrolysis, and collect the hydrolysate after centrifugation.
[0008] (2) The liquid obtained in step (1) is subjected to ultrafiltration to collect components with a molecular weight of less than 3000 Da.
[0009] (3) Freeze-dry the target component obtained in step (2).
[0010] (4) The dried product obtained in step (3) was subjected to LC-MS / MS detection and peptide sequence analysis and identification. Finally, the peptide with the sequence VTHFPDSRRPIS was screened.
[0011] Preferably, in step (1), the solid-liquid ratio of lyophilized leech powder to ultrapure water is 1:5, with units of g:mL.
[0012] Preferably, in step (1), the amount of enzyme added is 500 U / g, the temperature of enzymatic hydrolysis is 55℃, and the time of enzymatic hydrolysis is 4h.
[0013] A third objective of this invention is to provide an application of the polypeptide in the following two aspects: (1) Application of polypeptides in the preparation of drugs that promote wound healing.
[0014] (2) Application of peptides in the preparation of cosmetics that help repair the skin barrier.
[0015] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention is the first to discover a polypeptide VTHFPDSRRPIS that can promote wound healing.
[0016] (1) The polypeptide VTHFPDSRRPIS described in this invention has significant proliferation and migration-promoting effects on HaCaT cells at concentrations of 200 μg / ml, 100 μg / ml and 50 μg / ml, suggesting that the polypeptide has strong wound healing activity.
[0017] (2) The polypeptide VTHFPDSRRPIS described in this invention can significantly upregulate the phosphorylation expression level of EGFR at 200 μg / ml, 100 μg / ml and 50 μg / ml, indicating that the polypeptide can promote wound healing by activating the EGFR receptor. Attached Figure Description
[0018] Figure 1 For the assay of VTHFPDSRRPIS cell proliferation activity.
[0019] Figure 2 This is an experiment to promote cell healing using VTHFPDSRRPI.
[0020] Figure 3 The graph shows the determination and quantification of the effect of VTHFPDSRRPIS on EGFR protein. Detailed Implementation
[0021] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0022] Example 1 Preparation of polypeptides from Hirudo medicinalis The leech sample used in this embodiment was provided by Huazhi Pharmaceutical Co., Ltd., and was a lyophilized powder (300 mesh). The specific steps are as follows: (1) Add the freeze-dried leech powder to ultrapure water. The solid-liquid ratio of the freeze-dried leech powder to ultrapure water is 1:5 (g:mL). Extract with ultrasonic assistance at 25℃. Then, enzymatically hydrolyze the solution with alkaline protease at 55℃. The enzyme dosage is 3000U / g (g:mL) and the hydrolysis time is 4 h.
[0023] (2) After centrifugation, the enzymatic hydrolysate was collected and further ultrafiltration was used to obtain three different molecular weight components (i.e., FNZ-4 (Mw: >10kDa), FNZ-5 (Mw: 3-10 kDa) and FNZ-6 (Mw: 0-3kDa).
[0024] (3) Freeze-dry the target component obtained in step (2).
[0025] (4) Screening for HaCaT cell proliferation activity showed that the FNZ-6 component had very good proliferative activity. Therefore, the component with a molecular weight of 0-3 kDa was selected for the next experiment.
[0026] (5) Analyze the peptide mass spectrometry information in 0–3 kDa obtained in step (3) using liquid chromatography-tandem mass spectrometry (LC-MS / MS). Chromatographic conditions: Analytical column: 150 μm id × 150 mm, packed with Acclaim PepMapRPLC C18, 3 μm, 100 Å, mobile phase A is water containing 0.1% formic acid, mobile phase B is 80% acetonitrile containing 0.1% formic acid, flow rate 600 nL / min. The elution gradients were as follows: 0-2 min (4-8% B), 2-45 min (8-28% B), 45-55 min (28-40% B), 55-56 min (40-95% B), and 56-66 min (95% B). Mass spectrometry conditions were as follows: Level 1 mass spectrometry parameters: resolution 70000, AGC target: 3e6, maximum injection time 100 ms, scan range 100 to 1500 mass-to-charge ratio. Level 2 mass spectrometry parameters were as follows: resolution 17500, AGC target: 1e5, maximum injection time 50 ms, TopN: 20, NCE / stepped NCE: 28. For mass spectrometry results analysis, the Byonic database was searched using the software uniprotkb_taxonomy_Poecilobdellamanillensis. De novo sequencing was used to analyze the components of the identified peptide fragments. Among the identified peptides, peptide fragments with a "Score>200" were simultaneously screened. Then, the bioactivity prediction was performed using the computer bioinformatics tool BIOPEP-UWM (https: / / biochemia.uwm.edu.pl / ), retaining peptides with potential biological activity for wound healing, as shown in Table 1.
[0027] Table 1 Screening of peptides with potential biological activity for wound healing The bioactivity of the peptides in Table 1 was predicted using the computer bioinformatics tool BIOPEP-UWM (https: / / biochemia.uwm.edu.pl / ), and the results are shown in Table 2. The VTHFPDSRRPIS peptide has multiple wound healing-related activities, including antimicrobial peptide, hemolytic peptide, neuropeptide, and DPP-IV inhibitor, and has the potential to promote wound healing.
[0028] Table 2 Example 2 Predicted properties of absorption, distribution, metabolism, excretion, and toxicity of the polypeptides shown in Table 2 (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 (http: / / lmmd.ecust.edu.cn / admetsar1) was used to predict the ADMET properties of the peptides shown in Table 2, and the results are shown in Table 3. According to Table 3, the peptides shown in Table 2 have good solubility, extremely low risk of drug interactions, high local safety, and no central toxicity.
[0029] Table 3 uses SMILES codes to predict ADME characteristics of the peptides shown in Table 2. Example 3 The peptide VTHFPDSRRPIS was molecularly docked with EGFR. The molecular docking method is as follows: The 3D structure of the peptide was constructed using ChemDraw 20.0, and energy minimization was performed before converting it to "mol2" format. The EGFR structure was obtained from the RCSB protein database (http: / / www.rcsb.org), PDBID 1M17. Subsequently, the receptor protein EGFR 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. Molecular docking was performed using AutoDock Vina. All results were visualized and analyzed using PyMol and LigPlot. The optimal binding conformation of the peptide and EGFR was predicted based on the binding energy score. The interaction between the peptide and the active site of EGFR was constructed. The results are shown in Table 4. Seven binding conformations were established between VTHFPDSRRPIS and EGFR (GLY-850 (2.2 Å), VAL-852 (2.8 Å), VAL-852 (2.6 Å) and EGFR (GLY-850 (2.2 Å), VAL-852 (2.8 Å), VAL-852 (2.6 Å)). The VTHFPDSRRPIS molecules exhibit hydrogen bond interactions with EGFR, including Å2, ALA-698 (2.2 Å), LYS-721 (2.5 Å), THR-766 (2.0 Å), and MET-769 (2.5 Å). The binding energy between VTHFPDSRRPIS and EGFR is -7.3 kcal / mol, indicating a relatively stable bond and good binding activity. Table 4 shows the interaction between VTHFPDSRRPIS and EGFR.
[0030] Table 4. Interaction between VTHFPDSRRPIS and EGFR Example 4 The viability assay of HaCaT cells using a polypeptide with the sequence VTHFPDSRRPIS (synthesized by Shanghai Qiangyao Biotechnology Co., Ltd.) was performed using the following steps: (1) When the HaCaT cell coverage in the culture dish reaches about 70%, remove the old culture medium and wash the cells 2-3 times with 2 mL PBS along the wall of the culture dish. Then add 1 mL trypsin to digest the cells. After digestion, immediately add 2 mL of the corresponding complete culture medium (DMEM high glucose medium with 10% fetal bovine serum (FBS)) to stop the digestion. Pipe the cells to detach them, make a uniform cell suspension and count them.
[0031] (2) Cell plating and culture: Based on the requirement of 5×10³ cells per well in a 96-well plate, calculate the total volume of cell suspension required and add 90 µL of cell suspension to each well; after labeling the cell plate, place it in a cell culture incubator and culture for 24 h.
[0032] (3) Drug treatment: After culturing cells for 24 h, solutions containing 0, 50, 100 and 200 µg / mL peptides were added to the corresponding cell wells, with 3 replicates for each concentration. The cells were then cultured for another 24 h to allow the drug to take effect.
[0033] (4) Cell fixation and washing: After 24 h of drug treatment, 25 µL of 50% TCA (trichloroacetic acid) solution was added to each well to fix the cells. The cell plate was placed in a 4℃ refrigerator and left to stand for 1.5 h. After fixation, the cell plate was washed 5 times with ultrapure water and then placed in a fume hood to dry.
[0034] (5) Staining and destaining: After the cell plate is dried, add 100 µL of 0.4% SRB (sulfonylrhodamine B) staining solution to each well using a pipette and stain in the dark for 30 min. After staining, wash away excess SRB staining solution with 1% HAC (acetic acid) washing solution using a plate washer, and then put the cell plate into a fume hood to dry.
[0035] (6) Detection and result processing: Add 100 µL of 10 mM Tris solution to each well of the dried cell plate to dissolve the SRB dye. After shaking for 10 min, use an ELISA reader to detect the OD value of each well at a wavelength of 570 nm and record it in detail. Calculate the average OD value at each concentration. To ensure the accuracy and stability of the experimental results, each cell proliferation experiment should be repeated at least 3 times, and 3 replicate wells should be set for each concentration each time.
[0036] During wound healing, keratinocytes (HaCaT) play a central role, restoring the epidermal barrier function through proliferation, migration, and differentiation. Therefore, keratinocytes, which constitute the largest proportion of the epidermis, were selected to investigate the cell proliferation-promoting activity of the peptide VTHFPDSRRPIS at the in vitro level. The calculation results are as follows: Figure 1As shown, different concentrations of VTHFPDSRRPIS have an effect on cell proliferation. VTHFPDSRRPIS promotes the proliferation of HaCaT cells, indicating that this peptide has the potential to promote wound healing and skin barrier repair.
[0037] Example 5 The peptide with the sequence VTHFPDSRRPIS exhibits healing-promoting activity against HaCaT cells. (1) When the cell coverage in the culture dish reaches 70%, begin preparations for plating; mark the bottom of the 6-well plate with a marker, and then adjust the cell suspension concentration to ensure that the number of cells seeded in each well is 5 × 10⁶. 4 Add 1.8 mL of complete culture medium to each well.
[0038] (2) Place the 6-well plate with the cells in an incubator and incubate for 24 h until the cells adhere and grow to full size.
[0039] (3) After culturing for 24 h, first aspirate the original culture medium in the well; using a ruler as an aid, use a 200 µL pipette tip to draw a line evenly along the vertical direction of the culture dish, ensuring that the line width and depth are consistent throughout the process; then rinse with PBS solution to remove suspended cells and replace with serum-free culture medium (DMEM high glucose medium).
[0040] (4) Add polypeptide solutions containing different concentrations of 0, 50, 100 and 200 µg / mL to the corresponding cell wells and continue culturing.
[0041] (5) Place the 6-well plate under a fluorescence inverted microscope and take pictures at three time points: 0 h, 6 h and 12 h after scratching.
[0042] (6) Use ImageJ software to measure and analyze the cell migration recorded by photographs. Calculate the migration rate according to the formula “migration rate (%) = (initial area - 0 h / 6 h / 12 h area) / initial area × 100%”. To ensure the reliability of the results, the above experiments should be repeated at least 3 times.
[0043] The results are as follows Figure 2 The results showed that VTHFPDSRRPIS significantly enhanced the scratch healing activity of HaCaT cells, demonstrating that VTHFPDSRRPIS has strong activity in promoting wound healing and skin barrier repair.
[0044] Example 6 Effect of the peptide with the sequence VTHFPDSRRPIS on EGFR protein (1) When HaCaT cells reach 70% coverage, plate them: remove the old culture medium, rinse along the cell wall with 2 mL PBS; add 1 mL trypsin to digest for 4 min, count the cells, and adjust the cell suspension concentration to 3 × 10⁻⁶.6 Each cell / well was seeded at 1.8 mL / well in a 6-well plate and cultured for 24 h. Then, peptide solutions of different concentrations (0, 50, 100, and 200 µg / mL) were added, and the drug was allowed to act for 24 h.
[0045] (2) 24 h after drug treatment: the 6-well plate was transferred to the operating table, the culture medium was discarded, and the plate was washed with PBS 2-3 times. The plate was placed in an ice box, and 350 µL of protein lysis buffer containing mercaptoethanol was added to each well. The plate was repeatedly pipetted with a 1 mL pipette to remove the protein from the wall. The plate was placed on ice for 10 min to lyse. The lysate was transferred to a 1.5 mL centrifuge tube and sonicated until the liquid was no longer viscous (3 min each time). The plate was heated in a 98℃ metal bath for 10 min to denature the protein. The plate was centrifuged at 14000 rpm and 4℃ for 10 min. The supernatant was aliquoted and stored at -20℃.
[0046] (5) Preparation of separating gel and stacking gel: Prepare 5 mL of 8% separating gel and shake to mix well; slowly inject 4.5 mL of gel solution into the gap between the glass plates (avoid air bubbles), immediately add 1 mL of isopropanol solution to seal the air bubbles, let stand and wait for solidification, after the separating gel has completely solidified, pour it out and absorb the residual isopropanol; prepare 2 mL of 5% stacking gel according to the ratio and shake to mix well; after injecting 1.5 mL of stacking gel, insert the comb smoothly and wait for solidification.
[0047] (6) Sample loading and electrophoresis: Clamp the SDS-PAGE gel and glass plate together and place them in the electrophoresis tank. Add 1× electrophoresis buffer to cover the gel surface. Carefully remove the comb and heat the -20℃ refrigerated protein sample obtained in step (5) in a 98℃ metal bath for 5 minutes (preheat for 2-3 minutes to aid dissolution). After it becomes a homogeneous liquid, accurately add the protein marker and each protein sample to the corresponding well. Add 1× electrophoresis buffer to the water level line and turn on the power supply to 70V. After the sample enters the separating gel, adjust the voltage to 130V to accelerate separation. Stop electrophoresis when the sample migrates to about 0.5cm from the bottom of the glass plate and prepare for membrane transfer.
[0048] (7) Gel cutting and transfer: The 0.45 µm PVDF membrane was soaked in anhydrous methanol for 5 min to activate it; then the membrane was tightly attached to the SDS-PAGE gel and placed in the transfer apparatus; a constant current of 250 mA was set and the transfer was carried out for 1.5 h to transfer the protein from the gel to the membrane.
[0049] (8) Blocking and antibody incubation: After transfer, the PVDF membrane was blocked in 5% skim milk powder at room temperature for 1 h; the membrane strips were cut according to the molecular weight of the target protein and placed in primary antibodies (β-Tubulin, EGFR, p-EGFR) containing 2% skim milk powder and incubated overnight at 4°C.
[0050] (9) Washing and secondary antibody incubation: After primary antibody incubation, the membrane strip was washed 5 times with 1×TBST (5 min each time); then placed in secondary antibody (specifically binds to primary antibody (identifies the species source of primary antibody)) solution and incubated at room temperature for 1.5 h. After incubation, it was washed again with 1×TBST to reduce background.
[0051] (10) Development: Prepare the developer solution (A solution:B solution = 1:1, mix well, where A solution and B solution are reagents in the DAB colorimetric kit, purchased from Zhongshan Jinqiao, number: ZLI-9018); lay the washed membrane strip flat on plastic wrap, add the developer solution, and let it stand in the dark for 2 min; immediately develop it with a chemiluminescence analyzer and take a picture for storage.
[0052] The results are as follows Figure 3 As shown, VTHFPDSRRPIS significantly upregulated the phosphorylation level of EGFR protein in human immortalized epidermal keratinocytes (HaCaT), without significantly affecting the expression of total EGFR protein. EGFR, as a key receptor regulating epidermal cell function, requires phosphorylation for activation. Only activation through phosphorylation can initiate downstream signaling pathways, thereby promoting HaCaT cell proliferation, migration, and epithelialization, accelerating wound closure, and participating in skin barrier repair by enhancing keratinocyte differentiation and promoting the synthesis of filaggrin, keratin, and related barrier structure proteins. VTHFPDSRRPIS induced increased expression of phosphorylated EGFR (p-EGFR), suggesting that this peptide enhances the repair capacity of epidermal cells through activation of the EGFR signaling pathway, enabling it to play an important molecular mechanism in promoting wound healing and protecting the skin barrier.
[0053] 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: The amino acid sequence of the polypeptide is: Val-Thr-His-Phe-Pro-Asp-Ser-Arg-Arg-Pro-Ile-Ser.
2. The use of the polypeptide of claim 1 in the preparation of a wound-healing drug.
3. The use of the polypeptide of claim 1 in the preparation of cosmetics that help repair the skin barrier.