Self-assembled polypeptide with IGF-1 function and hair growth application thereof

Abstract

The invention relates to the technical field of biological medicine, and provides a self-assembly polypeptide with an IGF-1 function and hair growth application thereof, and the self-assembly polypeptide comprises an end-capping group, a self-assembly unit and an insulin-like growth factor C structural domain polypeptide IGF-1C which are connected in sequence. The self-assembled polypeptide with the IGF-1 function, provided by the invention, has a beta-folded secondary structure, and can simulate a binding mode of IGF-1 and an insulin-like growth factor receptor IGF-1R and induce conformational change of an IGF-1R dimer, so that phosphorylation of the IGF-1R is promoted, physiological functions of the IGF-1R are simulated, and growth of hair is promoted. The self-assembled polypeptide with the insulin-like growth factor 1 (IGF-1) function, provided by the invention, can be applied to an alopecia area in a skin smearing manner, and particularly shows an excellent hair growth promoting effect in a mouse physiological alopecia model and an androgenic alopecia model.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a self-assembling polypeptide with IGF-1 function and its application in hair growth. Background Art

[0002] Hair loss is a common clinical condition characterized by a disruption of the hair follicle cycle, caused by multiple factors including genetics, hormones, and the environment. Androgenetic alopecia (AGA) is the most common non-scarring form of hair loss, accounting for over 90% of all cases of hair loss. While the pathogenesis of androgenic alopecia is not fully understood, it is generally believed to be caused by the conversion of testosterone to dihydrotestosterone (DHT) via type II 5α-reductase. DHT, which is much more active than testosterone, overactivates the androgen receptor (AR) in hair follicles, leading to hair follicle atrophy and thinning of the hair shaft. Currently, the only FDA-approved treatments for hair loss are minoxidil and finasteride. However, minoxidil requires long-term use, and discontinuation of treatment can lead to renewed hair loss and adverse reactions such as allergic dermatitis and hirsutism. Long-term use of finasteride can cause side effects including sexual dysfunction, birth defects, and insulin resistance. Therefore, there is an urgent need for safer and more effective treatments for hair loss.

[0003] Insulin-like growth factor 1 (IGF-1), structurally similar to insulin, has been identified as a key cytokine regulating growth metabolism, playing a central role in cell proliferation and differentiation. IGF-1 is produced in hair follicles by dermal papilla cells. IGF-1 binds to the insulin-like growth factor 1 receptor (IGF-1R), which exists as a dimer, inducing a conformational change in the IGF-1R dimer, shifting the autoinhibitory state to an activated state. This in turn induces IGF-1R autophosphorylation, activating downstream signaling pathways and promoting hair follicle cell proliferation and migration. It plays a crucial role in regulating the hair cycle and hair shaft differentiation. Studies have shown that DHT inhibits hair follicle cell proliferation by suppressing IGF-1 production in dermal papilla cells, thereby hindering hair growth. This suggests that IGF-1 has significant potential for treating androgenic alopecia. However, clinical applications of IGF-1 protein face challenges such as high production costs, demanding transportation and storage conditions, as well as poor transdermal absorption and low bioavailability, which significantly limit its application. Therefore, developing a compound with IGF-1 functionality to replace IGF-1 protein in the treatment of hair loss is of great significance.

[0004] Peptides are a class of compounds between amino acids and proteins, composed of multiple amino acids linked by peptide bonds in a specific sequence. They offer advantages such as good biocompatibility, high stability, simple synthesis and purification, low production cost, small molecular weight, and high permeability, making them an excellent choice for replacing proteins. The self-assembly process of peptides is driven by non-covalent bonds. By altering the amino acid sequence of a peptide, its secondary structure and morphology can be manipulated, thereby improving its biological activity and pharmacokinetic properties, increasing drug penetration and retention at the site of action, and providing new insights into the development of novel, highly effective, and low-toxic biomaterials.

[0005] However, how to design and regulate the structure of the polypeptide so that it has the function of IGF-1 protein and can promote the proliferation of hair follicle cells and normalize the hair follicle cycle still faces huge challenges. Summary of the Invention

[0006] The present invention aims to provide a self-assembling polypeptide with IGF-1 function and its application in hair growth. The self-assembling polypeptide with IGF-1 function is prepared by sequentially linking a capping group, a self-assembling unit and an IGF-1C polypeptide derived from the C domain of insulin-like growth factor. The secondary structure of IGF-1C is regulated by adjusting the sequence of the capping group and the self-assembling unit, so that the secondary structure is converted into a β-fold, thereby better mimicking the spatial structure of IGF-1, enhancing binding to IGF-1R, promoting the autophosphorylation of IGF-1R and activating downstream pathways, thereby exerting a physiological function similar to that of IGF-1, and promoting the proliferation of hair follicle cells and hair growth.

[0007] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: The present invention provides a self-assembling polypeptide with IGF-1 function, wherein the self-assembling polypeptide comprises a capping group, a self-assembling unit and an insulin-like growth factor C domain polypeptide IGF-1C connected in sequence; The end-capping group is an acetyl group; the self-assembly unit is any one of GFFY, GFYF, GYFF, GFFYNKPT, GFYFNKPT, and GYFFNKPT.

[0008] Preferably, the amino acid sequence of the insulin-like growth factor C domain polypeptide IGF-1C is shown in SEQ ID NO.1.

[0009] Preferably, F and Y in the self-assembly unit are both L-amino acids or D-amino acids.

[0010] Preferably, the self-assembling polypeptide has a structure represented by any one of structural formulas I, II, III, IV or V: Formula I; Formula II; Formula III; Formula IV; Formula V.

[0011] The present invention also provides a bioactive solution containing the self-assembling polypeptide, wherein the solvent of the bioactive solution is water, and the concentration of the self-assembling polypeptide in the bioactive solution is 0.1-1000 μM.

[0012] Preferably, the bioactive solution is administered by skin application.

[0013] The present invention also provides the use of the self-assembling polypeptide or the bioactive solution in preparing a product for promoting hair growth.

[0014] Preferably, the hair growth product is a product for treating androgenic alopecia and alopecia areata.

[0015] The beneficial effects of the present invention are: (1) The self-assembling polypeptide with IGF-1 function provided by the present invention can mimic the structure of IGF-1 protein, efficiently bind to IGF-1R, and induce the autophosphorylation of IGF-1R, activating the downstream signaling pathway, thereby promoting the proliferation of hair follicle cells and promoting hair growth. It has demonstrated excellent hair growth-promoting effects in physiological hair loss models and androgenic alopecia models.

[0016] (2) The self-assembling polypeptide with IGF-1 function provided by the present invention has a small molecular weight, high transdermal absorption efficiency, good biocompatibility, and no negative impact on the body.

[0017] (3) The bioactive solution of the self-assembling polypeptide with IGF-1 function provided by the present invention has good stability. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The HPLC and mass spectrometry images of the self-assembling polypeptide Ac-GFFY-IGF-1C with IGF-1 function synthesized in Example 1 are shown, wherein the upper image is the HPLC chromatogram and the lower image is the primary mass spectrometry image; Figure 2 The HPLC and mass spectrometry images of the self-assembling polypeptide Ac-GFYF-IGF-1C with IGF-1 function synthesized in Example 2 are shown, wherein the upper image is the HPLC chromatogram and the lower image is the primary mass spectrometry image; Figure 3 The HPLC and mass spectrometry images of the self-assembling polypeptide Ac-GYFF-IGF-1C with IGF-1 function synthesized in Example 3 are shown, wherein the upper image is the HPLC chromatogram and the lower image is the primary mass spectrometry image; Figure 4 The HPLC and mass spectrometry images of the self-assembling polypeptide Ac-GFFYNKPT-IGF-1C with IGF-1 function synthesized in Example 4 are shown, wherein the upper image is the HPLC chromatogram and the lower image is the primary mass spectrometry image; Figure 5 The HPLC and mass spectrometry images of the self-assembling polypeptide Ac-GFYFNKPT-IGF-1C with IGF-1 function synthesized in Example 5 are shown, wherein the upper image is the HPLC chromatogram and the lower image is the primary mass spectrometry image; Figure 6 The HPLC and mass spectrometry images of the polypeptide Ac-IGF-1C synthesized in Comparative Example 1 are shown, wherein the upper image is the HPLC chromatogram and the lower image is the primary mass spectrometry image; Figure 7 This is a microscopic morphology of the polypeptide Ac-GFFY-IGF-1C synthesized in Example 1; Figure 8 This is a microscopic morphology of the polypeptide Ac-IGF-1C synthesized in Comparative Example 1; Figure 9 Circular dichroism spectra of the synthetic polypeptides of Example 1 and Comparative Example 1; Figure 10 This is a graph showing the binding force between the polypeptide Ac-GFFY-IGF-1C synthesized in Example 1 and the IGF-1R protein; Figure 11 This is a graph showing the binding force between the polypeptide Ac-IGF-1C synthesized in Comparative Example 1 and the IGF-1R protein; Figure 12 This is a dose-response graph of cell proliferation of the polypeptide Ac-GFFY-IGF-1C synthesized in Example 1; Figure 13 This is a confocal image of EdU fluorescence labeling; Figure 14 The figure is the result of western blot; Figure 15 This is a hair growth monitoring chart for the mouse physiological hair loss model; Figure 16 This is a hair growth monitoring chart for the mouse androgenic alopecia model; Figure 17 This is a graph for monitoring hair growth of the polypeptides of Examples 2 to 5. DETAILED DESCRIPTION

[0019] The present invention provides a self-assembling polypeptide with IGF-1 function, wherein the self-assembling polypeptide comprises a capping group, a self-assembling unit and an insulin-like growth factor C domain polypeptide IGF-1C connected in sequence; The capping group is preferably an acetyl group; the self-assembly unit is preferably any one of GFFY, GFYF, GYFF, GFFYNKPT, GFYFNKPT, and GYFFNKPT, and is further preferably any one of GFFY, GFYF, GYFF, GFFYNKPT, and GFYFNKPT.

[0020] In the present invention, the amino acid sequence of the insulin-like growth factor C domain polypeptide IGF-1C is preferably as shown in SEQ ID NO. 1; The amino acid sequence of SEQ ID NO. 1 is preferably: GYGSSSRRAPQT.

[0021] In the present invention, F and Y in the self-assembly unit are preferably both L-amino acids or D-amino acids.

[0022] In the present invention, the self-assembling polypeptide preferably has a structure represented by any one of structural formulas I, II, III, IV or V: Formula I; Formula II; Formula III; Formula IV; Formula V.

[0023] The present invention also provides a bioactive solution containing the self-assembling polypeptide. The solvent of the bioactive solution is preferably water. The concentration of the self-assembling polypeptide in the bioactive solution is preferably 0.1-1000 μM, more preferably 1-500 μM, and even more preferably 2-100 μM.

[0024] In the present invention, the bioactive solution is preferably administered by skin application.

[0025] The present invention also provides the use of the self-assembling polypeptide or the bioactive solution in preparing a product for promoting hair growth.

[0026] In the present invention, the hair growth product is preferably a product for treating androgenic alopecia and alopecia areata. The technical solutions provided by the present invention are described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0027] Example 1 Synthesis of polypeptide Ac-GFFYGYGSSSRRAPQT This embodiment provides a self-assembling polypeptide with IGF-1 function, whose structural formula is shown in Formula I. It is synthesized using the classic Fmoc-solid phase synthesis method. The resulting polypeptide is abbreviated as Ac-GFFY-IGF-1C. The polypeptide is synthesized by coupling from the C-terminus to the N-terminus.

[0028] Formula I.

[0029] The specific steps are as follows: 1) Weigh 430 mg (0.5 mmol) of 2-chlorotrityl resin into a solid phase synthesis tube, add 15 mL of DCM, and place on a shaker for 10 minutes to allow the 2-Cl-Trt resin to fully swell; 2) Use an ear bulb to completely squeeze out DCM from the solid phase synthesizer containing 2-Cl-Trt resin; 3) Coupling of the first amino acid: Dissolve 0.75 mmol of Fmoc-threonine (Fmoc-Thr-OH) in 10 mL of DCM, add 1.5 mmol of DIPEA, mix well, and add to the above-mentioned solid phase synthesizer. Incubate on a shaker at room temperature for 1 h. 4) Blocking: Use an ear bulb to blow out the reaction solution in the solid-phase synthesis tube, wash with DCM three times, each time using 10 mL, washing for 1 min, blow out the washing solution, add 20 mL of blocking solution (DCM:DIPEA:methanol = 17:1:2, volume ratio) to the solid-phase synthesis tube, and react at room temperature for 15 min; 5) Deprotection: Use an ear bulb to blow out the blocking solution in the solid-phase synthesis tube. Wash with DCM three times, 10 mL each time, for 1 min, then wash with DMF three times, 10 mL each time, for 1 min. Blow out the washing solution and add 15 mL of a DMF solution containing 20% piperidine (piperidine to DMF volume ratio of 1:4) to remove the Fmoc protecting group. React at room temperature for 30 min, then wash with DMF five times, 10 mL each time, for 1 min. 6) Amino acid coupling: Weigh 1 mmol each of the amino acid Fmoc-glutamine (Fmoc-Gln-OH) and HATU, add 2 mmol of DIPEA, dissolve completely in 10 mL of DMF, and add to the above solid phase synthesis tube. React at room temperature for 1 hour. 7) Repeat steps 5) and 6) to sequentially add amino acid and end-capping reactant acetic anhydride; then wash with DMF 3 times and DCM 3 times before proceeding to the next step; 8) Prepare 10 mL of a cutting solution containing 95% TFA, 2.5% TIS, and 2.5% H2O by volume and add it to the solid phase synthesis tube. React at room temperature for 1 hour. Collect the cutting solution in a round-bottom flask. Wash the solid phase tube three times with DCM, 10 mL each time, and collect the solution in a round-bottom flask. Remove the organic solvent using a rotary evaporator. Precipitate the polypeptide with glacial ether and filter to obtain a crude product. Then, separate and purify it using high performance liquid chromatography and freeze dry it in a freeze dryer to obtain the self-assembling polypeptide Ac-GFFY-IGF-1C with IGF-1 function in Example 1. The structural formula of the polypeptide in Example 1 is shown in Formula I. The purity and molecular weight were determined by high performance liquid chromatography and mass spectrometry. The results are shown in FIG. Figure 1 ,from Figure 1 It can be seen that the peptide was successfully synthesized and the purity was greater than 95%.

[0030] Example 2 Synthesis of polypeptide Ac-GFYFGYGSSSRRAPQT This example provides a self-assembling polypeptide with IGF-1 function, whose structural formula is shown in Formula II. It was prepared according to the Fmoc-solid phase synthesis method of Example 1. The obtained polypeptide is referred to as Ac-GFYF-IGF-1C. The purity and molecular weight were determined by high performance liquid chromatography and mass spectrometry. The results are shown in FIG. Figure 2 ,from Figure 2 It can be seen that the peptide was successfully synthesized and the purity was greater than 95%.

[0031] Formula II.

[0032] Example 3 Synthesis of polypeptide Ac-GYFFGYGSSSRRAPQT This example provides a self-assembling polypeptide with IGF-1 function, whose structural formula is shown in Formula III. It was prepared according to the Fmoc-solid phase synthesis method of Example 1. The obtained polypeptide is referred to as Ac-GYFF-IGF-1C. The purity and molecular weight were determined by high performance liquid chromatography and mass spectrometry. Figure 3 ,from Figure 3 It can be seen that the peptide was successfully synthesized and the purity was greater than 95%.

[0033] Formula III.

[0034] Example 4 Synthesis of polypeptide Ac-GFFYNKPTGYGSSSRRAPQT This example provides a self-assembling polypeptide with IGF-1 function, whose structural formula is shown in Formula IV. It was prepared according to the Fmoc-solid phase synthesis method of Example 1. The obtained polypeptide is referred to as Ac-GFFYNKPT-IGF-1C. The purity and molecular weight were determined by high performance liquid chromatography and mass spectrometry. Figure 4 ,from Figure 4 It can be seen that the peptide was successfully synthesized and the purity was greater than 95%.

[0035] Formula IV.

[0036] Example 5 Synthesis of polypeptide Ac-GFYFNKPTGYGSSSRRAPQT This example provides a self-assembling polypeptide with IGF-1 function, whose structural formula is shown in Formula V. It was prepared according to the Fmoc-solid phase synthesis method of Example 1. The obtained polypeptide is referred to as Ac-GFYFNKPT-IGF-1C. The purity and molecular weight were determined by high performance liquid chromatography and mass spectrometry. Figure 5 ,from Figure 5 It can be seen that the peptide was successfully synthesized and the purity was greater than 95%.

[0037] Formula V.

[0038] Comparative Example 1 Synthesis of polypeptide Ac-GYGSSSRRAPQT The polypeptide of Comparative Example 1 was prepared according to the Fmoc-solid phase synthesis method of Example 1. The obtained polypeptide is referred to as Ac-IGF-1C. The purity and molecular weight were determined by HPLC and mass spectrometry. Figure 6 ,from Figure 6 It can be seen that the peptide was successfully synthesized and the purity was greater than 95%.

[0039] Formula VI.

[0040] Experimental Example 1 Transmission Electron Microscopy Experiment 5.00 mg of the Ac-GFFY-IGF-1C polypeptide synthesized in Example 1 and the Ac-IGF-1C polypeptide synthesized in Comparative Example 1 were weighed, 500 μL of PBS solution was added, and samples were prepared after heating and cooling. The micromorphology was observed by transmission electron microscopy. Figure 7 and Figure 8 .

[0041] Figure 7 and Figure 8 It shows that the self-assembling polypeptide Ac-GFFY-IGF-1C with IGF-1 function prepared in Example 1 self-assembles into nanospheres after heating and cooling treatment, while Ac-IGF-1C cannot assemble under the same conditions, so no nanostructure is captured under the transmission electron microscope.

[0042] Experimental Example 2 Determination of secondary structure by circular dichroism

[0043] 500 μL of 50 μM peptide Ac-GFFY-IGF-1C and peptide Ac-IGF-1C solutions were added to the quartz dish for detection. The spectrum in the wavelength range of 190 nm-260 nm was collected. After deducting the background value of the solvent water, the final circular dichroism spectrum of the sample was obtained. The results are as follows: Figure 9 shown.

[0044] from Figure 9 It can be seen that the secondary structure of the polypeptide Ac-IGF-1C is mainly β-turn, and the secondary structure of the polypeptide Ac-GFFY-IGF-1C is mainly β-sheet.

[0045] Experimental Example 3 Microscale Thermophoresis (MST) Test of Peptide Binding Ability to IGF-1R First, 100 μg of IGF-1R protein was dissolved in 100 μL of ddH₂O to a concentration of 6.7 μM. Then, 10 μL of 210 μM Dye NT-647 NHS from the Monolith NT.115™ Protein Labeling Kit was mixed with the 100 μL IGF-1R protein solution and incubated in the dark for 30 minutes. The reaction mixture was added to a column equilibrated with PBSP, followed by 550 μL of PBSP. After the column was drained, 450 μL of PBSP was added for elution. The eluate was collected, which contained the fluorescently labeled IGF-1R protein at a concentration of 1.49 μM. The fluorescently labeled protein was diluted 10-fold with PBSP to obtain a solution with a concentration of 149 nM. The peptides Ac-GFFY-IGF-1C and Ac-IGF-1C were diluted to different concentrations, and then an equal volume of fluorescently labeled IGF-1R protein with a concentration of 149 nM was added to each of them. The final protein concentration was 75 nM. The mixture was mixed evenly and incubated at room temperature for 30 minutes. The peptide-protein mixture was aspirated with a capillary tube, and the binding ability of different peptides to IGF-1R protein was measured by microcalorimetry (Monolith NT.115). The results are shown in Figure 2. Figure 10 and Figure 11 shown.

[0046] Equilibrium dissociation constant (K d ) usually reflects the affinity of the compound to the target. The smaller the value, the stronger the affinity. Figure 10 and Figure 11 It can be seen that the K d The value was 27.7 nM, which was significantly lower than that of Ac-IGF-1C (K d The affinity of Ac-GFFY-IGF-1C for IGF-1R was increased by nearly 10 times.

[0047] Experimental Example 4: Detection of proliferation of primary mouse dermal papilla cells using Cell Counting Kit-8 (CCK-8)

[0048] Mouse primary dermal papilla cells were cultured at a rate of 2×10 3 The cells were seeded into a 96-well plate at a density of 100 μg / well and incubated in a 37°C incubator with 5% CO2 for 24 h. The culture medium was discarded and DMEM culture medium containing different concentrations of the peptide Ac-GFFY-IGF-1C was added and incubated for 48 h. The supernatant was discarded and DMEM culture medium containing 10% CCK-8 solution was added and incubated for 2 h. The absorbance was measured at 450 nm using a microplate reader. The results are shown in Figure 2. Figure 12 shown.

[0049] Depend on Figure 12 It can be seen that the dose-effect curve of the polypeptide Ac-GFFY-IGF-1C inducing the proliferation of mouse primary dermal papilla cells is bell-shaped. The optimal concentration of this material to promote cell proliferation is 30~125nM. Within this concentration range, Ac-GFFY-IGF-1C treatment increased the cell proliferation activity to 1.5 times that of the untreated group, showing good proliferation-promoting ability at the cellular level.

[0050] Experimental Example 5 EdU Cell Proliferation Kit to Detect Cell Proliferation

[0051] According to 1×10 5 Primary mouse dermal papilla cells were plated at a density of 100 μM in six confocal microplates. After complete attachment, the culture medium was replaced with DMEM containing the peptides Ac-IGF-1C and Ac-GFFY-IGF-1C, or with untreated DMEM, respectively. The peptide concentration was 100 nM, and the cells were incubated for 48 hours. An equal volume of 20 μM EdU working solution was added to the confocal microplates to a final EdU concentration of 10 μM. The cells were incubated for another 6 hours. After EdU labeling, the culture medium was discarded, and 1 mL of 4% paraformaldehyde was added for fixation at room temperature for 15 minutes. The fixative was discarded, and the cells were washed twice with 1 mL of PBS per well for 3 minutes each. The PBS was discarded, and the cells were incubated with 1 mL of PBS containing 0.3% Triton X-100 per well for 15 minutes at room temperature. The permeabilization solution was discarded, and the cells were washed twice with 1 mL of PBS per well for 3 minutes each. Prepare the Click reaction solution according to the requirements of the BeyoClick™ EdU-594 Cell Proliferation Assay Kit, add 500 μL of Click reaction solution to each well, and incubate at room temperature in the dark for 30 minutes. Aspirate the Click reaction solution and wash three times with PBS for 3 minutes each time. After aspirating the PBS, add 1 mL of 1× Hoechst 33342 solution to each well and incubate at room temperature in the dark for 15 minutes. Aspirate the 1× Hoechst 33342 solution and wash three times with PBS for 3 minutes each time. After staining, images were taken using a laser scanning confocal microscope (Leica TSC SP8) under uniform parameters. The results are shown in Figure 2. Figure 13 shown.

[0052] from Figure 13 It can be seen that there is no significant difference in the number of EdU-positive cells in the Ac-IGF-1C group and the Control group, indicating that simple Ac-IGF-1C has no obvious proliferation-promoting effect. Compared with the Control group and the Ac-IGF-1C group, the number of EdU-positive cells in the Ac-GFFY-IGF-1C group increased significantly, further indicating that the structurally modified Ac-GFFY-IGF-1C has a good cell proliferation-promoting effect.

[0053] Experimental Example 6: Western blotting technology to detect the effects of peptides on IGF-1R and downstream signaling pathways

[0054] This experiment set up three groups, namely the Control group (simple DMEM culture medium), Ac-IGF-1C group and Ac-GFFY-IGF-1C group. 5 Mouse primary dermal papilla cells were seeded into six-well plates at a density of 1:1 / well. After cells were fully attached, 100 nM of the corresponding drug was added to each group for 48 hours. The culture medium was discarded, and cells were washed three times with pre-chilled PBS. The culture medium was removed, and 80 μL of RIPA lysis buffer containing a protease inhibitor cocktail and a phosphatase inhibitor was added to each well. Lysis was carried out on ice for 10 minutes. The cell lysate was collected in a 1.5 mL EP tube and centrifuged at 14,000 rpm for 15 minutes at 4°C. The supernatant was collected and protein quantified using a BCA assay kit. The remaining samples were added with 4× loading buffer at a volume ratio of 3:1 between the cell lysate and 4× loading buffer. The samples were heated in a metal bath at 100°C for 10 minutes to obtain protein samples. SDS-PAGE polyacrylamide gel electrophoresis was performed using Blue Polymer precast gels, with a protein loading of 30 μg. The electrophoresis voltage was 150 V for 50 minutes. The gel was then cut, and the membrane was transferred from the gel to a PVDF membrane. The PVDF membrane was washed once with 1× TBST, incubated with rapid blocking solution at room temperature for 15 minutes, and washed three times with 1× TBST for 10 minutes each. The membrane was cut according to the marker's instructions, incubated with primary antibody at 4°C overnight, washed three times with 1× TBST for 10 minutes each, incubated with secondary antibody at room temperature for 2 hours, and washed three times with 1× TBST for 10 minutes each. After washing, the membrane was exposed and photographed. Figure 14 shown.

[0055] from Figure 14 It can be seen that compared with the Control group and Ac-IGF-1C group, the expression level of p-IGF-1R in the Ac-GFFY-IGF-1C group was significantly increased, indicating that the polypeptide Ac-GFFY-IGF-1C is similar to the IGF-1 protein and can activate IGF-1R and induce the phosphorylation of IGF-1R, thereby promoting the proliferation of mouse dermal papilla cells.

[0056] Experimental Example 7 Physiological hair loss model in mice

[0057] Male C57BL / 6 mice (6 weeks old) were used in this experiment. The back hair of the mice was shaved with a shaver, and the fine hair on the back skin was removed with a depilatory cream. Mice with pink back skin were randomly divided into six groups, each with four mice: the control group, the Ac-GFFY-IGF-1C 1 μM group, the Ac-GFFY-IGF-1C 2 μM group, the Ac-GFFY-IGF-1C 5 μM group, the Ac-IGF-1C 2 μM group, and the Ac-IGF-1C 5 μM group. Among them, the control group mice were smeared with aqueous solution, the Ac-GFFY-IGF-1C 1 μM group, Ac-GFFY-IGF-1C 2 μM group, and Ac-GFFY-IGF-1C 5 μM group mice were smeared with Ac-GFFY-IGF-1C polypeptide aqueous solution with concentrations of 1 μM, 2 μM, and 5 μM on their backs, respectively, and the Ac-IGF-1C 2 μM group and Ac-IGF-1C5 μM group mice were smeared with Ac-IGF-1C polypeptide aqueous solution with concentrations of 2 μM and 5 μM on their backs, respectively, once a day. The growth of the hair on the back of the mice was recorded on the 0th, 7th, and 10th day of administration. The results are shown in the figure. Figure 15 shown.

[0058] Depend on Figure 15 It can be seen that the growth cycle of hair follicles includes the growth phase, the decline phase and the resting phase. When the hair follicles are in the resting phase, the skin is pink, and when the hair follicles are in the growth phase, the skin is gray or black. Therefore, skin pigmentation can be used as a visual indicator of the transition from the resting phase to the growth phase of hair. Figure 16 As can be seen, compared to the control group, the Ac-IGF-1C peptide had almost no effect on promoting hair growth at a concentration of 2 μM, and had a weak effect at a concentration of 5 μM. However, the Ac-GFFY-IGF-1C peptide promoted hair growth in mice at concentrations of 1 μM, 2 μM, and 5 μM, with the 2 μM concentration showing the best effect. Therefore, the 2 μM concentration is preferred for subsequent animal experiments.

[0059] Experimental Example 8 Mouse Model of Androgenic Alopecia Male C57BL / 6 mice (6 weeks old) were used in this experiment. The dorsal hair of the mice was shaved with a shaver, and fine hair on the dorsal skin was removed using a depilatory cream. Mice with pink dorsal skin were randomly divided into four groups of five: the Blank group, the AGA group, the Ac-GFFY-IGF-1C group, and the Minoxidil group. The Blank group received no treatment. The AGA, Ac-GFFY-IGF-1C, and Minoxidil groups received an intraperitoneal injection of dihydrotestosterone (1 mg / 100 μL / mouse) on the day of hair removal (Day 0), followed by injections every two days thereafter. Starting on Day 1, mice in the Ac-GFFY-IGF-1C and Minoxidil groups were treated daily with a 2 μM aqueous solution of Ac-GFFY-IGF-1C peptide and a 5% minoxidil solution, respectively. Then, photos were taken on the 1st, 7th, 10th, 14th and 18th days to record the hair growth of the mice. Figure 16 shown.

[0060] from Figure 16 It can be seen that the skin of the Blank group mice began to turn black on the 7th day, new hair began to grow on the 10th day, and the new hair coverage rate reached 100% on the 14th day, indicating that the hair follicles on the back of the Blank group mice had entered the growth phase from the resting phase on the 7th day, while the skin on the back of the AGA group mice was still pink on the 7th day, and did not begin to turn black until the 14th day, indicating that dihydrotestosterone inhibited the process of the mouse hair follicles entering the growth phase from the resting phase, and also indicating that the androgenic alopecia model was successfully established. The back skin of the five mice in the Ac-GFFY-IGF-1C group began to turn black on the 10th day, and the new hair coverage rate of all mice reached 100% on the 18th day. Compared with the AGA group, it significantly accelerated the process of the mouse hair follicles entering the growth phase from the resting phase, and promoted the growth of mouse hair. At the same time, compared with the minoxidil group, only three mice had a new hair coverage rate of 100% on the 18th day. The peptide Ac-GFFY-IGF-1C showed a better therapeutic effect in the androgenic alopecia model.

[0061] Experimental Example 9 Evaluation of the Peptides in Examples 2 to 5 for Promoting Hair Growth

[0062] The mice used in this experiment were male C57BL / 6 (6 weeks old). The hair on the back of the mice was shaved with a shaver, and the fine hair on the back skin was removed with a depilatory cream. The mice with pink back skin were randomly divided into 5 groups, with 3 mice in each group, namely the Control group, Ac-GFYF-IGF-1C group, Ac-GYFF-IGF-1C group, Ac-GFYFNKPT-IGF-1C group and Ac-GFFYNKPT-IGF-1C group. Among them, the mice in the Control group were smeared with an aqueous solution, and the mice in the other groups were smeared with a corresponding polypeptide aqueous solution with a concentration of 2 μM on their backs, once a day. The growth of the hair on the back of the mice was recorded by taking photos on the 0th, 7th and 10th days of administration. The results are shown in the figure. Figure 17 shown.

[0063] from Figure 17 It can be seen that on the 7th day, the back skin of mice in the Ac-GFYF-IGF-1C group, Ac-GYFF-IGF-1C group, Ac-GFYFNKPT-IGF-1C group and Ac-GFFYNKPT-IGF-1C group began to blacken, and the coverage rate and degree of the black area were stronger than those in the Control group. On the 10th day, the new hair coverage rate of each polypeptide group was also higher than that of the Control group, indicating that these four polypeptides all have the effect of promoting hair growth in mice.

[0064] As can be seen from the above examples, the present invention provides a self-assembling polypeptide with IGF-1 function and its application in hair growth. The self-assembling polypeptide comprises a sequentially connected capping group, a self-assembling unit, and an insulin-like growth factor C domain polypeptide IGF-1C. The self-assembling polypeptide with IGF-1 function provided by the present invention has a β-pleated secondary structure and can mimic the binding mode of IGF-1 and the insulin-like growth factor receptor IGF-1R, inducing conformational changes in the IGF-1R dimer, thereby promoting IGF-1R phosphorylation, mimicking its physiological function, and promoting hair growth. The self-assembling polypeptide with insulin-like growth factor 1 (IGF-1) function provided by the present invention can be applied to hair loss areas by skin application, and has demonstrated excellent hair growth-promoting effects in a mouse model of physiological hair loss and an androgenic alopecia model.

[0065] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A self-assembling polypeptide having IGF-1 function, characterized in that: The self-assembling polypeptide comprises a capping group, a self-assembling unit and an insulin-like growth factor C domain polypeptide IGF-1C connected in sequence; The end-capping group is an acetyl group; the self-assembly unit is any one of GFFY, GFYF, GYFF, GFFYNKPT, GFYFNKPT, and GYFFNKPT.

2. The self-assembling polypeptide according to claim 1, characterized in that The amino acid sequence of the insulin-like growth factor C domain polypeptide IGF-1C is shown in SEQ ID NO.

1.

3. The self-assembling polypeptide according to claim 2, characterized in that F and Y in the self-assembly unit are both L-type amino acids or D-type amino acids.

4. The self-assembling polypeptide according to claim 3, characterized in that The self-assembling polypeptide has a structure shown in any one of structural formulas I, II, III, IV or V: Formula I; Formula II; Formula III; Formula IV; Formula V.

5. A biologically active solution containing the self-assembling polypeptide according to any one of claims 1 to 4, characterized in that: The solvent of the bioactive solution is water, and the concentration of the self-assembling polypeptide in the bioactive solution is 0.1-1000 μM.

6. The bioactive solution according to claim 5, characterized in that The bioactive solution is administered by skin application.

7. Use of the self-assembling polypeptide according to any one of claims 1 to 4 or the bioactive solution according to claim 5 in the preparation of a product promoting hair growth.

8. The use according to claim 7, characterized in that The hair growth product is a product for treating androgenic alopecia and alopecia areata.

Images