Use of the protein ambp in the preparation of a formulation for promoting hair follicle cell activity or for the prevention or treatment of hair loss

Abstract

The present application belongs to the field of biomedical and cosmetic technology, and particularly relates to the application of protein AMBP in the preparation of a preparation for promoting the activity of hair follicle cells or for preventing and treating alopecia. The present application first discovers that the protein AMBP has the activity of regulating the hair follicle hair papilla cells, hair matrix cells and outer root sheath cells, can promote the proliferation of the hair papilla cells, the hair matrix cells and the outer root sheath cells, and improve the ability of the outer root sheath cells to remove active oxygen; also has the effects of promoting the hair follicle cycle transition, promoting hair growth and hair thickening. Therefore, the protein AMBP can be applied to the preparation of a preparation for treating alopecia.

Description

Technical Field

[0001] This invention belongs to the fields of biomedicine and cosmetics, and specifically relates to the application of protein AMBP in the preparation of formulations for promoting hair follicle cell activity or for preventing and treating hair loss. Background Technology

[0002] Currently, hair loss treatment options mainly include hair transplantation, oral medications, and emerging PRP injections. Drug treatments have certain side effects, and their effectiveness often falls short of patient expectations. The effectiveness of hair transplantation is closely related to the condition of the hair follicles in the donor area, and the results of newer treatments vary from person to person (ZHOU Y, et al. Advances in microneedles research based on promoting hair regrowth [J]. Journal of Controlled Release, 2023, 353: 965-74.). Therefore, finding and developing new ingredients with preventative and therapeutic effects on hair loss is of great significance.

[0003] In mature individuals, hair follicles generally do not regenerate, but rather exhibit a cyclical pattern, including the anagen (growth) phase, catagen (regression) phase, and telogen (resting) phase. Hair grows rapidly during the anagen phase, stops growing during the catagen phase, and falls out during the telogen phase. New hair then grows back after entering the next anagen phase. Hair consists of two parts: the hair shaft and the hair root. The part exposed on the skin surface is the hair shaft, and the part embedded in the skin is the hair root. The tissue structure surrounding the hair root is the hair follicle (HF). The hair follicle is an accessory organ of the skin, distributed in most parts of the human skin. It is a complex micro-organ with a tissue structure consisting of two layers: an inner epithelial sheath and an outer connective tissue sheath (HAMIDA OB, et al. Hair Regeneration Methods Using Cells Derived from Human Hair Follicles and Challenges to Overcome [J]. Cells, 2025, 14(1): 7.). The upper end of the epithelial sheath is continuous with the skin epidermis, and the lower end wraps around the hair root and fuses with the hair root, swelling to form a hair bulb. The epithelial cells in the hair bulb are mainly hair matrix cells, which can differentiate into hair root and epithelial sheath cells. Therefore, hair matrix cells are the growth point of hair and the only source cells responsible for the actual synthesis and extension of hair. The activity and differentiation direction of hair matrix cells directly determine the thickness, length and growth rate of hair. Almost all types of hair loss are ultimately directly or indirectly manifested as damage to the function and weakening of activity of hair matrix cells (MA S, et al. Transcriptomic Analysis Reveals Candidate Ligand-Receptor Pairs and SignalingNetworks Mediating Intercellular Communication between Hair Matrix Cells and Dermal Papilla Cells from Cashmere Goats [J]. Cells, 2023, 12(12): 1645.).

[0004] Connective tissue protrudes from the base of the hair bulb to form the dermal papilla, which contains abundant capillaries, nerve endings, and dermal papilla cells. The dermal papilla cells are the signal command center of the hair follicle cycle. During the transformation of the hair follicle from the resting phase to the anagen phase, the dermal papilla cells induce the hair follicle to enter the anagen phase. At this time, the activity of alkaline phosphatase (ALP) is enhanced, and a series of growth factors and signaling molecules (such as Wnt, FGF, HGF, VEGF, etc.) are secreted, activating hair follicle stem cells and hair matrix cells, and starting a new growth cycle (MEHTA A, MOTAVAF M, RAZA D, et al. Revolutionary Approaches to Hair Regrowth: Follicle Neogenesis, Wnt / β-Catenin Signaling, and Emerging Therapies [J]. Cells, 2025, 14(11): 779.). Studies have found that human dermal papilla cells in the "bald area" of androgenetic alopecia patients show an earlier aging phenotype and lose their hair follicle induction function (PAPPALARDO A, et al. Restoration of hair follicle inductive properties by depletion of senescent cells [J]. AgingCell, 2025, 24(1): e14353.), therefore, regulating the vitality and secretory capacity of dermal papilla cells can promote the hair cycle and hair growth.

[0005] The outer root sheath cells of hair follicles are the main reservoir of hair follicle stem cells. They receive signals from dermal papilla cells and translate them into instructions for proliferation and differentiation, providing a cellular source for the periodic regeneration of hair follicles (AGRAMUNT J, PARKE B, MENA S, et al. Mechanical stimulation of human hair follicle outer rootsheath cultures activates adjacent sensory neurons [J]. Science Advances, 2023, 9(43): eadh3273.). Therefore, regulating and maintaining the vitality of dermal papilla cells, hair matrix cells, and outer root sheath cells is crucial for the treatment of hair loss.

[0006] Protein AMBP, also known as alpha-1-microglobulin / bikunin precursor, is a "precursor factory" that produces two proteins with distinct functions. It plays a central role in maintaining homeostasis, particularly in responding to oxidative stress and regulating inflammation. After intracellular proteolytic processing, this precursor generates two independent functional proteins: alpha-1-microglobulin and bikunin. These two proteins work collaboratively within the body. Alpha-1-microglobulin is primarily responsible for removing harmful substances from the body and protecting tissues. It is a powerful antioxidant that can directly reduce free radicals and bind to and degrade highly oxidizing heme and heme proteins, thereby protecting cells from oxidative damage. Studies have also shown that α1-microglobulin can inhibit antigen-induced lymphocyte proliferation, cytokine secretion, and neutrophil oxidative bursts, thus helping to regulate inflammatory responses and prevent excessive immune responses from damaging tissues (Kristiansson A, et al. Kidney Protection with the Radical Scavenger α1-Microglobulin (A1M) during Peptide Receptor Radionuclide and Radioligand Therapy. Antioxidants (Basel). 2021;10(8):1271.). Bikunin primarily inhibits the activity of serine proteases through its Kunitz domain. It stabilizes the extracellular matrix and prevents its excessive degradation, which is crucial for tissue remodeling and inflammation control (Ramadan, Sherif et al. Chemical Synthesis and Anti-Inflammatory Activity of Bikunin Associated Chondroitin Sulfate 24-mer. ACS Central Science vol. 6,6(2020): 913-920.). AMBP may affect the skin barrier by regulating keratinocyte function and may also be involved in the pathological process of skin-related autoimmune diseases, especially skin lesions related to inflammation and immune regulation. Currently, there are no literature reports on the regulatory effects of the protein AMBP on hair follicle-associated cells, dermal papilla cells, hair follicle matrix cells, and outer root sheath of hair follicles. Summary of the Invention

[0007] The purpose of this invention is to overcome the shortcomings and deficiencies of the prior art and to provide the application of protein AMBP in the preparation of formulations for promoting hair follicle cell activity or for preventing and treating hair loss.

[0008] The objective of this invention is achieved through the following technical solution: the application of protein AMBP in the preparation of formulations for promoting hair follicle cell activity or for preventing and treating hair loss. This invention is the first to discover that protein AMBP regulates the activity of dermal papilla cells, hair matrix cells, and outer root sheath cells in hair follicles, promoting the proliferation of these cells and enhancing the ability of outer root sheath cells to scavenge reactive oxygen species; it also promotes hair follicle cycle transition, hair growth, and hair thickening. Therefore, protein AMBP can be used to prepare formulations for treating hair loss.

[0009] The protein AMBP described is a secreted complex glycoprotein precursor, with the gene symbol AMBP, located in the q32 region of human chromosome 9. It is a single polypeptide chain that, after enzymatic processing, releases two functional proteins, α1-microglobulin and bismuth substantia nigra. The molecular weight of the α1-microglobulin after precursor processing is approximately 26 kDa.

[0010] The promotion of hair follicle cell activity refers to promoting the proliferation of at least one of the hair papilla cells, hair matrix cells, and outer root sheath cells, and / or enhancing the ability of outer root sheath cells to scavenge reactive oxygen species.

[0011] The preferred type of preparation is a topical liniment, topical patch, or injectable preparation.

[0012] The preferred dosage form of the topical liniment is a liquid, cream, or ointment.

[0013] The preferred topical patch is a transdermal patch.

[0014] The injectable formulation is preferably a skin injection formulation; more preferably, it is at least one of a microneedle injection formulation and a dot injection formulation.

[0015] The concentration of protein AMBP in the formulation is preferably 1-10000 ng / mL; more preferably 10-10000 ng / mL.

[0016] The present invention has the following advantages and effects compared with the prior art:

[0017] (1) The present invention uses CCK8 experiments to clarify that the protein AMBP has the effect of promoting the proliferation of hair follicle dermal papilla cells, hair matrix cells and outer root sheath cells, and determines the optimal concentration.

[0018] (2) The present invention discovered through ROS experiments that the protein AMBP has the ability to scavenge reactive oxygen species in the outer root sheath cells of hair follicles.

[0019] (3) The present invention found through mouse experiments that the protein AMBP has the effect of promoting hair follicle cycle transformation and hair growth thickening.

[0020] In summary, this invention is the first to discover a protein—AMBP—that regulates the function of dermal papilla cells, hair matrix cells, and outer root sheath cells in hair follicles. This compound enhances the cell vitality of these cells, promotes cell proliferation, strengthens the ability of outer root sheath cells to scavenge reactive oxygen species, and promotes the transition of the hair follicle cycle from the resting phase to the anagen phase, resulting in hair growth and thickening. Therefore, AMBP protein can be used in the preparation of formulations for treating hair loss and has significant application value. Attached Figure Description

[0021] Figure 1 This is a diagram showing the effect of protein AMBP in promoting the proliferation of dermal papilla cells.

[0022] Figure 2 This is a diagram showing the effect of the protein AMBP in promoting the proliferation of hair matrix cells.

[0023] Figure 3 This is a diagram showing the effect of protein AMBP in promoting the proliferation of outer root sheath cells in hair follicles.

[0024] Figure 4 The image shows the detection results of the ability of protein AMBP to enhance the scavenging of reactive oxygen species in the outer root sheath cells of hair follicles; where A is a photograph of reactive oxygen species staining in the outer root sheath cells of hair follicles after treatment with protein AMBP, and B is a statistical graph of the quantitative fluorescence of reactive oxygen species in the outer root sheath cells of hair follicles after treatment with protein AMBP.

[0025] Figure 5 This is a diagram showing the effect of protein AMBP in promoting the cycle transition of hair follicles on the back of mice.

[0026] Figure 6 The images show the effect of the protein AMBP on promoting hair growth and thickening on the back of mice; where A is a photograph, B is a statistical result of hair diameter, and C is a statistical result of hair length.

[0027] In the figure, * indicates a comparison with the control group. P <0.05, ** indicates P <0.01, *** indicates P <0.001, **** indicates P <0.0001. Detailed Implementation

[0028] The present invention will be further described in detail below with reference to the embodiments and accompanying drawings, but the embodiments of the present invention are not limited thereto.

[0029] Example 1

[0030] (1) Protein AMBP was dissolved in serum-free medium (DMEM high glucose medium, Gibco) to obtain protein AMBP solutions with concentrations of 1 ng / mL, 10 ng / mL and 100 ng / mL respectively.

[0031] (2) Human hair follicle papilla cells (purchased from NEWGAINBIO, IH1006, Wuxi Xinrun Biotechnology Co., Ltd.) were seeded in 96-well plates at a density of 2000 cells per well. When the cells reached 50%–60% confluence, they were starved in serum-free medium (DMEM high-glucose medium, Gibco) for 8 hours. The cells were then divided into a control group and a protein AMBP treatment group. The original medium was discarded. Protein AMBP solutions at concentrations of 1 ng / mL, 10 ng / mL, and 100 ng / mL were added to the protein AMBP treatment group, 100 μL / well, with three replicates for each concentration. The control group received only serum-free medium. The 96-well plates were incubated at 37°C in a 5% CO2 incubator for 24 hours for CCK8 assay. Absorption at 450 nm was measured using a microplate reader (BLJ BIO-RECH, China, DLJ-200). Data analysis was performed using SPSS 25 statistical software. For the measurement data, paired measurements were used for comparison at different time points. t Test. Data showing statistically significant differences are indicated by different asterisks (*). P <0.05,** P <0.01, *** P <0.001, **** P <0.0001. Experimental results are shown in [link to results]. Figure 1 The results showed that the addition of protein AMBP could promote the proliferation of hair follicle dermal papilla cells; when the concentration of protein AMBP was 10 ng / mL, it significantly promoted the proliferation of hair follicle dermal papilla cells.

[0032] Example 2

[0033] CCK8 assay was used to detect the effect of different concentrations of AMBP protein on the proliferation of human hair follicle matrix cells:

[0034] Human hair follicle matrix cells (purchased from Qingqi (Shanghai) Biotechnology Development Co., Ltd., BLUEFBIO) TMCells (BFN60803943) were seeded at a density of 2000 cells per well in 96-well plates. When the cells reached 50%–60% confluence, they were starved for 8 hours in serum-free medium (DMEM high-glucose medium, Gibco). The cells were then divided into a control group and a protein AMBP treatment group. The original medium was discarded. The protein AMBP treatment groups were treated with 100 μL / well of protein AMBP solution at concentrations of 1 ng / mL, 10 ng / mL, and 100 ng / mL, with three replicates for each concentration. The control group received only serum-free medium. The 96-well plates were incubated at 37°C in a 5% CO2 incubator for 24 hours before CCK8 assay. Absorption at 450 nm was measured using a microplate reader (BLJ BIO-RECH, China, DLJ-200). Data analysis was performed using SPSS 25 statistical software. For comparisons of measurements at different time points, paired measurements were used. t Test. Data showing statistically significant differences are indicated by different asterisks (*). P <0.05,** P <0.01, *** P <0.001. Experimental results are shown in [link to results]. Figure 2 . Figure 2 The results showed that the addition of protein AMBP could promote the proliferation of hair follicle matrix cells; when the concentration of protein AMBP was 10 ng / mL, it significantly promoted the proliferation of hair follicle matrix cells.

[0035] Example 3

[0036] CCK8 assay was used to detect the effect of different concentrations of AMBP protein on the proliferation of human hair follicle outer root sheath cells.

[0037] Human hair follicle outer root sheath cells (purchased from Applied Biological Materials (abm), T9233) were seeded at a density of 2000 cells per well in 96-well plates. When the cells reached 50%–60% confluence, they were starved for 8 hours in serum-free medium (DMEM high-glucose medium, Gibco). The cells were then divided into a control group and a protein AMBP treatment group. The original medium was discarded. The protein AMBP treatment groups were treated with 1 ng / mL, 10 ng / mL, and 100 ng / mL protein AMBP solution, 100 μL / well, with three replicates for each concentration. The control group received only serum-free medium. The 96-well plates were incubated at 37°C in a 5% CO2 incubator for 24 hours, and CCK8 assays were performed. Absorbance at 450 nm was measured using a microplate reader (BLJ BIO-RECH, China, DLJ-200). Data analysis was performed using SPSS 25 statistical software. For comparisons of measurements at different time points, paired measurements were used. tTest. Data showing statistically significant differences are indicated by different asterisks (*). P <0.05,** P <0.01, *** P <0.001, **** P <0.0001. Experimental results are shown in [link to results]. Figure 3 . Figure 3 The results showed that the addition of protein AMBP could promote the proliferation of outer root sheath cells of hair follicles; when the concentration of protein AMBP was 10 ng / mL, it significantly promoted the proliferation of outer root sheath cells of hair follicles.

[0038] Example 4

[0039] The effect of the protein AMBP on the ability of human hair follicle outer root sheath cells to scavenge reactive oxygen species was detected by a reactive oxygen species activity assay.

[0040] Human hair follicle outer root sheath cells (purchased from Applied Biological Materials (abm), T9233) were cultured at 5 × 10⁶ cells per well. 4 Cells were seeded at a density of 1000 g / L in 6-well plates. When the cells reached 50%–60% confluence, they were starved for 8 hours in serum-free medium (DMEM high-glucose medium, Gibco). Cells were then divided into a control group and a protein AMBP treatment group. The original medium was discarded. The protein AMBP treatment group received 1.5 mL of 10 ng / mL protein AMBP solution per well; the control group received only the same volume of serum-free medium. After 24 hours of culture, the original medium was discarded, and the cells were washed three times with PBS. 1.5 mL of DCFH-DA working solution (diluted 1:1000 with serum-free medium to a concentration of 10 μmol / L) was added to each well. The cells were incubated at 37°C for 20 minutes. The DCFH-DA working solution was discarded, and the cells were washed three times with 0.01 M PBS (pH 7.4) to remove any uninfiltrated DCFH-DA. The DCFH-DA working solution induces green fluorescence in outer root sheath cells with high reactive oxygen species content at an excitation wavelength of 488 nm. Fluorescence images were observed and captured using an inverted fluorescence microscope (Leica, Germany). Data analysis was performed using SPSS 25 statistical software. For measurement data, paired measurements were used for comparison at different time points. t Test. Data showing statistically significant differences are indicated by different asterisks (*). P <0.05,** P <0.01, *** P <0.001, **** P <0.0001. Reactive oxygen species detection results (see...) Figure 4)showed that treatment with protein AMBP could reduce the content of reactive oxygen species in outer root sheath cells of hair follicles and improve the ability of cells to scavenge reactive oxygen species.

[0041] Example 5

[0042] Experimental study on the effect of protein AMBP on hair follicle cycle using C57BL / 6 mouse hair follicle cycle model:

[0043] C57BL / 6 mice (purchased from the Experimental Animal Center of Southern Medical University, SCXK(Beijing) 2021-0059) were anesthetized by intraperitoneal injection of 2% pentobarbital (Sigma, St. Louis, MO, USA; w / v; 0.01 mL / g body mass). The dorsal hair shafts were removed using depilatory cream without damaging the hair roots, and the hair follicles on the dorsal skin of the mice were identified as being in the telogen phase by the pink skin color. After depilation, the mice were randomly divided into two groups of five mice each. In the treatment group, 100 μL of a protein AMBP solution with a concentration of 10 μg / mL (solvent: PBS) was applied to each mouse, and in the control group, 100 μL / mouse of PBS was applied. It was applied once a day for 7 days. On the 7th day, the dorsal skin of the mice was photographed with a digital camera. The experimental results are shown in Figure 5 . Figure 5 The results showed that treatment of the dorsal skin of mice with a protein AMBP solution at a concentration of 10 μg / mL significantly promoted the transition of the hair follicle cycle from the telogen phase to the anagen phase.

[0044] Example 6

[0045] Experimental study on the effect of protein AMBP on the hair length and diameter of the dorsal skin of mice using C57BL / 6 mouse hair follicle cycle model:

[0046] C57BL / 6 mice (purchased from the Experimental Animal Center of Southern Medical University, SCXK(Beijing) 2021-0059) were anesthetized by intraperitoneal injection of 2% pentobarbital (Sigma, St. Louis, MO, USA; w / v; 0.01 mL / g body mass). The dorsal hair shafts were removed using depilatory cream without damaging the hair roots, and the hair follicles on the dorsal skin of the mice were identified as being in the telogen phase by the pink skin color. After depilation, the mice were randomly divided into two groups of five mice each. In the treatment group, 100 μL of a protein AMBP solution with a concentration of 10 μg / mL (solvent: PBS) was applied to each mouse, and in the control group, 100 μL / mouse of PBS was applied. It was applied once a day for 24 days. On the 24th day, 40 hairs (8 hairs per mouse) were randomly plucked from the two treatment areas on the dorsal skin of the mice, photographed under a stereomicroscope, and the length and diameter of the hairs were measured using LitoDigital software. For the measured data, paired t tests were used for comparison of the measured values at different time points. For data with statistically significant differences, * indicatesP <0.05, *** P <0.01, **** P <0.001. Experimental results are shown in [link to results]. Figure 6 The results showed that treatment of mouse dorsal skin with a protein AMBP solution at a concentration of 10 µg / mL significantly promoted hair growth, manifested as an increase in hair diameter (see [link to study]). Figure 6 (A and B in the text) and the increase in hair length (see A and B in the text) Figure 6 (C in the figure), and it is statistically significant.

[0047] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. The application of protein AMBP in the preparation of formulations for the prevention and treatment of hair loss, characterized in that: The protein AMBP mentioned above is derived from humans.

2. The application according to claim 1, characterized in that: The aforementioned prevention and treatment of hair loss is achieved by promoting the activity of hair follicle cells; The promotion of hair follicle cell activity refers to promoting the proliferation of at least one of the hair papilla cells, hair matrix cells, and outer root sheath cells, and / or enhancing the ability of outer root sheath cells to scavenge reactive oxygen species.

3. The application according to claim 1, characterized in that: The preparation is of the type of topical liniment, topical patch, or injectable preparation.

4. The application according to claim 3, characterized in that: The dosage form of the topical liniment is liquid, cream, or ointment.

5. The application according to claim 3, characterized in that: The aforementioned external patch is a transdermal patch.

6. The application according to claim 3, characterized in that: The injectable preparation is a skin injection preparation.

7. The application according to claim 6, characterized in that: The skin injection preparation is at least one of microneedle injection preparation and dot injection preparation.

8. The application according to any one of claims 1 to 7, characterized in that: The concentration of protein AMBP in the formulation is 1–10,000 ng / mL.

9. The application according to claim 8, characterized in that: The concentration of protein AMBP in the formulation is 10–10,000 ng / mL.

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