Use of JWA polypeptide in preparation of drug for resisting androgenetic alopecia

Abstract

The present invention relates to use of a JWA polypeptide in the preparation of a drug for resisting androgenetic alopecia. An amino acid sequence of the polypeptide is shown as I or II: I: FPGSDRF-Z; II: X-FPGSDRF-Z, wherein an amino acid S is subjected to phosphorylation modification, and X and Z are an amino acid or an amino acid sequence, respectively. The described JWA polypeptide can directly target integrin molecules onto hair follicle cells and enter the cells to play a role in regulating and controlling hair follicle proliferation, promoting hair growth and the like; the telogen phase of the hair follicles is significantly shortened, and the anagen phase of the hair follicles is prolonged; the hair shaft and follicle volume is increased; and the expression level of the target cell integrin molecule αvβ1 can be improved, and the hair follicle stem cell signal channel MEK / ERK / E2F1 / SP1 / Wnt10a / 10b / β-catenin can be accurately activated.

Description

Application of JWA peptides in the preparation of anti-androgen alopecia drugs Technical Field

[0001] This invention relates to the application of JWA polypeptide in the preparation of anti-androgen alopecia drugs, belonging to the field of dermatological drug technology. Background Technology

[0002] Androgenetic alopecia (AGA), also known as male pattern baldness or female pattern baldness, is a hair loss disorder that occurs during and after puberty and is one of the most common types of hair loss in the world. [1,2] AGA is a chronic disease associated with multiple factors, including genetics and age, which can severely impact a patient's mental health and quality of life. [3,4] The incidence and prevalence of AGA increase with age, affecting 42% of women and 80% of men over 70 years of age. [5,6] A growing body of research indicates that AGA is closely related to aging. [7] Clinical symptoms of AGA include a decrease and change in hair thickness and density, lighter hair color, and increased oil production. [8] Hair loss patterns vary by gender. [9] In men, it manifests as "M-shaped alopecia," a progressive baldness that radiates from the temples to the crown, with reduced hair density and thickness in the mid-frontal scalp.

[0010] In women, the condition manifests as diffuse decreased hair density, primarily affecting the forehead and vertex scalp, while the hairline is usually unaffected.

[0011] The main pathological manifestation of AGA is the shortening of the hair follicle growth phase, leading to follicle miniaturization, which causes the coarser, pigmented terminal hairs to be gradually replaced by finer, depigmented vellus hairs.

[0012] .

[0003] The human body is covered with approximately 5 million hair follicles, which are miniature organs composed of various cells. From the inside out, they are divided into the hair shaft (HS), inner root sheath (IRS), and outer root sheath (ORS). The lower end of the hair follicle expands into the hair bulb, which contains the dermal papilla.

[0013] Hair follicles grow in a cyclical manner, consisting of three phases: the anagen (growth) phase, the catagen (transitional) phase, and the telogen (resting) phase.

[0014] The duration of each cycle is relatively constant; the cyclical process causes old hair shafts to fall off and new hair shafts to form.

[0015] The growth of new hair requires cells located in the outer root sheath of the hair follicle, i.e., the telogen effluvium, to enter the anagen phase. This anagen phase, aided by adjacent dermal papilla (DP) cells and the stem cell microenvironment, forms the hair bulb. The human scalp hair follicle cycle undergoes an active anagen phase lasting 2 to 6 years, followed by a brief transitional phase of 1-2 weeks (catagen phase), and then a resting phase lasting approximately 3 months.

[0016] The anagen phase represents the formation and growth of new hair, while the latter two phases include follicle regression and shedding.

[0017] The growth cycles of human hair follicles are asynchronous; at the same time, hair follicles are in different growth stages. Keratinocytes in the hair follicle epithelium proliferate rapidly during the anagen (growth) phase and undergo apoptosis during the catagen (regression) phase, leading to follicle degeneration. At this point, the hair follicle enters the telogen (resting) phase, and hair growth stops until signaling from dermal papilla cells (DPCs) activates the proliferation of hair follicle stem cells, which then refill the hair follicle epithelium, allowing the follicle to re-enter the anagen phase.

[0018] During this process, if signal activation fails, the hair follicle cannot re-enter the growth phase. The resting hair follicle becomes cavitary, the original hair shaft falls out, and no new hair grows, ultimately leading to baldness. In normal human scalp, approximately 85%–100% of hair follicles are in the growth phase, 0%–15% are in the resting phase, and only about 1% are in the regression phase.

[0019] .

[0004] AGA is a complex disease, and the following factors are generally believed to be involved in its pathogenesis: (1) Genetic factors play an important role in the pathogenesis of AGA.

[0020] It is estimated that androgen receptor (AR) gene variants can account for 40% of the total genetic risk and are considered to be at high risk for a single gene. (2) Androgens are the main mediators of AGA. Androgens stimulate premature termination of the growth phase in hair follicles and gradually increase the duration of the resting phase.

[0021] Testosterone (T) is the main form of androgen. Increased activity of type II 5α-reductase in the AGA-affected area leads to the conversion of more free T into dihydrotestosterone (DHT). DHT has an affinity for AR more than five times that of T.

[0022] Then, DHT and AR combine to form a complex that inhibits the proliferation of dermal papilla cells (DPCs) and maintains them in the resting phase, leading to follicle miniaturization. This causes the original terminal hair to gradually transform into fine and light-colored vellus hair.

[0023] The bald scalp has higher levels of 5α-reductase and androgen receptors. Overactivation of androgen receptors leads to follicle miniaturization through a gradually shortened growth phase.

[0024] (3) Aromatase, located in the keratinocytes of the dermal papilla cells and the outer root sheath, also participates in the metabolism of androgens. It can convert testosterone into estradiol and androstenedione into estrone, thereby reducing testosterone and DHT in the hair follicle, which may play a role in protecting the hair follicle.

[0021] (4) AGA is associated with the dysregulation of inflammatory cytokine expression. Dermal papillary cells of hair follicles secrete many cytokines, such as TGF-β1, IL-1α and TNF-α, which can induce premature termination of the hair follicle growth phase.

[0025] In addition, oxidative stress

[0026] ,senescence

[0027] Smoking [28,29] and ultraviolet radiation

[0030] Factors such as genetics and androgens can also contribute to the development of AGA. In conclusion, the increased prevalence of AGA is related to the combined effects of genetic factors, androgens, and environmental factors.

[0005] Because AGA is influenced by numerous factors and its pathogenesis is not fully understood, clinical efficacy remains a significant challenge. Currently, only two drugs are approved by the U.S. Food and Drug Administration (FDA) for the treatment of AGA: topical minoxidil.

[0031] and oral finasteride

[0032] Other treatments include low-dose oral minoxidil.

[0033] Dutasteride

[0034] Spironolactone

[0035] Platelet-rich plasma

[0036] Minoxidil also has some therapeutic effects. It has strong vasodilatory properties and was initially used to treat hypertension.

[0037] By increasing blood circulation, inducing vasodilation and overexpression of vascular endothelial growth factor (VEGF), it increases mitosis of hair matrix keratinocytes, promoting faster and thicker hair growth, and also prolonging the growth phase. [33,38] However, it can cause adverse reactions such as scalp itching, dermatitis, and excessive hair growth in the affected area.

[0039] Finasteride is a type II 5α-reductase inhibitor.

[0040] It irreversibly binds to enzymes, reducing DHT levels in serum and scalp by more than 60%.

[0041] Furthermore, finasteride induces the telogen phase to transition into the anagen phase in the hair cycle, but carries the risk of sexual dysfunction and depression.

[0039] Both of these medications are expensive and can cause dependence; once discontinued, hair loss will resume.

[0042] In conclusion, currently available treatments for AGA are very limited, and all have varying degrees of side effects, potentially causing secondary harm to patients' physical and mental well-being. Therefore, there is an urgent need to find an effective drug with minimal side effects to fill the clinical gaps in existing AGA treatments.

[0006] The JWA gene, also known as ARL6IP5 (GenBank: AF070523, 1998), is a typical, broad-spectrum environmental response gene and also an aging-related gene.

[0043] It is widely involved in cellular responses to various environmental physicochemical stimuli, such as oxidative stress and thermal stress.

[0044] JWA gene knockout mice exhibit aging phenotypes such as weight loss, shortened lifespan, skin atrophy, and hair loss. Cells lacking the JWA gene also show premature aging phenotypes. Previous studies have also found that JWA is an important molecule for intestinal epithelial renewal and post-injury repair, and its mechanism is not only related to anti-oxidative stress and DNA damage repair, but also to the activation of small intestinal pituitary stem cell proliferation and differentiation; conditional knockout of the JWA gene in the small intestinal epithelium can lead to premature aging phenotypes similar to those in mice with systemic knockout of the gene.

[0045] JP1 (sequence: FPGSDRFGGGG-RGD, S-site phosphorylation) is a small JWA polypeptide designed and synthesized based on the JWA protein coding sequence, based on previous basic research results. Previous studies have shown that JP1 can perform biological functions similar to the JWA protein, and its mechanism of action is related to the activation of the MAPK signaling pathway, suggesting that the JWA polypeptide can achieve some of the biological functions of the JWA protein through a similar mechanism.

[0007] The inventors' research group has achieved research results on the treatment of testosterone propionate (TP)-induced AGA with JWA peptides, and has applied for this invention patent based on these results.

[0008] The references mentioned in the background section are as follows:

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[0054] Summary of the Invention

[0055] The main objective of this invention is to address the problems existing in the prior art by proposing the application of JWA polypeptide in the preparation of anti-androgenic alopecia drugs. Whether administered systemically by injection or applied topically to the skin, it can promote the proliferation and activation of skin hair follicle stem cells, promote hair growth, effectively reverse the inhibitory effect of androgens on hair follicle cells, and provide a new clinical drug possibility for the treatment of androgenic alopecia.

[0056] The technical solution of this invention to solve its technical problem is as follows:

[0057] The use of a polypeptide, characterized in that the use is for preparing an anti-hair loss drug;

[0058] The amino acid sequence of the polypeptide is shown in I or II:

[0059] I: FPGSDRF-Z;

[0060] II: X-FPGSDRF-Z;

[0061] Among them, amino acid S is phosphorylated, and X and Z are amino acids or amino acid sequences, respectively;

[0062] X is selected from one of F, (R)9, (R)9-F, 6-aminohexanoic acid, 6-aminohexanoic acid-F, 6-aminohexanoic acid-(R)9, and 6-aminohexanoic acid-(R)9-F;

[0063] Z is selected from (G). n -RGD、A-(G) n -RGD is one of them, where n is an integer greater than or equal to 0, and the value of n ranges from 0 to 10.

[0064] Preferably, the anti-hair loss drug includes a drug for treating androgenetic alopecia.

[0065] Preferably, the function of the anti-hair loss drug is to target hair follicle tissue, promote the growth of hair follicles and hair shafts, shorten the resting phase of hair follicles, prolong the growth phase of hair follicles, and significantly reverse the inhibition of hair growth by androgens.

[0066] Preferably, the function of the anti-hair loss drug is to target integrin molecules into hair follicle cells and upregulate the expression of integrin molecules in the hair follicle cell membrane; the integrin molecules mainly include ανβ1.

[0067] Preferably, the function of the anti-hair loss drug is to promote the expression of nuclear transcription factor SP1 by activating the MEK / ERK / E2F1 signaling axis, thereby activating the Wnt / β-catenin signaling pathway of hair follicle stem cells.

[0068] Preferably, the N-terminus of the polypeptide is acetylated and the C-terminus is amidated.

[0069] Preferably, the amino acid sequence of the polypeptide is one of SEQ ID No. 1 to SEQ ID No. 39.

[0070] Preferably, in the polypeptide, the amino acid configuration of each amino acid in the FPGSDRF sequence is L-type or D-type.

[0071] Preferably, the drug includes a carrier, which is a pharmaceutically acceptable carrier.

[0072] Preferably, the dosage form of the drug is an injectable drug or a topical drug.

[0073] The polypeptides involved in this invention are part of a series of polypeptides described in Chinese invention patents CN201310178099X and CN103239710B. Through practical research, the inventors have confirmed that these polypeptides have therapeutic effects on androgenetic alopecia. They can directly target integrin molecules to hair follicle cells, entering the cells to regulate hair follicle proliferation and promote hair growth; significantly shorten the resting phase of hair follicles and prolong the anagen phase; increase hair shaft and follicle volume; and increase the expression level of the target cell integrin molecule αvβ1 and precisely activate the hair follicle stem cell signaling pathway MEK / ERK / E2F1 / SP1 / Wnt10a / 10b / β-catenin. Therefore, these polypeptides have promising applications in the preparation of corresponding drugs for androgenetic alopecia. Attached Figure Description

[0074] Figure 1 shows the design and initial screening results of the JP1-promoted hair growth model in AGA mice in Example 1 of this invention. (a) Schematic diagram of the animal model. (b) Appearance of the skin on the back of the mouse. (c) Trend graph of mouse weight.

[0075] Figure 2 shows the experimental results of JP1 promoting skin color change in AGA model mice in Example 2 of this invention. (a) Schematic diagram of skin color scoring. (b) Statistical graph of mouse skin color scoring. (c) Summary table of P-values ​​between groups at different time points.

[0076] Figure 3 shows the experimental results of JP1 promoting the early entry of hair follicles into the growth phase in AGA model mice in Example 3 of the present invention. (a) Longitudinal section of hair follicle H&E staining. (b) Cross section of hair follicle H&E staining. (c) Statistical graph of dermal thickness in mice. (d) Statistical graph of total number of hair follicles in mice.

[0077] Figure 4 shows the experimental results of the stability evaluation of JP1 in the coating solvent in Example 4 of the present invention. The figure shows the sum of the main peak area and peak area ratio of each JP1 group.

[0078] Figure 5 shows the experimental results of JP1 targeting the hair follicle site and evaluating its half-life in skin tissue in Example 5 of the present invention. Specifically, it shows the immunofluorescence staining of the FITC-JP1 entry pathway into the hair follicle and the corresponding H&E staining of the region.

[0079] Figure 6 shows the dose-dependent experimental results of JP1 promoting hair growth in Example 6 of the present invention. (a) Flowchart of animal model design. (b) Appearance of skin color changes on the back of mice at different time points during the model period. (c) Weight curve of model mice. (d and e) Statistical table of skin color scores on the back of mice and summary of P-values.

[0080] Figure 7 shows the experimental results of JP1 prolonging the time of hair follicles in the growth phase in Example 7 of the present invention. (a) Longitudinal section of hair follicle H&E staining. (b) Statistical diagram of dermal thickness in mice. (c) Cross section of hair follicle H&E staining on day 11. (d) Statistical diagram of the total number of hair follicles in mice on day 11. (e) Statistical diagram of the diameter of hair follicle bulb on day 11.

[0081] Figure 8 shows the experimental results of JP1 promoting hair follicle growth by activating the Wnt signaling pathway in Example 8 of this invention. (a) Expression levels of AR and SRD5A2 mRNA in the skin of mice in the control group, model group, JP1 treatment group, and minoxidil group. (b) KEGG enrichment map of upregulated genes in the 1% JP1 vs. model group. (c) Gene enrichment analysis using GSEA.

[0082] Figure 9 shows the experimental results of JP1 activating β-catenin expression in skin tissue in Example 9 of this invention. (a) β-catenin and LEF1 mRNA expression levels. (b) Western blot analysis of β-catenin protein expression level. (c) Quantitative analysis of β-catenin protein expression. (d) Representative images of β-catenin in each group detected by immunofluorescence staining. (e) Fluorescence intensity statistics.

[0083] Figure 10 shows the experimental results of JP1 increasing β-catenin expression level via Wnt10a / Wnt10b in Example 10 of the present invention. (a) Screening for Wnt ligands that regulate β-catenin and detecting mRNA expression levels. (b) Correlation between JWA mRNA and Wnt10a mRNA. (c) Correlation between JWA mRNA and Wnt10b mRNA.

[0084] Figure 11 shows the experimental results of JP1 potentially upregulating Wnt10a / Wnt10b expression via SP1 / MYC in Example 11 of this invention. (a) Western blot analysis of Wnt10a / Wnt10b protein expression levels in each group. (b) Relative quantitative analysis of Wnt10a protein expression in each group. (c) Relative quantitative analysis of Wnt10b protein expression in each group. (d) Online tool prediction of transcription factors that JP1 activates in Wnt10a / Wnt10b.

[0085] Figure 12 shows the experimental results of JP1 promoting increased SP1 expression in Example 12 of this invention. (a) Detection of SP1 and MYC mRNA expression levels. (b) Detection of SP1 protein expression levels by Western blotting. (c) Relative quantitative analysis of SP1 protein expression. (d) Representative images of SP1 in each group detected by immunofluorescence staining. (e) Statistical graph of fluorescence intensity.

[0086] Figure 13 shows the experimental results of JP1 activating the Wnt pathway through the E2F1-SP1 axis in Example 13 of this invention. (a) Predicting upstream transcription factors of SP1 using three online tools and taking the intersection of the transcription factors. (b) qPCR detection of FOXI1 mRNA expression level. (c) qPCR detection of FOXD3 mRNA expression level. (d) qPCR detection of E2F1 mRNA expression level. (e) Correlation between SP1 mRNA and E2F1 mRNA, data from GSE212301.

[0087] Figure 14 shows the experimental results of JP1 functioning through the MEK / ERK / E2F1 axis in Example 14 of the present invention. (a) Western blot analysis of protein levels of MAPK signaling pathway-related molecules pc-Raf, p-MEK, p-ERK, and E2F1. (b) Image J analysis of protein grayscale values.

[0088] Figure 15 shows the experimental results of MTA inhibiting SP1 in Example 15 of the present invention, which blocked the hair follicle growth-promoting effect of JP1 on AGA model mice. (a) Model design scheme diagram. (b) Mouse weight change curve during the experiment. (c) Appearance of the hair-loss area on the mouse skin during the experiment. (d) Skin color rating of mice in each group.

[0089] Figure 16 shows the histomorphological changes observed in mice in Example 16 of this invention, where MTA inhibition of SP1 blocked JP1 and promoted hair follicle growth in AGA model mice. (a) Longitudinal section of hair follicle stained with H&E. (b) Relative comparison of dermal layer thickness in mouse skin on day 12. (c) Statistical analysis of hair bulb diameter in mouse hair follicles on day 12. (d) Cross section of hair follicle stained with H&E. (e) Statistical analysis of the total number of hair follicles in mouse skin on day 12.

[0090] Figure 17 shows the experimental results of MTA blocking the effect of JP1 on the expression of downstream molecules of SP1 in Example 17 of the present invention. (a) Changes in the expression of key molecules in the JP1 mechanism pathway at the end of the immunoblotting model (day 12). (b) Changes in the protein levels of SP1, Wnt10a, Wnt10b, and β-catenin in mouse skin on day 4. (c) Changes in the protein levels of SP1, Wnt10a, Wnt10b, and β-catenin in mouse skin on day 8.

[0091] Figure 18 shows the experimental results of JP1 targeting integrin ανβ1 into hair follicle tissue in Example 18 of this invention. (a) mRNA expression levels of integrin α and integrin β subunits that can bind to RGD in the skin tissue of the control group and AGA mice. (b) Effect of JP1 intervention on the mRNA expression levels of each integrin α and β subunit. (c) Immunoblotting detection of protein expression levels of integrin αν and β1. (d) Image J statistical analysis of gray values ​​of integrin αν and β1.

[0092] Figure 19 shows representative results of immunofluorescence detection of integrin αν and β1 in Example 19 of this invention. (a) Representative distribution and expression level of integrin αν in each group after immunofluorescence staining. (b) Image J statistical analysis of fluorescence intensity of integrin αν in each group and comparison of its differences. (c) Representative distribution and expression level of integrin β1 in each group after immunofluorescence staining. (d) Image J statistical analysis of fluorescence intensity of integrin β1 in each group and comparison of its differences.

[0093] Figure 20 illustrates the molecular mechanism by which JP1 of the present invention promotes hair growth in a TP-induced AGA model. Detailed Implementation

[0094] The present invention will now be described in further detail with reference to the accompanying drawings and embodiments. However, the present invention is not limited to the examples given. The materials, methods, experimental model conditions, etc. used in each embodiment are appended after each embodiment. Unless otherwise specified, all materials used are conventional materials (such as commercially available products), and all experimental methods used are conventional experimental methods.

[0095] Example 1

[0096] The JP1 sequence used in this embodiment is FPGSDRF-RGD (SEQ ID No. 1), wherein amino acid S is phosphorylated; furthermore, the conformation of each amino acid in the FPGSDRF sequence is L-type or D-type. Note: The JP1 used in the following embodiments is the same as that in this embodiment, and will not be repeated below.

[0097] This embodiment investigates the effect of JP1 on hair growth.

[0098] In this embodiment, C57BL / 6 mice were used to establish the model according to the first batch of design schemes (Figure 1a). For specific experimental details, please refer to the "Experimental Methods" section below, "I. TP-induced AGA Model", which describes the experimental content conducted according to "Animal Model I".

[0099] In the experimental results:

[0100] (1) On day 10, the skin on the back of the control group mice turned black, while the model group did not change, indicating that the model was successfully established.

[0101] (2) JP1 promotes hair growth whether injected intraperitoneally or applied topically.

[0102] (3) The combination of 2% JP1 and minoxidil had a synergistic effect and was more effective than the JP1 or minoxidil monotherapy group (Figure 1b).

[0103] (4) There was no statistically significant difference in weight gain among the groups during the mouse model period (Figure 1c, P>0.05).

[0104] The results of this embodiment indicate that JP1 can promote hair growth in AGA mice.

[0105] Example 2

[0106] This embodiment scores the skin color change in the AGA experimental area of ​​the model mouse in Example 1, and the results are shown in Figure 2.

[0107] The color scoring reference standard for the hairless area on the back of the mouse is compared with that of Figure 2a.

[0108] In the experimental results:

[0109] (1) The hair growth index of the 2% JP1 group was significantly higher than that of the model group (P<0.001), and the hair growth index of the 2% JP1 (apply) group was also higher than that of the model group (P<0.05). However, the skin color of the mice in the 1% JP1 group had just begun to change and was not statistically significant compared with the model group (P>0.05). Note: The administration method for the 1% JP1 group and the 2% JP1 group was intraperitoneal injection, and the administration method for the 2% JP1 (apply) group was topical application. The same applies below.

[0110] (2) The hair growth index of the 2% JP1 (apply) group was also higher than that of the solvent-applied skin group (vehicle (apply) group), indicating that the solvent alone does not promote hair growth (Figures b and c in Figure 2).

[0111] The results of this embodiment show that JP1 significantly accelerates the change in skin color on the back of AGA mice.

[0112] Example 3

[0113] In this embodiment, the dermal thickness, number and size of hair follicles were observed after H&E staining of transverse and longitudinal sections of skin tissue from the AGA experimental area of ​​the model mouse in Example 1. The results are shown in Figure 3.

[0114] In the experimental results:

[0115] (1) The longitudinal section results showed that, compared with the model group and the solvent group, the dermal thickness of each JP1 treatment group increased and the hair follicle length was longer (Figure 3a). The dermal thickness is related to the growth cycle of the hair follicle. The dermal layer is thicker when the hair follicle is in the growth phase and gradually thins as the hair follicle enters the regression phase. The model group and the solvent control group were in the resting phase, while the other treatment groups had entered the growth phase (Figure 3c).

[0116] (2) Cross-sectional results showed that the total number of hair follicles in each treatment group was greater than that in the model group and the solvent control group (P<0.001, Figures b and d in Figure 3). The total number of hair follicles in the group treated with 2% JP1 and minoxidil was greater than that in the group treated with either of them alone (Figures b and d in Figure 3).

[0117] The results of this embodiment show that JP1 can shorten the resting phase of hair follicles, allowing them to enter the growth phase more quickly, and its effect is comparable to that of minoxidil.

[0118] Example 4

[0119] The results of Example 1 have demonstrated that the JP1 skin application group promotes hair growth in AGA mice, and the effect is similar to that of the intraperitoneal injection group.

[0120] This embodiment further explores the stability of JP1 in coating solvents. In this embodiment, a solvent group and a JP1 group were set up. The content and molecular weight of JP1 in the coating solvent were detected by high-performance liquid chromatography (HPLC) on days 1, 3, 7, and 14 after solution preparation for evaluation. The prepared solutions were stored in a 4°C refrigerator. Specific experimental details are provided in "Experimental Methods" section "II. JP1 Stability Experiment" below.

[0121] The results are shown in Figure 4: No JP1 degradation was found in the skin-applied solvent within 14 days, and the main peak area accounted for nearly 100%, indicating that, apart from impurities in the solvent, the peak area of ​​substances generated by the degradation of the drug to be tested was <0.5mAU, and no JP1 degradation was detected.

[0122] The results of this embodiment show that JP1 has good stability.

[0123] Example 5

[0124] This embodiment aims to elucidate the mechanism of JP1's penetration into the skin and hair follicles. Following the model design strategy described in Example 1, an AGA model was established for 14 days. Afterward, a 1% FITC-JP1 solution was applied to the hair-removed area on the back of the mice in a light-protected environment. Five observation time points were set: 0.5, 1, 2, 4, and 8 hours. Fresh skin tissue was prepared into frozen sections, and the penetration pathway of JP1 in the skin tissue and its accumulation in hair follicles were observed under an inverted fluorescence microscope. Specific experimental details are provided in "Experimental Methods" section "III. JP1 Permeability Experiment" below.

[0125] The results are shown in Figure 5: Compared with the skin structure shown by H&E staining, the drug basically covered the skin surface at 0.5h, and some of it had entered the deep layers of the skin. Then it gradually entered the hair follicles, reaching its peak at 4h. The fluorescence brightness was significantly reduced at 8h, indicating that JP1 can penetrate the skin and target the hair follicle tissue.

[0126] The results of this embodiment show that JP1 can penetrate the skin and target hair follicle tissue, exhibiting good transdermal properties.

[0127] Example 6

[0128] The animal model results of Example 1 have confirmed that JP1 can promote hair growth whether injected intraperitoneally or applied topically. To investigate whether there is a dose-response relationship between JP1 topically and AGA mice, this example establishes an AGA mouse model and uses different doses of JP1 for topical intervention.

[0129] In the experiment exploring the transdermal permeability of JP1 (Example 5), it was found that the FITC fluorescence accumulation of JP1-linked cells in the hair follicle was highest 4 hours after administration, while the amount of JP1 significantly decreased at 8 hours. To prolong the duration of action of JP1, the animal model design in this example adjusted the initial once-daily administration to twice-daily administration with an interval of 6 hours (Figure 6a). For specific experimental details, please refer to the experimental content of "I. TP-induced AGA model" under "Experimental Methods" below, which is conducted according to "Animal Model II".

[0130] The results showed that on day 11 after continuous intervention, the skin color of mice in the model group was orange, while the skin color of all JP1 groups and the minoxidil group had turned gray. The differences in skin color among the groups became more pronounced on day 15. On day 18, the 1% JP1 group had the highest hair coverage, followed by the 0.5% JP1 group, and the 0.1% JP1 group had the lowest (Figure 6, b). Skin color scores also showed differences (Figure 6, d and e). Meanwhile, there was no statistically significant difference in body weight among the groups during the experiment (Figure 6, c). These results suggest that topical administration of JP1 promotes TP-induced AGA hair follicle regeneration with a significant dose-response relationship.

[0131] The results of this embodiment indicate that JP1 promotes hair growth in a dose-dependent manner.

[0132] Example 7

[0133] In this embodiment, longitudinal sections of hair follicles from skin tissue samples of the model mouse from Example 6 were stained with H&E.

[0134] The results (Figures 7a to 7e) showed that on day 11, the model group had just entered the anagen phase, while the JP1 intervention group was in the mid-anagen phase, with longer hair follicles and larger hair bulbs; the 1% JP1 group had the largest hair bulbs. On day 18, all groups were in the anagen phase. On day 25, all groups gradually transitioned to the catagen phase. On day 32, the model group entered the telogen phase, while the hair follicles in the medium- and high-dose JP1 groups re-entered the anagen phase. Changes in dermal layer thickness were consistent with the longitudinal section results. Transverse section results showed that, compared to the model group, the total number of hair follicles in the JP1 intervention group was significantly increased in a dose-dependent manner; the hair bulb diameter was also significantly larger than that in the model group.

[0135] The results of this embodiment show that there were significant differences in the hair follicle growth cycle among the groups after intervention with JP1 or minoxidil; both drugs can accelerate the entry of hair follicles into the anagen phase and shorten the telogen phase, while also prolonging the time that hair follicles are in the anagen phase. This is also the pathological evidence for the dose-dependent hair growth promotion of JP1.

[0136] Example 8

[0137] This embodiment uses skin tissue from the mouse model in Example 6.

[0138] To elucidate the molecular mechanism by which JP1 promotes hair follicle growth, this example first detected the mRNA levels of AR and SRD5A2 in the skin tissues of each model mouse. The results showed that there was no statistically significant difference in mRNA levels among the groups (Figure 8a, P>0.05).

[0139] To explore the mechanism of JP1's hair growth promotion at the protein level, this embodiment performed proteomics and high-throughput sequencing of protein phosphorylation modifications on the skin tissues of the model group and the 1% JP1 group mice. KEGG signaling pathway analysis of the sequencing data showed that the main difference between the two groups was enriched in the Wnt signaling pathway (Figure 8b). GSEA analysis also showed that the Wnt / β-catenin signaling pathway was enriched in the skin tissues of JP1-treated mice (Figure 8c).

[0140] The results of this embodiment indicate that JP1 promotes hair follicle growth by activating the Wnt signaling pathway.

[0141] Example 9

[0142] This embodiment verifies the regulatory effect of JP1 on β-catenin.

[0143] β-catenin is the most critical indicator in the Wnt signaling pathway and a key point in controlling hair follicle formation within this pathway.

[0144] The results (Figures 9a to 9e) showed that, compared to the model group, the mRNA levels of β-catenin and its nuclear transcription factor Lef1 in skin tissue were significantly increased in the JP1 group (Figure 9a). Western blot analysis indicated that JP1 intervention increased β-catenin expression in a dose-response relationship; minoxidil also exerted its effect through this pathway, but the effect was slightly weaker than that of the JP1 group (Figures 9b and 9c). The immunofluorescence staining results were consistent with the trends observed in the Western blot analysis (Figures 9d and 9e).

[0145] Example 10

[0146] This embodiment verifies that JP1 regulates the upstream Wnt ligand of β-catenin.

[0147] The Wnt family can be divided into two types: one involved in the β-catenin-mediated classical signaling pathway, including Wnt1, Wnt2, Wnt2b, Wnt3, Wnt3a, Wnt7a, Wnt7b, Wnt8, Wnt8b, Wnt10A, and Wnt10b; and the other non-classical signaling pathway independent of β-catenin, including Wnt4, Wnt5a, Wnt6, and Wnt11. Previous studies have identified Wnt ligands primarily involved in androgenetic alopecia as including Wnt1, Wnt3a, Wnt4, Wnt5a, Wnt7b, Wnt10a, and Wnt10b.

[0148] To identify the upstream Wnt ligand that JP1 regulates β-catenin, this embodiment used the intersection of Wnt-related ligands from the classical Wnt signaling pathway and AGA-related Wnt ligands to screen for relevant Wnt ligands, including Wnt1, Wnt3a, Wnt7b, Wnt10a, and Wnt10b. In this embodiment, the mRNA of these Wnt molecules was detected by qPCR in skin tissue from the mouse model of Example 6.

[0149] The results show:

[0150] (1) In the JP1 group, the expression of Wnt10a and Wnt10b was increased and showed a dose-response relationship (P<0.05); while the mRNA levels of other Wnt molecules were not statistically significant among the groups (P>0.05, Figure 10a).

[0151] (2) Further analysis of the correlation between Wnt10a / Wnt10b and JWA in the alopecia area tissue of AGA patients in the public database revealed that the expression levels of JWA mRNA and Wnt10a mRNA were significantly positively correlated (P<0.001, r=0.7626, Figure 10b), and the expression levels of JWA mRNA and Wnt10b mRNA were also significantly positively correlated (P<0.005, r=0.6876, Figure 10c).

[0152] The results of this embodiment show that JP1 increases the expression level of β-catenin by Wnt10a / Wnt10b.

[0153] Example 11

[0154] This embodiment predicts the upstream transcription factor that JP1 regulates Wnt / β-catenin.

[0155] In this embodiment, a Western blot experiment was performed on the skin tissue of the model mouse from Example 6. The results confirmed that JP1 significantly upregulated the protein expression of Wnt10a and Wnt10b in a dose-dependent manner (Figures a to c of Figure 11).

[0156] To demonstrate the mechanism by which JP1 upregulates Wnt10a / Wnt10b expression, this embodiment used two online tools, JASPAR (https: / / jaspar.elixir.no / ) and Animal TFDB (https: / / guolab.wchscu.cn / AnimalTFDB4 / / # / ), to predict the transcription factors of Wnt10a / Wnt10b. After taking the intersection of these predictions, two candidate transcription factors, SP1 and MYC, were identified (Figure 11d). The results suggest that JP1 may upregulate Wnt10a / Wnt10b expression through SP1 / MYC.

[0157] Example 12

[0158] This embodiment verifies that SP1 is a transcription factor that regulates Wnt / β-catenin by JP1.

[0159] Based on the online tool prediction results of Example 11, this example uses qPCR to detect the mRNA levels of SP1 and MYC in the skin tissue of the model mice from Example 6. The results show:

[0160] (1) SP1 mRNA was highly expressed in the JP1 group, and the expression level in the 1% JP1 group was significantly higher than that in the 0.1% JP1 group (P<0.05). However, there was no statistically significant difference in MYC mRNA levels among the groups (P>0.05), suggesting that JP1 may activate the nuclear transcription factors Wnt10a and Wnt10b (Figure 12a).

[0161] (2) Immunoblotting results showed that SP1 protein expression level increased in the JP1 group (Figures b and c in Figure 12).

[0162] (3) Similarly, the immunofluorescence results were consistent with the protein immunoblotting results (Figure 12, d and e).

[0163] The results of this embodiment indicate that JP1 can promote the increase of SP1 expression.

[0164] Example 13

[0165] To further demonstrate the upstream regulatory mechanism of JP1 activation of SP1 expression, this embodiment first used three online tools to predict transcription factors: JASPAR (https: / / jaspar.elixir.no / ), PROMO (https: / / alggen.lsi.upc.es / cgibin / promo_v3 / promo / promoinit.cgi?dirDB=TF_8.3), and Animal TFDB (https: / / guolab.wchscu.cn / AnimalTFDB4 / / # / ) (Figure 13a). The intersection of the predicted data yielded four transcription factors that can bind to the SP1 promoter region: E2F1, AR, FOXD3, and FOXI1 (Figure 13a).

[0166] In this embodiment, the mRNA levels of the above four transcription factors were further detected in the skin tissue of the mouse model from Example 6. The results showed that:

[0167] (1) After JP1 intervention, the mRNA expression level of E2F1 increased (P<0.01), while the expression of other molecules did not change significantly (P>0.05), suggesting that E2F1 regulates SP1, thereby causing changes in downstream molecules (Figures b and c of Figure 13).

[0168] (2) Further analysis of the results from the GEO database (GSE212301) showed that there was a significant positive correlation between SP1 mRNA and E2F1 mRNA (Figure 13, d), suggesting that JP1 activates the Wnt pathway through the E2F1-SP1 axis, thereby promoting hair growth on the back of AGA mice.

[0169] The results of this embodiment show that JP1 activates the Wnt pathway through the E2F1-SP1 axis.

[0170] Example 14

[0171] Previous studies have confirmed that JP1 is a phosphorylated small peptide kinase that regulates downstream molecules and signaling pathways by promoting the phosphorylation of interacting molecules. Literature reports that E2F1 is a downstream molecule of the MAPK signaling pathway; its expression increases when PKC / Raf / MEK / ERK signaling molecules in the MAPK pathway are activated. Previous studies have found that JWA participates in regulating MEK-ERK activity in the MAPK signaling pathway, but has no effect on upstream Raf.

[0172] Accordingly, this embodiment examined the protein levels of pc-Raf, p-MEK, p-ERK, and E2F1 in the skin tissue of the model mice in Example 6. The results showed that under JP1 intervention, the expression levels of p-MEK and p-ERK in the MAPK signaling pathway were increased (P<0.05, Figure 14a), and showed a dose-response relationship (Figure 14b); however, the pc-Raf protein level did not change significantly. This suggests that JP1 is involved in activating the activity of p-MEK and p-ERK, but does not affect the activity of upstream Raf.

[0173] The results of this embodiment show that JP1 functions through the MEK / ERK / E2F1 axis.

[0174] Example 15

[0175] To verify that JP1 promotes AGA hair follicle regeneration by activating the Wnt / β-catenin signaling pathway through the MAPK / E2F1 / SP1 signaling axis, this embodiment further constructed an AGA mouse model that specifically inhibits SP1 (Figure 15a). For specific experimental details, please refer to the experimental content of "I. TP-induced AGA model" in "Experimental Methods" below, which is conducted according to "Animal Model III".

[0176] The weight of the mice was measured every 2 days during the model period. The results showed that there was no significant difference in weight change among the groups (P>0.05, Figure 15b).

[0177] Analysis of the skin color on the backs of mice in each model group showed that the skin of mice in the 1% JP1 group turned gray from day 8, while the skin of mice in the 1% JP1+MTA group did not change. On day 10, the skin of mice in the 1% JP1 group turned significantly dark gray (P<0.001), while only a small number of areas in the 1% JP1+MTA group showed color change (Figure 15c). These results suggest that MTA inhibition of SP1 can block the hair follicle growth-promoting effect of JP1 on AGA model mice (Figure 15d).

[0178] The results of this embodiment indicate that MTA inhibition of SP1 can block the hair follicle growth-promoting effect of JP1 on AGA model mice.

[0179] Example 16

[0180] To further confirm at the tissue morphology level that JP1 promotes hair follicle growth on the back of AGA model mice through the Wnt / β-catenin signaling pathway regulated by SP1, this example performed H&E staining on skin tissues of the model mice in Example 15 at different time points (days 4, 8, and 12 after intervention).

[0181] Longitudinal sections of skin tissue showed that on day 4, the hair follicle morphology was consistent across all groups. On day 8, compared with the 1%JP1+MTA group, the hair follicles in the 1%JP1 group extended downwards, and the hair bulbs swelled, indicating that the hair follicles entered the anagen phase earlier after JP1 intervention (Figure 16a). The 1%JP1 group significantly increased dermal thickness and hair bulb diameter (P<0.001), while the 1%JP1+MTA group and the model group had thinner dermal layers and smaller hair bulb diameters (Figures 16b and c).

[0182] The results of cross-section of skin tissue showed that the total number of hair follicles in the 1%JP1 group was significantly higher than that in the model group and the 1%JP1+MTA group on days 8 and 12 (P<0.001, Figure 16, d and e).

[0183] The results of this embodiment indicate that MTA inhibition of SP1 can block the histomorphological changes that promote hair follicle growth in AGA model mice by JP1.

[0184] Example 17

[0185] In this embodiment, relevant molecular immunoblotting detection was performed on the back skin tissue of mice in each group of the AGA model in Example 15 above. The results showed that: after inhibiting SP1, there was no significant effect on the expression level of upstream molecules MEK / ERK / E2F1 of SP1, while the expression levels of SP1 and its downstream molecules were inhibited, thereby blocking the growth-promoting effect of JP1 on hair follicles (Figure 17a).

[0186] The results of the back skin appearance images in Example 15 showed that differences in skin color began to appear among the groups on day 8 (Figure 15, c). To verify the changes in key molecules of the hair follicle growth signaling pathway regulated by JP1 at different time points in the model, the immunoblotting results of this example showed that the expression levels of the above key molecules did not change significantly in the control group, model group, and 1% JP1 group on day 4 of intervention; however, on day 8, the protein expression levels of four molecules in the 1% JP1 group increased significantly, consistent with the changes in skin color (Figure 17, b and c).

[0187] The results of this embodiment show that MTA can block the effect of JP1 on the expression of downstream molecules of SP1.

[0188] Example 18

[0189] Having clarified the phenotypic and molecular mechanism by which the JP1 targeting peptide effectively promotes hair follicle growth in AGA model mice, this example further investigated the dynamic process of the JP1 active peptide, labeled with FITC and linked to the targeting integrin RGD, penetrating the skin and targeting hair follicles, as well as the exact molecules it binds to, using skin tissue from the model mice of Example 6. It is known that the RGD target can bind to αⅡb, αv, α5, and α8 of the integrin α isoform and β1, β3, β5, β6, and β8 of the integrin β isoform. The results of this example, detecting the mRNA expression levels of the integrin subunits that RGD can bind in skin tissue, showed that αν expression was significantly highest in both the normal control group and TP-induced AGA mouse skin tissue; β1 expression was significantly highest in the integrin β isoform (Figure 18a). This suggests that in AGA model mouse skin tissue, the effect of JP1 targeting hair follicle tissue is mainly achieved through integrin ανβ1.

[0190] This embodiment further examined the mRNA expression levels of RGD-targeting molecules in the skin tissues of mice in the AGA model control group, model group, and JP1 treatment group. The results showed that the expression levels of integrin αν and β1 in the skin tissues of mice in the TP model group were significantly decreased, while the expression levels of integrin αν and β1 were significantly increased after JP1 intervention (P<0.01), followed by β5. At the end of the model, the expression levels of integrin αν and β1 in the JP1 intervention group had recovered to the control group levels (P<0.01, Figure 18b).

[0191] In this embodiment, Western blotting was used to further verify the protein levels of integrin αν and β1. The results also showed that JP1 reversed the downregulated expression levels of integrin αν and β1 by TP (P<0.01, Figures c and d in Figure 18). It is worth noting that although the above series of model results showed that minoxidil and JP1 had similar effects on promoting hair follicle growth, they had no significant effect on the expression levels of integrin αν and β1 inhibited by TP (Figures c and d in Figure 18).

[0192] The results of this embodiment indicate that JP1 primarily targets integrin ανβ1 to enter hair follicle tissue.

[0193] Example 19

[0194] To verify whether JP1-targeted integrin molecules are localized to hair follicles in skin tissue, this example used immunofluorescence staining of skin tissue from the model mice of Example 6 to detect the expression levels and tissue localization and distribution of integrin αν and integrin β1. The results showed that the expression of integrin αν (Figures a and b in Figure 19) and integrin β1 (Figures c and d in Figure 19) was increased in all JP1 intervention groups, and both were localized to hair follicles. The changes in target molecule expression in each group were consistent with the trend of protein expression levels detected by Western blotting.

[0195] The results of this embodiment show that in the AGA model, JP1 not only targets integrin ανβ1 into hair follicle cells, but also reverses the integrin ανβ1 expression level in hair follicle cells suppressed by TP.

[0196] Based on the results of the above embodiments, as shown in Figure 20, the molecular mechanism by which JP1 promotes hair follicle growth in the AGA model includes:

[0197] (1) JP1, as a functional polypeptide fragment of the JWA gene, enters the hair follicle target cells through RGD targeting integrin ανβ1 after local application, upregulates the expression of integrin ανβ1, and thus increases the content of JP1 polypeptide in the hair follicle target cells, thereby promoting hair follicle growth through positive feedback effect.

[0198] (2) JP1 activates the MEK / ERK / E2F1 signaling axis, which in turn promotes the expression of SP1 and activates Wnt10a / Wnt10b. It binds to the Frizzled receptor family and LRP5 / LRP6 co-receptors, increases the aggregation of free β-catenin into the nucleus, binds to the LEF / TCF transcription complex, activates downstream signals such as c-myc, and plays a role in promoting AGA hair follicle growth.

[0199] Example 20

[0200] This embodiment is to verify the anti-AGA effects of each JWA peptide other than JP1.

[0201] In this embodiment, each JWA polypeptide (SEQ ID No. 2 to SEQ ID No. 39 in sequence, note: Acp is the abbreviation for 6-aminocaproic acid) shown in the table below was tested according to Examples 6 to 12. The amino acid S of each JWA polypeptide was modified by phosphorylation.

[0202] Due to space limitations, specific experimental data are not listed in this embodiment. The obtained experimental data show that the results of the detection of each JWA peptide according to Examples 6 to 15 are basically consistent with JP1.

[0203] in conclusion

[0204] Based on the research results represented by the above embodiments, this invention successfully constructed a TP-induced androgenetic alopecia mouse model. Whether through intraperitoneal injection or topical application, the series of JWA peptides, represented by JP1, can dose-dependently promote dorsal hair growth in the TP-induced AGA mouse model. Mechanistically, the aforementioned JWA peptides activate the p-MEK / p-ERK / E2F1 axis, promoting the expression of nuclear transcription factor SP1, thereby activating the Wnt pathway. Wnt10a and Wnt10b bind to receptors on the cell membrane, causing a large amount of free β-catenin to accumulate and enter the nucleus, activating downstream signaling. This achieves the effect of regulating the hair follicle cycle and promoting hair growth. Furthermore, the aforementioned JWA peptides target integrin ανβ1 into hair follicle target cells, upregulating the expression level of integrin ανβ1 suppressed by TP, and improving the hair follicle microenvironment. This invention not only provides scientific evidence for the treatment of AGA with the aforementioned JWA peptides but also elucidates a new molecular mechanism by which the aforementioned JWA peptides activate hair follicle stem cells to promote hair growth.

[0205] The materials, methods, experimental model conditions, etc. used in the above embodiments are as follows.

[0206] I. Experimental polypeptides

[0207] The JWA peptide series, represented by JP1, was synthesized by GL Biochem (Shanghai) Ltd. and Hybio Pharmaceutical Co., Ltd. (Shenzhen, China). It has a purity >98% and is water-soluble. FITC-JP1 was synthesized by Zhejiang Paitai Biotechnology Co., Ltd. (Hangzhou, China), and its purity >98% was confirmed by high-performance liquid chromatography. JWA peptide lyophilized powder can be stored long-term at -20℃.

[0208] II. Reagents and Kits Used in the Experiment

[0209] Specific information on the reagents used in this invention is shown in Table 1.

[0210] Table 1: Specific information on reagents used in this invention

[0211] III. Antibodies used in the experiment

[0212] Specific information about the antibodies used in this invention is shown in Table 2.

[0213] Table 2: Specific information on the antibodies used in this invention

[0214] IV. Primer Synthesis

[0215] The primers used in this study were synthesized by Shanghai Jierui Biotechnology Co., Ltd., and their specific sequences are shown in Table 3.

[0216] Table 3: Specific primer sequences used in this study

[0217] V. Prepare reagents yourself

[0218] 1. PBS solution: Weigh 3.5g Na2HPO4·12H2O, 8g NaCl, 0.2g KCl and 0.2g KH2PO4 and dilute to 1000ml with double-distilled water. After autoclaving, store at 4℃.

[0219] 2. RIPA lysis buffer: Weigh 5g sodium deoxycholate, 0.5g SDS and 4.39g NaCl, place them in 25mL 1M Tris-HCl (pH=7.6) and 5mL Triton X-100, dissolve with double-distilled water and bring the volume to 500ml, store at 4℃.

[0220] 3. 5× Protein Loading Buffer (5×SDS): Weigh 0.5g SDS and 25mg bromophenol blue, then add 5ml glycerol and 5ml 1M Tris-HCl (pH=6.8) buffer, aliquot into 1ml vials and store at -20℃. Before use, add 50μl β-me (mercaptoethanol) to each vial and mix well.

[0221] 4. Stacking gel (top gel) buffer: Weigh 121.1g of Tris powder and dissolve it in double-distilled water. Adjust the pH to 6.8 with concentrated hydrochloric acid, and finally bring the volume to 1000ml with double-distilled water.

[0222] 5. Separating gel (lower gel) buffer: Weigh 181.5g of Tris powder and dissolve it in double-distilled water. Adjust the pH to 8.8 with concentrated hydrochloric acid, and then bring the volume up to 1000ml with double-distilled water.

[0223] 6. 30% Acrylamide solution: Weigh 290g of acrylamide and 10g of methylenebisacrylamide, dissolve in double-distilled water and bring the volume to 1000ml, store in the dark at 4℃.

[0224] 7. 1× Electrophoresis Buffer: Weigh 3.03g Tris, 14.4g glycine, and 1g SDS into a beaker, dissolve in double-distilled water, and bring the volume to 1000ml.

[0225] 8. 1× Transfer Buffer: Weigh 2.4g Tris and 11.5g glycine, add 200mL methanol, and double distill to a final volume of 1000mL.

[0226] 9. 1×TBST buffer: Weigh 24.2g Tris and 80g sodium chloride into a beaker, dissolve in double-distilled water, adjust the pH to 7.6 with concentrated hydrochloric acid, and bring the volume to 1000ml with double-distilled water. Store at room temperature to obtain 10×TBS. Measure 100ml of 10×TBS, add it to 900ml of double-distilled water, and add 1mL of Tween-20. Mix well before use.

[0227] 10. Blocking solution (5% skim milk powder): Weigh 10g of skim milk powder and add 200mL of 1×TBST and mix thoroughly.

[0228] 11. Eluent: Dissolve 15g glycine and 1g SDS in double-distilled water, add 10mL Tween-20, adjust the pH to 2.2 with concentrated hydrochloric acid, and bring the volume to 1000ml with double-distilled water.

[0229] 12. JP1 Intraperitoneal injection solvent: physiological saline.

[0230] 13. JP1 skin coating solvent: 10% physiological saline + 40% anhydrous ethanol + 50% propylene glycol; 50 μl / animal / time.

[0231] 14. Modeling drug: Testosterone propionate TP (prepared by equal dilution method) was diluted with soybean oil to a concentration of 25 mg / ml of testosterone propionate injection to 2.5 mg / ml. The solution was shaken thoroughly for 2-3 minutes. The dosage was 5 mg / kg / day, 50 μl / animal, once daily. Testosterone propionate TP (25 mg / kg) was purchased from Sichuan Jinke Pharmaceutical Co., Ltd.

[0232] 15.5% Minoxidil: Purchased from Zhejiang Wansheng Pharmaceutical Co., Ltd., for topical application, 100 μl / animal, once daily.

[0233] 16. Mithramycin A (MTA): SP1 inhibitor, purchased from MCE. Solvent: 10% DMSO + 40% PEG300 + 5% Tween 80 + 45% physiological saline.

[0234] Experimental methods

[0235] I. TP-induced AGA model

[0236] The mice used in this study were 5-week-old male C57BL / 6J mice (18–20g), all purchased from Jiangsu Jicui Yaokang Biotechnology Co., Ltd. All experimental animals were housed in an SPF-grade barrier environment at the Nanjing Medical University Experimental Animal Center, and all related animal experimental procedures were approved by the Animal Ethics Committee of Nanjing Medical University. The control group received no treatment, while mice in other groups received daily subcutaneous injections of TP on their backs. After two weeks, the hair on the backs of the mice was shaved using an electric shaver in an area of ​​2cm × 4cm, and any remaining hair was removed with Veet depilatory cream. AGA model mice continued to receive subcutaneous injections of TP in the shaved area on their backs until the end of the experiment. Treatment groups began receiving medication on the first day after hair removal.

[0237] Animal Model 1:

[0238] Except for the normal control group (n=3), all mice subcutaneously injected with TP were randomly divided into the following groups (n=3): model group, 1% JP1 and 2% JP1 (intraperitoneal injection), 1% JP1 and 2% JP1 (topical application), 5% minoxidil group, and 2% JP1 (intraperitoneal) and 5% minoxidil combination group. Mice were euthanized after 35 days of continuous administration.

[0239] Animal Model Two:

[0240] Except for the normal control group (n=20), all mice subcutaneously injected with TP were randomly divided into the following groups (n=20): model group, 0.1% JP1, 0.5% JP1, 1% JP1 (all administered topically), and 5% minoxidil group. During the experiment, changes in skin color on the backs of the mice were observed, and photographs were taken at key time points. Mice were euthanized in four batches at the key time points (n=5 per group each time).

[0241] Animal Model 3:

[0242] Except for the normal control group (n=20), all mice subcutaneously injected with TP were randomly divided into the following groups (n=20): model group, 1% JP1 (skin coating), and 1% JP1 + MTA group. During the experiment, changes in skin color on the backs of the mice were observed, and photographs were taken at key time points. Mice were euthanized in batches at key time points (n=5 per group each time). MTA was administered at a dose of 0.3 mg / kg / day, once daily, intraperitoneally, 100 μl per injection.

[0243] Mice were weighed every two days during the experiment. Skin color was assessed using skin scoring maps.

[0046] Mice were euthanized after the experiment, and skin tissue from the hairless area on their backs was collected. Some tissues were fixed with 4% paraformaldehyde solution and prepared as paraffin sections for H&E or immunofluorescence staining, while other tissues were stored at -80°C for related molecular expression detection (qRT-PCR, Western Blot, IP, etc.).

[0244] II. JP1 Stability Test

[0245] The stability of JP1 dissolved in the coating solvent was tested at different time points. JP1 was prepared using the coating solvent, and a solvent control group and a 10 mM JP1 group were set up. The prepared solutions were stored at 4°C. High-performance liquid chromatography (HPLC) was used to detect the JP1 at five time points: 0, 1, 3, 7, and 14 days. A standard curve was plotted with peak area on the ordinate and JP1 concentration on the abscissa.

[0246] III. JP1 Permeability Test

[0247] Fifteen male C57BL / 6J mice were injected with TP on their backs for two weeks, and then their backs were shaved. On the first day after shaving, 1% FITC-JP1 solution was applied topically to the shaved area in the dark, and the mice were divided into five groups (n=3) at 0.5h, 1h, 2h, 4h and 8h.

[0248] Back skin was harvested at the appropriate time points and fixed in 4% paraformaldehyde. After 48 hours of fixation, the skin tissue was dehydrated for 2 days each in 20% and 30% sucrose solutions. After gradient dehydration, sections (20 μm) were prepared using a cryostat. Fluorescent sections were photographed using a Zeiss inverted fluorescence microscope.

[0249] IV. Protein Extraction and Concentration Determination

[0250] 1. Protein Extraction: Weigh 70 mg of skin tissue into a 1.5 mL centrifuge tube, add 500 μl of RIPA tissue lysis buffer containing protease inhibitors and phosphatase inhibitors, place the tissue on an ice box, first use a tissue homogenizer to pulverize the tissue, then use an ultrasonic homogenizer to obtain a tissue suspension, and then place the suspension in a 4°C rotary fan for lysis for 2 h. Finally, set the centrifuge to 12,000 × g, 4°C, and centrifuge for 15 min, collecting the supernatant as total protein.

[0251] 2. BCA kit for protein concentration determination:

[0252] a) Preparation of standard curve: Take 10 μl of BSA standard protein with a concentration of 5 mg / mL and add 90 μl of physiological saline to prepare BSA standard protein with a final concentration of 0.5 mg / mL. Add 0, 1, 2, 4, 8, 12, 16, and 20 μl to each well of a 96-well plate, respectively, and then add physiological saline to bring the total concentration of each well to 20 μl.

[0253] b) Prepare protein samples: Add 1 μl of protein sample to each well, then add 19 μl of physiological saline.

[0254] c) Preparation of BCA working solution: Prepare BCA working solution according to the ratio of solution A: solution B = 50:1. Add 200 μl of working solution to each well and incubate in a 37°C incubator in the dark for 30 min.

[0255] d) Protein concentration determination: After 30 min, the absorbance of the microplate reader was set to 572 nm, and the protein sample concentration was calculated according to the protein concentration standard curve.

[0256] V. Western Blotting of Proteins

[0257] 1. Boiling protein samples: The total protein content of each tissue is 50 μg. Calculate the corresponding protein sample volume, add physiological saline to make up to 16 μl, then add 4 μl of 5×SDS loading buffer, heat at 100℃ for 5 min, cool to room temperature and store at -20℃.

[0258] 2. SDS-PAGE Gel Preparation: First, clean and leak-check the assembled glass plates. Prepare a lower separating gel of appropriate concentration based on the molecular weight of the protein to be detected. Use a lower concentration separating gel for high molecular weight proteins and a higher concentration for low molecular weight proteins; typically, a 10% separating gel is used. In a centrifuge tube, add the appropriate amounts of double-distilled water, 30% acrylamide solution, lower gel buffer, 10% SDS, 10% AP, and TEMED sequentially. Mix well and pour the mixture into the gaps between the glass plates. Add anhydrous ethanol to prevent the lower gel from drying out. Let it stand at room temperature until the lower gel solidifies for approximately 40 minutes, then drain the anhydrous ethanol. Next, prepare the upper gel. After adding the upper gel, insert a 1.0 mm sample comb into the upper gel, avoiding air bubbles. Let it solidify for approximately 30 minutes.

[0259] 3. Protein loading: Fix the gel onto the electrophoresis rack, fill the electrophoresis tank with electrophoresis buffer, remove the sample comb from the upper gel, add 10 μl of protein sample to each well using a pipette with a volume of 10 μl, and finally add 2 μl of molecular weight marker.

[0260] 4. Electrophoresis: Electrophoresis is performed under constant voltage conditions. The voltage is 80V for about 30 minutes. Then the voltage is adjusted to 120V until the electrophoresis is completed.

[0261] 5. Transfer: Place the PVDF membrane, activated with methanol solution, into the transfer buffer. Cut the desired protein according to molecular weight, and place the following components in sequence: white transfer clip, sponge, filter paper, PVDF membrane, gel, filter paper, sponge, and white transfer clip. Avoid generating air bubbles during the process. Place two ice boxes in the wet transfer tank, then place the tank on top of the ice. Finally, add the pre-cooled wet transfer buffer. Transfer at a constant current of 220 mA for 90 minutes.

[0262] 6. Sealing: Immerse the PVDF membrane in 5% skim milk (prepared with TBST) at room temperature for 1 hour. After sealing, wash once with TBST.

[0263] 7. Primary antibody incubation: Place the cut protein bands in the corresponding primary antibody and incubate overnight at 4°C on a shaker. TBST washing 3

[0264] Each session lasts 10 minutes.

[0265] 8. Secondary antibody incubation: Incubate with the corresponding secondary antibody at room temperature for 1 hour. Wash with TBST 3 times, 10 minutes each time.

[0266] 9. Protein band development: Prepare the luminescent solution at a 1:1 ratio in the dark. Use filter paper to absorb the TBST on the protein bands. Apply the luminescent solution evenly to the bands and finally develop them using a luminescence imaging system.

[0267] VI. Tissue RNA Extraction and Reverse Transcription

[0268] 1. Sample preparation: Take 15-20 mg of mouse skin tissue and place it in a 1.5 ml centrifuge tube. Quickly add 600 μl of pre-cooled lysis buffer and homogenize using an electric homogenizer. Gently pipette the homogenate 10 times and let it stand at room temperature for 3-5 min. Then centrifuge at 14,000 × g for 5 min and transfer the supernatant to a new centrifuge tube using a pipette with an appropriate volume.

[0269] Note: The dosage of mouse skin tissue should generally not exceed 30 mg. Excessive dosage may lead to incomplete lysis and a decreased yield. After lysis and centrifugation, some gelatinous material may remain at the bottom of the centrifuge tube; this should be transferred as supernatant to the next step. Discarding this gelatinous material will result in a yield decrease of approximately 30-50%. The gelatinous material will disappear after adding the binding buffer. Centrifugation is to remove any obvious lumps that are not fully homogenized. If viscous material remains after lysis, increase the number of pipetting cycles until the solution is completely clear. For samples rich in RNase, DTT should be added to a final concentration of 40 mM or β-mercaptoethanol to a final concentration of 1%.

[0270] 2. Add an equal volume of binding solution to the lysis buffer and gently invert to mix 3-5 times. Precipitation is normal.

[0271] 3. Transfer the mixture (including precipitate) to a purification column, centrifuge at 12,000×g for 30 seconds, and discard the liquid in the collection tube.

[0272] Note: When the volume of the lysis buffer is greater than 300 μl, the total volume will exceed the capacity of the purification column after adding an equal volume of binding buffer. In this case, the mixture should be passed through the column in two stages: pass half of the mixture through the column, and then repeat step 3 once with the remaining mixture. For some special samples, such as when the solution does not pass through completely, the centrifugation time can be extended to 1-2 minutes, or the centrifugation force can be increased to 16,000 × g.

[0273] 4. Add 600 μl of washing buffer I, centrifuge at 12,000 × g for 30 sec, and discard the liquid in the collection tube.

[0274] 5. Add 600 μl of washing buffer II, centrifuge at 12,000 × g for 30 sec, and discard the liquid in the collection tube.

[0275] 6. Repeat step 5 once.

[0276] 7. Centrifuge at the highest speed (approximately 14,000-16,000 × g) for 2 minutes to remove residual liquid.

[0277] 8. Place the RNA purification column in the Beyotime RNAeasy container. TM Add 30-50 μl of elution buffer to the RNA elution tube provided in the Animal RNA Extraction Kit (Centrifugal Column Type), incubate at room temperature for 2-3 minutes, and centrifuge at the highest speed for 30 seconds. The resulting solution is the purified RNA.

[0278] Note: The elution buffer needs to be added to the center of the purification column to ensure complete absorption. If the room temperature is low, preheat the elution buffer at 37°C for a short time. Adding the eluted solution back to the original purification column and centrifuging again can increase the yield by approximately 10-30%; alternatively, eluting once with fresh elution buffer after the first elution will yield approximately 15-40% of the RNA eluted in the first elution.

[0279] 9. Use a NanoDrop2000 nucleic acid and protein analyzer to detect RNA concentration and purity. Dilute the obtained RNA concentration to 1 μg / μl with DEPC water.

[0280] 10. Reverse transcribe the RNA into cDNA according to the instructions of the reverse transcription kit, and then perform subsequent experiments or store at -20℃ for later use.

[0281] VII. Real-time quantitative PCR experiment

[0282] 1. Dilute the specific gene amplification primers to 10 μM with ddH2O, prepare the qPCR reaction system according to the kit instructions, and add them sequentially to the 384-well plate.

[0283] The reaction system is as follows:

[0284] 2. Seal the 384-well plate with sealing film, centrifuge at 3000 rpm for 1 min at room temperature, and then perform qPCR amplification. Preheat the ABI 7900HT PCR instrument under the following thermal cycling conditions:

[0285] 3. Using GAPDH as an internal reference, 2 -ΔΔCT The relative mRNA expression level of a gene was calculated using a relative quantitative method.

[0286] 8. H&E staining

[0287] Basic process of preparing HE-stained paraffin sections:

[0288] 1. Sampling and Fixation: After the experiment, skin tissue was taken from the hairless area on the back of the mouse and placed in a 10ml EP tube. 4% paraformaldehyde was added to the EP tube, and the entire tissue was submerged in it to denature and coagulate the proteins of the skin tissue, thus maintaining the original morphology and structure of the cells.

[0289] 2. Dehydration and Clearing: Using ethanol of varying concentrations, from low to high, as the dehydrating agent, the tissue block is gradually dehydrated in the following order: 75% ethanol for 5 minutes, 85% ethanol for 5 minutes, 95% ethanol for 5 minutes, anhydrous ethanol I for 5 minutes, and anhydrous ethanol II for 5 minutes. The tissue block is then placed in xylene, a clearing agent soluble in both ethanol and paraffin, to clear it, replacing the ethanol in the tissue block with xylene.

[0290] 3. Paraffin embedding: Place the transparent skin tissue block in molten paraffin and keep it warm in a paraffin bath. After the paraffin has completely penetrated the tissue block, embedding is performed: Pour molten paraffin into a pre-prepared container, quickly pick up the paraffin-impregnated tissue block and place it into the container. Allow it to cool and solidify into a block.

[0291] 4. Sectioning and mounting: Fix the embedded paraffin block onto a microtome and cut it into thin sections of approximately 5-8 μm. The cut sections will be wrinkled, so place them in heated water to flatten them, and finally mount them onto glass slides and dry them in a 45°C oven.

[0292] 5. Dewaxing and Staining: H&E staining increases the color differences between different parts of tissue cell structures, facilitating observation. Hematoxylin (H) is a basic dye that stains the cell nucleus and ribosomes blue-purple; structures stained with hematoxylin are basophilic. Eosin (E) is an acidic dye that stains the cytoplasm red or pale red; structures stained with eosin are eosinophilic. Before staining, tissue sections are sequentially immersed in xylene I for 30 min, xylene II for 30 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 95% ethanol for 5 min, 85% ethanol for 5 min, 75% ethanol for 5 min, and PBS for 5 min.

[0293] 6. H&E staining: After distilling, immerse the sections in hematoxylin solution for several minutes. Separate the sections in acid and ammonia solution for several seconds each. Rinse with running water for 1 hour, then immerse in distilled water briefly. Dehydrate in 70% and 90% alcohol for 10 minutes each. Stain with alcohol-eosin staining solution for 2-3 minutes.

[0294] 7. Dehydration and clearing: After staining, the tissue blocks are gradually dehydrated by applying anhydrous ethanol I for 5 minutes and anhydrous ethanol II for 5 minutes, and then the sections are cleared by xylene.

[0295] 8. Sealing: Drip resin onto the transparent section and cover with a coverslip. After the resin has dried slightly, attach a label.

[0296] IX. Immunohistofluorescence staining experiment

[0297] 1. Drying: Place the skin tissue slices in an oven at 60°C for 2 hours.

[0298] 2. Dewaxing: Place the tissue sections in xylene I for 30 min, xylene II for 30 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 95% ethanol for 5 min, 85% ethanol for 5 min, 75% ethanol for 5 min, and PBS for 5 min in sequence.

[0299] 3. Antigen retrieval: After antigen retrieval, the slides are placed in citrate antigen retrieval buffer and then washed three times with PBS on a shaker.

[0300] 4. Blocking: Block with goat serum for 1 hour, then wash once with PBS.

[0301] 5. Primary antibody incubation: Circle the tissue with a histochemical pen, add about 50 μl of primary antibody to the tissue, and incubate in a humidified chamber overnight at 4°C. Wash 3 times with PBS.

[0302] 6. Secondary antibody incubation: Add approximately 50 μl of the corresponding fluorescent secondary antibody, incubate in the dark at room temperature for 2 hours. Wash 3 times with PBS.

[0303] 7. Photographs: DAPI-containing anti-fluorescence quenching mounting medium was dropped onto the tissue, covered with a coverslip, and incubated at room temperature in the dark for 5 minutes. Molecular expression was observed using a Zeiss LSM900 confocal microscope, and fluorescence intensity was quantified using ImageJ.

[0304] 10. Analysis of Public Bioinformatics Databases

[0305] 1. Use the GEO database (https: / / www.ncbi.nlm.njh.gov / geo / ) to query the correlations between molecules needed for the study of hair loss areas and non-hair loss areas in AGA patients, in order to further support the experimental results.

[0306] 2. Animal TFDB (https: / / guolab.wchscu.cn / AnimalTFDB4 / / # / ), PROMO (https: / / alggen.lsi.upc.es / cgibin / promo_v3 / promo / promoinit.cgi?dirDB=TF_8.3), and JASPAR (https: / / jaspar.elixir.no / ) predict transcription factors.

[0307] 3. Proteomics and protein phosphorylation sequencing

[0308] Appropriate amounts of skin tissue from the model group and the 1% JP1 group (n=3) were weighed into a mortar pre-cooled with liquid nitrogen and ground thoroughly into powder. The samples were then added to lysis buffer and sonicated. After centrifugation at 12,000×g for 10 min at 4℃, protein concentration was determined using BCA. Following trypsin digestion, analysis was performed using liquid chromatography-mass spectrometry. KEGG enrichment analysis was performed on the identified proteins, with Log2 Fold enrichment > 1 defined as significantly upregulated.

[0309] XI. Statistical Analysis

[0310] Quantitative data in this study are expressed as mean ± standard deviation. Student's t-test was used to test differences between two groups. ANOVA (one-way analysis of variance) was used for statistical analysis of multiple quantitative data, and Pearson correlation analysis was used for correlation analysis. *P<0.05, **P<0.01, ***P<0.001 indicated statistical significance, and nsP>0.05 indicated no statistical significance. PSS 20.0 and GraphPad Prism 8.0.1 were used for analysis and graphing.

[0311] In addition to the embodiments described above, the present invention may have other implementations. All technical solutions formed by equivalent substitution or equivalent transformation fall within the protection scope claimed by the present invention.

Claims

1. The use of a polypeptide, characterized in that, The intended use is for preparing anti-hair loss drugs; The amino acid sequence of the polypeptide is shown in I or II: I: FPGSDRF-Z; II: X-FPGSDRF-Z; Among them, amino acid S is phosphorylated, and X and Z are amino acids or amino acid sequences, respectively; X is selected from one of F, (R)9, (R)9-F, 6-aminohexanoic acid, 6-aminohexanoic acid-F, 6-aminohexanoic acid-(R)9, and 6-aminohexanoic acid-(R)9-F; Z is selected from (G). n -RGD、A-(G) n -RGD is one of them, where n is an integer greater than or equal to 0, and the value of n ranges from 0 to 10.

2. The use according to claim 1, characterized in that, The aforementioned anti-hair loss drugs include medications for treating androgenetic alopecia.

3. The use according to claim 1, characterized in that, The functions of the anti-hair loss drug are: to target hair follicle tissue, promote the growth of hair follicles and hair shafts, shorten the resting phase of hair follicles, prolong the growth phase of hair follicles, and significantly reverse the inhibition of hair growth by androgens.

4. The use according to claim 1, characterized in that, The function of the anti-hair loss drug is to target integrin molecules into hair follicle cells and upregulate the expression of integrin molecules in the hair follicle cell membrane; the integrin molecules mainly include ανβ1.

5. The use according to claim 1, characterized in that, The function of the anti-hair loss drug is to promote the expression of nuclear transcription factor SP1 by activating the MEK / ERK / E2F1 signaling axis, thereby activating the Wnt / β-catenin signaling pathway of hair follicle stem cells.

6. The use according to any one of claims 1 to 5, characterized in that, The peptide has an N-terminus modified by acetylation and a C-terminus modified by amidation.

7. The use according to any one of claims 1 to 5, characterized in that, The amino acid sequence of the polypeptide is one of SEQ ID No. 1 to SEQ ID No.

39.

8. The use according to any one of claims 1 to 5, characterized in that, In the polypeptide, the amino acids of the FPGSDRF sequence are configured in either L-type or D-type.

9. The use according to any one of claims 1 to 5, characterized in that, The drug includes a carrier, which is a pharmaceutically acceptable carrier.

10. The use according to any one of claims 1 to 5, characterized in that, The drug is available in either injectable or topical form.

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