Use of nervonic acid in the preparation of products for promoting hair regrowth

Abstract

The application provides application of nervonic acid in preparation of a product for promoting hair regeneration, and relates to the biological field. The application of nervonic acid in preparation of the product for promoting hair regeneration includes that the concentration of the nervonic acid in the product is 3%-10%, at least one of the following is included in promoting hair regeneration: activating Wnt signal pathway in hair follicle stem cells, promoting expression of beta-catenin, LEF1 and PCNA, and further including promoting hair follicle organoid development in vitro and improving hair follicle regeneration capacity. It is found that the nervonic acid can act on the hair follicle stem cells, effectively promotes the hair follicle from the resting phase to the growth phase by activating the Wnt / beta-catenin and other regeneration pathways, and up-regulating the expression of PCNA, LEF1 and other proliferation-related factors, and promotes hair regeneration. The technology retains the high efficiency and safety of the nervonic acid regulation, further expands the new application scene of the nervonic acid in the field of hair regeneration, and has significant conversion value and broad market prospect in the field of clinical treatment of alopecia.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically, to the application of nervonic acid in the preparation of products that promote hair regeneration. Background Technology

[0002] Abnormal hair regeneration is one of the core causes of hair loss, severely impacting an individual's appearance and mental health. The hair growth cycle is a crucial physiological process for maintaining normal hair metabolism, primarily consisting of the anagen (growth) phase, catagen (transitional) phase, and telogen (resting) phase. Its dynamic balance is mainly regulated by signaling pathways and key regulatory factors within the hair follicle microenvironment. Hair follicle stem cells, as the core functional cells maintaining the hair follicle cycle and hair regeneration, are influenced by internal factors such as genetics, hormone levels, and aging, as well as external environmental factors such as dietary imbalances, stress, medication, ultraviolet radiation, and environmental pollution. Impaired activity of hair follicle stem cells can hinder hair regeneration and worsen hair loss symptoms.

[0003] Currently, common interventions for hair regeneration mainly include drug therapy, physical therapy, and surgical hair transplantation. Among them, commonly used chemical drugs such as minoxidil and finasteride can promote hair growth to a certain extent, but they have obvious limitations: minoxidil is prone to causing local adverse reactions such as scalp irritation, itching, dryness, and flaking. Some patients may also experience mild systemic discomfort such as dizziness and fatigue. Long-term topical use may also lead to decreased scalp tolerance. Finasteride may cause sexual dysfunction and has a risk of birth defects. Long-term use can also easily lead to drug resistance. Physical therapies such as low-energy laser therapy and platelet-rich plasma (PRP) injection have problems such as high treatment costs, large individual differences in efficacy, and the need for long-term continuous intervention. Although surgical hair transplantation can quickly improve local hair loss, it has drawbacks such as limited donor area resources, postoperative infection risk, and greater trauma.

[0004] Nervonic acid is a very long-chain monounsaturated fatty acid, chemically named cis-15-tetracosanoic acid. It was first isolated from shark oil by Japanese scholars in 1926, hence its alternative name, shark acid. Nervonic acid possesses excellent biocompatibility and safety, and has demonstrated unique advantages in areas such as nerve repair, immune regulation, metabolic regulation, and skin repair. However, its role, mechanism, and administration methods in regulating hair follicle stem cells, hair follicle cycle transition, and promoting hair regeneration have not yet been publicly reported, remaining a novel and unexplored application area. This invention discovers that nervonic acid exhibits therapeutic potential in promoting hair regeneration and growth, potentially avoiding the adverse reactions and limitations of traditional interventions, and providing a novel treatment strategy and candidate for hair-related diseases such as androgenetic alopecia and alopecia areata. Summary of the Invention

[0005] The purpose of this invention is to provide the application of nervonic acid in the preparation of products that promote hair regeneration. It is the first discovery that nervonic acid shows therapeutic potential in promoting hair regeneration and growth, which is expected to avoid the adverse reactions and application limitations of traditional intervention methods, and provide a new treatment strategy and candidate solution for hair-related diseases such as androgenetic alopecia and alopecia areata.

[0006] The technical problem solved by this invention is achieved by the following technical solution.

[0007] On the one hand, embodiments of this application provide the application of nervonic acid in the preparation of products that promote hair regeneration, wherein the concentration of nervonic acid in the products is 3%-10%.

[0008] Furthermore, promoting hair regeneration includes activating the Wnt signaling pathway in hair follicle stem cells and promoting the expression of at least one of β-catenin, LEF1, and PCNA.

[0009] Furthermore, promoting hair regeneration also includes promoting the development of hair follicle organoids in vitro and enhancing the regenerative capacity of hair follicles.

[0010] Furthermore, the above-mentioned product consists of nervonic acid and pharmaceutically acceptable salts or stereoisomers, as well as a pharmaceutically acceptable carrier.

[0011] Furthermore, the dosage forms of the above products include any one of the following: lotion, ointment, patch, film-forming agent, gel, microneedle, aerosol, or spray.

[0012] On the other hand, embodiments of the present invention also provide the application of the above-mentioned nervonic acid in the preparation of anti-hair loss products.

[0013] Compared with the prior art, the embodiments of the present invention have at least the following advantages or beneficial effects: 1. This invention reveals for the first time that nervonic acid can act on hair follicle stem cells, and can effectively promote the transformation of hair follicles from the resting phase to the growth phase by activating regeneration pathways such as Wnt / β-catenin and upregulating the expression of proliferation-related factors such as PCNA and LEF1, thereby promoting hair regeneration; 2. This invention also reveals that nervonic acid can effectively promote the development of hair follicle organoids in vitro and enhance the effect of hair regeneration after transplantation; the pharmaceutical preparation based on nervonic acid not only retains the high efficiency and safety of nervonic acid regulation, but also further expands its new application scenarios in the field of hair regeneration, and has significant transformational value and broad market prospects in the clinical treatment of hair loss. Attached Figure Description

[0014] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation of the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0015] Figure 1 This is a diagram showing the hair growth pattern on the backs of two groups of mice in Example 1 of the present invention; Figure 2 These are H&E staining images of the skin on the backs of two groups of mice in Example 1 of this invention; Figure 3 These are IF staining images of the skin on the backs of two groups of mice in Example 1 of this invention; Figure 4 This is a diagram showing the hair growth of two groups of mice after transplantation of hair follicle organoids in Example 2 of the present invention. Detailed Implementation

[0016] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0017] It should be noted that, unless otherwise specified, the embodiments and features described in this application can be combined with each other. The present invention will now be described in detail with reference to specific embodiments.

[0018] Example 1 This embodiment uses mice as the research subject, and firstly, it verifies in detail the effect of nervonic acid (NA) on the fur on the back of mice. The specific steps are as follows: 1) Preparation of nervonic acid working solution: Weigh 100 mg of nervonic acid powder (produced by Shanghai Haoyuan Biomedical Technology Co., Ltd., catalog number HY-N2526), ​​add 2.73 mL of DMSO (dimethyl sulfoxide) to prepare a 100 mM stock solution (concentration of approximately 36.66 mg / mL). For injection, dilute the 100 mM stock solution 20 times with PBS to prepare a 5 mM nervonic acid working solution (concentration of approximately 1.83 mg / mL) for later use.

[0019] 2) Six 7-8 week old female C57BL / 6 mice with identical growth environments were selected. They were anesthetized, and the hair on their backs was carefully shaved to expose the skin. Symmetrical circular areas for injection were then marked on both sides of the mice's backs with a red marker. Three mice were subcutaneously injected with 50 μL of PBS buffer as the control group, while the other three mice were subcutaneously injected with 50 μL of nervonic acid working solution at a concentration of 5 mM (1.83 mg / mL), designated as the NA treatment group. Injections were given every other day for a total of four times (the experiment was set up with three replicates). Skin color changes and hair growth on the backs of the mice were observed daily after injection. Twenty-one days after injection, skin samples were collected from the backs of the control and nervonic acid-treated mice for H&E staining and IF staining, respectively. The experimental results are as follows: Figure 1 - Figure 3 As shown. By Figure 1 As can be seen, before the intervention (Day 0), the shaved areas on the backs of mice in both the control group and the NA-treated group showed a uniform pink color, indicating that the hair follicle cycle of the mice was in the resting phase. However, after 21 days of intervention (Day 21), no new hair grew in the shaved areas of the control group mice, and the hair follicles were in a low-activation resting state. In contrast, the NA-treated group mice had grown a large area of ​​dense black hair in the shaved areas on their backs, and the hair coverage and density were significantly higher than those in the control group. This suggests that nervonic acid treatment can effectively activate resting hair follicles and significantly promote the hair regeneration process in mice.

[0020] Additionally, please refer to Figure 2 H&E staining results showed that the control group mice had sparse hair follicles on their backs, most of which were in the telogen phase, with short follicles located in the superficial dermis and no obvious hair shaft formation. The nervonic acid treatment group showed a significant increase in the number of hair follicles, with most follicles entering the anagen phase. This was characterized by a significant increase in follicle length, intact and enlarged hair bulbs, and follicles extending downwards into the deep dermis. Obvious melanin deposition was visible within the hair shaft, and the dermal blood vessel density increased. These findings suggest that nervonic acid can effectively activate the transformation of hair follicles from the telogen phase to the anagen phase, significantly promoting hair regeneration.

[0021] Further reference Figure 3 ,in, Figure 3 (A) is a diagram showing the results of immunofluorescence (IF) staining. Figure 3(B) shows the results of quantitative analysis. Immunofluorescence staining results showed that the expression levels of β-catenin, LEF1, and PCNA in the skin of control mice were extremely low, with only a small number of positive cells; while the nervonic acid-treated group showed significantly enhanced β-catenin, LEF1, and PCNA signals in the hair follicle region, with a large number of positive cells concentrated in the hair bulb and hair follicle stem cell region. Quantitative analysis results showed that the number of β-catenin, LEF1, and PCNA cells in the nervonic acid-treated group was significantly higher than that in the control group (P<0.001 or P<0.0001), suggesting that nervonic acid can promote hair follicle cell proliferation by activating the Wnt / β-catenin signaling pathway, thereby promoting hair regeneration.

[0022] Example 2 This embodiment further verifies the effect of nervonic acid on the dorsal fur of mice. The specific scheme is as follows: 1) Similar to the steps in Example 1, first prepare the nervonic acid working solution by diluting the stock solution 100 times with culture medium to prepare a 1 mM working solution (concentration approximately 366.62 μg / mL) for later use. Prepare complete culture medium: 5 mL fetal bovine serum (FBS), 500 μL penicillin-dextrose antibody, and 45 mL DMEM culture medium.

[0023] 2) Select several newborn CD1 mice, spray their bodies with 75% alcohol and wash them repeatedly three times. Sacrifice the mice, carefully remove the skin from their backs with sterile forceps, and place it in a new sterile culture dish. Then, slowly add 0.025% trypsin solution to the culture dish containing the back skin sample. Seal the culture dish and place it in a 4°C refrigerator overnight. On the second day, separate the dermis and epidermis and place them in different culture dishes for later use.

[0024] 3) Use sterile scissors to cut the dermal tissue into small pieces (about 1 mm³), pour in 1 mL of 0.35% type I collagenase solution, gently shake the culture dish to mix the dermal tissue and collagenase thoroughly, and then place the culture dish in a 37°C water bath for about 20 minutes to heat and digest. During dermal tissue digestion, the epidermal tissue was minced as finely as possible using sterile scissors. 15 mL of complete culture medium was added, and the mixture was gently pipetted to mix, avoiding excessive force that could damage the cells. The cell suspension was then filtered through a 70-mesh filter to remove any unminced tissue fragments, and the filtered epidermal cell suspension was collected. After centrifugation using a low-temperature centrifuge (4°C, 1100 rpm, 5 min), the supernatant was slowly discarded. 15 mL of complete culture medium was added to the centrifuge tube, and the suspension was gently resuspended. The tube was then centrifuged again under the same conditions for 5 min, and the mixture was gently pipetted to mix again to obtain the epidermal cell suspension, which was then placed on ice for later use.

[0025] After dermal cell digestion, 10 mL of complete culture medium was added to terminate the digestion. The mixture was gently pipetted and filtered through a 70-mesh filter. The filtered dermal cell suspension was centrifuged (4°C, 1100 rpm, 5 min). After centrifugation, the supernatant was slowly discarded, and complete culture medium was added to the centrifuge tube to a final volume of 15 mL. The mixture was gently pipetted and then centrifuged again under the same conditions for 5 min to further remove impurities and dead cells. After centrifugation, the supernatant was discarded, and 15 mL of complete culture medium was added. The mixture was gently pipetted and then stored on ice for later use.

[0026] 4) Mix the dermal cell suspension and epidermal cell suspension at a ratio of 9:1, centrifuge at 4℃ and 1100 rpm for 5 min, discard the supernatant to obtain a well-mixed dermal-epidermal cell cluster. Add approximately 20 μL of the mixed dermal-epidermal cells to the upper chamber of each insert cell, and add an appropriate amount of culture medium to the lower chamber. The treatment group's culture medium is diluted with 1 mM nervonic acid working solution, while the control group is given an equal volume of culture medium without nervonic acid. After cell seeding, culture for 3-5 days to obtain hair follicle organoids.

[0027] 5) Select several healthy nude mice with the same growth environment and age, and divide them into experimental group and control group. After anesthetizing the nude mice in both groups, make a circular skin incision in the marked area on the back of the nude mice, remove a small piece of full-thickness back skin to form a circular wound, and then gently place the pre-treated hair follicle organoids on the wound and suture. On the 14th day after surgery, remove the bandage and gauze on the back of the nude mice, and observe and record the hair regeneration in the transplanted area on the back.

[0028] Experimental results are as follows Figure 4 As shown in the figure, compared with the control group, the hair in the transplanted area of ​​mice treated with nervonic acid was denser and longer, which directly shows that nervonic acid can promote hair growth.

[0029] Based on Examples 1 and 2, it is evident that nervonic acid can significantly promote hair follicle cell proliferation and in vitro hair follicle organoid development. According to this property, those skilled in the art can prepare products that promote hair regeneration primarily using nervonic acid. These products can be formulated with pharmaceutically acceptable carriers and presented in the form of lotions, creams, patches, films, gels, microneedles, aerosols, or sprays.

[0030] In summary, the embodiments of the present invention provide the application of nervonic acid in the preparation of products that promote hair regeneration. It reveals for the first time that nervonic acid can act on hair follicle stem cells and can effectively promote the transformation of hair follicles from the resting phase to the growth phase by activating regeneration pathways such as Wnt / β-catenin and upregulating the expression of proliferation-related factors such as PCNA and LEF1, thereby promoting hair regeneration. This invention also reveals that nervonic acid can effectively promote the development of hair follicle organoids in vitro and enhance the effect of hair follicle regeneration. The pharmaceutical preparation based on nervonic acid not only retains the high efficiency and safety of nervonic acid regulation, but also further expands its new application scenarios in the field of hair regeneration. It has significant transformational value and broad market prospects in the clinical treatment of hair loss.

[0031] The embodiments described above are some, but not all, embodiments of the present invention. The detailed description of the embodiments of the present invention is not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

Claims

1. The application of nervonic acid in the preparation of products that promote hair regeneration, characterized in that, The concentration of nervonic acid in the product is 3%–10%.

2. The application according to claim 1, characterized in that, The promotion of hair regeneration includes activating the Wnt signaling pathway in hair follicle stem cells and promoting the expression of at least one of β-catenin, LEF1, and PCNA.

3. The application according to claim 2, characterized in that, The promotion of hair regeneration also includes promoting the development of hair follicle organoids in vitro and enhancing the hair follicle regeneration capacity.

4. The application according to claim 1, characterized in that, The product comprises the nervonic acid and a pharmaceutically acceptable salt or stereoisomer, as well as a pharmaceutically acceptable carrier.

5. The application according to claim 4, characterized in that, The dosage form of the product includes any one of the following: lotion, ointment, patch, film coating, gel, microneedle, aerosol or spray.

6. The application of nervonic acid as described in claim 1 in the preparation of anti-hair loss products.

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