Application of platelet reaction protein 4 in preparation of medicine for treating alopecia

Abstract

The invention relates to application of a platelet reaction protein 4 in preparation of an alopecia medicine, and belongs to the field of medical biology. The invention aims to solve the technical problems of limited curative effect, obvious side effect and narrow application range of the existing alopecia treatment medicine. The invention provides an application of a platelet reaction protein 4 (THBS4) in preparation of an alopecia medicinal preparation, which can promote hair follicles to be converted from a resting period to a growing period by regulating and controlling a mechanical microenvironment around the hair follicles, enhancing the interaction between fibroblasts and hair follicle stem cells and activating regeneration signal channels in hair papilla and hair bud areas. The traditional Chinese medicine composition breaks through the limitation that existing treatment only aims at specific alopecia types, and can be widely applied to various alopecia diseases such as androgenetic alopecia, pressure alopecia and alopecia after chemotherapy. And compared with the traditional medicine, the traditional Chinese medicine composition has the advantages of quick response, no skin irritation reaction and improved hair follicle regeneration efficiency, and provides a brand new solution for safe and efficient treatment of alopecia.

Description

Technical Field

[0001] The present invention belongs to the field of medicine and biology, and relates to the application of thrombospondin 4 in the preparation of alopecia drugs. Background Art

[0002] Hair growth in humans and animals progresses through three distinct phases: growth, catagen, and telogen. Hair loss is a common condition affecting hundreds of millions of people worldwide. It not only impacts personal appearance but also negatively impacts psychological well-being, social interactions, and career advancement. The mechanisms of hair loss remain unclear and are thought to be caused by a variety of factors, including, but not limited to, genetics, aging, stress, childbirth, cancer treatment, burns, hormonal imbalances, medications, infections, and infectious disease states.

[0003] The mechanism of hair loss primarily involves a shortened growth phase and a prolonged resting phase. Current drug treatments have limited effectiveness in regulating the regeneration of the hair follicle cycle. Hair follicle stem cells (HFSCs) at the hair bulge play a crucial role in activating the hair follicle cycle, while the interaction between fibroblasts and HFSCs is also a key factor influencing hair follicle regeneration.

[0004] Currently, common hair loss treatments include medication (such as minoxidil and finasteride), hair transplant surgery, laser therapy, and traditional Chinese medicine. However, these methods have certain limitations. For example, minoxidil stimulates the proliferation and differentiation of hair follicle epithelial cells, promotes angiogenesis, and causes hair follicles to transition from the resting phase to the growth phase, but it can cause adverse reactions such as erythema, itching, desquamation, and delicate skin. Drug therapy is only effective for androgenic alopecia, while hair transplant surgery is risky and cannot increase the number of hair follicles. Therefore, it is necessary to provide a safe, stable, effective, and low-side-effect drug for the prevention and treatment of hair loss. Summary of the Invention

[0005] In light of this, the present invention increases the interaction between fibroblasts and hair follicle stem cells by in vitro injection of the extracellular matrix (ECM) molecule thrombospondin 4 (THBS4), regulating the cyclical activation of hair buds, dermal papillae, and other areas, thereby promoting the transition of hair follicles from the catagen phase to the anagen phase, thereby treating various hair loss-related diseases caused by various factors. This treatment is suitable for hair growth anywhere on the body, not just the scalp, eyebrows, eyelashes, and other facial areas, and is used to treat hair loss caused by various reasons.

[0006] The purpose of the present invention is to provide an application of thrombospondin 4 in the preparation of a hair loss pharmaceutical preparation.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] The present invention provides an application of thrombospondin 4 in the preparation of a drug for preventing hair loss;

[0009] Furthermore, the use of thrombospondin 4 in the preparation of a drug for treating hair loss;

[0010] Furthermore, the use of thrombospondin 4 in the preparation of a drug for promoting hair growth;

[0011] Preferably, the amino acid sequence of thrombospondin 4 is shown in SEQ ID NO: 1;

[0012] Preferably, the dosage of thrombospondin 4 is 1-500 ng / mL;

[0013] Preferably, the drug comprises a pharmaceutically acceptable carrier and / or excipient;

[0014] Preferably, the drug is in the form of powder injection, injection solution, microspheres, microcapsules, liposomes, microemulsions, tablets, pills, capsules, granules or powders.

[0015] The beneficial effects of the present invention are:

[0016] The present invention uses localized thrombospondin 4 (THBS4) injection into the scalp in vitro to alter the mechanical properties surrounding the hair follicles, increase the interaction between fibroblasts and hair follicle stem cells, regulate the cyclical activation of areas such as the hair bud and dermal papilla, promote the entry of hair follicles into the growth phase, and effectively promote hair regeneration. The treatment is applicable to hair growth anywhere on the body, not just the scalp, eyebrows, eyelashes, and other facial areas. It is used to treat all types of hair loss, including but not limited to malnutrition, postoperative hair loss, post-pregnancy hair loss, certain medications, hot oil treatments (perms, etc.), stress, traction alopecia, androgenic alopecia, non-scarring annular alopecia, and all other forms of hair loss. Compared to existing medications and treatments, the present invention offers significant therapeutic effects and a wide range of treatment options. It treats hair loss-related diseases caused by a variety of factors, including androgenic alopecia and stress-induced alopecia. It is also more affordable, has a shorter treatment cycle, and is simple to operate, reducing the financial and psychological burden on patients.

[0017] Other advantages, objects, and features of the present invention will be described in part in the following description and, in part, will be apparent to those skilled in the art upon examination of the following description or may be learned from practice of the present invention. The objects and other advantages of the present invention may be realized and obtained through the following description. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention will be described in detail below with reference to the accompanying drawings, in which:

[0019] Figure 1 This image shows the effect of THBS4 on hair growth. Compared to the PBS control group, THBS4 treatment promoted hair regeneration. This image shows the regeneration of hair follicles on the back of mice after shaving and treatment with THBS4 recombinant protein.

[0020] Figure 2 The results of H&E staining of the hair follicles on the back of mice after treatment with THBS4 recombinant protein;

[0021] Figure 3 Immunofluorescence staining of hair follicles on the back of C57BL / 6 mice shows the regeneration of hair follicles on the back after shaving the mice and applying THBS4 recombinant protein.

[0022] Figure 4 The results of H&E staining of the back hair follicles of androgenic alopecia model mice after treatment with THBS4 recombinant protein;

[0023] Figure 5 The results of H&E staining of the back hair follicles of androgenic alopecia model mice treated with minoxidil hair growth solution and THBS4 recombinant protein.

[0024] Figure 6 This is a diagram of the skin organoid transplantation model and effect, which shows a skin organoid transplantation experiment after treatment with THBS4 recombinant protein. DETAILED DESCRIPTION

[0025] The following describes the embodiments of the present invention by means of specific examples, and those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the details in this specification can also be modified or changed in various ways based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that the illustrations provided in the following embodiments are only schematic illustrations of the basic concept of the present invention, and the following embodiments and features in the embodiments can be combined with each other without conflict.

[0026] Among them, the accompanying drawings are only for illustrative purposes and represent only schematic diagrams rather than actual pictures, and should not be understood as limiting the present invention. In order to better illustrate the embodiments of the present invention, some parts of the accompanying drawings may be omitted, enlarged or reduced, and do not represent the dimensions of actual products. For those skilled in the art, it is understandable that some well-known structures and their descriptions may be omitted in the accompanying drawings.

[0027] The same or similar numbers in the drawings of the embodiments of the present invention correspond to the same or similar parts; in the description of the present invention, it should be understood that if there are terms such as "upper", "lower", "left", "right", "front", "back", etc. indicating directions or positional relationships, they are based on the directions or positional relationships shown in the drawings. They are only for the convenience of describing the present invention and simplifying the description, and do not indicate or imply that the device or element referred to must have a specific direction, be constructed and operate in a specific direction. Therefore, the terms describing the positional relationship in the drawings are only used for illustrative purposes and cannot be understood as limiting the present invention. For ordinary technicians in this field, the specific meanings of the above terms can be understood according to specific circumstances.

[0028] The THSB4 recombinant protein used in the following examples was purchased from Wuhan Cloud-Clone Technology Co., Ltd. (Cat. No. RPD824Mu01). Its amino acid sequence is shown in SEQ ID NO: 1. The THSB4 recombinant protein was lyophilized powder and dissolved in 2 mL of PBS to prepare a stock solution at a concentration of 100 μg / mL before use and stored at -80°C.

[0029] Example 1 Hair growth experiment

[0030] After 7-8 week old C57BL / 6 female mice were anesthetized, the hair on the back was carefully shaved to expose the skin, and then a red marker was used to mark symmetrical circular areas on the left and right sides of the mouse's back for injection. Three mice were subcutaneously injected with 50μL PBS solution as a control group, and the other three mice were subcutaneously injected with 50μL THSB4 recombinant protein at a concentration of 0.1μg / mL-100μg / mL. The injection was once every other day, for a total of three injections. After the injection, the skin color changes and hair growth on the back of the mice were observed every day. The treated areas on the back of the mice were photographed on the 15th day. The results are as follows Figure 1 After the mice were killed, the drug-treated skin on the back was trimmed into strips of 1.5 cm long and 1 cm wide, flattened on a fixing plate, fixed in 4% paraformaldehyde, then dehydrated with gradient alcohol, embedded in paraffin, and made into paraffin sections. After H&E staining, the sections were observed under a microscope. The results are as follows: Figure 2 .

[0031] Immunofluorescence staining observation: After drug treatment, the mouse skin tissue was made into paraffin sections, immunofluorescence staining was performed for histology and hair growth related molecules such as Vimentin, KRT14, LEF1, etc., and confocal microscopy was used to take pictures. The results are as follows Figure 3 .

[0032] Example 2 Hair regeneration experiment based on mouse androgenic alopecia model

[0033] 5-6 week old male C57BL / 6 mice were randomly divided into model group, control group and experimental group, with 4 mice in each group. The experiment was divided into modeling phase (day -14 to day -1) and experimental phase (day 0 to day 16). During the modeling phase, 2 mg / kg of DHT was injected subcutaneously into the neck of the three groups of mice every day; during the experimental phase, the back area of all mice was shaved (4 × 1.5 cm 2 ), the experimental group was injected with 50 μL of THSB4 recombinant protein every other day at a concentration of 0.1 μg / mL-100 μg / mL, and the control group was injected with the same volume of PBS for a total of 8 injections. The model group was not treated. During the experiment, the shaved area on the back of the mice was photographed every 2 days (from D0 to D16) to record hair regeneration. The time point of skin graying was recorded (the change in skin color from pink to gray indicates the transition of hair follicles from the resting phase to the growth phase). Hair coverage was calculated (Image J analysis):

[0034]

[0035] On day 17 of the experiment, mice were killed by cervical dislocation and skin samples were taken from the shaved area of the back. 8 μm thick paraffin sections were prepared. H&E and immunofluorescence staining were performed to observe the morphology, number, and growth cycle (telogen / growth phase ratio) of hair follicles. Figure 4 .

[0036] Example 3 Comparative Experiment on the Effects of Minoxidil and THBS4 in Hair Regeneration

[0037] 5-6 week old male C57BL / 6 mice were divided into control group, minoxidil group and THBS4 treatment group, with 4 mice in each group. First, the mouse androgenic alopecia model was established. The three groups of mice were injected subcutaneously with 2 mg / kg DHT in the neck every day for 15 days. During the experimental period, the hair on both sides of the back of all mice was shaved (4×1.5 cm 2 ), the control group applied 300 μL of PBS solution on the back of the mice, the THBS4 treatment group applied 300 μL of THSB4 recombinant protein (concentration of 0.1 μg / mL-100 μg / mL) on the back of the mice, and the minoxidil treatment group applied 300 μL of minoxidil hair growth solution on the back of the mice, once a day for a total of 7 days. Every two days, photos were taken to record the hair growth on the back of the mice. After injection, the skin color changes and hair growth on the back of the mice were observed every day to record the time point when the skin turned gray (the change in skin color from pink to gray indicates that the hair follicles transition from the resting phase to the growth phase). Calculate the hair coverage (Image J analysis):

[0038]

[0039] On day 15 of the experiment, mice were killed by cervical dislocation and skin samples were taken from the shaved area of the back. 8 μm thick paraffin sections were prepared. H&E and immunofluorescence staining were performed to observe the morphology, number, and growth cycle (telogen / growth phase ratio) of hair follicles. Figure 5 .

[0040] Example 4 Skin organoid transplantation experiment

[0041] After weighing the nude mice, the dosage of anesthetic was calculated, and then the mice were anesthetized with 1% anesthetic to ensure that they remained painless and unconscious throughout the experiment. The organoids (control group and THBS4 recombinant protein treatment group) were taken out of the incubator and gently placed on the ice box. The skin of the anesthetized 7-8 week old female nude mice was disinfected, and the body was wiped three times with iodine. Use scissors to make fine cuts along the natural morphology of the organoid cell clusters, and be sure to maintain cell integrity. Use sharp tweezers to clamp the skin on the back of the nude mouse, and cut a wound of the same size as the cell on the dorsal side of the nude mouse. The cell surface of the organoid was gently placed on the wound, and the organoid was sutured to the mouse skin. Apply an appropriate amount of antibacterial agent around the transplant site to prevent infection. Subsequently, the transplant site was covered with gauze and elastic bandage to provide appropriate support and protection. One week after the transplant surgery, the bandage was removed, and the hair growth at the transplant site on the back of the nude mouse was continuously observed. Photos were taken for subsequent analysis and research. The results are as follows. Figure 6 .

[0042] This example experimentally demonstrates that THBS4 can enhance the proliferation of dermal papilla cells, thereby promoting the transition of hair follicles from the resting phase to the growing phase, which is beneficial for hair regeneration. By increasing THBS4 expression, dermal papilla cell activity can be effectively enhanced, promoting hair follicle regeneration. In skin organoid transplantation experiments, the addition of a certain concentration of THBS4 recombinant protein significantly improved hair follicle regeneration. Based on this, the present invention demonstrates that subcutaneous injection of a certain dose of THBS4 protein (1-500 ng / mL) can effectively promote hair regeneration.

[0043] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions, which should all be included in the scope of the claims of the present invention.

Claims

1. Application of thrombospondin 4 in the preparation of drugs for preventing hair loss.

2. Application of thrombospondin 4 in the preparation of drugs for treating hair loss.

3. Application of thrombospondin 4 in the preparation of drugs for promoting hair growth.

4. Application of thrombospondin 4 in the preparation of hair growth promoting organoids.

5. The use according to any one of claims 1 to 4, characterized in that: The amino acid sequence of thrombospondin 4 is shown in SEQ ID NO:

1.

6. The use according to any one of claims 1 to 4, characterized in that: The thrombospondin 4 dosage is 1-500 ng / mL.

7. The use according to any one of claims 1 to 4, characterized in that: The medicine comprises a pharmaceutically acceptable carrier and / or excipient.

8. The use according to any one of claims 1 to 4, characterized in that: The medicine is in the form of powder injection, injection solution, microsphere, microcapsule, liposome, microemulsion, tablet, pill, capsule, granule or powder.

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