Application of cholesterol in preparation of hair growth promoter

Abstract

The invention discloses an application of cholesterol in preparation of a hair growth promoter, and proves that the cholesterol does not directly promote hair follicle stem cell proliferation, but enhances sympathetic nerve activity by increasing tyrosine hydroxylase phosphorylation level so as to promote hair follicle stem cell proliferation, thereby providing a new strategy for treating alopecia. And the natural biocompatibility of cholesterol is utilized, so that the side effect risk of the medicine on other tissues and organs is reduced, and the safety of long-term use and the compliance of patients are ensured.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to the application of cholesterol in the preparation of a hair growth promoter. Background Art

[0002] Hair loss affects millions of people worldwide. Only two drugs, minoxidil and finasteride, are approved by the FDA, but their efficacy is limited and often accompanied by side effects. The existing methods for treating hair loss have the following main shortcomings: 1) Minoxidil: It is an external medication for treating hair loss. It can improve hair growth by increasing the duration of the hair follicle growth phase and promoting blood circulation in the scalp. However, minoxidil may cause side effects such as scalp irritation and allergic reactions. 2) Finasteride: It is an oral medication that inhibits 5α-reductase, reduces the level of dihydrotestosterone (DHT) in the body, and promotes hair regeneration. It is mainly used to treat male pattern baldness. However, finasteride may cause side effects such as sexual dysfunction, and long-term use is risky. 3) Laser therapy: It uses low-energy lasers to irradiate hair follicles to stimulate hair follicle growth. The effect is relatively mild, but it needs to be used for a long time and is costly. 4) Hair transplant surgery: Healthy hair follicles are transplanted to the hair loss area through surgery. The effect is more significant and lasting, but the cost of the surgery is high and there are certain surgical risks.

[0003] Therefore, providing a new hair growth promoter to solve the problems of limited efficacy, obvious side effects, unclear mechanism and insufficient targeting of existing hair growth drugs is an urgent problem to be solved in this field. Summary of the invention

[0004] The purpose of the present invention is to provide the use of cholesterol in the preparation of a hair growth promoter in view of the deficiencies in the prior art.

[0005] To achieve the above object, the technical solution adopted by the present invention is:

[0006] Provided is the use of cholesterol in preparing a hair growth promoter.

[0007] Furthermore, cholesterol is the only active ingredient of the hair growth promoter.

[0008] Furthermore, the hair growth promoter also includes a pharmaceutically acceptable excipient.

[0009] Furthermore, the hair growth promoter is a liquid preparation.

[0010] Furthermore, the hair growth promoter is in the form of an injection.

[0011] The present invention adopts the above technical solution, and has the following technical effects compared with the prior art:

[0012] The present invention proves that cholesterol does not directly promote the proliferation of hair follicle stem cells, but rather promotes the proliferation of hair follicle stem cells by increasing the phosphorylation level of tyrosine hydroxylase and thereby enhancing the activity of sympathetic nerves, thus providing a new strategy for treating hair loss. In addition, the natural biocompatibility of cholesterol is utilized to reduce the risk of side effects of drugs on other tissues and organs, ensuring the safety of long-term use and patient compliance. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The results show that cholesterol can promote hair growth in mice; (A) is a schematic diagram of the experiment; (B) after removing the hair from the back skin of 7-week-old C57BL / 6 mice (the hair follicles are in the resting phase), equal amounts of anhydrous ethanol and cholesterol were injected subcutaneously; 21 days after the injection, the difference in hair regeneration area between the control group and the cholesterol group could be observed; the percentage of skin darkening area was measured (n=3); (C) is the mouse skin color scoring index; (D) is the quantification of the mouse skin color score in (B) based on the mouse skin color score in (C); (E) is hematoxylin and eosin (H&E) staining of the skin of the control group and the cholesterol group, scale bar = 50 μm (n=3); *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; P values ​​were determined by unpaired t-test (B), two-way ANOVA (D) or chi-square test (E).

[0014] Figure 2The results show that cholesterol activates hair follicle stem cells and sympathetic nerves; (A) is K14 / Ki67 immunofluorescence staining performed on the 18th day, and the results show that cholesterol treatment can promote the proliferation of hair follicle stem cells, scale bar = 50 μm (n = 3); (B) is the CCK-8 experiment to detect the proliferation of hair follicle stem cells in the control group or cholesterol-treated cells after stimulating the cells for 48 hours; (C) is the q-PCR result showing the expression changes of genes related to hair follicle stem cell proliferation; (DF) are fluorescent images of anti-CD45, anti-CD31 and anti-TH (red) and K14 (green) staining of different groups; quantification of CD45, CD31 and TH positive cells per unit area; scale bar = 50 μm (DF, n = 3); (H) is immunofluorescence detection showing that cholesterol induces phosphorylation of tyrosine hydroxylase in PC12 cells, scale bar = 20 μm; (I) is Western Blotting was used to evaluate the effect of cholesterol induction on THpSer40 expression in PC12 cells (n=3); (J) Skin tissues of experimental animals were collected and Western blotting was used to analyze the differences in TH pSer40 between the experimental group and the control group; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; P values ​​were analyzed by unpaired t-test (A, DF, and J), one-way ANOVA (H and I), and two-way ANOVA (B).

[0015] Figure 3 The sympathetic nervous system affects hair growth; (A) is a schematic diagram of the experimental process; (B) is a quantification of the percentage of darkened back skin area in mice treated with 6-OHDA and control mice (n=3); (C) is based on Figure 1 The mouse skin color scores in C quantify the mouse skin color scores in (A); (D) is the fluorescent image of anti-TH staining in different groups; (E) is the H&E staining of the skin of the control group and 6-OHDA group, scale bar = 50 μm; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001; P values ​​were analyzed by unpaired t-test (B, D), two-way ANOVA (C) or chi-square test (E).

[0016] Figure 4 The results show that cholesterol promotes the proliferation of hair follicle stem cells and hair growth by activating sympathetic nerves; (A) shows that after blocking the dorsal sympathetic nerves of mice with 6-OHDA, cholesterol and control solvent were injected subcutaneously to observe the changes in dorsal hair growth (n=3); (B) shows that Figure 1Mouse skin color scores in C were quantified; (C) H&E staining of the skin of the control group and cholesterol group, scale bar = 50 μm; (D) Fluorescence images of anti-Ki67 staining of different groups (n = 3); ns not significant, P values ​​were determined by unpaired t-test (A, D), two-way ANOVA (B) or chi-square test (C).

[0017] Figure 5 The results show that cholesterol activates sympathetic nerves by phosphorylating tyrosine hydroxylase via PKA; (A) Western blotting was used to evaluate the changes in TH pSer40 expression in cholesterol-treated PC12 cells after the addition of HA-1004 (n=3); (B) After shaving, HA-1004 was injected subcutaneously into the back skin of mice, and then the previous experiment was repeated ( Figure 1 A)(n=3); (C) is based on Figure 1 The mouse skin color score in C quantifies the mouse skin color score; (D) is K14 / Ki67 immunofluorescence staining performed on the 18th day, and the results show that HA-1004 treatment promotes the proliferation of hair follicle stem cells, scale bar = 50 μm (n = 3); (E) HE-stained sections were obtained from the injection area of ​​the back skin of each group of mice on the 18th day for analysis, scale bar = 50 μm (n = 3); *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, ns not significant; P values ​​were analyzed by independent sample t-test (AB, DE) or two-way analysis of variance (C).

[0018] Figure 6 Cholesterol was shown to promote hair growth in a dose-dependent manner; after removing the dorsal skin hair, 7-week-old C57BL / 6 mice were subcutaneously injected with graded concentrations of cholesterol. DETAILED DESCRIPTION

[0019] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments, but is not intended to be limiting of the present invention. It should be noted that the embodiments and features in the embodiments of the present invention may be combined with each other without conflict.

[0020] 1. Experimental Methods

[0021] 1.1 Animals

[0022] C57BL / 6 mice were purchased from Slack Laboratory Animal Company. Six-week-old C57BL / 6 male mice were used in the experiment, and their hair cycle was re-entered into the growth phase by shaving the back hair. In the self-control experiment, the back skin of mice was subcutaneously injected with anhydrous ethanol (2.5mmol / L) and cholesterol (2.5mmol / L, dissolved in anhydrous ethanol), respectively. Before the experimental procedures, all mice were anesthetized by intraperitoneal injection of 4% chloral hydrate solution, and measures were taken to ensure stable body temperature. After the experiment, the mice were humanely killed by cervical dislocation, and the skin samples were immediately stored at -80°C for further analysis. The body weight of the mice was measured once before treatment and then every other day. All animal experiments were performed according to the protocol established by the Animal Experiment Committee of Tongji University and in accordance with the guidelines of Tongji University School of Medicine.

[0023] 1.2 Cell culture

[0024] In this experiment, hair follicle stem cells isolated from mouse skin hair follicle tissue were used and cultured. The culture medium was a mixture of FBS, growth supplements, penicillin and streptomycin. The cells were placed in a cell culture incubator at 37°C, 5% CO2 and saturated humidity for static culture.

[0025] PC12 cells (a subclone derived from the PC12 rat pheochromocytoma cell line) were cultured in RPMI1640 basal medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin to ensure nutrient supply and prevent bacterial contamination. Culture was carried out at 37°C and 5% CO2, and the culture medium was replaced every two days.

[0026] 1.3 Western blot

[0027] Proteins in PC12 cells of different treatment groups were extracted by using buffers such as 1% phenylmethylsulfonyl fluoride (Solarbio) and radioimmunoprecipitation assay (RIPA) protein lysis buffer to obtain total proteins. Then, these solutions were centrifuged at 13,000 × g for 5 minutes at 4°C to obtain protein fractions, after which the protein content was quantified and finally the relevant proteins were detected. Different proteins were measured and screened using BCA protein analysis kit (Solarbio) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), respectively. In the subsequent process, 20 micrograms of protein were filtered through 8% or 10% SDS-PAGE and then transferred to polyvinylidene difluoride membranes. The membranes were blocked (5% skim milk, room temperature, 1 hour) and incubated with primary antibodies overnight at 4°C to detect relevant proteins. The primary antibodies used included: tyrosine hydroxylase (TH) (Proteintech, Cat#25859-1-AP), tyrosine hydroxylase phosphorylation (PTH) (CellSignaling Technology, Cat#2791S). After incubation, the membrane was washed with 1× Tris-buffered saline buffer containing Tween 20. After that, a 1-hour secondary antibody incubation was performed. These antibodies were labeled with horseradish peroxidase and included specific anti-rabbit secondary antibodies and specific anti-mouse secondary antibodies (1:5000 dilution ratio; Bioworld, Nanjing, China). Super-enhanced ECL reagent (BioSharp, Hefei, China) was used to visualize the protein bands, and the Fluor Chem FC3 system (Protein-Simple, San Jose, CA, USA) was used to analyze the protein bands. On day 21, the experimental mice were killed and dorsal skin samples were collected and frozen in liquid nitrogen, ground into powder, incubated in cold RIPA buffer, centrifuged at 16,000 rpm for 20 min at 4 degrees Celsius, and the supernatant was collected and determined by immunoblotting.

[0028] 1.4 Histological examination and immunofluorescence staining

[0029] On the 18th day after drug intervention, skin samples from the back of mice were taken for cell morphological analysis. As described in previous studies, the method was as follows: the samples were fixed with 4% paraformaldehyde for 24 h, followed by routine dehydration and transparency, and embedded in paraffin. Continuous longitudinal sections of 5 μm thickness were cut from the prepared paraffin blocks for subsequent morphological observation. Hair follicle tissue sections were routinely stained with hematoxylin-eosin (HE) to evaluate skin morphological changes and hair regeneration. For tissue immunofluorescence staining, sections were incubated with Ki67 antibody (Cat#GB121141-100, Servicebio; 1:500 dilution), cytokeratin 14 antibody (Cat#ab1196-95, Abcam; 1:500 dilution), TH antibody (Cat#45648S, Cell Signaling Technology; 1:500 dilution), CD31 antibody (Cat#553370, BD Biosciences; 1:500 dilution), phosphotyrosine hydroxylase (Ser40) antibody (Cat#2791S, Cell Signaling Technology; 1:500 dilution), or CD45 antibody (Cat#GB14038-50, Servicebio; 1:500 dilution) at 4°C overnight, followed by staining with secondary antibodies for 2 hours at room temperature.

[0030] For cell immunofluorescence staining, cells were washed three times with PBS and fixed with 4% paraformaldehyde for 15 minutes at room temperature, then rinsed with PBS and exposed to PBS containing 0.1% Triton X-100 and 5% BSA for 1 hour. Anti-β3 tubulin (1:500 dilution) diluted in PBS was incubated overnight at 4°C, followed by staining with secondary antibodies for 2 hours at room temperature. Imaging was performed using an OLYMPUS microscope.

[0031] 1.5 Real-time quantitative PCR

[0032] Total RNA was extracted from tissues or cells using the RNAsimple Total RNA Extraction Kit (Tiangen) (DNA was removed by DNA-free DNase treatment and removal reagent), and then reverse transcribed using the FastQuant RT Reverse Transcription Kit (Tiangen). Real-time quantitative PCR was performed using the SuperReal SYBR Green Kit (Tiangen) and the LightCycler 96 instrument (Roche). The relative expression of the target gene was calculated and normalized to β-actin.

[0033] 1.6 Sympathetic nerve blockade

[0034] Sympathetic nerve ablation: 6-hydroxydopamine hydrochloride (6-OHDA, Sigma 162957) solution was prepared by dissolving 6-OHDA in 0.1% ascorbic acid (dissolved in 0.9% NaCl). 0.6 mg of 6-OHDA was dissolved in 100 μl of 0.1% ascorbic acid and injected subcutaneously into mice at P18 or P19. Control animals were injected with vehicle (100 μl of 0.1% ascorbic acid). Skin was analyzed one week after ablation.

[0035] 1.7 Cell proliferation assay

[0036] The cell proliferation experiment was performed using CCK-8 assay. Hair follicle stem cells were seeded in 96-well plates (6 × 10 3 Cells were cultured with standard culture medium for 24 hours, and then cultured with fresh culture medium containing the corresponding drug concentration for another 72 hours. The culture medium was then replaced and CCK-8 reagent diluted in DMEM (1:9) was used to culture at 37°C for 2 hours. The optical density value of each well was measured by a microplate reader (SpectraMAX190, Molecular Technologies, Japan) at a wavelength of 1:1.

[0037] 1.8 Statistical analysis

[0038] Statistical analysis was performed using Prism software, using unpaired two-tailed Student's t test. Statistical significance is indicated by asterisks (P<0.05[*], P<0.01[**], P<0.0001[***]). Data are presented as mean ± SEM (standard error).

[0039] 2. Experimental results

[0040] 2.1 Cholesterol promotes hair growth

[0041] In order to explore the effect of cholesterol on hair development, the present invention first determined a method for evaluating hair growth. In order to eliminate individual differences between mice, all mice were depilated during the regression period so that all hair could grow synchronously. Focusing on the study of the effect of local cholesterol changes in hair follicle tissue on hair growth, when selecting the drug injection method, compared with intraperitoneal injection or mouse tail vein injection, local fixed-point subcutaneous injection can bypass the systemic circulation and directly act on the target tissue, avoiding systemic clearance and side effects, while also maximizing drug concentration. Next, we depilated 7-week-old male C57BL / 6 mice, injected anhydrous ethanol into the control group, and injected cholesterol into the subcutaneous tissue of the experimental group once every 2 days ( Figure 1 A and Figure 6 We observed that cholesterol promotes hair growth ( Figure 1B). It has been reported that as hair grows, mouse skin can gradually change from pink to white, gray, gray, and dark gray. Changes in mouse skin color can be used as a sensitive indicator for evaluating hair regeneration. Therefore, we also used the mouse skin color rating scale to quantitatively score hair growth and development ( Figure 1 C). The results showed that the score curve of the cholesterol side of the mice was higher than that of the control side, and a significant difference was observed on the 21st day ( Figure 1 D) In ​​order to observe the histological changes of hair follicles after cholesterol stimulation, skin tissues on both sides of mice were taken for hematoxylin-eosin staining on the 22nd day after injection. Figure 1 As shown in E, compared with the skin tissue on the control side, the number of hair follicles on the cholesterol side increased significantly, and more hair follicles were in the growth phase ( Figure 1 E).

[0042] 2.2 Cholesterol activates hair follicle stem cells and sympathetic nerves

[0043] The above conclusions have shown that cholesterol can promote hair growth. The hair growth process is a cycle of three periods: growth phase, regression phase and resting phase. Hair follicle stem cells have the ability to self-renew and differentiate into a variety of cells, playing a vital role in the hair growth cycle. When the hair enters the growth phase, it can quickly activate and divide to promote hair regeneration. To further observe the effect of cholesterol on hair follicle stem cells, skin tissues were taken from both sides of the back of mice, and the proliferation marker Ki67 was studied by immunofluorescence staining. The results showed that cholesterol can promote the proliferation of hair follicle stem cells ( Figure 2 A). To explore whether cholesterol directly promotes the proliferation of hair follicle stem cells, we used cholesterol to stimulate hair follicle stem cells. Through cell proliferation experiments and q-PCR methods, we found that the proliferation-related genes between the control group and the experimental group did not show statistical differences ( Figure 2 BC). Over the past few decades, increasing evidence has shown that immune cells, blood vessels, and sympathetic nerves play a key role in promoting hair growth. Through immunofluorescence experiments, we observed that compared with the control group, the sympathetic nervous system was activated after cholesterol injection, while no significant differences in blood vessels and inflammatory cells were observed ( Figure 2 DF). In addition, we observed that after cholesterol stimulation, the phosphorylation activity of the sympathetic nervous system in PC12 cells gradually increased. Similarly, when extracting cholesterol-injected skin tissue, we observed the same phenomenon, proving that cholesterol acts by activating the sympathetic nervous system ( Figure 2 HJ).

[0044] 2.3 Cholesterol promotes hair follicle stem cell proliferation and hair growth by activating the sympathetic nervous system

[0045] 6-OHDA is a selective sympathetic nerve toxin that can effectively ablate sympathetic nerves while preserving the remaining nerves in the skin. To explore the effects of sympathetic nerves on hair growth and development, we intradermally injected 6-OHDA to eliminate the sympathetic nerves in the entire back skin of mice when hair follicle stem cells were in the quiescent phase. Immunofluorescence experiments were performed to ensure effective elimination of sympathetic nerves. Compared with control mice, blocking sympathetic nerves slowed down the hair growth rate of mice ( Figure 3 AE). At the same time, blocking the sympathetic nerves inhibited the effect of cholesterol in promoting hair growth, indicating that the activation of the sympathetic nervous system plays a key role in the process of cholesterol promoting hair growth. The above results all show that cholesterol promotes hair growth by activating the sympathetic nerves ( Figure 4 AD).

[0046] 2.4 Cholesterol activates sympathetic nerves through PKA phosphorylation of tyrosine hydroxylase

[0047] Existing studies have shown that cholesterol is particularly effective in inducing phosphorylation of tyrosine hydroxylase at Ser40, because this site can directly change the synthesis of catecholamines and have the greatest effect on TH activity. HA-1004 is a PKA kinase inhibitor. Previous studies have found that HA-1004 can inhibit the phosphorylation of tyrosine hydroxylase at Ser40. In order to explore whether cholesterol induces phosphorylation of tyrosine hydroxylase through the PKA pathway, we first verified the inhibitory effect of this inhibitor (HA-1004) on tyrosine hydroxylase phosphorylation in PC12 cells ( Figure 5 A). The inhibitor (100 μl, 1000 ng / mL) was injected subcutaneously into the back of mice to inhibit sympathetic nerve activity. One week later, we injected the same dose of cholesterol and control solvent into the back of mice at the same site and observed a significant slowdown in hair growth. 40 days after injection, there was no significant difference in hair growth between the two groups ( Figure 5 BC). Figure 5 As shown in DF, cholesterol failed to further promote the proliferation of HFSCs after inhibitor treatment. These results suggest that cholesterol promotes hair growth by activating the PKA signaling pathway, thereby promoting the phosphorylation of tyrosine hydroxylase and subsequently activating the sympathetic nervous system.

[0048] The above description is only a preferred embodiment of the present invention, and does not limit the implementation mode and protection scope of the present invention. For those skilled in the art, it should be aware that all solutions obtained by equivalent substitutions and obvious changes made using the contents of the specification and illustrations of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of cholesterol in the preparation of hair growth promoter.

2. The use according to claim 1, It is characterized in that Cholesterol is the only active ingredient of the hair growth promoter.

3. The use according to claim 2, It is characterized in that The hair growth promoter further comprises a pharmaceutically acceptable adjuvant.

4. The use according to claim 1, It is characterized in that The hair growth promoter is a liquid preparation.

5. The use according to claim 4, It is characterized in that The dosage form of the hair growth promoter is injection.

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