Application of fasting inducible factor Angptl4 protein as molecular target in treatment or prevention of alopecia stress

Abstract

The invention discloses application of a fasting inducible factor Angptl4 protein as a molecular target in treatment or prevention of stress alopecia, the expression of the Angptl4 protein is up-regulated to improve alopecia and hair follicle atrophy caused by slow stress, and the expression of the Angptl4 protein can be improved by intermittent fasting or injection of the Angptl4 protein; furthermore, the Angptl4 protein disclosed by the invention improves the alopecia caused by slow pressure by activating a PI3K-AKT signal channel. The application disclosed for the first time is a potential target of the Angptl4 protein for preventing and treating pressure alopecia, a new idea is provided for preventing and treating pressure alopecia, and a new possibility is provided for preparing a medicine for preventing and treating pressure alopecia.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and specifically relates to the application of fasting-inducing factor Angptl4 protein as a molecular target in treating or preventing stress-induced hair loss. Background Art

[0002] Angiopoietin-like protein 4 (Angptl4) protein is a secreted glycoprotein that plays a physiological role in lipid metabolism and is expressed in a variety of cells, including adipocytes, hepatocytes, cardiomyocytes, endothelial cells, and macrophages. Under fasting conditions, Angptl4 expression is upregulated and then secreted into the peripheral blood circulation, inhibiting the activity of endogenous lipoprotein lipase (LPL), playing an important role in regulating lipid metabolism and distribution; in addition, Angptl4 is involved in tumorigenesis, angiogenesis, vascular permeability, and stem cell regulation.

[0003] In contemporary society, hair loss is becoming an urgent and growing public health issue. With the accelerated pace of life and the surge in competitive pressure, the problem of hair loss caused by mental stress has become increasingly prominent and has become a major hidden pain that plagues modern people. Stress-induced hair loss refers to hair loss caused by an individual experiencing long-term psychological stress or emotional tension. This type of hair loss is usually not permanent, but if stress persists, hair loss may worsen. Symptoms include sudden thinning of hair, receding crown or hairline, and decreased overall hair density.

[0004] The cause of stress-induced hair loss is related to the body's physiological response to stress. When the human body is stressed, it secretes more stress hormones, such as cortisol. These hormones can interfere with the normal life cycle of hair follicles, causing the hair follicles to enter the resting phase prematurely, thus causing hair loss. The latest research shows that increased levels of corticosterone (the stress hormone corresponding to human cortisol) inhibit the activation of hair follicle stem cells and prolong the resting phase of hair follicles, thereby affecting the normal growth of hair.

[0005] For a long time, traditional methods of preventing and treating hair loss have mostly relied on topical treatments - from the essence of the ancient herbal arborvitae leaves to microneedle therapy under cutting-edge technology. Although these attempts are innovative and intelligent, they have encountered bottlenecks in practical applications. In addition, due to the diversity of human characteristics, different individuals may experience different degrees of treatment reactions, including but not limited to side effects, when using the same medication or treatment method. Faced with this situation, there is an urgent need to explore a set of more universal, simpler and more effective treatment methods that do not damage physical health to deal with stress-induced hair loss, in order to fundamentally reduce the physical and mental burden of patients.

[0006] In view of this, new hair loss prevention and treatment methods that are universal and cost-effective are particularly important, but there are currently no targeted methods for preventing and treating stress-induced hair loss. Summary of the invention

[0007] The first objective of the present invention is to address the deficiencies of the prior art and provide a method for using the fasting-inducing factor Angptl4 protein as a molecular target in the preparation of a drug for preventing or treating stress-induced hair loss.

[0008] Preferably, upregulation of Angptl4 protein expression improves hair loss and hair follicle atrophy caused by chronic stress.

[0009] Preferably, the concentration of Angptl4 protein in the blood is increased by intermittent fasting (IF) or injection of Angptl4 protein.

[0010] Preferably, Angptl4 protein improves hair loss caused by chronic stress by activating the PI3K-AKT signaling pathway.

[0011] The second object of the present invention is to provide a pharmaceutical composition for treating hair loss, wherein the active ingredient of the pharmaceutical composition at least includes Angptl4 protein.

[0012] Preferably, pharmaceutically acceptable excipients are also included.

[0013] The active ingredients of the pharmaceutical composition may also include other drugs for treating hair loss.

[0014] Preferably, the pharmaceutical composition is introduced into the body tissue by the following methods: injection, spraying, infiltration, absorption, physical mediation, chemical mediation, or introduced into the body tissue after being mixed or wrapped with other substances.

[0015] Preferably, the pharmaceutical composition adopts any pharmaceutically acceptable dosage form.

[0016] The third object of the present invention is to provide a non-therapeutic method for preventing or improving stress-induced hair loss, wherein the method specifically comprises: utilizing intermittent fasting to increase the expression of Angptl4 protein, thereby preventing or improving hair loss caused by chronic stress.

[0017] Preferably, the intermittent fasting is: fasting for 16 hours a day and eating freely within 8 hours, for at least 2 months.

[0018] Preferably, intermittent fasting can also improve hair follicle density and the number of hair follicle stem cells in vivo.

[0019] Compared with the prior art, the present invention has the following beneficial effects: The present invention constructs mice with systemic knockout of Angptl4, establishes a model, simulates slow stress conditions in vitro, and conducts various tissue, cell, and molecular experiments to prove that the expression of Angptl4 protein is upregulated during intermittent fasting, and that Angptl4 protein can improve hair loss and hair follicle atrophy caused by slow stress; it also proves that Angptl4 protein improves hair loss caused by slow stress by activating the PI3K-AKT signaling pathway, and is a potential target for preventing and treating stress-induced hair loss. In summary, the present invention discloses for the first time that fasting and the fasting-induced factor Angptl4 have a protective effect on hair loss, providing a new idea for preventing and treating stress-induced hair loss. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 These are the genotype identification results and knockout effect verification of Angptl4 knockout mice, where A: genotype identification of Angptl4 knockout mice; BD: qPCR verification of the knockout effect of Angptl4 in mouse liver, white fat, and skin; EG: WB verification of the knockout effect of Angptl4 in mouse white fat, brown fat, liver, and lung.

[0021] Figure 2 These are the experimental results of Angptl4 protein protecting hair follicles under slow stress conditions, where A: a direct image of the hair of mice in the Angptl4 protein injection group and the control group under slow stress conditions; B: ELISA determination of Angptl4 protein concentration in the serum of the two groups of mice; C: HE staining of the skin hair follicles of the two groups of mice; D: Immunofluorescence detection of hair follicle stem cell markers in the two groups of mice.

[0022] Figure 3 These are the experimental results of Angptl4 protein regulating the hair follicle cycle and prolonging the resting phase of hair follicles, where A: the hair growth status of mice in the systemic Angptl4 protein knockout group and the control group 3 weeks after hair was shaved; B: ELISA determination of Angptl4 protein concentration in the serum of the two groups of mice; C: HE staining of the skin hair follicles of the two groups of mice; D: Immunofluorescence detection of hair follicle stem cell markers in the two groups of mice.

[0023] Figure 4 The experimental results show that intermittent fasting can reverse hair follicle atrophy caused by chronic stress and prevent hair loss caused by chronic stress, where A: a direct image of the hair of mice in the intermittent fasting group and the control group 4 weeks after the chronic stress model was established; B: ELISA determination of the Angptl4 protein concentration in the serum of the two groups of mice; C: HE staining of the skin hair follicles of the two groups of mice; D: Immunofluorescence detection of hair follicle stem cell markers in the two groups of mice.

[0024] Figure 5The improvement effect of Angptl4 protein on hair loss in mice under slow stress through the PI3K-AKT signaling pathway, where A: PCA analysis of the Angptl4 protein injection group and the control group; B: Volcano plot analysis of the differential proteins between the two groups of mice; C: KEGG analysis of the differential proteins between the two groups of mice; D: GO analysis of the differential proteins between the two groups of mice; E: Detection of PI3K-AKT signaling pathway activity in hair follicles of mice injected with Angptl4 protein and the control group. DETAILED DESCRIPTION

[0025] The technical solution of the present invention is further explained below in conjunction with the accompanying drawings and embodiments, but the experimental conditions and setting parameters therein should not be regarded as limiting the basic technical solution of the present invention.

[0026] Example 1 1. Construction of Angptl4 whole-body knockout mouse model (1) Angptl4 whole body knockout mice: Angptl4 is a secretory glycoprotein that is mainly secreted from the liver and adipose tissue into the blood. In order to exclude the influence of Angptl4 protein secreted by systemic tissues on mouse hair follicles, Angptl4 knockout mice were constructed in this example.

[0027] Three-week-old C57BL / 6 female mice were superovulated, and fertilized eggs were collected after checking the vaginal plug, and sgRNA target 1-ggtgctatgcgcggctactgcgg (SEQ ID NO.1) and sgRNA target 2-ggagtagacaagacttcgagggg (SEQ ID NO.2) were designed. sgRNA and Cas9 mRNA were microinjected into the oviduct of surrogate mothers to construct Angptl4 whole-body knockout mice.

[0028] 2. Mouse tail DNA extraction and genotype identification (1) Take peripheral blood from the mice to be tested, add 5ul of anticoagulant to every 1.5ml of blood, and let it stand at room temperature for 1h; (2) 25°C, 5000 rpm, centrifugation for 15 min. (3) Let stand at room temperature for 1 hour and take the supernatant; (4) Add 100ul of acid lysis solution and react at 96℃ for 2h; (5) Add 100 µl of alkali solution to stop the reaction; (6) Perform PCR amplification according to the settings in Table 1 and repeat for 35 cycles; Table 1 PCR amplification procedure (7) Prepare 1.5% agarose gel (1.5 g agarose + 100 ml 1×TAE buffer + 5 μl GelRed) and perform electrophoresis at 140 V for 36 min. Take photos using a gel imaging system.

[0029] 3. qPCR analysis (1) Mix cDNA with gene-specific primers and SYBR green PCR Master Mix (Sigma Aldrich) on ice throughout the process; (2) Run on the Applied Biosystems 7900HT Fast Real-Time PCR System.

[0030] (3) The primer sequences are as follows: ANGPTL4: forward primer: 5′-TCCTGGACCACAAGCACCTAGAC-3′ (SEQ ID NO.3); Reverse primer: 3′-CGGTTGAAGTCCACTGAGCCATC-5′ (SEQ ID NO.4) GAPDH: forward primer: 5′-GCACCGTCAAGGCTGAGAAC-3′ (SEQ ID NO.5); Reverse primer: 3′-TGGTGAAGACCGCCAGTGGA-5′ (SEQ ID NO.6) 4. WB analysis (1) Prepare protein samples for immunoblotting by mixing 0.1 M Tris-HCl (pH 7.0), 4% SDS (sodium dodecyl sulfate), 20% glycerol, 1 mM DTT (dithiothreitol) and protease inhibitors.

[0031] (2) subjecting the sample to polyacrylamide gel electrophoresis; (3) Transfer the protein from the gel to a PVDF membrane (Millipore), stain with bromophenol blue, and perform electrophoresis at 4°C and 100 V. (4) Incubate the membrane in 5% skim milk for 1 h and then rinse three times, 10 min each time; (5) Continue to incubate the membrane with the primary antibody (containing 5% bovine serum albumin) at 4°C overnight and rinse three times, 10 min each time; (6) Incubate with secondary antibody (1:3000) for 1 hour, rinse three times, add developer for development, and image with an imager to obtain the results.

[0032] Figure 1 The genotype identification results and knockout effect verification of Angptl4 whole-body knockout mice were obtained. The verification results of qPCR and WB respectively showed that Angptl4 was completely knocked out at the transcription level and protein expression level, and Angptl4 whole-body knockout mice were successfully constructed.

[0033] Example 2: Angptl4 protein protects hair follicles from damage caused by chronic stress and improves hair growth in mice This example selects the Angptl4 protein injection group and the control group mice under slow pressure conditions to directly observe the hair growth status and measure the Angptl4 protein concentration, etc., including the following steps: 1. Slow stress modeling in mice (1) C57 mice were randomly divided into two groups and subjected to the following two stressors in a random manner every day for 9 weeks: tilted or isolated cages, crowded stocking density; humid housing environment, and rapid changes in light.

[0034] (2) At week 5, the experimental group was intravenously injected with Angptl4 recombinant protein at a dose of 5 ug / kg every two days, and the control group was injected with the same volume of normal saline.

[0035] 2. Observe the hair growth after modeling (1) Observe the hair condition of mice two weeks after 4 weeks of slow stress: The mice were originally shaved, and then tracked to complete a complete growth cycle. The progress of the hair growth cycle was recorded through standardized photos at the beginning of each experiment and every week thereafter.

[0036] (2) Observe the hair condition of the two groups after injection of Angptl4 protein and continued culture until the 9th week; 3. ELISA determination of Angptl4 protein content in two groups of mice (1) Centrifuge the blood sample at 3000 rpm / min for 20 min and aspirate the supernatant; (2) Preparation of standard curve using standard substances; (3) Add 40ul of diluent and 10ul of sample to the wells, seal the plate and incubate at 37°C for 30min, spin dry the liquid, add 50ul of enzyme-labeled reagent to the wells, and incubate at 37°C for 30min; (4) Color development: Add 100 μl of color developer (A:B=1:1), mix well, and incubate at 37°C in the dark for 10 min. (5) Add 50 μl of stop solution to terminate the reaction, measure the absorbance at 450 nm, and perform analysis.

[0037] 4. Observation of hair follicle density in two groups of mice (1) After dewaxing, tissue sections were fixed with citrate for 16 hours and washed with PBS; (2) Add 3% hydrogen peroxide solution, incubate for 10 min at room temperature, and wash with PBS; (3) Block with sheep serum at room temperature for 30 min, add primary antibody, and incubate at 4°C overnight; (4) Add secondary antibody, incubate at room temperature for 1 h, and wash with PBS; (5) DAB staining, hematoxylin counterstaining, neutral resin sealing, and observation under a microscope.

[0038] 5. Immunofluorescence detection of the number of hair follicle stem cells in two groups of mice (1) Tissue sections were dewaxed, fixed at room temperature for 15 min, and washed with PBS; (2) Block with sheep serum at room temperature for 30 min, add primary antibody, and incubate at 4°C overnight; (3) Add secondary antibody, incubate at 37°C for 1 h, and seal the slides with DAPI.

[0039] Figure 2 The results showed that Angptl4 protein has a protective effect on hair follicle cells, reverses hair follicle atrophy caused by chronic stress, and improves hair loss caused by chronic stress: under chronic stress conditions, the hair of mice injected with Angptl4 recombinant protein and the control group showed significant differences at 9 weeks ( Figure 2 A) The concentration of Angptl4 in the serum of the experimental group was significantly different from that of the control group ( Figure 2 B) Under slow pressure, HE staining of the skin hair follicles of mice injected with Angptl4 recombinant protein and the expression of hair follicle stem cell marker K15 showed that the hair follicle density of the experimental group was higher ( Figure 2 C-2D).

[0040] Example 3: Angptl4 regulates the hair follicle cycle In this example, Angptl4 knockout mice and control mice were selected for hair removal and then the Angptl4 protein in serum and the hair follicle density were measured. Figure 3 shown.

[0041] The following steps are involved: 1. Depilate the Angptl4 knockout mice and littermate control mice to induce a new hair follicle cycle. The growth phase is determined by the darkening of the mouse hair. Shave the back skin of the mouse with electric scissors and record the skin color changes and hair recovery of the mouse. Once the hair has recovered to about 90% of the back skin, shave the mouse again to monitor the entry into the next growth phase, and repeat 5 times;

[0042] 2. Observe the hair growth of the two groups of mice after three weeks of feeding; 3. ELISA was used to determine the Angptl4 protein content in the serum of the two groups of mice; 4. Immunohistochemistry was used to determine the hair follicle density of the two groups of mice; 5. Immunofluorescence detection of the number of hair follicle stem cells in the two groups of mice; Figure 3 The results showed that Angptl4 protein regulates the hair follicle cycle: the Angptl4 content in the serum of knockout mice was significantly lower than that of the control mice in the same litter, and the hair growth of Angptl4 KO mice was significantly slower than that of the control mice. HE staining showed that the density of hair follicles was significantly reduced, indicating that Angptl4 knockout prolonged the resting phase of hair follicles. Fluorescence staining with hair follicle stem cell marker protein K15 showed that the number of hair follicle stem cells decreased. The above results indicate that Angptl4 inhibits the proliferation of hair follicle stem cells, thereby prolonging the resting phase of hair follicles.

[0043] Example 4: Intermittent fasting reverses hair follicle atrophy caused by chronic stress and prevents hair loss In this example, C57 mice were randomly divided into groups, and after slow stress modeling, different treatments were performed on the experimental group and the control group mice to observe the effects of intermittent fasting on hair growth and hair follicle density of mice under slow stress. The results are as follows: Figure 4 shown.

[0044] 1. C57 mice were randomly divided into two groups and subjected to slow stress modeling. Four weeks after modeling, the mice in the experimental group were subjected to intermittent fasting (Fasting), i.e., fasting for 16 hours and free feeding within 8 hours. The intermittent control mice used in this example were free to feed for 24 hours (ND); 2. Observe the hair growth of the two groups of mice after 9 weeks of feeding; 3. ELISA was used to determine the Angptl4 protein content in the serum of the two groups of mice; 4. Immunohistochemistry was used to determine the hair follicle density of the two groups of mice; 5. Immunofluorescence detection of the number of hair follicle stem cells in the two groups of mice; Figure 4 The effect of intermittent fasting on improving hair loss and hair follicle atrophy under slow stress. After 4 weeks of slow stress modeling, both groups showed stress-induced hair thinning. After 5 weeks of intermittent fasting in the experimental group, the hair thinning phenotype of the experimental group mice was improved compared with that of the control group mice. The Angptl4 content in the serum of the experimental group mice was significantly higher than that of the control group; the hair follicle density and the number of hair follicle stem cells were significantly higher than those of the control group. The results showed that intermittent fasting reversed the hair follicle atrophy caused by slow stress and prevented the hair loss caused by slow stress.

[0045] Experimental Example 5: Angptl4-mediated improvement of mouse hair growth under slow stress is related to the PI3K-AKT signaling pathway In this example, skin samples (n=3) of mice injected with Angptl4 recombinant protein and control group under slow pressure were collected for RNA-seq sequencing analysis.

[0046] The results are as follows Figure 5 As shown: Through KEGG pathway enrichment analysis, it was found that the pathway enriched in the differentially expressed protein TOP1 in the group injected with Angptl4 recombinant protein was the PI3K-AKT signaling pathway. Through WB analysis, it can be seen that the PI3K-AKT signaling pathway of mouse hair follicles was significantly activated after the injection of Angptl4 recombinant protein, thereby promoting hair follicle growth. The above results show that Angptl4 recombinant protein improves the problem of hair loss in mice caused by chronic stress through the PI3K-AKT signaling pathway.

[0047] The above are only some preferred embodiments of the present invention, and the protection scope of the present invention is not limited to the above embodiments. Those skilled in the art who make modifications or equivalent substitutions based on their understanding of the technical solution of the present invention without departing from the spirit or scope of the technical invention solution should be included in the protection scope of the present invention.

Claims

1. Application of fasting-induced factor Angptl4 protein as a molecular target in the preparation of drugs for preventing or treating stress-induced hair loss.

2. The use according to claim 1, characterized in that: Upregulation of Angptl4 protein expression improves hair loss and hair follicle atrophy induced by chronic stress.

3. The use according to claim 2, characterized in that: The concentration of Angptl4 protein in the blood is increased by intermittent fasting or injection of Angptl4 protein.

4. The use according to any one of claims 1 to 3, characterized in that: Angptl4 protein improves hair loss caused by chronic stress by activating the PI3K-AKT signaling pathway.

5. A pharmaceutical composition for treating hair loss, characterized in that: The active ingredient of the pharmaceutical composition at least includes Angptl4 protein.

6. The pharmaceutical composition according to claim 5, characterized in that The pharmaceutical composition also includes pharmaceutically acceptable excipients.

7. The pharmaceutical composition according to claim 5, characterized in that The pharmaceutical composition is introduced into the body tissue by the following methods: injection, spraying, infiltration, absorption, physical mediation, chemical mediation, or being mixed or wrapped with other substances before being introduced into the body tissue.

8. The pharmaceutical composition according to claim 5, characterized in that It is characterized in that The pharmaceutical composition adopts any pharmaceutically acceptable dosage form.

9. A method for preventing or improving stress-induced hair loss for non-therapeutic purposes, characterized in that: The method specifically comprises: utilizing intermittent fasting to increase the expression of Angptl4 protein, thereby preventing or improving hair loss caused by chronic stress, and improving hair follicle density and hair follicle stem cell content.

10. The method according to claim 9, characterized in that The intermittent fasting specifically includes: fasting for 16 hours a day and eating freely within 8 hours, for at least 2 months.

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