Application of protein nano-selenium hydrogel in preparation of medicine for preventing and treating androgenetic alopecia

Abstract

The invention belongs to the technical field of biological medicines, and discloses application of protein nano-selenium hydrogel in preparation of a medicine for preventing and treating androgenetic alopecia. The protein nano-selenium hydrogel is used for preventing and treating androgenetic alopecia, and compared with existing inorganic nano-selenium, the protein nano-selenium hydrogel has higher antioxidant activity, better drug effect and good safety. Pharmacodynamic studies show that compared with inorganic nano-selenium, the protein nano-selenium has a better curative effect in an androgenetic alopecia mouse model, melanin pigmentation occurs earlier, and hair follicle cells can be activated to enter a hair growth period earlier.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine, and particularly relates to use of a protein nano-selenium hydrogel in preparing a drug for preventing and treating androgenic alopecia. Background Art

[0002] Androgenetic alopecia (AGA) is a common progressive hair loss type in clinical practice. It is an androgen-dependent genetic disease characterized by a progressive decrease in hair density. AGA occurs in both men and women, and the incidence rate in men is higher than that in women. Male AGA mainly occurs in the frontal and temporal regions and the top of the head, showing a typical "horseshoe" shape. Female AGA is characterized by sparse hair on the top of the head, and the hairline does not move back. Patients also have oily scalp, increased dandruff and itching. According to studies, the incidence of AGA is related to race. Caucasians have a higher incidence rate, while Asians and blacks have a lower incidence rate. The incidence rate in China and South Korea is not much different. Secondly, the incidence rate of AGA is positively correlated with age. The probability of getting sick at the age of 30 is 30%, the probability of getting sick at the age of 50 is 50%, and the probability of getting sick at the age of 70 is 80%. In recent years, due to the influence of the environment, work pressure, and bad habits such as lifestyle and diet, the incidence trend of AGA has become younger. AGA is a chronic progressive disease. Although it is not life-threatening, it is a disfiguring disease that has a serious impact on the patient's self-image, social interaction, and psychological and emotional health.

[0003] There are many treatments for AGA, including FDA-approved minoxidil and finasteride for the treatment of male and female pattern hair loss, but their mechanisms of action and routes of administration are different. 2+Calcium channel openers are effective for 80%-90% of hair loss patients, but they can also cause adverse reactions, such as dry and itchy skin, redness of the hair and skin, excessive hair on the face of women, and contact dermatitis in a few cases. Finasteride, a member of the azosteroid family, is used orally to treat male-onset AGA. Long-term use of finasteride can affect male sexual function, including decreased libido and erectile dysfunction. Local injection of platelet-rich plasma (PRP) improves microcirculation around hair follicles and promotes hair follicle regeneration. These growth factors may stimulate stem cells located in the bulge area of ​​the hair papilla and promote the growth of hair follicles; however, the use of PRP to treat AGA is still in its early stages. Androgen receptor inhibitors inhibit the transcription and translation of target genes by inhibiting the complex formed by the binding of androgens and androgen receptors in hair papilla cells, thereby inhibiting the formation of proteins with specific functions and affecting hair growth; its adverse reactions are mainly hyperkalemia, menstrual disorders, decreased libido, breast pain, etc. Hair transplantation is a surgical method for the clinical treatment of AGA hair regeneration. It is a free transplantation method that transplants hair from a certain part of the patient to another part of the body or an allogeneic part through a surgical approach; but there are requirements for the transplanted hair. Generally, it will fall off two months after the operation, and obvious effects can only be seen in 6-9 months. The operation has high requirements for the operator, is expensive, and the survival rate of transplanted hair follicles is not high. Low-frequency energy laser treatment, as an auxiliary treatment for AGA, uses low-frequency laser to penetrate the epidermis to improve local blood circulation in the scalp, reduce hair follicle inflammation, and increase intracellular ATP to provide nutrition, but its mechanism of action is still unclear. In summary, it is very necessary to find a class of highly effective and low-toxic AGA treatment drugs. Summary of the invention

[0004] In order to overcome the shortcomings and deficiencies in the above-mentioned prior art and avoid the side effects of existing commercially available drugs, the purpose of the present invention is to provide a use of a protein nano-selenium hydrogel in the preparation of a drug for preventing and treating androgenic alopecia, and the use of the protein nano-selenium hydrogel is safe and effective.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] A use of a protein nano-selenium hydrogel in preparing a drug for preventing and treating androgenic alopecia. The preparation steps of the protein nano-selenium hydrogel include: reducing albumin with a protein reducing agent at 37°C, then adding a sodium selenite solution containing chitosan or a sodium selenate solution containing chitosan, stirring and reacting for 3 to 12 hours at room temperature or 4°C, dialyzing or centrifuging to remove unreacted substances, and obtaining protein nano-selenium particles; then, adding the protein nano-selenium particles to a hydrogel matrix raw material, and preparing the protein nano-selenium hydrogel under sufficient stirring and the action of a cross-linking agent or a cross-linking inducer (triethanolamine to change pH or environmental changes).

[0007] The protein reducing agent is tris(2-carboxyethyl)phosphine (TCEP), and dithiothreitol, β-mercaptoethanol, cysteine ​​or glutathione can also be used.

[0008] The raw material of the hydrogel matrix is ​​carbomer gel, hyaluronic acid powder, chitosan, sodium alginate or gelatin.

[0009] The albumin may be one or more of human serum albumin, recombinant human serum albumin, bovine serum albumin, ovalbumin, donkey serum albumin, transferrin or similar disulfide bond-rich proteins.

[0010] The protein nano-selenium hydrogel is prepared specifically according to the following steps:

[0011] (1) dissolving albumin in a phosphate buffer solution containing a protein reducing agent at a pH of 5.0 to 9.0, wherein the final concentration of albumin is 0.01 to 200 mg / mL, and the final concentration of the protein reducing agent is 0.1 to 60 mM; reacting at 37° C. for 90 to 110 min, stirring, and obtaining a homogeneous protein solution with a spatially unfolded structure; then, adding a sodium selenite solution containing chitosan or a sodium selenate solution containing chitosan to the homogeneous protein solution with a spatially unfolded structure; The final concentration is 0.05-5 mg / mL, the final concentration of sodium selenite or sodium selenate is 0.1-30 mM, and the reaction is stirred at room temperature or 4°C for 3-12 hours to obtain a crude protein nano-selenium solution; the crude protein nano-selenium solution is placed in a dialysis bag and dialyzed overnight in a low-temperature PBS solution at 0-20°C, or the crude protein nano-selenium solution is centrifuged at 8000-30000 rpm for 10-60 minutes and repeatedly resuspended with deionized water and centrifuged several times to finally obtain protein nano-selenium particles;

[0012] (2) adding carbomer gel into water and stirring to fully swell it, then adding the protein nano-selenium particles obtained in step (1) and deionized water, and adding triethanolamine to adjust the pH under full stirring until it becomes a hydrogel, thereby obtaining a protein nano-selenium hydrogel;

[0013] Alternatively, water is added to the hyaluronic acid powder and stirred thoroughly to make it swell, and then the protein nano-selenium particles obtained in step (1) and deionized water are added, and finally cross-linking agents carbodiimide and N-hydroxysuccinimide are added to cross-link the hyaluronic acid and stirred thoroughly to form a hydrogel, thereby obtaining a protein nano-selenium hydrogel;

[0014] Alternatively, chitosan is dissolved in acetic acid, and then the dissolved chitosan is added to water, followed by the protein nano-selenium particles obtained in step (1) and deionized water, and then cross-linking agent glutaraldehyde is added and stirred thoroughly to form a protein nano-selenium hydrogel.

[0015] Alternatively, a sodium alginate solution is added with a crosslinking agent CaCl2 and stirred to prepare a sodium alginate hydrogel, and then the protein nano-selenium particles obtained in step (1) and deionized water are added to prepare a protein nano-selenium hydrogel;

[0016] Alternatively, gelatin is ultrasonically dispersed in deionized water at 60°C to induce cross-linking of gelatin molecules. When the gelatin molecules are completely formed into a hydrogel, the protein nano-selenium particles obtained in step (1) and deionized water are added to prepare a protein nano-selenium hydrogel.

[0017] The dialysis molecular retention of the dialysis bag in step (1) is not less than 1000.

[0018] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0019] (1) The present invention uses protein nano-selenium hydrogel to prevent and treat androgenic alopecia, which has stronger antioxidant activity, higher safety and effectiveness than existing inorganic nano-selenium hydrogels. Pharmacodynamic studies have shown that the protein nano-selenium hydrogel of the present invention has better therapeutic effects in the androgenic alopecia mouse model than inorganic nano-selenium hydrogels, with earlier melanin deposition and the ability to activate hair follicle cells to enter the hair growth phase earlier.

[0020] (2) Se has a very narrow safe dosage range in the human body and is prone to toxicity due to overdose. Although inorganic nano-selenium particles improve the bioavailability and biological activity of selenium and improve the safety of Se supplementation, they are still susceptible to oxidation and aggregation, which limits their widespread application. Human serum albumin (HSA) is a natural, non-toxic, non-immunogenic, biodegradable, and biocompatible drug carrier that can be encapsulated in proteins through various drug binding sites present in HSA molecules and transported to the treatment site, thereby improving the effective utilization of drugs. To this end, the present invention uses protein nano-selenium particles (HSA-Se) constructed using protein assembly technology, and studies have found that it has better biological activity and safety than inorganic nano-selenium (Nano-Se).

[0021] (3) Preliminary studies of the present invention have found that after protein nano-selenium particles (HSA-Se) and inorganic nano-selenium (Nano-Se) are prepared into hydrogels, the protein nano-selenium hydrogel (HSA-Se-Gel) has a significant promoting effect on AGA hair growth compared with the inorganic nano-selenium hydrogel (Nano-Se-Gel); the study found that the protein nano-selenium hydrogel (HSA-Se-Gel) has simulated enzyme activity, which can simulate the activity of CAT and SOD to remove ROS in the microenvironment around the hair follicles, inhibit the damage of excessive ROS to hair follicle cells, and promote hair regeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1These are the morphology and characterization pictures of protein nano-selenium particles (HSA-Se) and inorganic nano-selenium particles (Nano-Se), where A is the naked eye morphology of HSA-Se and Nano-Se particles; and B is the transmission electron microscopy of HSA-Se and Nano-Se particles.

[0023] Figure 2 These are the morphology and characterization pictures of protein-only hydrogel (Gel), protein nano-selenium hydrogel (HSA-Se-Gel), inorganic nano-selenium hydrogel (Nano-Se-Gel), and blank carbomer gel (Matrix-Gel), where A is the morphology observed by the naked eye; B is the transmission electron microscope picture; CJ are the rheological analysis of the gel, including amplitude sweep test, frequency sweep test and linear viscoelastic area.

[0024] Figure 3 It is the in vitro biosafety of protein nano-selenium hydrogel (HSA-Se-Gel) and inorganic nano-selenium hydrogel (Nano-Se-Gel), where A is the HSF cell biosafety evaluation; B is the HaCaT cell biosafety evaluation.

[0025] Figure 4 is the biosafety evaluation of mice after treatment with each gel group, wherein AD is the number of red blood cells (RBC), white blood cells (WBC) and platelets (PLT) and the total level of hemoglobin (HGB) of mice treated with Nano-Se-Gel (Se 20μg / g), Nano-Se-Gel (Se 40μg / g), Nano-Se-Gel (Se80μg / g), HSA-Se-Gel (Se 20μg / g), HSA-Se-Gel (Se 40μg / g), HSA-Se-Gel (Se 80μg / g), Minoxidil on the 19th day; EH is the serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), creatinine (SCr) and urea nitrogen (BUN) levels of mice on the 19th day after treatment in each group; I is the weight change of mice in each treatment group on the 19th day; J is the H&E sections of the main organs of mice in each treatment group after the 19th day.

[0026] Figure 5It is an evaluation chart of the antioxidant capacity of different forms of Se, where A is the DPPH scavenging rate of HSA-Se, Nano-Se and VC (2.5μg / ml); B is the DPPH scavenging rate of HSA-Se, Nano-Se and VC at different concentrations in 4h; C is the efficiency of HSA-Se, Nano-Se and VC (20μg / ml) in scavenging superoxide anions; D is the concentration-dependency of HSA-Se in scavenging superoxide anions; E is the scavenging effect of HSA-Se, Nano-Se and VC (10μg / ml) on hydroxyl radicals; F is the concentration-dependency of HSA-Se in scavenging hydroxyl radicals.

[0027] Figure 6 The figure shows the effect of HSA-Se and Nano-Se on reducing H2O2-induced oxidative stress in HSF cells. A shows the cell activity after cells were treated with H2O2 at different concentrations for 2 hours. BC shows the protective effect of HSA-Se and Nano-Se at different concentrations on oxidative stress damage in cells induced by H2O2. D shows the expression of ROS in HSF cells after treatment with HSA-Se and Nano-Se at different concentrations. E shows the quantification of ROS fluorescence intensity in HSF cells.

[0028] Figure 7 This is the evaluation of hair regeneration in AGA mice by HSA-Se and Nano-Se, where A is the overall hair regeneration in AGA mice; B is the length of new hair; C is the diameter of new hair; D is the weight of new hair / area of ​​hair loss; E is the skin color score of AGA mice; F is the H&E section of AGA mouse skin; and G is the MDA content in the skin of AGA mice. DETAILED DESCRIPTION

[0029] The present invention is further described in detail below in conjunction with embodiments and drawings, but the embodiments of the present invention are not limited thereto.

[0030] Example 1: Preparation of protein nano-selenium (HSA-Se) and inorganic nano-selenium (Nano-Se) and their hydrogels (HSA-Se-Gel and Nano-Se-Gel)

[0031] (1) Preparation of protein nanoselenium (HSA-Se):

[0032] Human serum albumin was dissolved in a phosphate buffer solution (PBS) containing a protein reducing agent tri(2-carboxyethyl)phosphine (TCEP) at a pH of 5.0, the final concentration of albumin was 10 mg / mL, and the final concentration of the protein reducing agent was 20 mM; the mixture was reacted at 37°C for 100 min with stirring to obtain a homogeneous protein solution with a spatially unfolded structure; then, a sodium selenite solution containing chitosan was added to the homogeneous protein solution with a spatially unfolded structure, the final concentration of chitosan was 0.05 mg / mL, and the final concentration of sodium selenite was 5 mM, and the mixture was stirred at 4°C for 12 h to obtain a crude protein nano-selenium solution; the crude protein nano-selenium solution was placed in a dialysis bag and dialyzed overnight in a low-temperature PBS solution at 0 to 20°C to finally obtain protein nano-selenium particles (HSA-Se).

[0033] (2) Preparation of inorganic nano-selenium (Nano-Se):

[0034] Weigh 46.05 mg of glutathione (GSH) and 30 mg of human serum albumin (BSA) and pour them into 7.0575 ml of water. After stirring evenly, add 0.3225 mL of 20 mg / mL Na2SeO3 solution, and finally add 120 μL of 1 mol / L NaOH solution and stir evenly to prepare an inorganic nano-selenium particle (Nano-Se) solution.

[0035] (3) Preparation of HSA-Se and Nano-Se carbomer hydrogels:

[0036] First, 3 g of Carbomer 934 was added to 100 mL of water and stirred thoroughly to make it swell, thereby preparing a 3% Carbomer hydrogel. Then, 13 mL of 3% Carbomer 934 was taken and added with 25.675 mL of deionized water and stirred to make it swell fully, and 1.33 g of HSA-Se obtained in step (1) and Nano-Se obtained in step (2) were added. At the same time, no substance was added as a blank group, and a cross-linking inducer (triethanolamine) was added under sufficient stirring to prepare a gel containing only protein (Gel), a 60 μg / g protein nano-selenium carbomer hydrogel (HSA-Se-Gel), an inorganic nano-selenium carbomer hydrogel (Nano-Se-Gel), and a blank Carbomer gel (Matrix-Gel).

[0037] (4) Preparation of HSA-Se and Nano-Se chitosan hydrogels:

[0038] First, weigh 1.42 g of chitosan, add 2% acetic acid solution and stir thoroughly to dissolve, so that the chitosan is fully swollen; then add 1.33 g of HSA-Se obtained in step (1) and Nano-Se obtained in step (2)

[0039] Stir thoroughly and evenly; finally, add 2% glutaraldehyde and continue stirring to obtain 60 μg / g protein nano-selenium chitosan hydrogel (HSA-Se-Gel) and inorganic nano-selenium chitosan hydrogel (Nano-Se-Gel).

[0040] (5) Preparation of HSA-Se and Nano-Se hyaluronic acid hydrogels:

[0041] First, hyaluronic acid sodium salt was fully dissolved with 0.01M HCl, and 100mg of acidic HA was activated with 251mg of carbodiimide (EDC) and 151mg of N-hydroxysuccinimide (NHS) in 50mL of DMSO for 24h. Then 1.33g of HSA-Se obtained in (1) and Nano-Se obtained in step (2) were added and stirred thoroughly. 60μg / g protein nano-selenium hyaluronic acid hydrogel (HSA-Se-Gel) and inorganic nano-selenium hyaluronic acid hydrogel (Nano-Se-Gel) were obtained.

[0042] Example 2: Characterization of protein nano-selenium (HSA-Se) and inorganic nano-selenium (Nano-Se)

[0043] The naked eye morphology of protein nano-selenium (HSA-Se) and inorganic nano-selenium (Nano-Se) obtained in Example 1 is as follows: Figure 1 As shown in A; protein nano-selenium (HSA-Se) and inorganic nano-selenium (Nano-Se) particles were observed by transmission electron microscopy. Figure 1 As shown in B.

[0044] Example 3: Characterization of protein nanoselenium (HSA-Se) and inorganic nanoselenium (Nano-Se) hydrogels

[0045] The morphologies of the protein-only gel (Gel) obtained in step (3) of Example 1, protein nano-selenium hydrogels (HSA-Se-Gel, protein nano-selenium carbomer hydrogels) of different concentrations, inorganic nano-selenium hydrogels (Nano-Se-Gel, inorganic nano-selenium carbomer hydrogels) of different concentrations, and blank carbomer gel (Matrix-Gel) were observed. Figure 2 As shown in A, HSA-Se-Gel and Nano-Se-Gel were found to be orange-red, and the higher the concentration, the darker the color, while Matrix-Gel was transparent white. The morphology of protein nano-selenium (HSA-Se) and inorganic nano-selenium (Nano-Se) hydrogels was observed by transmission electron microscopy, and it was found that they had a loose and porous structure, as shown in Figure 2 As shown in B. The amplitude scanning, frequency scanning and linear viscoelastic region measurement by rheometer show that the elasticity of each gel is greater than the viscosity, and it is easy to spread and stay on the skin, and is suitable for skin external use, such as Figure 2Shown by CJ.

[0046] Example 4: In vitro biosafety evaluation of protein nano-selenium (HSA-Se) and inorganic nano-selenium (Nano-Se) obtained in Example 1

[0047] Methods: HSF cells (5×10 3 Each well was inoculated with 100 μL of serum-free medium containing 20 μL of MTT, and 100 μL of SDS was added to each well 4 hours later. After 24 hours of attachment, different concentrations of HSA-Se (0.01953125, 0.0390625, 0.078125, 0.15625, 0.3125, 0.625, 1.25, 2.5, 5, 10 μM) and Nano-Se (0.01953125, 0.0390625, 0.078125, 0.15625, 0.3125, 0.625, 1.25, 2.5, 5, 10 μM) were added, with 5 replicates per group. After 24 hours, the culture medium was discarded, and 100 μL of serum-free medium containing 20 μL of MTT was added to each well. After 4 hours, 100 μL of SDS was added to each well. After 24 hours of dissolution of the blue-purple crystalline formazan, the absorbance was measured at 570 nm. The cell survival rate was calculated, cell survival rate % = (OD value of the drug administration group - OD value of the blank group) / OD value of the blank group × 100%.

[0048] Conclusion: HSF and HaCaT cells were treated with different concentrations of HSA-Se and Nano-Se for 24 h, and cell activity was detected by MTT. It was found that HSA-Se and Nano-Se had no obvious cytotoxicity below 10 μM (such as Figure 3 A and B).

[0049] Example 5: Effects and in vivo safety of the protein nano-selenium hydrogel (HSA-Se-Gel, protein nano-selenium carbomer hydrogel) and inorganic nano-selenium hydrogel (Nano-Se-Gel, inorganic nano-selenium carbomer hydrogel) obtained in step (3) of Example 1 on the AGA mouse model

[0050] 1. Build the model:

[0051] C57BL / 6 male mice aged 7 weeks and weighing about 20g were used as experimental subjects; the mice were randomly divided into groups, and the 2×3cm area of ​​the back skin of the mice was depilated in turn with a razor and depilatory cream. All mice were artificially induced to enter the resting phase of hair, and the skin was pink. 1mL of 0.5% (w / v) testosterone ethanol solution (testosterone was dissolved in 50% ethanol) was applied daily.

[0052] 2. Grouping and medication:

[0053] The mice were randomly divided into groups and each group was treated with the following drugs: Normal, blank carbomer gel (Gel), Model, Minoixdil (5%, 50 mg / g), HSA-Se-Gel (Se 20 μg / g), HSA-Se-Gel (Se 40 μg / g), HSA-Se-Gel (Se 80 μg / g), Nano-Se-Gel (Se 20 μg / g), Nano-Se-Gel (Se 40 μg / g) and Nano-Se-Gel (Se 80 μg / g). Testosterone was applied every morning, and 0.2 mL of each drug was applied 4 hours later.

[0054] 3. Observation:

[0055] (1) Hair growth: Take photos every day to record the time when the skin of AGA mice darkens and the hair growth. On the 19th day of the experiment, remove the newly grown hair of AGA mice with a razor and record the diameter, length and weight of the newly grown hair of each group of AGA mice.

[0056] (2) Oxidation indicators in mouse skin: MDA: On the 19th day after depilation, mice were killed, and the skin from the depilated area on the back of the mice was weighed and recorded, and stored in a -80°C refrigerator. The grinding adapter and steel beads were pre-frozen in a -20°C refrigerator overnight. The skin in the -80°C refrigerator was cut into pieces, placed in a 2mL special centrifuge tube, and 2 No. 5 steel beads were added. PBS (500μL) was added. Note: The weight ratio of skin tissue to PBS is 1:9. Place it in a tissue grinder and grind at 65HZ for 1 min, and carefully remove the steel beads. Place it in a refrigerated centrifuge and freeze it for 30 minutes (4°C, 15000rpm), and take the supernatant. The MDA content was determined according to the instructions of the MDA kit.

[0057] (3) Biosafety: On day 19, the heart, liver, spleen, lung, and kidney of the mice were collected, fixed with 4% paraformaldehyde, and stained with H&E.

[0058] 4. Conclusion:

[0059] AGA mice were treated with HSA-Se-Gel (Se 20μg / g), HSA-Se-Gel (Se 40μg / g), HSA-Se-Gel (Se 80μg / g), Nano-Se-Gel (Se 20μg / g), Nano-Se-Gel (Se 40μg / g), and Nano-Se-Gel (Se 80μg / g) percutaneously for 19 days. The mice were then killed for blood routine tests, liver and kidney function tests, and weight changes. Pathological sections were taken of the heart, liver, spleen, lungs, and kidneys of each group of mice. The results showed that HSA-Se-Gel had good biosafety ( Figure 4 AJ).

[0060] Example 6: In vitro antioxidant evaluation of protein nano-selenium (HSA-Se) and inorganic nano-selenium (Nano-Se) obtained in Example 1

[0061] 1. Experimental methods

[0062] DPPH method for free radical determination:

[0063] (1) Prepare 0.1 mM DPPH ethanol solution: Dissolve 0.002 g DPPH in 50 mL anhydrous ethanol and store in a dark place.

[0064] (2) Prepare 0.5 mg / mL VC solution.

[0065] (3) Prepare sample solutions: inorganic nano-selenium (0.625, 1.25, 2.5, 5 μg / mL) and protein nano-selenium (0.625, 1.25, 2.5, 5 μg / mL).

[0066] (4) The sample solution and VC solution were mixed with the DPPH ethanol solution respectively (in a dark place), and placed in a dark place at room temperature. The UV absorbance was measured at 517 nm after 0.5 h, 1 h, 2 h, and 4 h.

[0067] (5) Group setting: Sample group (A i ): sample solution (100 μL) + DPPH ethanol solution (100 μL); blank group (A j ): sample solution (100 μL) + anhydrous ethanol (100 μL); control group (A0): deionized water (100 μL) + DPPH ethanol solution (100 μL); 3 parallel samples in each group.

[0068] (6) Result calculation: DPPH clearance rate % = [1-(A i -A j ) / A0]×100%;DPPH residual rate %=(A i -A j ) / A0×100%.

[0069] Determination of hydroxyl free radicals by saffron red T:

[0070] (1) Prepare sample solutions HSA-Se and Nano-Se (2.5, 5, and 10 μg / mL), with 3 parallel samples in each group.

[0071] (2) Prepare 25% ethanol solution as a control.

[0072] (3) Prepare 0.30% H2O2 solution; 0.40mmol / L MnSO4 solution; 0.20mol / LH2SO4; and 0.030% Safranine Red T solution.

[0073] (4) Add 0.16 mL of MnSO4 solution, 0.07 mL of H2SO4 solution, 0.08 mL of Safranine T solution, and 0.12 mL of H2O2 solution into a 1.5 mL centrifuge tube. Dilute to 1 mL mark with distilled water, shake well, and place in a dark place for 10 min. Measure the absorbance at 518 nm with distilled water as reference, and calculate the difference △A between the two absorbances.

[0074] (5) Add different concentrations of HSA-Se and Nano-Se to the above system, and perform the same operation as above to measure the absorbance A of the antioxidant solution. s ; Safranine T and sulfuric acid were used as blank system, and its absorbance was A0; the hydroxyl radical reaction system without adding antioxidants had its absorbance as A; 3 parallel samples were set in each group.

[0075] (6) The hydroxyl radical scavenging rate can be expressed as: R = (A s -A) / (A0-A)×100%.

[0076] Pyrogallol method for determination of superoxide anion:

[0077] (1) Prepare sample solutions HSA-Se and Nano-Se (5, 10, 20 μg / mL), with 3 parallel samples in each group.

[0078] (2) Prepare 7.567 mg / mL of pyrogallol (dissolved in 1 mM HCl), Tris-HCl, 1 mM HCl, and 0.1 M HCl.

[0079] (3) Add the sample, Tris-HCl, and pyrogallol in sequence, and measure the absorbance at a wavelength of 319 nm, every 30 seconds for 240 seconds. Tris-HCl and 1 mM HCl are used as blank systems.

[0080] 2. Conclusion

[0081] The total antioxidant capacity of HSA-Se and Nano-Se was evaluated by DPPH method. The results showed that the total antioxidant capacity of HSA-Se was strong, concentration-dependent, and significantly better than that of Nano-Se ( Figure 5 AB). The pyrogallol method was used to evaluate the scavenging ability of different selenium forms and VC on superoxide anions. The study found that HSA-Se and the positive control VC greatly reduced the generation of superoxide anions, while Nano-Se had almost no effect, suggesting that HSA-Se is significantly better than Nano-Se in scavenging superoxide anions ( Figure 5CD), and showed concentration dependence. In addition, the scavenging rate of hydroxyl free radicals by HSA-Se and Nano-Se showed that the scavenging rate of hydroxyl free radicals by HSA-Se was significantly higher than that of Nano-Se ( Figure 5 EF), and its clearance rate is positively correlated with concentration.

[0082] Example 7: In vitro cell experiment on protein nano-selenium (HSA-Se) and inorganic nano-selenium (Nano-Se) obtained in Example 1

[0083] 1. H2O2 induces oxidative stress

[0084] (1) HSF cells (5×10 3 Each well was inoculated with 100 μL of serum-free medium containing 20 μL of MTT. After 4 hours, 100 μL of SDS was added to each well. After 24 hours of dissolution of the blue-purple crystalline formazan, the absorbance was measured at 570 nm.

[0085] (2) HSF cells (5×10 3 Each well was inoculated with 20 μL of MTT and 100 μL of SDS per well. After 24 hours of attachment, different concentrations of HSA-Se (0.01953125, 0.0390625, 0.078125, 0.15625, 0.3125, 0.625, 1.25, 2.5, 5 μM) and Nano-Se (0.01953125, 0.0390625, 0.078125, 0.15625, 0.3125, 0.625, 1.25, 2.5, 5 μM) were added, with 5 replicates per group. After 24 hours of treatment, 800 μM H2O2 was added. After 2 hours of treatment, the culture medium was discarded, 20 μL of MTT was added to each well, and 100 μL of SDS was added to each well after 4 hours. After 24 hours, the absorbance was measured at 570 nm.

[0086] 2. Detection of ROS in HSF cells - DCFH-DA fluorescence probe method

[0087] (1) HSF cells (1×10 5 / well) were inoculated into a 12-well plate. After 24 hours of attachment, 100μM H2O2 was added for 1 hour, and then HSA-Se (1.25μM, 0.3125μM) Nano-Se (1.25μM, 0.3125μM) was added for 24 hours.

[0088] (2) The cells were removed, washed once with PBS, and 10 μM DCFH-DA fluorescent probe was added. The cells were incubated at 37°C for 30 min, and fluorescence was photographed under an inverted fluorescence microscope.

[0089] 3. Conclusion:

[0090] After treating HSF cells with 100, 200, 400, 600, and 800 μM H2O2 for 2 h, the cell viability was examined using MTT, and it was found that the cell mortality rate was about 50% at 800 μM. Subsequent experiments used 800 μM H2O2 as the concentration for inducing oxidative stress damage in HSF cells ( Figure 6 A). The cells were first damaged with 800 μM H2O2 for 2 h, and then treated with different concentrations of HSA-Se. The results showed that HSA-Se began to protect the cells at 1.25 μM. HSA-Se has a protective effect on H2O2-induced cell oxidative stress damage ( Figure 6 In addition, after pretreatment with different concentrations of HSA-Se and Nano-Se for 24 hours, H2O2 was added to damage the cells for 2 hours. The results showed that HSA-Se had a stronger protective effect against cell oxidative stress damage than Nano-Se ( Figure 6 C). We also detected the intracellular ROS level using the DCFH-DA fluorescent probe. First, after treatment with 100μM H2O2, HSA-Se (0.3125, 1.5μM) and Nano-Se (0.3125, 1.5μM) were given. After incubating the cells with the DCFH-DA fluorescent probe, it was found that HSA-Se could significantly reduce the ROS level in the cells. Although the fluorescence intensity of Nano-Se was lower than that of the model group, HSA-Se had a more significant effect in reducing intracellular ROS ( Figure 6 DE).

[0091] Example 8: Effects of the protein nano-selenium hydrogel (HSA-Se-Gel, protein nano-selenium carbomer hydrogel) and inorganic nano-selenium hydrogel (Nano-Se-Gel, inorganic nano-selenium carbomer hydrogel) obtained in step (3) of Example 1 on the AGA mouse model

[0092] 1. Build the model:

[0093] C57BL / 6 male mice aged 7 weeks and weighing about 20g were used as experimental subjects. The mice were randomly divided into groups and depilated with a razor and depilatory cream on the 2×3cm area of ​​the mouse back skin. All mice were artificially induced to enter the resting phase of hair and the skin was pink. By applying 0.5%

[0094] (w / v) testosterone ethanol solution 1 mL (testosterone is dissolved in 50% ethanol).

[0095] 2. Grouping and medication:

[0096] The mice were randomly divided into groups and each group was treated with the following drugs: Normal, blank carbomer gel (Gel), Model, Minoixdil (5%, 50 mg / g), HSA-Se-Gel (Se 20 μg / g), HSA-Se-Gel (Se 40 μg / g), HSA-Se-Gel (Se 80 μg / g), Nano-Se-Gel (Se 20 μg / g), Nano-Se-Gel (Se 40 μg / g), Nano-Se-Gel (Se 80 μg / g). Testosterone was applied every morning, and 0.2 mL of each drug was applied 4 hours later.

[0097] 3. Observation:

[0098] (1) Hair growth: Take photos every day to record the time when the skin of AGA mice darkens and the hair growth. On the 19th day of the experiment, remove the newly grown hair of AGA mice with a razor and record the diameter, length and weight of the newly grown hair of each group of AGA mice.

[0099] (2) Oxidation indicators in mouse skin: MDA: On the 19th day after depilation, mice were killed, and the skin from the depilated area on the back of the mice was weighed and recorded, and stored in a -80°C refrigerator. The grinding adapter and steel beads were pre-frozen in a -20°C refrigerator overnight. The skin in the -80°C refrigerator was cut into pieces, placed in a 2mL special centrifuge tube, and 2 No. 5 steel beads were added. PBS (500μL) was added. Note: The weight ratio of skin tissue to PBS is 1:9. Place it in a tissue grinder and grind at 65HZ for 1 min, and carefully remove the steel beads. Place it in a refrigerated centrifuge and freeze it for 30 minutes (4°C, 15000rpm), and take the supernatant. The MDA content was determined according to the instructions of the MDA kit.

[0100] 4. Conclusion:

[0101] C57BL / 6 mice were used to establish an AGA model. The mice were divided into groups and treated with carbomer gel (Gel) containing only protein, Model, Minoixdil (5%, 50 mg / g), HSA-Se-Gel (Se 20 μg / g), HSA-Se-Gel (Se 40 μg / g), HSA-Se-Gel (Se 80 μg / g), Nano-Se-Gel (Se 20 μg / g), Nano-Se-Gel (Se 40 μg / g), and Nano-Se-Gel (Se 80 μg / g). The Normal group was not treated. On the first day of depilation, the skin of all mice showed pink color, and no obvious vellus hair was generated. On the 9th day, the blank group began to show melanin deposition on the skin, while the other groups did not show melanin deposition. On the 11th day, the blank group had obvious hair growth. In addition, the HSA-Se-Gel (Se 20μg / g) group, the HSA-Se-Gel (Se 40μg / g) group, and the HSA-Se-Gel (Se 80μg / g) group all showed signs of hair growth on the 11th day. The mice's skin began to darken earlier than the Nano-Se-Gel group, while the positive drug minoxidil did not show melanin deposition until 13 days. On the 19th day, the regenerated hair coverage rate of the HSA-Se-Gel group reached more than 90%, but the Nano-Se-Gel (Se 20μg / g) group and the Nano-Se-Gel (Se40μg / g) group did not reach a hair coverage rate of more than 90% ( Figure 7 A). The newly grown hair was removed with a razor, and the hair length, hair diameter, and hair weight of each group of mice were measured. The results showed that the hair length, diameter, and weight of the HSA-Se-Gel group were better than those of the Nano-Se-Gel group, and the hair regeneration effect of the HSA-Se-Gel (Se 40μg / g) group was better than that of the HSA-Se-Gel (Se 20μg / g) group and the HSA-Se-Gel (Se 80μg / g) group ( Figure 7 The skin scores of mice also showed that the melanin deposition time of the mice in the HSA-Se-Gel group was earlier than that in the Nano-Se-Gel group, indicating that the HSA-Se-Gel group entered the hair growth phase earlier ( Figure 7 In addition, H&E sections of AGA mouse skin ( Figure 7F) It can be seen that compared with the model, the number of hair follicles in the HSA-Se-Gel group increased significantly, and the complete hair follicle structure was visible, and most of the hair follicles were in the growth stage or early regression stage; the miniaturization of hair follicles and the atrophy of hair follicles in the model group were significantly improved after the administration of HSA-Se-Gel; and most of the hair follicles in the HSA-Se-Gel group were in the subcutaneous fat layer, and the thickness of the skin was thickened after the administration of HSA-Se-Gel. Although the Nano-Se-Gel group was improved compared with the model group, the effect was far less than the therapeutic effect of the HSA-Se-Gel group on AGA. The MDA content in the mouse skin after administration was lower than that in the model group ( Figure 7 G).

[0102] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principles of the present invention shall be equivalent replacement methods and shall be included in the protection scope of the present invention.

Claims

1. Use of a protein nano-selenium hydrogel in the preparation of a drug for preventing and treating androgenic alopecia, Features: The preparation steps of the protein nano-selenium hydrogel include: reducing albumin with a protein reducing agent at 37°C, then adding a sodium selenite solution containing chitosan or a sodium selenate solution containing chitosan, stirring and reacting at room temperature or 4°C for 3 to 12 hours, dialyzing or centrifuging to remove unreacted substances to obtain protein nano-selenium particles; then adding the protein nano-selenium particles to a hydrogel matrix raw material, and preparing the protein nano-selenium hydrogel under sufficient stirring and the action of a cross-linking agent or a cross-linking inducer.

2. Use of a protein nano-selenium hydrogel according to claim 1 in preparing a drug for preventing and treating androgenic alopecia, Features: The protein reducing agent is tris(2-carboxyethyl)phosphine.

3. Use of the protein nano-selenium hydrogel according to claim 1 in preparing a drug for preventing and treating androgenic alopecia, Features: The raw material of the hydrogel matrix is ​​carbomer gel, hyaluronic acid powder, chitosan, sodium alginate or gelatin.

4. Use of the protein nano-selenium hydrogel according to claim 1 in preparing a drug for preventing and treating androgenic alopecia, Features: The protein nano-selenium hydrogel is prepared specifically according to the following steps: (1) dissolving albumin in a phosphate buffer solution containing a protein reducing agent at a pH of 5.0 to 9.0, wherein the final concentration of albumin is 0.01 to 200 mg / mL, and the final concentration of the protein reducing agent is 0.1 to 60 mM; reacting at 37° C. for 90 to 110 min, stirring, and obtaining a homogeneous protein solution with a spatially unfolded structure; then, adding a sodium selenite solution containing chitosan or a sodium selenate solution containing chitosan to the homogeneous protein solution with a spatially unfolded structure; The final concentration is 0.05-5 mg / mL, the final concentration of sodium selenite or sodium selenate is 0.1-30 mM, and the reaction is stirred at room temperature or 4°C for 3-12 hours to obtain a crude protein nano-selenium solution; the crude protein nano-selenium solution is placed in a dialysis bag and dialyzed overnight in a low-temperature PBS solution at 0-20°C, or the crude protein nano-selenium solution is centrifuged at 8000-30000 rpm for 10-60 minutes and repeatedly resuspended with deionized water and centrifuged several times to finally obtain protein nano-selenium particles; (2) adding the carbomer gel into water and stirring to fully swell it, then adding the protein nano-selenium particles obtained in step (1) and deionized water, and adding triethanolamine under full stirring to induce the carbomer to cross-link into a hydrogel, thereby obtaining a protein nano-selenium hydrogel; Alternatively, water is added to the hyaluronic acid powder, and the mixture is stirred thoroughly to make it swell, and then the protein nano-selenium particles obtained in step (1) and deionized water are added, and finally carbodiimide is added to cross-link the hyaluronic acid to increase its hydrophilicity, and the mixture is stirred thoroughly to form a hydrogel, thereby obtaining a protein nano-selenium hydrogel; Alternatively, chitosan is dissolved in acetic acid, and then the dissolved chitosan is added to water, followed by the addition of the protein nano-selenium particles obtained in step (1) and deionized water, and the mixture is allowed to stand in a closed environment containing ammonia water for 12 hours to allow cross-linking between molecules until a hydrogel is formed, thereby obtaining a protein nano-selenium hydrogel. Alternatively, sodium alginate solution is added with the crosslinking agent CaCl 2 Stirring to prepare sodium alginate hydrogel, and then adding the protein nano-selenium particles obtained in step (1) and deionized water to prepare protein nano-selenium hydrogel; Alternatively, gelatin is ultrasonically dispersed in deionized water at 60° C. to induce its molecular action to completely form a gel, and the protein nano-selenium particles obtained in step (1) and deionized water are added to prepare a protein nano-selenium hydrogel.

5. The use according to claim 4, Features: The dialysis molecular retention of the dialysis bag in step (1) is not less than 1000.

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