Preparation method of stem cell exosome and application thereof in medicines and cosmetics

Abstract

The application provides a preparation method of stem cell exosomes and application of the stem cell exosomes in medicines and cosmetics. The application also provides an anti-aging and anti-oxidation polypeptide CY3-4-A polypeptide, which is obtained by screening and identifying grass carp skin, has good anti-oxidation properties, and can treat skin photoaging properties through mouse anti-skin photoaging property identification. The polypeptide can also have good anti-wrinkle effect when being loaded on mesenchymal stem cell exosomes to prepare a gel, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biology, more particularly to a preparation method of stem cell exosome and its application in pharmaceuticals and cosmetics. BACKGROUND

[0002] Skin, as a tissue covering the surface of the whole body, directly contacts with the external environment. It is an organ that protects the human body from injury, infection and dehydration, and realizes body temperature regulation and sensory perception, accounting for about 16% of body weight, and is the first line of defense between the human body and the external environment. Therefore, skin becomes the first organ to suffer damage from the outside world and appear aging. Skin is mainly composed of closely combined epidermis and dermis, which has important barrier protection function. The epidermis is the outermost layer of the human body, mainly providing defense against external damage, and its self-renewal ability decreases with age. The dermis plays a crucial role in the structure of body tissues, and its constituent connective tissue is formed by fibroblasts, which are mainly responsible for the synthesis of elastin and collagen, keeping the skin elastic and vigorous. With the passage of time and long-term exposure to solar ultraviolet radiation, the decline in skin structure and function has similar histopathological features, such as decreased skin elasticity, decreased sebaceous gland and sweat gland secretion, degeneration of elastic fibers, decreased collagen content, and deposition of peroxidized lipids and denatured proteins in the skin.

[0003] Exosomes are nanoscale vesicles of 40-100 nm released by various types of cells into the extracellular space, and this vesicle is widely distributed in various body fluids. The biological production process of exosomes includes their origin from endosomes, and then interacting with other intracellular vesicles and organelles to produce the final contents of exosomes. The biological molecules carried by exosomes include nucleic acids, proteins, lipids, amino acids and metabolites, which can reflect the source cells. Therefore, exosomes are not only a window for researchers to understand the changes in the state of cells or tissues, but also a diagnostic marker for detecting various diseases in biological fluids. In addition, due to the cell specificity of exosomes, they can promote intercellular communication with adjacent cells by transferring bioactive compounds, so their application in the biomedical field has greatly increased. Because of the potential application of exosomes in clinical diagnosis and treatment, the research in various fields of medicine is gradually deepening and expanding. Mesenchymal stem cell-derived exosomes refer to 50-200 nm vesicles secreted into the extracellular space when mesenchymal stem cell-derived multivesicular bodies and plasma membranes fuse. At present, mesenchymal stem cell-derived exosomes are attracting attention due to their potential in the field of anti-aging. Studies have found that these exosomes can restore the physiological function of photoaging damaged skin by reducing the expression of matrix metalloproteinases, increasing the production of collagen, regulating intracellular signal pathway transduction, and intercellular communication. Other related studies have shown that adipose tissue-derived mesenchymal stem cell-derived exosomes play an important role in immune regulation, umbilical cord-derived mesenchymal stem cell-derived exosomes show outstanding performance in tissue damage repair, and bone marrow-derived mesenchymal stem cell-derived exosomes show strong regenerative capacity.

[0004] Mesenchymal stem cells (MSCs) have been found to increase collagen synthesis, thicken the dermis and subcutaneous fat layers, and inhibit the proliferation and synthesis of melanocytes, thereby alleviating skin aging. Related articles have reported that the use of HUCMSCs can promote skin regeneration and restore vitality, have anti-aging / wrinkle effects, inhibit skin pigmentation, and have other biological functions. Previous studies have also shown that mesenchymal stem cell-derived exosomes (MSC-exosomes) can replace MSCs to exert similar biological performance, and their function is stronger, which is expected to become an ideal carrier for future development. Previous studies have found that HUCMSCs-derived exosomes prepared by ultrasonic waves can be used to increase the skin extracellular matrix and enhance skin vitality. By isolating exosomes from cells grown in a three-dimensional culture system and exploring their ability to regulate HaCaT keratinocyte photoaging, it was found that HUCMSCs-derived exosomes can enhance normal HaCaT cell proliferation and migration, while inhibiting UVB-induced damage to these cells. These exosomes also reduce HaCaT cell apoptosis and senescence in photoaged HaCaT cells, increase collagen type I expression, and reduce matrix metalloproteinase (MMP1) expression. The increase in collagen is a key factor in maintaining skin vitality and youthfulness. This study confirms that human umbilical cord mesenchymal stem cell-derived exosomes have great potential in treating skin aging and provides a new strategy for clinical anti-aging. To further explore whether HUCMSCs-ex can prevent acute skin photodamage caused by ultraviolet radiation, a rat model of acute photodamage (ultraviolet radiation-induced skin tissue redness, scaling, and inflammatory cell infiltration for three consecutive days) was established, and it was found that subcutaneous injection of HUCMSCs-ex in vivo can protect skin cells from ultraviolet radiation-induced DNA damage, inflammation, and apoptosis, significantly reducing skin inflammation and promoting skin cell regeneration. The main 14-3-3zeta protein delivered by HUCMSCs-ex exerts a cell-protective function by regulating the SIRT1-dependent antioxidant pathway.

[0005] Exosomes have high proliferation rates, extensive pluripotency, do not produce teratomas or other tumors, and are low immunogenic and safe, making them widely used in skin aging or skin anti-aging applications. However, there are still some problems in clinical applications, such as insufficient therapeutic effect, and the therapeutic effect of a single exosome drug needs to be further improved. SUMMARY

[0006] The application provides an anti-aging antioxidant polypeptide, which is named CY3-4-A polypeptide, and the amino acid sequence is shown as SEQ ID NO: 1.

[0007] Furthermore, the polypeptide is obtained by isolation, screening and identification from the skin of grass carp.

[0008] Specifically, the polypeptide can be artificially synthesized.

[0009] Specifically, the polypeptide can also be conservatively substituted by amino acids, but still retains the corresponding polypeptide activity.

[0010] The term "amino acid" refers to naturally occurring and non-naturally occurring amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally encoded amino acids are the 20 common amino acids found in proteins (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine and valine) as well as pyrrolysine and selenocysteine. Amino acid analogs refer to compounds that have the same basic chemical structure (i.e., an alpha carbon bonded to a hydrogen, a carboxyl, an amino and an R group) as a naturally occurring amino acid, such as homo-serine, norleucine, methionine sulfoxide, methionine methylsulfonium. These analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but otherwise function in a manner similar to the naturally occurring amino acids. Reference to an amino acid includes, for example, naturally occurring proteinogenic L-amino acids, D-amino acids; chemically modified amino acids, such as amino acid variants and derivatives; naturally occurring non-proteinogenic amino acids, such as beta-alanine, ornithine, and the like; and chemically synthesized compounds having properties known in the art to be characteristic of amino acids. Examples of non-naturally occurring amino acids include, but are not limited to, alpha-methyl amino acids (e.g., alpha-methyl alanine), D-amino acids, histidine-like amino acids (e.g., 2-amino-histidine, beta-hydroxy-histidine, homo-histidine, alpha-fluoromethyl-histidine, and alpha-methyl-histidine), amino acids with additional methylene groups in the side chain ("homo" amino acids), and amino acids with a sulfonic acid group in place of the carboxylic acid function in the side chain (e.g., cysteine).

[0011] Further, the present application provides a pharmaceutical composition for delaying skin aging, which comprises the CY3-4-A polypeptide of the present application.

[0012] With respect to the composition of the present application, preferably, the composition comprises one or more excipients. For example, the excipients can further improve the stability of the polypeptide of the present application during storage, ensuring a longer shelf life and stable biological activity level. Preferably, the excipients include pH buffers, stabilizers, fillers, tonicity modifiers, and the like. Particularly preferred are excipients suitable for freeze-drying.

[0013] Suitable excipients are known to the person skilled in the art. For example, the composition preferably contains a pH buffer, preferably selected from the group consisting of glycine, histidine, glutamic acid, succinic acid, phosphate, acetate and aspartic acid. The composition further preferably comprises a bulking agent, preferably selected from the group consisting of mannitol, glycine, sucrose, dextran, polyvinylpyrrolidone, carboxymethylcellulose, lactose, sorbitol, trehalose or xylitol. The composition preferably comprises a stabilizer selected from the group consisting of sucrose, trehalose, mannose, maltose, lactose, glucose, raffinose, cellobiose, gentiobiose, isomaltose, arabinose, glucosamine, fructose, mannitol, sorbitol, glycine, arginine hydrochloride, polyhydroxyl compounds (including polysaccharides such as dextran, starch, hydroxyethyl starch, cyclodextrin, N-methylpyrrolidine, cellulose and hyaluronic acid), sodium chloride. The composition can also comprise a surfactant, preferably selected from the group consisting of sodium dodecyl sulfate, dioctyl sodium succinate, dioctyl sodium sulfonate, chenodeoxycholic acid, sodium N-dodecylsarcosinate, lithium dodecyl sulfate, sodium 1-octanesulfonate, sodium hydrous cholate, sodium deoxycholate, sodium glycocholate, benzalkonium chloride or benzethonium chloride, cetylpyridinium chloride, cetyltrimethylammonium bromide, CHAPS, CHAPSO, SB3-10, SB3-12, digitonin, Triton X-100, Triton X-114, lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 40, 50 and 60, glycerol monostearate, polysorbate 20, 40, 60, 65 and 80, soybean lecithin, DOPC, DMPG, DMPC and DOPG; sucrose fatty acid ester, methylcellulose and carboxymethylcellulose.

[0014] Advantageously, the composition comprises at least 0.01 mg of the polypeptide, preferably at least 0.1 mg, more preferably at least 1 mg, more preferably at least 5 mg, more preferably at least 10 mg. The composition contains the polypeptide in a concentration of at least 0.0001 % (weight / weight, w / w), preferably at least 0.001 % w / w, more preferably at least 0.01 % w / w, more preferably at least 0.1 % w / w, more preferably at least 1 % w / w, more preferably at least 10 % w / w.

[0015] Advantageously, the composition is a solid composition (at 25 °C and atmospheric pressure), preferably a lyophilized composition. In another preferred embodiment, the composition is a liquid composition (at 25 °C and atmospheric pressure). Preferably, the composition contains the polypeptide in a concentration of at least 0.001 mg / mL, preferably at least 0.01 mg / mL, more preferably at least 0.1 mg / mL, more preferably at least 0.5 mg / mL, most preferably at least 2 mg / mL.

[0016] It is further preferred that the polypeptide comprised in the composition according to the application can be produced by biological cells, in particular isolated from a host, such as a bacterium, e.g. E. coli.

[0017] The polypeptide according to the application can be used for various therapeutic applications, such as in skin anti-aging. Thus, in preferred embodiments, the composition is a pharmaceutical composition, preferably comprising one or more excipients which are pharmaceutically acceptable when administered to an individual, in particular a mammal, especially a human. Preferred excipients are known to the person skilled in the art, such as water, preferably water for injection, physiological saline, Ringer's solution, dextrose solution, buffers, Hank's solution, vesicle forming compounds (e.g. lipids), fixed oils, ethyl oleate, 5% dextrose in saline, substances that enhance isotonicity and chemical stability, buffers, and preservatives. Other suitable excipients include any compound which, when administered to a patient, does not induce the production of antibodies harmful to the patient. Examples are well-tolerated proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, and amino acid copolymers. The pharmaceutical composition is preferably suitable for parenteral administration, in particular intravenous administration. The pharmaceutical composition can be administered in the form of injectable dosage units, such as solutions, suspensions or emulsions, formulated with the above-mentioned pharmaceutically acceptable excipients. All preferred embodiments described above for the composition according to the application, in particular relating to the concentration and amount of the polypeptide, are also preferred when the composition is a pharmaceutical composition.

[0018] A typical dosage can be in the range of, for example, 0.001 to 1000 μg; although dosages below or above this exemplary range are envisioned within the scope of the application. The dosage can be about 0.1 μg / kg to about 100 mg / kg of total body weight per day (e.g., about 5 μg / kg, about 10 μg / kg, about 100 μg / kg, about 500 μg / kg, about 1 mg / kg, about 50 mg / kg, or a range defined between any two of the foregoing values) of total body weight per day, preferably about 0.3 μg / kg to about 10 mg / kg (e.g., about 5 μg / kg, about 1 μg / kg, about 50 μg / kg, about 150 μg / kg, about 300 μg / kg, about 750 μg / kg, about 1.5 mg / kg, about 5 mg / kg, or a range defined between any two of the foregoing values) of total body weight per day, more preferably about 1 μg / kg to 1 mg / kg (e.g., about 3 μg / kg, about 15 μg / kg, about 75 μg / kg, about 300 μg / kg, about 900 μg / kg, or a range defined between any two of the foregoing values) of total body weight per day, and even more preferably about 0.5 to 10 mg / kg of body weight per day (e.g., about 2 mg / kg, about 4 mg / kg, about 7 mg / kg, about 9 mg / kg, or a range defined between any two of the foregoing values) per day. The efficacy of the treatment or prevention can be monitored by periodic assessment of the treated patient. For repeated administrations of doses over several days or longer, depending on the condition, the treatment is repeated until the disease symptoms are ameliorated to the desired extent. However, other dosage regimens can be useful, and are within the scope of the application. The desired dose can be delivered by a single bolus injection of the composition, by multiple bolus injections of the composition, or by continuous infusion of the composition.

[0019] The polypeptide-containing compositions of the application can be administered to a mammal using standard administration techniques, including oral, intravenous, intraperitoneal, subcutaneous, pulmonary, transdermal, intramuscular, intranasal, buccal, sublingual, or via a suppository. The compositions are preferably adapted for parenteral administration. As used herein, the term "parenteral" includes intravenous, intramuscular, subcutaneous, rectal, vaginal, and intraperitoneal administration. More preferably, the compositions are delivered to the mammal using the peripheral system via intravenous, intraperitoneal, or subcutaneous injection.

[0020] Further, the present application provides an anti-wrinkle cosmetic.

[0021] Optionally, the cosmetic is in the form of a gel.

[0022] The preparation method of the gel is prepared according to the following formula using the preparation method in the art: 1-5% of acrylic acid (ester) / C10-30 alkyl alcohol acrylate cross-linked polymer, 0.1-0.2% of disodium EDTA, 2-8% of glycerol, 1-5% of butanediol, 0.1-0.5% of PHENONIP, 1-10% of exosomes loaded with polypeptides, 2-8% of sodium hyaluronate, and then the pH is adjusted to 5.6 with a 10% potassium hydroxide solution, and the balance is water.

[0023] Further, the preparation method of the exosomes loaded with polypeptides is as follows: the exosomes and the polypeptides are added into 1 mL of 50 mM trehalose PBS solution respectively, mixed, then added into an electroporation dish, and placed into an electroporation instrument, and the reaction conditions are as follows: voltage 120 V, capacitance 155 mu F, discharge time 1 ms, and discharge times 2; after the electroporation, the suspension is placed into a cell incubator for incubation for 1 hour, and then centrifuged at 100000 g for 80 min twice, and the supernatant is carefully discarded, and the precipitate is resuspended with PBS to obtain the exosomes loaded with polypeptides.

[0024] Specifically, the loading mode can also be other modes commonly used in the art.

[0025] Beneficial effects

[0026] The application provides a preparation method of stem cell exosomes and application of the stem cell exosomes in medicines and cosmetics. The application also provides an anti-aging and antioxidant polypeptide CY3-4-A polypeptide, which is screened and identified from grass carp skin and has good antioxidant properties. The polypeptide can treat skin photoaging, as proved by mouse skin photoaging identification. The polypeptide loaded on mesenchymal stem cell exosomes and prepared into a gel has good anti-wrinkle effect and wide application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0027] Figure 1 Result graph of skin grade score of mice after polypeptide administration

[0028] Figure 2 Result graph of the influence of the polypeptide on the Hyp level in the skin of mice after administration DETAILED DESCRIPTION

[0029] Specific embodiments of the application will be described in greater detail below with reference to the accompanying drawings. Although specific embodiments of the application are shown in the drawings, it should be understood that the application can be implemented in various forms and should not be limited by the embodiments described herein. On the contrary, these embodiments are provided so that the application can be more thoroughly understood and the scope of the application can be fully conveyed to those skilled in the art.

[0030] Example 1 Preparation of Anti-aging antioxidant polypeptides

[0031] Fresh grass carp skin was continuously stirred in 0.1 mol / L NaOH solution for 2 hours (solid-to-liquid ratio 1:15 (g / mL), stirring speed: 120 r / min) to remove impurities and proteins. This process was repeated once. The skin was then soaked in 10% isopropanol solution (solid-to-liquid ratio 1:15 (g / mL)) for 5 hours to remove fat. This process was repeated once. After soaking, the skin was rinsed 5 times with water. 0.05 mol / L acetic acid solution (solid-to-liquid ratio 1:12 (g / mL)) was added and stirred for 5 hours. The skin was washed with distilled water until neutral, and then extracted with 10 times its weight of hot water (45℃) for 5 hours. The extract was centrifuged at 5000 r / min for 15 minutes, and the supernatant was concentrated by rotary evaporation to obtain the initial extract. Protein was extracted, and an appropriate amount of the initial extracted protein was used to prepare a 4% protein solution. Papain and neutral protease (enzyme activity ratio of 1:1) were added to achieve an enzyme activity of 6000 U / g. Enzymatic hydrolysis was carried out at pH 7.2 and 54℃ for 5 hours. After cooling, the solution was centrifuged at 4000 r / min for 15 min. The supernatant was collected and sequentially passed through ultrafiltration membranes with molecular weight cutoffs of 5 kDa and 3 kDa. The fractions above and below the membranes were collected, freeze-dried, and designated as CY1 (>5 kDa), CY2 (3-5 kDa), and CY3 (<3 kDa). The hydroxyl radical (·OH) scavenging capacity of each fraction was determined, and the CY3 fraction showed the best antioxidant capacity. The CY3 fraction was separated and purified using an SP Sephadex C-25 cation exchange column (2.6 cm × 30 cm). The equilibration buffer was 20 mmol / L acetate buffer (pH = 4.0). A linear gradient elution was performed using acetate buffer containing NaCl (0–1 mol / L) at a rate of 2 mL / min. The detection wavelength was 220 nm. Five main peaks were collected and named CY3-1, CY3-2, CY3-3, CY3-4, and CY3-5, respectively. Further analysis using hydroxyl radical (·OH) scavenging capacity assays revealed that the CY3-4 fraction exhibited the best antioxidant capacity. The CY3-4 fraction was purified using a reversed-phase high-performance liquid chromatography (RP-HPLC) C18 semi-preparative column (9.4 mm × 250 mm) with a gradient elution of 5%–30% acetonitrile for 30 min at a rate of 1 mL / min and a column temperature of 30 °C. Two strongest peaks were collected at 220 nm. The fraction with the strongest hydroxyl radical (·OH) scavenging ability was identified as CY3-4-A. This fraction was then analyzed using LC-Q-TOF-Ms / Ms spectrometry from Thermo Fisher Scientific. The structure of the CY3-4-A peptide was obtained by comparison with a peptide mass spectrometry library using Max Quant 1.5.2.8. The precise molecular weight of the peptide was determined by primary mass spectrometry, and the amino acid sequence was determined by secondary mass spectrometry, as shown in SEQ ID NO: 1. The peptide was further synthesized by Shanghai Sangon Biotech Co., Ltd., and the concentration was adjusted to 2 mg / mL for later use.

[0032] Example 2 Antioxidant efficacy identification of CY3-4-A polypeptide

[0033] (1) Determination of superoxide anion radical scavenging capacity: 4.5 mL of phosphate buffer (0.05 mol / L) at pH 8.0 was heated at 25°C for 20 min, 1 mL of sample was mixed with 0.4 mL of pyrogallol (25 mmol / L) solution, and reacted at 25°C for 5 min, 1 mL of HCl (1 mol / L) was added to terminate the reaction, and the absorbance A1 was determined at 420 nm. The positive group did not add pyrogallol solution, the absorbance A2 was determined, and the blank group did not add sample, the absorbance A3 was determined. VC was used as a control, and the calculation formula was: superoxide anion radical scavenging rate = (1 - (A1-A2) / A3) x 100%. In the group, the concentration of polypeptide was 0.1 mg / mL, and the concentration of VC in the positive control group was also 0.1 mg / mL. (2) Determination of reducing capacity: 1 mL of sample with different concentrations was taken, deionized water was used as a blank, 2.5 mL of phosphate buffer (0.2 mol / L) at pH 6.6 and 2.5 mL of 1% potassium ferricyanide solution were sequentially added, mixed, and reacted at 50°C for 20 min, then 2.5 mL of 10% trichloroacetic acid solution was added, mixed, and centrifuged at 4000 r / min for 10 min. The supernatant was collected, 2.5 mL of supernatant and distilled water were taken, and then 0.5 mL of 0.1% ferric trichloride solution was added, and mixed rapidly. The absorbance at 700 nm was determined, and VC was used as a control. In the group, the concentration of polypeptide was 0.1 mg / mL, and the concentration of VC in the positive control group was also 0.1 mg / mL. (3) Determination of copper ion chelating capacity: 2 mL of sodium acetate buffer (50 mmol / L) at pH 6.0, 50 μL of each sample and CuSO4 solution (5 mmol / L) were sequentially added to a centrifuge tube, mixed uniformly, and placed at room temperature for 30 min. Then 50 μL of pyrocatechol violet solution (4 mmol / L) was added, mixed, and placed for 30 min. The absorbance A1 was determined at 632 nm, the absorbance A0 was determined with water as a blank, and EDTA was used as a control. The calculation formula was as follows: Cu 2＋ chelating rate =

(A0-A1) / A0

[0034] Table 1 Antioxidant detection results of each group

[0035]

[0036] As can be seen from Table 1, CY3-4-A polypeptide has good superoxide anion radical scavenging capacity, reducing capacity and copper ion chelating properties, indicating that it has good antioxidant properties.

[0037] Example 3 Anti-skin photoaging property identification of CY3-4-A polypeptide

[0038] SPF grade 8-week-old female KM mice (body weight 22-26 g) were purchased from Hunan Slike Jingda Experimental Animal Co., Ltd. (item number hnslkjd002), and were raised at a temperature of 18-22°C, a humidity of 50%-60%, and a light cycle of 12 h light / dark alternation, and were fed with ordinary maintenance feed.

[0039] After the mice were adaptively fed for 1 week, they were randomly divided into 10 per group, and all the mice were allowed to drink water and eat food freely. The positive control group was administered tretinoin, the model group and the blank group were not administered drugs, and were administered an equal amount of normal saline by gavage.

[0040] After the mice were adaptively fed for 1 week, their back fur was shaved off using a depilatory razor, and the exposed area on the back was 3 cm x 4 cm. The newly grown fur was shaved off every day to keep the back skin exposed all the time. An LED ultraviolet lamp [ultraviolet intensity: UVA (365 nm) = 360 μW·cm −2 and UVB (310 nm) = 30 μW·cm −2 ] was used to irradiate the back skin of the mice every day. The minimum erythema dose was 120 mJ·cm −2 , i.e. 5 min of irradiation, before the formal experiment started. Half of the minimum erythema dose (2.5 min·d −1 ) was used for the first to second weeks, and then 30 s was added every 2 weeks, i.e. 3 min·d −1 for the third to fourth weeks, 3.5 min·d −1 for the fifth to sixth weeks, 4 min·d −1 for the seventh to eighth weeks, for a total of 8 weeks, and the total irradiation dose was 4384.2 mJ·cm −2 .

[0041] Specifically, the blank group was not irradiated with ultraviolet light, and was administered an equal amount of normal saline by gavage, with the gavage time being the same as the irradiation time, for a total of 8 weeks.

[0042] The model group was irradiated with ultraviolet light according to the above method, and was administered an equal amount of normal saline by gavage, with the gavage time being the same as the irradiation time, for a total of 8 weeks.

[0043] The positive control group was irradiated with ultraviolet light according to the above method, and was administered tretinoin by gavage, with the daily gavage dose being 10 mg / kg, for a total of 8 weeks of administration;

[0044] The CY3-4-A polypeptide treatment group was irradiated with ultraviolet light according to the above method, and was administered tretinoin by gavage, with the daily gavage dose being 10 mg / kg, for a total of 8 weeks of administration;

[0045] Mice in each group were euthanized after being anesthetized with carbon dioxide. Their back skin was photographed and its grading was assessed. The grading criteria for skin wrinkles were as follows:

[0046] 0 points: No wrinkles or looseness, no thickening, fine vertical stripes running down the back, appearing or disappearing with movement;

[0047] 1 point: The skin is mostly composed of vertical fine lines, with a small number of horizontal fine lines, which may appear or disappear with movement;

[0048] 2 points: There are still a few vertical fine lines, and most of them are horizontal fine lines. They appear or disappear with movement, and the skin is slightly thickened.

[0049] 3 points: All vertical fine lines have disappeared, and a small number of horizontal shallow wrinkles have appeared. These wrinkles do not appear or disappear with movement, and the skin is slightly thickened.

[0050] 4 points: Persistent deep horizontal wrinkles and sagging, redness, and noticeably thickened skin;

[0051] 5 points: Persistent presence of numerous horizontal, rough, deep wrinkles and sagging, appearance of small red spots, and noticeable thickening of the skin;

[0052] 6 points: Severe wrinkles, large areas of erythema, development of tumors or lesions, and abnormal skin proliferation.

[0053] The results are as follows Figure 1 As shown.

[0054] from Figure 1 As can be seen, the skin score of the blank group was basically 0, while the skin score of the model group was (5.61±0.25) points, the score of the positive control group was (1.95±0.15) points, and the score of the CY3-4-A peptide treatment group was (1.12±0.09) points. From these scoring results, it can be seen that CY3-4-A peptide has a good effect on reducing skin grade scores, which indicates that CY3-4-A peptide has good anti-photoaging properties.

[0055] For each group, 0.1g of mouse skin tissue from the corresponding LED UV lamp irradiation site was collected. The tissue was minced and mixed with 9 volumes of phosphate buffer (pH 7.2). The mixture was homogenized thoroughly on ice using a handheld tissue homogenizer and centrifuged at 5000 rpm for 20 min. The supernatant was then refrigerated at 4°C for analysis. Skin antioxidant capacity was assessed by detecting SOD, GSH-Px, and MDA levels; the results are shown in Table 2.

[0056] Table 2 Results of skin antioxidant capacity in each group (# indicates significant difference compared with the model group)

[0057]

[0058] As shown in Table 2, the SOD and GSH-Px activities in the positive control group and the CY3-4-A peptide treatment group were significantly increased and the MDA activity was significantly decreased compared with the model group (P<0.05). The results indicate that the CY3-4-A peptide can significantly improve the antioxidant enzyme activity in the skin of photoaged mice, thereby enhancing the skin's ability to inhibit ROS-induced oxidative stress.

[0059] Furthermore, the levels of Hyp in the skin were further measured in each group to assess skin matrix damage. This measurement was performed using a mouse hydroxyproline (Hyp) ELISA kit. Results are as follows: Figure 2 As shown.

[0060] from Figure 2 It can be seen that the positive control group and the CY3-4-A peptide treatment group significantly increased the Hyp content compared with the model group. In particular, the Hyp content in the CY3-4-A peptide treatment group reached (5734±142) μg / g, which was significantly higher than that in the model group (4123±403) μg / g (P<0.05). This indicates that CY3-4-A peptide can improve the anti-photoaging properties of the skin by increasing Hyp activity, and has broad application prospects.

[0061] Example 4: Anti-wrinkle experiment using CY3-4-A peptide combined with stem cell exosomes.

[0062] The 3rd to 6th generation of logarithmic growth mouse adipose-derived mesenchymal stem cells (Shanghai Chunmai Biological Technology Co., Ltd., Catalog No. CM-2118) were cultured with 10% FBS DMEM culture medium until the cell density reached 70%-80%, the original culture medium was aspirated, washed with PBS, and replaced with 10% exosome-free FBS-containing DMEM culture medium for continuous culture at 37°C, 5% CO2 incubator for 45-50 hours, and the supernatant culture medium was collected; the supernatant culture medium was centrifuged at 3500g for 15 min to remove cells and cell debris, and the supernatant was transferred to a 100 kDa molecular weight ultrafiltration tube and centrifuged again at 1500-2500g for 30 min, and the concentrated liquid was collected; the exosome extraction reagent (Geneseed Exosome (Exosome) Extraction Kit) was added to the concentrated liquid, gently inverted and mixed, and placed at 4°C overnight; the mixture was centrifuged at 4°C, 1500g for 30 min, the supernatant was carefully discarded, PBS was added and centrifuged at 1500g for 5 min, and the residual liquid was aspirated, thereby obtaining the mouse adipose stem cell exosomes. The particle size of ADSC-Exo was analyzed by NTA method, and the results showed that the average size was about 152.8 nm, which basically met the size of exosomes. By transmission electron microscopy method, the morphology of ADSC-Exo presented a disc-shaped exosome morphology, and the expression of three marker proteins TSG101, CD9 and CD81 of exosomes was identified by western blot method, and the results showed that the three marker proteins in ADSC-Exo were expressed, which confirmed that the identified was exosomes.

[0063] The exosomes were loaded with CY3-4-A polypeptide, and the preparation method was as follows: 100 μg of exosomes and 20 μl of 1 mg / mL SEQ ID NO: 1 polypeptide DMSO solution were added to 1 mL of 50 mM trehalose PBS solution; after mixing, it was added to a 4 mm electroporation dish, and then placed in an electroporation instrument according to the following reaction conditions: voltage 120V, capacitance 155 μF, discharge time 1 ms, and discharge times 2; after electroporation, the suspension was placed in a cell incubator for incubation for 1 hour, followed by 100000g ultracentrifugation twice, each for 80 min, and the supernatant was carefully discarded, and the precipitate was resuspended with PBS to obtain polypeptide-loaded exosomes.

[0064] The polypeptide-loaded exosomes were prepared into experimental group gel form: the gel was prepared according to the following formula using conventional preparation methods in the art: 0.8% acrylic acid (ester) / C10-30 alkyl acrylate cross-linked polymer, 0.05% disodium EDTA, 4% glycerol, 2.5% butanediol, 0.25% PHENONIP, 2% polypeptide-loaded exosomes, 4% sodium hyaluronate, then adjust the pH to 5.6 with 10% potassium hydroxide solution, and the balance is water.

[0065] The preparation method of the gel is as follows: 0.8% of acrylic acid (ester) / C10-30 alkyl acrylate cross-linked polymer, 0.05% of disodium EDTA, 4% of glycerol, 2.5% of butanediol, 0.25% of PHENONIP, 4% of sodium hyaluronate, and then the pH is adjusted to 5.6 by using a 10% potassium hydroxide solution, and the balance is water.

[0066] Healthy women aged 35-55 were selected as the subjects, and pregnant or lactating women, people with facial skin prone to allergies, and people with facial skin with serious epidermal features such as scratches, birthmarks, pimples, and acne marks were excluded. The subjects had visible fine lines or wrinkles at the corners of the eyes, and no facial freckles or acne. They were randomly divided into the example group and the comparative example group, with 30 people in each group.

[0067] Test method: half-face test. Example group: the subjects evenly applied 0.5 mL of the experimental gel to the left half of the face at the same time every morning, and the right half of the face was treated with an equal amount of water. The experiment was conducted for 4 consecutive weeks, and no other skin care products were used during the experiment. Comparative example group: the subjects evenly applied 0.5 mL of the control gel to the left half of the face at the same time every morning, and the right half of the face was treated with an equal amount of water. The experiment was conducted for 4 consecutive weeks, and no other skin care products were used during the experiment.

[0068] The test was conducted before the experiment started and after the experiment ended. Before the test, the subjects cleaned their faces with water and wiped off the moisture. They sat in a constant temperature and humidity environment (25±1℃, relative humidity 70±10%) for 30 minutes, and then the keratin layer water content was detected. The results showed that the keratin layer water content treated with the experimental gel reached (139.54±18.46) A.U, the keratin layer water content treated with the control gel reached (115.38±22.32) A.U, and the water content of the skin treated with water reached (112.18±28.53) A.U. From this result, it can be seen that the exosomes loaded with polypeptides of the present application can effectively improve the moisturizing property of the skin.

[0069] Further, the change of facial wrinkles of the subjects was evaluated, and the results showed that the fine lines of the skin treated with the experimental gel almost completely disappeared, and the deep wrinkles also became significantly shallower, while the fine lines of the skin treated with the control gel were slightly reduced, and the number change was not very obvious, especially the deep wrinkles did not show obvious improvement, which indicated that the exosomes loaded with polypeptides of the present application could effectively improve the anti-wrinkle property of the skin.

[0070] The preferred embodiments of the application described herein are considered to be the best mode contemplated by the inventors. On the basis of the foregoing description, those skilled in the art can, and will, devise variations of the preferred embodiments of the application that lie within the conceptual scope of the applications. Those skilled in the art will appreciate that the conception, together with practical properties of the application, can be applied in a variety of other alternative embodiments. Any and all modifications, variations or equivalent arrangements that fall within the scope of the claimed application should be considered within the scope of the application.

Claims

1. An anti-aging and antioxidant polypeptide, characterized in that... The polypeptide is a CY3-4-A polypeptide, and its amino acid sequence is shown in SEQ ID NO:

1.

2. Use of the anti-aging and antioxidant polypeptide of claim 1 in the preparation of a pharmaceutical composition for anti-photoaging of the skin.

3. Use of the anti-aging and antioxidant polypeptide as described in claim 1 in the preparation of cosmetics for skin anti-wrinkle purposes.

4. The use as described in claim 3, wherein the polypeptide is loaded onto adipose-derived mesenchymal stem cell exosomes.

5. As described in claim 4, the specific loading method is to add exosomes and SEQ ID NO:1 peptide to 50mM trehalose PBS solution; after mixing, add to a 4mm electroporation dish, place in an electroporator to puncture, and then place the suspension in a cell culture incubator for 1 hour, followed by ultracentrifugation to obtain the loaded peptide exosomes.

6. The use as described in claim 5, wherein the cosmetic is in gel form.

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