Method for preventing, reducing or mitigating senescence-related changes, injuries or symptoms of skin

Abstract

Provided are a method for preventing, reducing or mitigating senescence-related changes, injuries or symptoms of skin, and a use of a platelet factor 4 in preparation of a composition for use in the method.

Description

Methods for preventing, reducing or improving aging-related changes, damage or signs of skin Technical Field

[0001] The present invention relates to methods for preventing, reducing or ameliorating aging-related changes, damage or signs in the skin and compositions comprising platelet factor 4 for use in such methods. Background Art

[0002] Aging is defined as a gradual process of physiological decline, leading to physical weakness, age-related diseases, and ultimately death. Cellular senescence is closely related to physiological processes such as tumor suppression, tissue repair embryogenesis, and body aging, and is a cellular state in these physiological processes (see non-patent literature 1). Cellular senescence often occurs in response to endogenous or exogenous stress, and its prominent features are: 1) permanent cell cycle arrest; 2) senescence-related secretory phenotype (i.e., a general term for proinflammatory cytokines and chemokines, growth regulatory factors, angiogenic factors, and matrix metalloproteinases (MMPs) secreted by senescent cells); 3) macromolecular damage (such as DNA damage); 4) metabolic disorders (such as weakened mitochondrial function and blocked ATP synthesis) (see non-patent literature 2). Similar to other organs, skin is not only affected by intrinsic aging (such as age), but also accelerated by environmental factors.

[0003] Fibroblasts are the primary component of connective tissue, which connects all histologically distinct skin cells and tissues into a functional organ. Fibroblasts in the skin synthesize and arrange the extracellular matrix (ECM) and maintain close intercellular communication with neighboring cells and tissues. Therefore, cellular senescence of skin fibroblasts may directly or indirectly lead to alterations in skin homeostasis or skin aging. It has been reported that senescent fibroblasts enter a state of prolonged proliferation arrest and release the pro-inflammatory senescence-associated secretory phenotype (SASP) into the surrounding environment, inducing local chronic inflammation, reducing the release of important growth factors, and accelerating the degradation of the extracellular matrix, thereby contributing to tissue decline and skin aging (see Non-Patent Document 3).

[0004] Mature platelet factor 4 (PF4, also known as CXCL4) is a small molecule polypeptide containing 70 amino acids synthesized by megakaryocytes. Platelet factor 4 is usually stored in platelet a granules and forms non-covalently linked tetramers under physiological conditions (see non-patent document 4). The main physiological function of platelet factor 4 is to promote blood coagulation. In addition, platelet factor 4 has been reported to be involved in various physiological processes such as inflammatory response and immune regulation. However, the relationship between platelet factor 4 and aging is still in a very early stage of exploration. Related studies have focused on platelet factor 4 as an inflammatory-related factor and its potential to improve cognitive function in aging. See, for example, non-patent documents 5 to 7.

[0005] To date, it is unclear whether platelet-derived factor 4 is involved in other physiological and / or pathological changes associated with aging, and there are no reports that platelet-derived factor 4 can directly affect the activity of skin fibroblasts.

[0006] References

[0007] Non-patent document 1: Di Micco, R., et al., Cellular senescence in ageing: from mechanisms to therapeutic opportunities. Nat Rev Mol Cell Biol, 2021.22(2):p.75-95;

[0008] Non-patent literature 2: Gorgoulis, V., et al., Cellular Senescence: Defining a Path Forward. Cell, 2019. 179(4): p. 813-827;

[0009] Non-patent document 3: Meinhard Wlaschek et al., Connective Tissue and Fibroblast Senescence in Skin Aging. J Invest Dermatol. 141(4S):985-992;

[0010] Non-patent document 4: Kowalska, MA, L. Rauova, and M. Poncz, Role of the platelet chemokine platelet factor 4 (PF4) in hemostasis and thrombosis. Thromb Res, 2010.125(4):p.292-6;

[0011] Non-patent literature 5: Schroer, AB, et al., Platelet factors attenuate inflammation and rescue cognition in aging. Nature, 2023. 620(7976): p. 1071-1079;

[0012] Non-patent document 6: Park, C., et al., Platelet factors are induced by longevity factor klotho and enhance cognition in young and aging mice. Nat Aging, 2023.3(9):p.1067-1078;

[0013] Non-patent document 7: Leiter, O., et al., Platelet-derived exerkine CXCL4 / platelet factor 4 rejuvenates hippocampal neurogenesis and restores cognitive function in aged mice. Nat Commun. 2023.14(1):p.4375.

[0014] Summary of the Invention

[0015] Through in-depth research, the inventors of this application discovered for the first time that platelet factor 4 can directly act on skin fibroblasts, effectively preventing and delaying the fibroblasts from entering a senescent state, and even reversing senescent fibroblasts to a youthful state, thereby preventing and delaying skin aging or restoring the skin to a youthful state.

[0016] Therefore, one object of the present invention is to provide the use of platelet factor 4 for preparing a composition for preventing, reducing or improving aging-related changes, damage or signs in the skin.

[0017] Another object of the present invention is to provide a method for preventing, reducing or improving aging-related changes, damage or signs of skin, comprising administering to a subject a composition comprising an effective amount of platelet factor 4.

[0018] Another object of the present invention is to provide use of platelet factor as the sole active ingredient in preparing a composition for repairing skin damage.

[0019] Another object of the present invention is to provide a composition comprising an effective amount of a platelet factor as the sole active ingredient for use in the uses and methods disclosed herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] FIG1 is a bar graph showing that platelet factor 4 enhances skin collagen release in Example 1: FIG1A shows a standard curve of collagen, and FIG1B shows the differences in collagen secretion by cells under different culture conditions;

[0021] Figure 2 shows the results of β-galactosidase staining of skin fibroblasts in Example 2. Figure 2A is an example of a staining photograph taken using an optical microscope; the scale bar is 20 μm. Figure 2B is a bar graph showing the results of statistical analysis of the grayscale values ​​of the staining in the control group (NC) and the experimental group (PF4) using ImageJ.

[0022] Figure 3 shows the results of β-galactosidase staining of skin fibroblasts in Example 3. Figure 3A is an example of a staining photograph taken using an optical microscope; the scale bar is 20 μm. Figure 3B is a bar graph showing the results of statistical analysis of the grayscale values ​​of the staining in the control group (NC) and the experimental group (PF4) using ImageJ.

[0023] FIG4 shows the age of skin fibroblasts assessed based on DNA methylation of aging-related sites in Example 4. DETAILED DESCRIPTION

[0024] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only intended to illustrate the present invention and are not intended to limit the scope of protection of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and the various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0025] I. Definition

[0026] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art. It should be understood that the terms used in the examples herein are intended to describe specific embodiments and are not intended to limit the scope of the invention. Throughout the specification and claims, the singular forms "a," "an," and "the" include the plural forms unless otherwise expressly indicated. When numerical ranges are given in the examples, it should be understood that both endpoints of each numerical range, as well as any value between the endpoints, may be used unless otherwise indicated.

[0027] Unless expressly provided or obvious from the context, otherwise as used herein, the term "about" should be understood to be within the normal tolerance range in the field, for example, within 2 standard deviations of the mean. "About" can be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02% or 0.01% of the value. Unless otherwise obvious from the context, all numerical values ​​provided herein are modified by the term "about".

[0028] As used herein, the term "senescence" refers to the process by which cells gradually decline in their functional activities, such as proliferation and differentiation, and physiological functions, over time. Senescent cells undergo morphological and metabolic changes, chromatin remodeling, changes in gene expression, cell cycle arrest, and secretion of the senescence-associated secretory phenotype (SASP). During the aging process, the skin develops a series of aging characteristics that many consider undesirable. These aging characteristics manifest as various changes, damage, or signs in the skin's structure and / or appearance, including but not limited to the appearance of fine lines and deep wrinkles, increased skin fragility, atrophy, decreased thickness and / or tautness, sagging, decreased firmness, fullness, flexibility, and / or softness, deterioration of skin texture, reduction or loss of barrier function, changes in contour appearance, changes in gloss and / or brightness, and decreased moisture. Each of these characteristics increases with age, and therefore "associated with aging" has the same meaning as "age-related" in this context. Herein, the aging characteristics of the skin are primarily caused by a decrease in the functional activity of dermal fibroblasts.

[0029] As used herein, the term "prevent" and synonymous variants thereof refer to delaying the appearance and occurrence of skin aging characteristics to any extent. As used herein, the term "reduce" and synonymous variants thereof refer to reducing the frequency of appearance of skin aging characteristics, or reducing the severity. As used herein, the term "improve" and synonymous variants thereof refer to reducing the number and / or severity of existing skin aging characteristics. Non-limiting examples of preventing, reducing or improving skin aging characteristics include, for example, maintaining and restoring skin elasticity; maintaining and restoring skin youthfulness; improving procollagen and / or collagen production; regenerating collagen; proliferating collagen; delaying fibroblast aging; repairing fibroblast damage; preventing, alleviating or improving dermatological signs of chronological aging, photoaging, hormonal aging and / or actinic aging; preventing, alleviating or improving skin cell damage caused by ultraviolet radiation; preventing, alleviating or improving the appearance of fine lines and / or wrinkles; reducing or improving skin fragility; preventing, alleviating or improving glycosaminoglycans and / or collagen. Loss of primordial protein; prevention, alleviation or improvement of skin atrophy; prevention, alleviation or improvement of decreased skin thickness and / or tightness; prevention, alleviation and / or improvement of skin sagging; improvement of skin firmness, fullness, flexibility and / or softness; improvement of skin texture and / or promotion of restoration of skin texture; improvement of skin barrier function; improvement of the appearance of skin contour; restoration of skin radiance and / or brightness; replenishment of components in the skin reduced due to aging and / or menopause; increase skin cell proliferation and / or reproduction; increase skin cell metabolism reduced due to aging and / or menopause; improvement of skin moisture; strengthening of skin thickness; or any combination thereof. In some embodiments, preventing, alleviating or improving the appearance of fine lines and / or wrinkles can be selected from reducing the noticeability of facial lines and wrinkles, facial wrinkles on the cheeks, forehead, vertical wrinkles between the eyes, horizontal wrinkles above the eyes and around the mouth, marionette lines, and particularly deep wrinkles or folds; preventing, alleviating and / or reducing the appearance and / or depth of fine lines and / or wrinkles; improving the appearance of infraorbital and / or periorbital lines; reducing the appearance of crow's feet.

[0030] In some embodiments, the present invention also provides the use of platelet factor 4 as the sole active ingredient in the preparation of a composition for repairing skin damage. In some embodiments, the skin damage is selected from acute or chronic wounds or scars, traumatic wounds caused by surgery, burns, radiation or accidents, and a tendency to spontaneous or induced contusions on the limbs.

[0031] As used herein, the term "platelet factor 4" broadly refers to natural PF4, its analogs, or active fragments thereof from any source (e.g., humans or other non-human primates). The term encompasses variants in which one or more amino acid residues in natural PF are replaced or deleted, or one or more amino acid residues are added to the sequence of natural PF4, and the variants do not significantly change the biological activity of the resulting product compared to natural PF4. Variants of PF4 can be naturally occurring variants, such as splice variants or allelic variants, or can be prepared using artificial synthesis and / or site-directed mutagenesis technology or other known techniques suitable for this purpose. In some embodiments, the platelet factor 4 disclosed herein is human platelet factor 4. In a preferred embodiment, the platelet factor 4 disclosed herein has an amino acid sequence as shown in SEQ ID No: 1 (EAEEDGDLQCLCVKTTSQVRPRHITSLEVIKAGPHCPTAQLIATLKNGRKICLDLQAPLYKKIIKKLLES):

[0032] As used herein, conservative amino acid substitutions are well known to those of ordinary skill in the art. The following six groups are examples of amino acids that are considered to be conservative substitutions for each other:

[0033] 1) Alanine (A), serine (S), threonine (T);

[0034] 2) Aspartic acid (D), glutamic acid (E);

[0035] 3) Asparagine (N), glutamine (Q);

[0036] 4) Arginine (R), Lysine (K);

[0037] 5) Isoleucine (I), Leucine (L), Methionine (M), Valine (V); and

[0038] 6) Phenylalanine (F), tyrosine (Y), tryptophan (W).

[0039] In certain embodiments, the platelet factor 4 disclosed herein may have an amino acid sequence that is more than 75% identical to the amino acid sequence shown in SEQ ID NO: 1, and has substantially the same biological activity as SEQ ID NO: 1, for example, it may have 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity.

[0040] Platelet factor 4 exists in the composition in various states. It can be a separate component, or it can form a complex with other proteins or other substances to more stabilize its activity, such as a fusion protein. It can also exist in a state separated from other components and mixed before use. When it is in a state separated from other components, it can be a dry powder or an aqueous solution. In some embodiments, the content of platelet factor 4 in the composition disclosed herein is 2 ng / ml or more. For example, the content of platelet factor 4 can be 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40 , 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93 3, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134 4, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 250, 300, 350, 400, 450 ,500,550,600,650,700,750,800,850,900,1000,1100,1200,1300,1400,1 500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, 5000ng / ml.In a preferred embodiment, the content of platelet factor 4 in the composition disclosed herein is 5 to 5000 ng / ml. In a more preferred embodiment, the content of platelet factor 4 in the composition disclosed herein is 10 to 200 ng / ml.

[0041] In some embodiments of the present invention, the compositions disclosed herein further comprise a pharmaceutically, cosmetically, dermatologically, or physiologically acceptable excipient. In a preferred embodiment, the excipient is a polymer.

[0042] In some embodiments of the present invention, the compositions disclosed herein are in the form of a gel, tincture, cream, ointment, lotion, or aerosol spray.

[0043] In some embodiments of the present invention, the composition disclosed herein is selected from skin care products, cosmetics, medicines, or additives to skin care products, cosmetics, and medicines. In a preferred embodiment, the skin care products are selected from facial cleansers, toners, plain creams, facial masks, lotions, facial creams, eye creams, essences, isolation creams, isolation sprays, and sunscreens. The skin care products disclosed herein are prepared by mixing appropriate amounts of activated platelet factor 4, moisturizers, tonics, thickeners, pearlescent agents, spices, and water. In a preferred embodiment, the cosmetics are selected from concealer pens, foundation creams, BB creams, pressed powders, loose powders, eye shadow palettes, blushes, highlighters, and lip glosses. The cosmetics disclosed herein are prepared by mixing and processing various raw materials (e.g., oily raw materials, powdery raw materials, colloid raw materials, surfactants, solvent raw materials, flavors and fragrances, dyes, pigments, etc.), and adding appropriate amounts of activated platelet factor 4 thereto. In a preferred embodiment, the medicines disclosed herein are selected from sprays, creams, creams, or gels. The drug can be applied by rubbing, spraying, wet compressing, coating, washing, or fumigation.

[0044] As used herein, "administering" includes administering to a localized skin area of ​​a subject. In preferred embodiments, the localized skin area to which the application is applied is a skin area prone to developing wrinkles or an aging skin area. In more preferred embodiments, the localized skin area to which the application is applied is the skin on the face, neck, hands, trunk, and limbs of the subject, or any combination thereof. In certain embodiments, the aging skin area to which the compositions disclosed herein are applied experiences a condition selected from the group consisting of fine or coarse wrinkles, decreased skin thickness, and decreased elasticity and firmness.

[0045] As used herein, "subject" refers to humans and non-human animals, as long as their skin structure contains fibroblasts. In some embodiments, the subject is selected from humans, horses, dogs and cats. In preferred embodiments, the subject is human.

[0046] As described above, the uses, methods, and compositions (e.g., agents, skin care products, cosmetics, medicines, etc.) disclosed herein have the following beneficial effects:

[0047] Based on the fact that platelet factor 4 can reduce inflammation and improve cognitive function in aging mice, this application first discovered that the use of exogenous platelet factor 4 protein can delay or even reduce the aging of skin fibroblasts, thereby promoting the release of collagen by skin fibroblasts, alleviating skin aging, enhancing skin repair function, and maintaining and restoring skin elasticity and youthfulness. This application's discovery of the new function of platelet factor 4 fills a gap in the field of skin anti-aging and has great application prospects in the medical and beauty industries.

[0048] Test purpose and materials

[0049] Unless otherwise specified, the experimental methods, detection methods, and preparation methods disclosed herein all employ conventional techniques in the art and related fields. In addition to the specific methods, equipment, and materials used in the examples, any prior art methods, equipment, and materials similar or equivalent to those described in the examples may be used to implement the present invention, based on the knowledge of the prior art by those skilled in the art and the disclosure of this invention.

[0050] Platelet factor 4 can be obtained commercially, for example, from Peprotech, Cat. No. 300-16, or from Sigma-Aldrich, Cat. No. SRP3142.

[0051] Skin fibroblasts #1 and #2 were used in this application with the approval of the Ethics Committee of Huashan Hospital Affiliated to Fudan University. Skin fibroblast #1 was derived from the skin of a 67-year-old female, and skin fibroblast #2 was derived from the skin of a 40-year-old male.

[0052] The culture medium for skin fibroblasts is: DMEM + 10% FBS + 1% GlutaMax + 10ng / ml bFGF, where the components are:

[0053] DMEM: Gbico, Cat.No.11965-092,

[0054] GlutaMax(100X): Gbico, Cat.No.35050-061,

[0055] FBS: Sigma, Cat.No..F8318-500ML,

[0056] b-FGF: Peprotech, Cat.No.100-18B,

[0057] Example 1:

[0058] This example describes the effect of platelet factor 4 on the ability of skin fibroblasts to produce collagen.

[0059] A collagen standard curve was measured using a Procollagen Type I C-Peptide (PIP) EIA kit (purchased from TAKARA, Cat. No. MK101) according to the manufacturer's instructions. A 640 ng / ml solution of Procollagen Type I C-Peptide (PIP) was prepared and serially diluted 2-fold to obtain solutions of 320, 160, 80, 40, 20, and 10 ng / ml, respectively. Ultrapure ddH2O without PIP was used as a blank control. The serially diluted solutions were incubated with anti-PIP antibody at 37°C for 3 hours. The cells were then washed with wash buffer (DPBS, SH30028.02). The substrate, TMBZ (3,3',5,5'-tetramethylbenzidine), was added and allowed to react at room temperature for 15 minutes. The reaction was then terminated by adding stop solution. Absorbance at OD450 nm was measured using a microplate reader (Thermo Scientific, Model 1530). Prism software was used to determine the best-fit curve, with absorbance as the vertical axis and PIP concentration (ng / ml) as the horizontal axis. The results are shown in Figure 1A.

[0060] In a 24-well plate, 0.8 × 10 5 Skin fibroblasts #1 were seeded at 100 cells / well and cultured in the aforementioned skin fibroblast culture medium, with 1 ml per well. Immediately after seeding, platelet factor 4 (PF4) was added to the culture medium of the experimental group (n=3) to a final concentration of 100 ng / ml; an equal amount of aqueous solvent was added to the control group (n=3). After 3 days of culture in a 37°C, 5% CO2 incubator, the supernatant was collected and analyzed by ELISA as described above. The concentration of type I procollagen C-peptide in the cell culture medium of each well of the control and experimental groups was determined using a standard curve. The results are shown in Figure 1B.

[0061] Figure 1B shows that compared with the control group without platelet factor 4, the collagen secretion of the experimental group increased by 7.74 times to 2074ng / ml. This indicates that platelet factor 4 can significantly increase the ability of skin fibroblasts to synthesize and secrete collagen.

[0062] Example 2:

[0063] This example describes the effect of platelet factor 4 on the senescent state of skin fibroblasts.

[0064] In a 24-well plate, 0.8 × 10 5 Skin fibroblasts #1 and #2 were seeded at 100 cells / well, and 1 ml of skin fibroblast culture medium was added to each well. Platelet factor 4 (PF4) was added to the culture medium of the experimental group (n=2) to a final concentration of 100 ng / ml; an equal amount of solvent water was added to the control group (n=2). Culture was carried out in an incubator at 37°C and 5% CO2 for 3 days. Then, each well was stained for β-galactosidase using a kit (purchased from Beyotime, product number C0602) according to the manufacturer's instructions. Specifically, the culture medium was aspirated, washed once with PBS or HBSS, and 1 ml of fixative was added, and fixed at room temperature for 15 minutes. Then, the fixative was aspirated, the cells were thoroughly washed with PBS or HBSS, and 1 ml of staining working solution was added to each well. After incubation at 37°C overnight, the staining results were observed and counted under an ordinary light microscope. The final staining results are shown in Figure 2.

[0065] In Figure 2A, blue staining indicates the presence of active β-galactosidase in the cells, which is a hallmark enzyme that increases with the aging process of cells. A large number of blue-stained cells were observed in the two control group cells. In contrast, the number of cells that could be stained blue was significantly reduced in the experimental group cells to which platelet factor 4 was added, and the blue color also became significantly lighter. In Figure 2B, the grayscale values ​​of the staining of random fields in the control and experimental groups were statistically analyzed using ImageJ software, and it was found that the reduction in β-galactosidase staining presented by the two cell lines was statistically significant. This shows that platelet factor 4 can significantly inhibit or delay the entry of skin fibroblasts into the senescent state and reduce the degree of fibroblast aging.

[0066] Example 3:

[0067] This example describes the effect of platelet factor 4 on skin fibroblasts that have already undergone aging damage. This example uses an ultraviolet irradiation model (see, for example, Florence Debacq-Chainiaux et al., UV, stress and aging. Dermato-Endocrinology 4: 3, 236-240; Alessio De Magis et al., UV-induced G4 DNA structures recruit ZRF1 which prevents UV-induced senescence. Nat Commun. 2023 Oct 23; 14(1): 6705; M Helenius et al., Attenuation of NF-kappaB signaling response to UVB light during cellular senescence. Exp Cell Res. 1999 Apr 10; 248(1): 194-202) as an example of aging skin fibroblasts. It is known to those skilled in the art that the results will also be applicable to other aging damage models.

[0068] In a 24-well plate, 0.1 × 10 5 Skin fibroblasts #1 and #2 were seeded at 100 cells / well, and 1 ml of skin fibroblast culture medium was added to each well. The cells were irradiated simultaneously at 22°C using a 30W UV lamp for 15 minutes. After irradiation, platelet factor 4 (PF4) was added to the culture medium of the experimental group (n=2) to a final concentration of 200 ng / ml; an equal amount of solvent water was added to the control group (n=2). The cells were cultured in a 37°C, 5% CO2 incubator for one day. The senescence status of the skin fibroblasts was then assessed using the same β-galactosidase staining method as in Example 2. The results are shown in Figure 3.

[0069] In Figure 3A, compared to the control group, skin fibroblasts cultured with platelet-derived factor 4 showed almost no blue staining. In Figure 3B, grayscale values ​​of random fields of view were calculated using ImageJ software, demonstrating statistically significant reductions in β-galactosidase staining in both cell lines. This suggests that platelet-derived factor 4 can repair aging damage to skin fibroblasts caused by external or internal pro-aging factors.

[0070] Example 4:

[0071] This example describes the effects of platelet factor 4 on epigenetics and cell age in skin fibroblasts.

[0072] In a 24-well plate, 0.1 × 10 5 Skin fibroblasts #1 and #2 were seeded in 100 cells / well, and 1 ml of skin fibroblast culture medium was added to each well. Platelet factor 4 (PF4) was added to the culture medium of the experimental group (n=2) to a final concentration of 200 ng / ml; an equal amount of solvent water was added to the control group (n=2). Culture was carried out in an incubator at 37°C and 5% CO2 for 3 days. Then, the culture medium was discarded and the fibroblasts were collected after trypsin digestion. Total DNA was extracted using a kit (QIAGEN, catalog number: 69504) according to the manufacturer's instructions. Then, the age of the cultured cells was assessed using a known method (see, for example, non-patent document 8: Atef Zaguia, Deepak Pandey et al., DNA Methylation Biomarkers-Based Human Age Prediction Using Machine Learning. Computational Intelligence and Neuroscience, Vol. 2022, Article ID 8393498). Specifically, the extracted genomic DNA was converted with bisulfite, and the DNA methylation status of 77 age-related sites (including cg22736354, cg19283806, cg18473521, cg02228185, cg06493994, cg19761273, cg01820374 and cg09809672, etc.) was measured. The cell age was then determined based on the methylation status of these sites using the machine learning and AI algorithms described in non-patent literature 8. The results are shown in Figure 4.

[0073] In Figure 4, compared with the skin fibroblasts used as a control, the age of cells cultured under the conditions of adding platelet factor 4 was significantly reduced, indicating that platelet factor 4 can effectively reverse the aging of skin fibroblasts and restore them to a younger state.

[0074] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form or substance. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the method of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention. Any equivalent changes, modifications and evolutions made by technicians familiar with this profession without departing from the spirit and scope of the present invention by using the technical content disclosed above are all equivalent embodiments of the present invention; at the same time, any equivalent changes, modifications and evolutions made to the above embodiments based on the essential technology of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. Use of platelet factor 4 in the preparation of a composition for preventing, reducing or improving changes, damages or signs associated with aging in the skin.

2. The use according to claim 1, wherein The aging-related changes, damages or signs of the skin are caused by a decrease in the functional activity of skin fibroblasts.

3. The use according to any one of the preceding claims, wherein The prevention, reduction or improvement of aging-related changes, damage or signs of skin is selected from the group consisting of: Maintain and restore skin elasticity; Maintain and restore skin youthfulness; Improve the production of procollagen and / or collagen; Regenerate collagen; Proliferate collagen; Delay fibroblast aging; Repair fibroblast damage; Prevent, reduce or improve dermatological signs of chronological, photoaging, hormonal and / or actinic aging; Prevent, reduce or improve skin cell damage caused by ultraviolet radiation; Prevent, reduce or improve the appearance of fine lines and / or wrinkles; Reduce or improve skin fragility; prevent, reduce or improve the loss of glycosaminoglycans and / or collagen; prevent, reduce or improve skin atrophy; prevent, reduce or improve the decrease of skin thickness and / or tightness; Prevent, reduce and / or improve skin sagging; Improve skin firmness, fullness, flexibility and / or softness; improve skin texture and / or promote skin texture restoration; Improve skin barrier function; improve the appearance of skin contours; restore skin radiance and / or brightness; replenish components in the skin that are reduced due to aging and / or menopause; increase skin cell proliferation and / or reproduction; increase skin cell metabolism that is reduced due to aging and / or menopause; improve skin moisture; strengthen skin thickness; or any combination thereof.

4. The method of claim 3, wherein the prevention, alleviation or improvement of the appearance of fine lines and / or wrinkles is selected from the group consisting of reducing the noticeability of facial fine lines and wrinkles, facial wrinkles on the cheeks, forehead, vertical wrinkles between the eyes, horizontal wrinkles above the eyes and around the mouth, marionette lines and in particular deep wrinkles or folds; preventing, alleviating and / or reducing the appearance and / or depth of fine lines and / or wrinkles; improving the appearance of suborbital and / or periorbital fine lines; reducing the appearance of crow's feet.

5. The use according to any one of the preceding claims, wherein The composition further comprises a pharmaceutically, cosmetically, dermatologically, or physiologically acceptable excipient, preferably a polymer.

6. The use according to any one of the preceding claims, wherein The composition is in the form of a gel, tincture, cream, ointment, lotion or aerosol spray.

7. The use according to any of the preceding claims, wherein the composition is selected from skin care products, cosmetics, medicines, or additives to skin care products, cosmetics or medicines; Preferably, the skin care product is selected from facial cleanser, toner, natural cream, facial mask, lotion, facial cream, eye cream, essence, isolation cream, isolation spray or sunscreen; Preferably, the cosmetics are selected from concealer pencils, foundation creams, BB creams, pressed powders, loose powders, eye shadow palettes, blush, highlighter powders or lip glosses; Preferably, the drug is selected from a spray, an ointment, a cream or a gel.

8. The use according to any one of the preceding claims, wherein The effective amount of platelet factor 4 contained in the composition is 2 ng / ml or more, preferably 5 to 5000 ng / ml, and more preferably 10 to 200 ng / ml.

9. The use according to any one of the preceding claims, wherein The administration includes administration to a localized skin area of ​​the subject, preferably a skin area prone to developing wrinkles or aging, more preferably the skin on the face, neck, hands, trunk and limbs of the subject or any combination thereof.

10. Use according to any of the preceding claims, wherein the aged skin area suffers from a condition selected from the group consisting of fine or coarse wrinkles, reduced skin thickness, reduced elasticity and firmness.

11. The use according to any one of the preceding claims, wherein The platelet factor 4 is human platelet factor 4, preferably a platelet factor 4 having an amino acid sequence as shown in SEQ ID NO:

1.

12. The use according to any one of the preceding claims, wherein The subject is a mammal selected from the group consisting of humans, horses, dogs and cats.

13. A method of preventing, reducing or ameliorating aging-related changes, damage or signs of skin comprising administering to a subject a composition comprising an effective amount of platelet factor 4.

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