CN113144192A

CN113144192A - Application of MAPK/ERK pathway inhibitors in antagonizing skin aging and radiation-induced premature skin aging

Info

Publication number: CN113144192A
Application number: CN202010813757.8A
Authority: CN
Inventors: 张亮; 于瑶
Original assignee: Shanghai Institute of Nutrition and Health of CAS
Current assignee: Shanghai Institute of Nutrition and Health of CAS
Priority date: 2020-01-22
Filing date: 2020-08-13
Publication date: 2021-07-23
Also published as: WO2021147740A1; CN113853218B; CN113853218A

Abstract

The invention relates to application of MAPK/ERK pathway inhibitor in antagonizing skin aging and radiation-induced skin premature aging. The invention provides the use of a MAPK/ERK pathway inhibitor in the preparation of a composition or formulation for the prevention or treatment of skin aging (or anti-skin aging). The invention also provides methods of preventing, antagonizing, or delaying skin aging/premature aging by inhibiting the MAPK/ERK pathway, as well as compositions and formulations for resisting skin aging.

Description

Application of MAPK/ERK pathway inhibitor in antagonizing skin aging and radiation-induced skin premature aging

Technical Field

The invention relates to the field of medicine, in particular to application of MAPK/ERK pathway inhibitors in antagonizing skin aging and radiation-induced skin premature aging.

Background

Aging of skin epithelial tissues (including epidermis, hair follicles, sebaceous glands, sweat glands, etc.) is the most prominent feature of human aging. The main manifestations of skin aging include wrinkle formation, reduced callus/regenerative capacity, hair whitening/thinning, hair follicle atrophy, etc. Epithelial stem cells in the skin (mainly including hair follicle stem cells and epidermal stem cells) are the basis for homeostatic maintenance of skin epithelial tissue and wound repair.

Aging of skin epithelial tissue, a tissue that is constantly self-renewing throughout life, is closely associated with the failure of its stem cells, but the specific molecular mechanisms that lead to such stem cell failure are not fully understood.

DNA damage is widely recognized as a major factor in the aging/premature aging of multiple tissues. In the presenile patients with genome stability related gene mutation and the corresponding mouse genetic models, typical skin aging phenotypes such as hair loss and albinism appear early.

In normal human skin tissue, exogenous radioactive stimuli are a major source of DNA damage. Among them, Ultraviolet (UV) rays in sunlight are well known causes of DNA damage, and can accelerate skin aging and cause photoaging of the skin. In addition, Ionizing Radiation (IR) commonly used in tumor radiotherapy can also induce a great amount of DNA damage, leading to premature aging of skin cells in the radiotherapy area, and further leading to long-term skin healing dysfunction, which is one of the most common complications in the tumor radiotherapy process, and no effective treatment means is available at present.

In recent years, studies have further demonstrated that DNA damage can directly induce the failure of skin epidermis and hair follicle stem cells. Therefore, the method for inhibiting the skin epithelial stem cell failure caused by DNA damage has wide application prospect in skin anti-aging beauty and prevention and treatment of radioactive skin damage. However, there is currently no satisfactory method of skin anti-aging, nor is there any specific treatment that enters the clinical stage.

Therefore, there is an urgent need in the art to develop substances effective in preventing, antagonizing, or delaying skin aging.

Disclosure of Invention

It is an object of the present invention to provide a substance and method which is effective in preventing, antagonizing or delaying skin aging.

In a first aspect of the invention, there is provided the use of an inhibitor of the MAPK/ERK pathway for the preparation of a composition or formulation for the prevention or treatment of skin ageing (or anti-skin ageing).

In another preferred embodiment, the composition comprises a pharmaceutical composition and a daily chemical composition.

In another preferred embodiment, the daily chemical composition comprises shampoo, shower gel, skin care product, hair care product and/or cosmetic.

In another preferred embodiment, the composition is administered transdermally, or transdermally.

In another preferred embodiment, the composition is a topical composition.

In another preferred embodiment, the composition or formulation is for one or more (or all) applications selected from the group consisting of:

(a) reducing the rate of hair whitening or reducing the production of whitened hair;

(b) improving the skin healing ability or promoting wound healing;

(c) promoting migration of keratinocytes;

(d) promoting epithelial stem cell function and/or slowing the reduction of epithelial stem cells; and

(e) improving hair regeneration ability.

In another preferred embodiment, said skin aging has one or more of the following indices selected from the group consisting of:

(l) Loss of skin epithelial stem cells;

(m) hair whitening;

(n) reduced hair regrowth ability; and

(o) decreased epithelial callus function.

In another preferred embodiment, the epithelial stem cell is selected from the group consisting of: hair follicle stem cells, epidermal stem cells, stem cells of skin epithelial tissue, or a combination thereof.

In another preferred embodiment, the skin aging further comprises: an increase in one or more representative senescent cell markers or markers, and/or a decrease in one or more representative young cell markers or markers.

In another preferred embodiment, the representative markers or markers of aging cells include (but are not limited to): SA- β -galactosidase, P16, cell proliferation capacity.

In another preferred embodiment, the representative markers or markers of young cells include (but are not limited to): H3K9me3, collagen gene COL17A 1.

In another preferred embodiment, the skin aging comprises: DNA damage-induced skin aging, radiation-induced premature skin aging, natural skin aging, and/or skin photoaging.

In another preferred embodiment, the skin aging is induced by an inducing factor selected from the group consisting of: ionizing radiation, gamma irradiation, ultraviolet light, radiation therapy, excessive exposure to light, chemical mutagens, natural skin aging caused by natural proliferation of skin cells, or combinations thereof.

In another preferred embodiment, said skin aging is due to age.

In another preferred embodiment, the MAPK/ERK pathway inhibitor inhibits the phosphorylation of MEK 1/2.

In another preferred embodiment, the MAPK/ERK pathway inhibitor inhibits phosphorylation of ERK 1/2.

In another preferred embodiment, the MAPK/ERK pathway inhibitor is a specific inhibitor of phosphorylation of ERK 1/2.

In another preferred embodiment, the MAPK/ERK pathway inhibitor is selected from the group consisting of: trametinib (Trametinib), U0126, MEK162, SCH772984, lixertinib, AZD0364, KO-947, HH2710, PD0325901, PD184352, Rifamtinib (Refametinib), Carbotinib, AS-701255, AS-701173, pimasetib (Pimasertib), RDEA436, RO4987655, RG7420, or a combination thereof.

In another preferred embodiment, the MAPK/ERK pathway inhibitor is selected from the group consisting of: u0126, Trametinib (Trametinib), MEK162, SCH772984, or a combination thereof.

In another preferred embodiment, the MAPK/ERK pathway inhibitor is trametinib, SCH772984, or a pharmaceutically acceptable salt thereof.

In another preferred embodiment, the administration of the MAPK/ERK pathway inhibitor comprises: oral or non-oral administration.

In another preferred embodiment, the non-oral administration includes (but is not limited to) painting.

In a second aspect of the present invention, there is provided a composition or formulation useful for preventing or treating skin aging, the composition or formulation comprising: (a) a physiologically acceptable carrier; and (b) an effective amount of an active ingredient which is a MAPK/ERK pathway inhibitor.

In another preferred embodiment, the MAPK/ERK pathway inhibitor is selected from the group consisting of: a small molecule compound, an antibody, a nucleic acid, or a combination thereof.

In another preferred embodiment, the MAPK/ERK pathway inhibitor is trametinib, or a pharmaceutically acceptable salt thereof.

In another preferred embodiment, the composition or formulation further comprises (c) other anti-aging active ingredients.

In another preferred embodiment, the other anti-aging active ingredients are selected from the group consisting of: MicroRNA31/miR-31 inhibitor (see CN 2019111758754).

In another preferred embodiment, the active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants which may be required if desired.

In another preferred embodiment, the composition or formulation further comprises a skin penetration enhancer.

In another preferred embodiment, the physiologically acceptable carrier is selected from the group consisting of: water, saline, liposomes, lipids, proteins, protein-antibody conjugates, peptides, nanogels, cellulose and its derivatives, gelatin, talc, solid lubricants, calcium sulfate, vegetable oils, polyols, emulsifiers, wetting agents, colorants, flavors, stabilizers, antioxidants, preservatives, pyrogen-free water, or combinations thereof.

In another preferred embodiment, the composition or formulation is in a dosage form for external use (topical).

In another preferred embodiment, the dosage form of the composition or the preparation comprises ointment, cream, patch, liniment, spray, micro-injection and the like.

In another preferred embodiment, the composition or formulation is administered in a manner selected from the group consisting of: topical, transdermal and transdermal administration.

In another preferred embodiment, the composition contains 0.05-10mg/ml (or mg/g), preferably 0.1-5mg/ml (or mg/g), more preferably 0.2-2mg/ml (or mg/g) of Trametinib.

In another preferred embodiment, the composition or formulation is a liquid formulation prepared by dissolving Trametinib in a solvent.

In another preferred embodiment, the solvent is selected from the group consisting of: water, C1-C6 alcohol, DMSO, or a combination thereof.

In another preferred embodiment, the C1-C6 alcohol is selected from the group consisting of: ethanol, propylene glycol, propanol, butanol, glycerol, or combinations thereof.

In another preferred embodiment, the solvent comprises 50 parts by weight of ethanol, 30 parts by weight of water, 20 parts by weight of propylene glycol and 0-5 parts by weight (preferably 2-4 parts by weight) of DMSO.

In a third aspect of the present invention, there is provided a method for in vitro screening of potential substances for anti-skin aging comprising the steps of:

(1) providing a test group and a control group, wherein a test substance is added to a test system in the test group, and a control substance is added to a test system in the control group, and the control substance comprises a negative control substance;

(2) detecting the levels of MAPK/ERK pathways in the test group and the control group respectively; if the level of MAPK/ERK pathway is significantly reduced in the test group compared with the negative group, it indicates that the test substance is a potential substance for resisting skin aging.

In another preferred embodiment, the experimental conditions of the test group and the control group are the same or substantially the same, except that the added test substance and the added control substance are different.

In another preferred embodiment, the level of the MAPK/ERK pathway is the phosphorylation level of MEK1/2 and/or the phosphorylation level of ERK 1/2.

In another preferred embodiment, the level of the MAPK/ERK pathway is the level of MAPK/ERK kinase activity, expression (including protein level or mRNA level).

In another preferred example, the step (1) further comprises, after or before, the steps of:

(1a) the test systems of the test group and the control group were exposed to a DNA damage inducing environment, respectively, to induce cell senescence or induce DNA damage.

In another preferred embodiment, in step (1a), the treatment is performed by a method selected from the group consisting of: irradiation treatment, chemical mutagens, high temperature, intense shaking, induction of pathological stress, or combinations thereof.

In another preferred embodiment, the irradiation treatment is selected from the group consisting of: ionizing radiation, gamma irradiation, UVA/UVB irradiation, radiation therapy, excessive exposure to sunlight, or combinations thereof.

In another preferred embodiment, the detection system is a cell system.

In another preferred embodiment, the cells comprise skin cells, preferably epithelial cells, more preferably epithelial stem cells.

In another preferred example, the method further comprises the steps of:

(3) respectively detecting the DNA damage tolerance capability of the epithelial stem cells in the test group and the control group; if the epithelial stem cells of the test group have a significantly improved ability to tolerate DNA damage compared to the control group, it is suggested that the test substance is a potential substance for anti-skin aging.

In another preferred example, in step (3), the DNA damage amplitude D1 in the test group and the DNA damage amplitude D0 in the control group are compared, and if the ratio of (D0-D1)/D0 is greater than or equal to 5%, preferably greater than or equal to 10%, preferably greater than or equal to 20%, preferably greater than or equal to 50%, more preferably greater than or equal to 80%, and most preferably greater than or equal to 100%, the test substance is suggested to be a potential substance for resisting skin aging.

In a fourth aspect of the present invention, there is provided a method for preventing or treating skin aging, comprising the steps of: administering to a subject in need thereof a MAPK/ERK pathway inhibitor, or a composition or formulation comprising a MAPK/ERK pathway inhibitor.

In another preferred embodiment, the composition or formulation comprises: inhibitors of the MAPK/ERK pathway as active ingredients.

In another preferred embodiment, the skin aging comprises skin aging caused by DNA damage and radiation-induced skin premature aging.

In another preferred embodiment, the skin aging is induced by an inducing factor selected from the group consisting of: ionizing radiation, gamma irradiation, ultraviolet light, radiation therapy, overexposure, chemical mutagens, or combinations thereof.

In another preferred embodiment, said administration is before, during or after induction by said induction factor.

In another preferred embodiment, the application is to skin without wound, or in the absence of skin wound (including topical, oral, or injection (e.g., intravenous, intramuscular) application).

In a fifth aspect of the present invention, there is provided a method of diagnosing/detecting skin aging, comprising the steps of:

(a) providing a sample to be detected, wherein the sample to be detected is skin cells or skin tissues; and

(b) and detecting L1 of the MAPK/ERK pathway level in the sample to be detected, and comparing with a reference value L0, if the MAPK/ERK pathway level of the sample to be detected is significantly higher than the reference value, indicating that the skin aging process of the sample to be detected is fast and/or the skin aging degree is high, wherein the reference value L0 is the MAPK/ERK pathway level in normally aging skin cells.

In another preferred embodiment, the sample to be tested is skin epithelial tissue or skin epithelial cells.

In another preferred embodiment, the expression "significantly higher" means that: the increase in the expression level of the corresponding miRNA-31 (i.e., the ratio of (L1-L0)/L0) is greater than or equal to 10%, preferably greater than or equal to 20%, preferably greater than or equal to 50%, preferably greater than or equal to 100%, more preferably greater than or equal to 150%, and most preferably greater than or equal to 200% compared to the negative control sample.

In another preferred embodiment, the level of the MAPK/ERK pathway is the phosphorylation level of ERK 1/2.

In a sixth aspect of the invention, there is provided the use of a level of the MAPK/ERK pathway or a test agent thereof in the manufacture of a test agent or test kit for assessing skin aging.

In another preferred embodiment, the skin is human skin.

In another preferred embodiment, the kit further comprises an instruction manual, and the instruction manual describes the method in the fifth aspect of the present invention.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

Fig. 1 shows the establishment of a model of local skin premature aging induced by local ionizing radiation. Which comprises the following steps:

1A, an experimental flow chart of a mouse skin local premature senility model (local skin premature senility mouse) induced by local ionizing radiation. Yellow is the irradiated area. nord: a non-irradiated region; rad: the irradiation zone is the same as below.

One round of phenotypic analysis of topical skin progeria mice-photographs representative of dorsal skin hair. Scale bar 1 cm.

One round of phenotypic analysis of local skin progeria mice-statistical plot of dorsal cortex full-thickness wound healing rate. Horizontal axis: days from wound healing, vertical axis: the rate of healing. **: p <0.01, n ═ 3, two-tailed t-test.

One round of phenotypic analysis of local precocious skin aging mice-keratinocyte migration callus rate experiments on dorsal skin tissue mass (Exo-plant). Left panel: photomicrographs, black skin tissue mass body, white line indicating the leading edge of the migrating keratinocyte layer. Scale bar 500 μm. Right panel: area statistics of the migrating keratinocyte layer: **: p <0.01, n ═ 3, two-tailed t-test.

One round of phenotypic analysis of local cutaneous progeria mice-relative ratio statistics of epithelial hair follicle stem cells in different regions of the dorsal cortex (based on nord region). **: p <0.01, n ═ 3, two-tailed t-test.

Two rounds of phenotypic analysis of topical skin progeria mice-photographs representative of dorsal skin hair. Visible irradiated areas where hair growth is significantly sparse resulting in a pink epidermis portion being bare. Scale bar 1 cm. Error bars (Error Bar) in all the above figures represent Standard errors (Standard Error).

Figure 2 shows that the oral MAPK/ERK pathway inhibitor Trametinib effectively antagonizes the intra-dermal aging phenotype. Which comprises the following steps:

experimental scheme (premature skin aging drug antagonism experiment) for testing the anti-aging effect of drugs (Trametinib or solvent control) in the model of the premature skin aging induced by the local irradiation. nord: a non-irradiated region; rad: and (4) an irradiation area. + S solvent treatment group. + T: trametinib treatment group, the same below.

Two rounds of phenotypic analysis in the experiment of antagonism of drugs for premature skin aging-photographs representative of the dorsal skin. IR: topical skin senilism mice that received topical irradiation. Ctrl: control mice were not irradiated. It can be seen that the irradiated area (Rad) hair of the IR + S group whitens substantially completely, while the pink cuticle is exposed because the hair is significantly thin. The mice in the IR + T group had only a small portion of hair whitened in the irradiated area and no cuticle denudation. Scale bar 1 cm.

Two rounds of phenotypic analysis of IR group mice in the premature skin aging drug antagonism experiment-hair relative density statistics in different areas of the dorsal skin. Based on the nord + S group. **: p <0.01, n ═ 3, two-tailed t-test.

Two rounds of phenotypic analysis of the dorsal cortex irradiation zone (Rad) of IR group mice in the premature skin aging drug antagonism experiment-percentage of hair whitening statistics. **: p <0.01, n ═ 3, two-tailed t-test.

One round of phenotypic analysis of IR group mice in the premature skin aging drug antagonism experiment-statistics of relative proportion of hair follicle stem cells in the epithelium in different areas of the dorsal skin. **: p <0.01, n ═ 3, two-tailed t-test.

One round of phenotypic analysis of IR group mice in the premature skin aging drug antagonism experiment-representative photographs of the healing process of full-thickness callus in different areas of the dorsal cortex. d 0: day 0 post-trauma. d 9: day 9 post-trauma.

Statistical analysis of the healing rate of the callus processes represented by the F plots. Horizontal axis represents days of callus: p <0.01, n ═ 3, x: p <0.05, two-tailed t-test.

Representative photographs of a round of phenotypic analysis of IR group mice in the premature skin aging drug antagonism experiment-ex vivo keratinocyte migration experiment (Exo-plant) of skin tissue mass from different dorsal skin areas. The white dotted line represents the leading edge of the migrating keratinocyte layer, and the black area in the lower left corner is the bulk of the skin tissue mass. Scale bar 500 μm.

Statistical analysis of the relative coverage of the keratinocyte layer in the in vitro keratinocyte migration experiment represented by the H-plot. **: p <0.01, n ═ 3, two-tailed t-test.

Error bars (Error Bar) in all figures represent Standard errors (Standard Error).

Figure 3 shows that the application of the MAPK/ERK pathway inhibitor Trametinib effectively antagonizes the intra-dermal aging phenotype. Which comprises the following steps:

A. schematic experimental procedure for Trametinib or solvent control surface smear intervention in the local irradiation induced mouse model of cutaneous progeria as described above. Mice received one-time topical irradiation at around 7 weeks of age (P50). Drug (Trametinib or control solvent) treatment was started simultaneously. The mode of drug treatment is one back smear once every 3 days for a total of 5 times for 15 days. Five days after irradiation wax depilation was performed to induce hair regrowth. nord: a non-irradiated region; rad: and (4) an irradiation area. + S solvent treatment group. + T: trametinib treatment group, the same below. One round of phenotype was performed before P83 dehairing again. Two rounds of phenotypic analysis were performed at P111.

B. One round of phenotype, in vitro callus capacity analysis of skin tissue blocks. Statistics are the relative size of the keratinocyte layer migration area of the skin tissue mass (exoplan) at day 6 of in vitro culture. Similar to the previously described fig. ii.

C. One round of phenotype, in vivo full-thickness wound healing rate analysis (n-3), was similar to the previous panel, panel two G. Both refer to the difference between Rad + S and Rad + T.

D. One round of phenotype, epithelial hair follicle stem cell ratio analysis. Similar to the previously described fig. two E.

E. Photographs of the dorsal skin of two-round phenotype, + S/+ T-intervened irradiated early-aging model mice (LIR) and normal control mice (Ctrl). Similar to the second panel B described above.

F. Two-round phenotype, white-hair ratio statistics (n-3) for Rad in panel E above, similar to panel two D above.

G. Two-round phenotype, relative hair density statistics (n-3) for Rad/nord regions of LIR mice in panel E above, similar to panel two C above.

**: p <0.01,: p <0.05, two-tailed t-test. All Error bars represent standard errors (standard Error)

Figure 4 shows that the application of the MAPK/ERK pathway inhibitor Sch772984 effectively antagonizes the intra-dermal senescence phenotype. Which comprises the following steps:

A. experimental protocol schematic for Sch772984 or solvent control oral administration in the aforementioned model of local irradiation induced premature skin aging in mice. Mice received one-time topical irradiation at around 7 weeks of age (P50). Drug (either Sch772984 or control solvent) treatment was started simultaneously. The mode of drug treatment was oral once every 3 days for a total of 5 times for 15 days. Five days after irradiation wax depilation was performed to induce hair regrowth. nord: a non-irradiated region; rad: and (4) an irradiation area. + S solvent treatment group. + sch: group treated by Sch772984, the same applies below. One round of phenotype was performed before P83 dehairing again. Two rounds of phenotypic analysis were performed at P111.

C. One round of phenotype, epithelial hair follicle stem cell ratio analysis. Similar to the previously described fig. two E.

D. Photographs of the dorsal skin of two-round phenotype, + S/+ sch-intervened irradiated early-senescence model mice (LIR) and normal control mice (Ctrl). Similar to the second panel B described above.

E. Two-round phenotype, white-hair ratio statistics (n-3) for Rad in panel E above, similar to panel two D above.

F. Two-round phenotype, relative hair density statistics (n-3) for Rad/nord regions of LIR mice in panel E above, similar to panel two C above.

**: p <0.01,: p <0.05, two-tailed t-test. All Error bars represent standard errors (standard errors).

FIG. 5 shows a schematic of the MAPK/ERK signaling pathway.

Detailed Description

The inventor of the invention has conducted extensive and intensive studies, and through a large number of screens, firstly discovers that the activation of MAPK/ERK signal channels is closely related to the depletion of skin hair follicle stem cells under the stimulation of DNA damage and the subsequent skin premature senility phenotype. Experiments show that a series of in vivo skin aging/premature aging phenotypes can be significantly inhibited by treatment with a targeted drug that antagonizes the MAPK/ERK signaling pathway, and the treatment mainly comprises: loss of skin hair follicle stem cells, hair whitening, reduced hair regeneration capability, and reduced epidermal callus function. Thus, the present study has for the first time unexpectedly found that the MAPK/ERK signaling pathway has a key role in promoting skin aging and premature aging, which is distinct from the concept that this signaling pathway has a role in promoting skin cell growth/survival as generally recognized in the art. More importantly, the research of the invention shows that the inhibition of MAPK/ERK pathway activity is an effective means for antagonizing skin aging and radiation-induced skin premature aging. The intervention means developed on the basis can be applied to the prevention and treatment of radiation skin premature senility and the delay of natural skin aging, and has wide application prospect.

Term(s) for

In order that the disclosure may be more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meaning given below, unless explicitly specified otherwise herein. Other definitions are set forth throughout the application.

The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined. For example, as used herein, the expression "about 100" includes 99 and 101 and all values in between (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

As used herein, the term "comprising" or "includes" can be open, semi-closed, and closed. In other words, the term also includes "consisting essentially of …," or "consisting of ….

Sequence identity is determined by comparing two aligned sequences along a predetermined comparison window (which may be 50%, 60%, 70%, 80%, 90%, 95% or 100% of the length of the reference nucleotide sequence or protein) and determining the number of positions at which identical residues occur. Typically, this is expressed as a percentage. The measurement of sequence identity of nucleotide sequences is a method well known to those skilled in the art.

Skin aging and DNA damage

As used herein, the terms "skin aging", "skin aging" are used interchangeably to refer to the aging of the epithelial tissue of the skin. In addition, the term includes normal skin aging, as well as premature or overly rapid skin aging (i.e., premature skin aging).

Aging of skin epithelial tissues (including epithelium, hair follicle, sebaceous gland, sweat gland) is the most prominent feature of human aging. Mainly manifested by wrinkle formation, reduced callus/regenerative capacity, hair whitening/thinning, hair follicle atrophy, and the like. Skin epithelial stem cells (mainly including hair follicle stem cells and epidermal stem cells) are the basis for skin epithelial tissue homeostasis maintenance and wound repair. The epithelial stem cells include hair follicle stem cells, epidermal stem cells, other stem cells belonging to the epithelial tissue of the skin, or a combination thereof. Wherein the epidermal stem cells maintain epithelial tissue homeostasis, while the hair follicle stem cells support hair growth and regeneration and are capable of repairing the epithelium in traumatic conditions. There are also some stem cells within the skin epithelium that are not well defined, such as the stem cells at the interface of the hair follicle and the epidermis, collectively referred to as skin epithelial stem cells. Aging of skin epithelial tissue, a tissue that is constantly self-renewing throughout life, is closely associated with the failure of its stem cells, but the specific molecular mechanisms that lead to such stem cell failure are not fully understood.

In a preferred embodiment of the invention, said skin ageing has one or more of the following indices selected from the group consisting of: (l) Loss of skin epithelial stem cells; (m) hair whitening; (n) reduced hair regrowth ability; and (o) decreased epithelial callus function.

In addition, skin aging also includes: an increase in one or more representative senescent cell markers or markers, and/or a decrease in one or more representative young cell markers or markers. Representative markers or markers of aging cells include (but are not limited to): SA- β -galactosidase, P16, cell proliferation capacity; representative markers or markers for young cells include (but are not limited to): H3K9me3, collagen COL17a 1.

DNA damage is widely recognized as a major factor in tissue aging/premature aging. In skin tissue, both exogenous and endogenous DNA damage inducing factors can lead to significant premature aging phenomena. In patients with premature aging with genomic stability-related gene mutations and their corresponding mouse genetic models, the typical skin aging phenotype, including hair loss and albinism, appears early. On the other hand, Ultraviolet (UV) rays in sunlight are well known causes of DNA damage and can also significantly accelerate skin aging, leading to the common symptoms of photoaging in the skin. Besides ultraviolet rays, Ionizing Radiation (IR) commonly used in tumor radiotherapy is a strong DNA damage cause, can induce skin cells in a radiotherapy area to senilism, and causes long-term skin callus dysfunction, so as to bring about the risk of generating refractory ulcer, and is one of the most common complications in tumor radiotherapy. In recent years, studies have further demonstrated that DNA damage is a direct cause of failure of the skin epithelium and epithelial stem cells. Inhibiting the skin epithelial stem cell failure caused by DNA damage has wide application prospect in skin anti-aging beauty treatment and prevention and treatment of radioactive skin damage. However, no specific treatment means for entering the clinical stage is available at present.

The invention provides a technical approach for effectively resisting skin aging and premature senility by inhibiting the senescence-promoting effect of DNA damage on skin epithelial stem cells through an inhibitor of targeted miR-31.

Disease model construction

In a preferred embodiment of the present invention, in order to simulate skin aging and DNA damage caused by radiation, each group of mice was subjected to gamma ray local irradiation treatment as in example 1, and then, in order to examine the hair regeneration ability of the mice, Wax depilation (Wax) treatment was used to induce hair regeneration, after which the skin phenotype was observed for about 28 days (4 weeks). The experimental flow chart is shown in fig. 1A.

MAPK/ERK pathway inhibitors

In the present invention, the term "active ingredient" or "active ingredient for skin anti-aging" refers to an antagonist or inhibitor of MAPK/ERK pathway inhibitor.

As used herein, the terms "MAPK/ERK pathway", "MAPK/ERK signaling pathway" are used interchangeably to refer to the mitogen-activated protein kinase/extracellular regulated protein kinase (MAPK/ERK) cascade pathway. A schematic of the MAPK/ERK pathway can be seen in FIG. 5.

Mitogen-activated protein kinase (MAPK) signaling pathways are involved in cellular events such as growth, differentiation and stress responses. Four parallel MAPK channels have been identified to date: ERK1/ERK2, JNK, p38, and ERK 5. These channels are a linear kinase cascade because MAPKKK phosphorylates and activates MAPKK, and MAPKK phosphorylates and activates MAPK. To date, seven MAPKK homologs (MEK1, MEK2, MKK3, MKK4/SEK, MEK5, MKK 6, and MKK7) and four MAPK families (ERK1/2, JNK, p38, and ERK5) have been identified. Activation of these channels regulates the activity of a variety of substrates through phosphorylation. MEK catalyzes the phosphorylation of MAPK substrates ERK1 and ERK 2. Preferably, the MAPK/ERK pathway in the present invention refers to a cascade pathway consisting of RAF-MEK1/2-ERK 1/2.

In the present invention, representative MAPK/ERK pathway inhibitors include (but are not limited to): a small molecule compound, an antibody, a nucleic acid, or a combination thereof. Preferred examples include compounds and antibodies that specifically target proteins of the MAPK/ERK pathway, particularly MAPK/ERK kinases.

As used herein, the terms "MAPK/ERK inhibitor", "MAPK/ERK pathway inhibitor" are used interchangeably and refer to a molecule capable of interfering with MAPK activity, and in particular ERK activity. The MAPK/ERK inhibitor may be an anti-MAPK/ERK antibody that specifically binds to and inhibits the activity of a MAPK/ERK protein. In addition, the MAPK/ERK inhibitor may be an inhibitory nucleic acid. The inhibitory nucleic acid may be an aptamer that specifically binds to a MAPK/ERK protein and inhibits its activity. Other inhibitory nucleic acids can bind to MAPK/ERK transcripts and inhibit their translation or degrade them. Typically, such inhibitory nucleic acids may be antisense nucleic acids, morpholino oligonucleotides, inhibitory RNA molecules, such as siRNA or microrna or ribozymes. In addition, the MAPK/ERK inhibitor may be a small molecule that binds to MAPK/ERK and inhibits its activity. Such small molecule inhibitors of MAPK/ERK can be obtained by well known screening procedures or molecular modeling methods aimed at identifying compounds that bind to the active site of the MAPK/ERK kinase domain.

Some representative inhibitors of MAPK/ERK signaling pathways may be found in WO2008079814, WO2008140553, WO2009064675, and the like.

Preferably, the MAPK/ERK inhibitor described in the present invention is a MEK inhibitor selected from: u0126, GSK1120212 (Trametinib), MEK162, SCH772984, lixertinib, AZD0364, KO-947, HH2710, or combinations thereof. More preferably, the MAPK/ERK inhibitor is GSK1120212 (Trametinib), MEK162, or SCH 772984.

One representative MAPK/ERK signaling pathway inhibitor is Trametinib (Trametinib); CAS: 871700-17-3; molecular weight: 615.39, respectively; chinese culture name: n- [3- [ 3-cyclopropyl-5- [ (2-fluoro-4-iodophenyl) amino group]-3, 4, 6, 7-tetrahydro-6, 8-dimethyl-2, 4, 7-trioxopyridino [4, 3-d]Pyrimidin-1 (2H) -yl]Phenyl radical]An acetamide; the chemical formula is as follows: c₂₆H₂₃FIN₅O₄. The English name is TRAMETINIB, also called GSK1120212, GSK 212 or JTP 74057. Such inhibitors are disclosed in WO 2005/121142.

A representative MAPK/ERK signaling pathway inhibitor is SCH772984: CAS:942183-80-4, available from Selleck (S7101), formula C₃₃H₃₃N₉O₂Is a specific ERK1/2 small molecule inhibitor.

Other suitable MAPK/ERK signaling pathway inhibitors include (but are not limited to): PD0325901 (Pfizer, pfeir, inc.) (disclosed in WO 02/06213); PD184352 (fevery); rafatinib (refectib) (also known as RDEA119 or Bay 86-9766); cabitinib, also known as XL518, and is commercially available from Roche (Roche) under the trade name; AS-701255 (Merck Serono); AS-701173 (Merck Cetorono); pimasetib (pimasetib), also known AS-703026 or MSC1936369B (merck snow lanno corporation); RDEA436 (ardea biosciences, algeria); RO4987655, also known as RG7167 (roche); and/or RG7420, also known as GDC-0623 (roche).

Compositions or formulations

The invention provides a composition or preparation (including cosmetics, daily chemical products and the like) for preventing or treating skin aging, which comprises the following components: (a) acceptable carriers (including pharmaceutically acceptable carriers, physiologically acceptable carriers, cosmetically acceptable carriers); and (b) an effective amount of an active ingredient which is a MAPK/ERK pathway inhibitor.

In the present invention, the effective amount of the active ingredient may vary depending on the mode of use, the degree of skin aging, and the like. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials).

As used herein, the term "effective amount" or "effective dose" refers to an amount that produces a function or activity in, and is acceptable to, a human and/or an animal.

As used herein, an ingredient of the term "pharmaceutically acceptable" is one that is suitable for use in humans and/or mammals without undue adverse side effects (such as toxicity, irritation, and allergic response), i.e., at a reasonable benefit/risk ratio. The term "pharmaceutically acceptable carrier" refers to a carrier for administration of a therapeutic agent, including various excipients and diluents.

The term "physiologically acceptable carrier" refers to a carrier for an active ingredient, including: water, saline, liposome, lipidSubstances, proteins, protein-antibody conjugates, peptide substances, cellulose, nanogels, cellulose and its derivatives (e.g., sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (e.g., stearic acid, magnesium stearate), calcium sulfate, vegetable oils (e.g., soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g., propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (e.g., sodium carboxymethyl cellulose, sodium ethyl cellulose, sodium lauryl sulfate, etc.), and the like

) A wetting agent (e.g., sodium lauryl sulfate), a coloring agent, a flavoring agent, a stabilizer, an antioxidant, a preservative, pyrogen-free water, or a combination thereof. The choice of carrier should be matched with the mode of administration, which is well known to those skilled in the art.

The pharmaceutical composition of the present invention contains a safe and effective amount of the active ingredient of the present invention and a pharmaceutically acceptable carrier. Such vectors include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical composition of the invention can be prepared into injections, oral preparations (tablets, capsules, oral liquids), transdermal agents and sustained-release agents. For example, by a conventional method using physiological saline or an aqueous solution containing glucose and other adjuvants. The pharmaceutical composition is preferably manufactured under sterile conditions.

The effective amount of the active ingredient of the present invention may vary depending on the mode of administration and the severity of the disease to be treated, etc. The selection of a preferred effective amount can be determined by one of ordinary skill in the art based on a variety of factors (e.g., by clinical trials). Such factors include, but are not limited to: pharmacokinetic parameters of the active ingredient such as bioavailability, metabolism, half-life, etc.; the severity of the disease to be treated by the patient, the weight of the patient, the immune status of the patient, the route of administration, and the like. In general, satisfactory results are obtained when the active ingredient of the invention is administered at a daily dose of about 0.00001mg to 50mg per kg of animal body weight (preferably 0.0001mg to 10mg per kg of animal body weight). For example, divided doses may be administered several times per day, or the dose may be proportionally reduced, as may be required by the urgency of the condition being treated.

Another class of compositions or products in the present invention are cosmetic or household products comprising an active ingredient of the invention (targeted inhibition of miR-31).

In the present invention, the composition or formulation further preferably contains a transdermal enhancer.

In the present invention, the dosage form of the cosmetic composition or preparation includes (but is not limited to): solid preparation, liquid preparation, gel preparation, and semisolid preparation. Some preferred dosage forms are ointment, cream, patch, liniment, spray, micro-injection, solution, shampoo, etc.

In the present invention, representative products (or daily chemical products) include (but are not limited to): shampoo, shower gel, skin care products, hair care products and facial masks.

The main advantages of the invention include:

(a) the invention surprisingly proves that irradiation stimulation leads to the exhaustion of epithelial stem cells of the skin by activating the MAPK/ERK signal pathway, thereby leading to the subsequent skin aging, so that the MAPK/ERK signal pathway can be used as a target for preventing or treating skin aging and premature senility.

(b) MAPK/ERK pathway inhibitors are effective in combating or delaying skin aging (especially premature aging).

(c) MAPK/ERK pathway inhibitors are effective in preventing the depletion of stem cells and thus the subsequent aging of the skin.

(d) Under the condition of skin premature senility caused by irradiation and the like, the subsequent skin aging can be effectively prevented by inhibiting the activation of the MAPK/ERK pathway in the early stage without long-term continuous medication, so that the MAPK/ERK pathway can be prevented from being inhibited for a long time, and the safety is realized.

(e) The invention surprisingly proves for the first time that different MAPK/ERK signal channel inhibitors can achieve the same effect of antagonizing skin aging and radiation-induced skin premature aging.

(f) The application form is wide, and different application modes (oral administration or smearing) of MAPK/ERK signaling pathway inhibitors can achieve the same effect of antagonizing skin aging and radiation-induced skin premature aging.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

Materials and methods

1) Animal model

An animal model of local irradiation induced premature skin aging was made using 7-week-old C57BL/6 wild-type mice as experimental mice. All C57BL/6 wild-type mice were purchased from shanghai slaike laboratory animals, llc.

2) Local irradiation of gamma rays

Gamma irradiation was performed according to standard experimental procedures at the radiology institute of the medical school of the university of counterdenier. When local irradiation is carried out, an anesthetized mouse is placed in a gap between an upper lead plate and a lower lead plate in an irradiation bin, the upper lead plate and the lower lead plate are the same, the lead plates are perpendicular to a radioactive source, two lead plates are respectively provided with a vertically aligned small hole with the diameter of 3 cm, the upper half part of the mouse is placed in the small hole, the lead plates cover the head part and the lower half part of the mouse, and the four limbs of the mouse are properly fixed by using an adhesive tape for irradiation. The irradiation dose was 10 Gy. All irradiation experiments utilized 7 week old C57BL/6 wild type mice.

3) Trametinib oral treatment

Trametinib (CAS:871700-17-3), available from Selleck (S2673), formula C₂₆H₂₃FIN₅O₄. In mouse experimentsTrametinib powder was dissolved in DMSO to make 15mg/ml stock solution, which was taken orally according to a 1: 25 dilutions were diluted in Corn oil (Sigma, C8267) and given orally at a dose of 1mg/kg per mouse, once every three days, for a total of five times. The control group was orally administered the same solvent.

4) Trametinib smearing treatment

Trametinib (CAS:871700-17-3), available from Selleck (S2673) of formula C₂₆H₂₃FIN₅O₄. Trametinib powder was dissolved in DMSO to make 15mg/ml stock solution, and the mice were shaved on their backs with a razor, topically applied according to a 1: a25 dilution was made in 50% ethanol, 30% water and 20% propylene glycol (Sangon, A501754-0500) solvent, and each mouse was applied at a dose of 1mg/kg once every three days for a total of five times. The control group was smeared with the same solvent.

5) Flow analysis of mouse dorsal skin hair follicle stem cells

Mice were shaved after sacrifice using a carbon dioxide lethal device and the dorsal skin was removed, the dermal adipose tissue was removed with a scalpel, washed with PBS, and digested in Trypsin-EDTA (ThermoFisher,25200056) overnight at 4 ℃. The following day the cells were scraped with a razor blade, cell suspensions were collected and stained on ice in PFE (PBS + 1% FBS + 1. mu.M EDTA) staining solution for CD34 (1: 100, eBioscience,11-0341-82), CD49f (1: 100, biolegend, 313602). After staining was complete the cells were washed and the viable cell dye Helix NP NIR (biolegend,425301) was added. Stained cells were detected on a flow analyzer (BD, CytoFLEX LX). Flow detection of CD34⁺CD49f⁺The live cell population was hair follicle stem cells, and the percentage of hair follicle stem cells in each group was analyzed by flow analysis software FlowJo 10.0, and compared to the percentage of untreated sites in wild-type mice.

6) Mouse dorsal skin callus test

After anesthetizing with isoflurane, the mice were shaved and the back was perforated with an 8mm hole punch to create wounds. The adhesive surgical towel (3M, SP1107) prevents the wound from deforming, the adhesive surgical towel is taken down after three days, the images are taken respectively before the wound is created and after the wound is created for 0, 4,7, 9, 12 and 15 days, the wound area is calculated according to a proportional scale, and the ratio of the wound area of the mice at different time points in each group to the area of the wound of the mice in each group at each time point is taken to obtain the wound healing percentage. Each group of data was recorded until the callus was completely finished.

7) Keratinocyte migration callus assay of in vitro cultured skin tissue blocks (Exo-plants)

After the mice were sacrificed, the hair was removed with depilatory cream, and the adipose tissue was removed with a surgical knife after 75% alcohol sterilization. After washing in PBS, skin pieces were obtained by punching with a 1mm punch, 1-2ul of matrix (Corning,356237) was added to each well, and the skin pieces were attached and incubated in a coating of a 24-well plate (Corning, CLS3527) coated with 1mg/ml fibronectin (Millipore, FC010-1 mg). After 7 days of culture, the cells were photographed under a microscope, and the area of the cells migrating from the edge of the skin block was measured using image statistics software Imagej, with the skin block as the center point. The area of each group is respectively taken as the ratio of the area of the wild type untreated part.

8) Mouse Hair regeneration Capacity test

The existing hair is completely removed by wax depilation, so that hair regrowth is activated and the rate of regrowth is followed by photography. The method of repeated hair regrowth ability test is to repeat the process by performing a second wax depilation after the first regrowth of the hair is completed. During the Hair regeneration process, the Hair was photographed and recorded by a camera (Canon) and a Hair detector (hairand Scale), respectively.

9) Sch772984 oral administration

Sch772984(CAS:942183-80-4), available from Selleck (S7101), formula C₃₃H₃₃N₉O₂Is a specific ERK1/2 small molecule inhibitor. In mouse experiments, the Sch772984 powder was dissolved in DMSO to make 15mg/ml stock solutions, which were taken orally according to a 1: 25 dilutions were diluted in corn oil (Sigma, C8267) and administered orally at 2mg/kg per mouse, once every three days, five times in total. The control group was orally administered the same solvent.

Example 1

Establishment of skin senilism research model

In this example, a model for the study of premature skin aging was established in mice by induction of local ionizing radiation. The method comprises the following steps: referring to fig. 1A, adult mice of about 7 weeks old were subjected to one-time gamma-ray local irradiation of an area of about 2cm diameter of the upper back, and hair regrowth was initiated by wax depilation after 5 days.

Specifically, mice received local irradiation at around 7 weeks of age (P50). Five days later Wax depilation (Wax) was performed to induce hair regrowth. The skin phenotype after completion of the first round of hair regrowth was observed 28 days later (4 weeks) (one round phenotype), or the second round of hair regrowth was induced again by wax depilation, and the skin phenotype after completion of the second round of hair regrowth was observed 4 weeks later (two round phenotype).

The results show that the irradiated areas of the upper back of the mouse can show a series of typical early skin aging phenotypes: the method mainly comprises the following steps:

1) fresh hair was visibly whitened (fig. 1B).

2) The skin wound healing ability is reduced. Including a decrease in wound healing rate in vivo (FIG. 1C) and a decrease in keratinocyte migration rate of an in vitro cultured skin tissue mass (Exo-plant) (FIG. 1D).

3) The number of hair follicle stem cells decreased in vivo (fig. 1E).

4) The ability of the hair to repeatedly regenerate was exhausted (fig. 1F).

Example 2

Oral MAPK/ERK signaling pathway inhibitor (Trametinib) antagonist for preventing skin aging and radiation-induced skin premature aging

To verify that inhibition of the MAPK/ERK signaling pathway is an effective means to antagonize intra-dermal aging and radiation-induced premature skin aging, an FDA-approved MEK1/2 small molecule inhibitor, Trametinib, was used as an example.

In this example, short-term drug treatment was introduced in the model of premature skin irradiation aging in order to verify the effectiveness of inhibiting the MAPK/ERK signaling pathway in antagonizing skin aging.

The test method is shown in FIG. 2A. Mice received one-time topical irradiation at around 7 weeks of age (P50). On the day of irradiation, oral treatment with the drug (Trametinib or control solvent) was started simultaneously. The mode of drug treatment was oral once every 3 days for a total of 5 times for 15 days. Five days after irradiation wax depilation was performed to induce hair regrowth. The skin phenotype after completion of the first round of hair regrowth was observed 28 days later (4 weeks) (one round phenotype), or the second round of hair regrowth was induced again by wax depilation, and the skin phenotype after completion of the second round of hair regrowth was observed 4 weeks later (two round phenotype).

As a result:

the first round of hair regrowth was complete in mice after 3 weeks of drug withdrawal (i.e., after 4 weeks of irradiation). At this point a portion of the mice were sampled for analysis of the dorsal skin phenotype (one round of phenotype). Another portion of the mice were wax dehaired again to induce a second hair regrowth and analyzed for the dorsal skin phenotype after 4 weeks (two-round phenotype, which is primarily representative of hair re-regrowth ability).

The results show that the irradiated area skin of the Trametinib treated group exhibited a significantly reduced skin aging phenotype compared to the irradiated area skin of the solvent control treated group, mainly reflected in:

1) the hair whitening phenomenon was significantly alleviated (fig. 2B, 2D).

2) Hair thinning caused by the decline of hair regrowth ability was significantly alleviated (fig. 2B, 2C).

3) The phenomenon of depletion of the number of hair follicle stem cells was significantly alleviated (fig. 2E).

4) The decline of the in vivo skin healing ability was significantly alleviated (fig. 2F, 2G).

5) The phenomenon of keratinocyte migration decline in the experiments of in vitro culture of skin tissue blocks (Exo-plant) was significantly alleviated (fig. 2H, 2I).

Example 3 application of MAPK/ERK Signaling pathway inhibitor (Trametinib) antagonists to skin aging and radiation-induced premature skin aging

The method and procedure of example 2 were repeated except that only oral administration was replaced with topical application of the skin, and the outline of this experimental procedure is shown in fig. 3A.

The results show that the skin of the irradiated area of the group topically applied Trametinib showed a significantly alleviated skin aging phenotype compared to the skin of the irradiated area of the solvent-control treated group, similar to the effect of the oral administration described previously.

The method specifically comprises the following steps:

1) the skin callus decline phenotype was significantly alleviated (fig. 3B, 3C);

2) the phenomenon of skin hair follicle stem cell depletion was significantly alleviated (fig. 3D);

3) skin hair whitening was significantly alleviated with the hair thinning phenotype (fig. 3E-G).

Example 4 oral MAPK/ERK Signaling pathway inhibitors (II)Sch772984) Antagonist of intrinsic skin aging and radiation-induced premature skin aging

The experimental procedure and procedures of example 2 were repeated except that oral administration of Trametinib was replaced with oral administration of Sch772984 (the outline of the experimental procedures is shown in fig. 4A).

The results show that the irradiated skin of the oral Sch772984 group showed a significantly alleviated skin aging phenotype compared to the irradiated skin of the oral solvent control treated group, similar to the effect of the aforementioned oral Trametinib.

The concrete expression is as follows: 1) the skin callus decline phenotype was significantly alleviated (fig. 4B);

2) the decrease in the proportion of skin epithelial hair follicle stem cells was significantly alleviated (fig. 4C);

3) skin hair whitening was significantly alleviated with the hair thinning phenotype (fig. 4D-F).

Discussion of the related Art

The MAPK/ERK signaling pathway is a highly evolutionarily conserved classical cell growth and differentiation regulation signaling pathway. The core of the protein kinase is a protein kinase cascade pathway which is composed of three protein kinases, namely MAPKKK (RAF), MAPKK (MEK1/2) and MAPK (ERK), in sequence. Cells typically receive a stimulation signal through surface receptors (receptor tyrosine kinases (RTKs), integrins, ion channels, etc.) and activate small molecule GTP-binding proteins (RAS, RAP1, etc.) through dockerin (SHC, GRB2, CRK, etc.) and guanylate exchange factor (SOS, C3G, etc.), thereby activating the above-described core cascade. The activated ERK dimers regulate target proteins in the cytoplasm and also regulate multiple transcription factor activities by phosphorylation in the nucleus.

MAPK/ERK signaling pathways are generally thought to have effects in promoting cell proliferation and survival, and are a broad cancer-promoting signaling pathway, whose over-activation is directly associated with the development and progression of various tumors. For example: the activating mutation of RAF can promote the generation of melanoma and metastatic non-small cell lung cancer, the BCR/ABL fusion gene promotes the generation of chronic granulocytic leukemia by activating MAPK/ERK signaling pathway, and the generation and development of breast cancer and prostate cancer are closely related to the abnormal activation of MAPK/ERK signaling. In addition, various cells can significantly activate MAPK/ERK signaling pathways under ionizing radiation stimulation, which has the effect of promoting cell survival after irradiation and is considered as a protective mechanism for cells against radiation.

In the skin, the MAPK/ERK signaling pathway is also a classical growth-promoting/carcinogenic pathway. Aberrant activation of the MAPK/ERK pathway mediated by mutations in the RAS gene can cause skin stem cells to become cancerous and thus induce the development of squamous cell carcinoma. In the development of skin cancer, increasing the activity of MAPK/ERK signaling pathways leads to higher proliferation and migration of cancerous skin stem cells, thereby exacerbating the malignancy of skin cancer. In normal skin tissue, the MAPK/ERK pathway has the effect of promoting callus. Physical trauma can significantly activate the MAPK/ERK pathway, while decreasing its activity slows wound healing. Ultraviolet stimulation can also significantly activate MAPK/ERK signaling pathways in epidermal cells to cause epidermal hyperplasia, but human skin shows significantly down-regulated MAPK/ERK signaling pathway activity in the aging process. Prior to the present invention, no relationship between the MAPK/ERK pathway and skin aging has been reported, and there is no clear evidence that the MAPK/ERK pathway is functionally related to natural aging or photoaging of skin in vivo.

The research results of the invention prove unexpectedly for the first time that: inhibition of the MAPK/ERK pathway is an effective means to antagonize skin aging and radiation-induced skin damage. Meanwhile, the inventor also proves that the skin premature senility phenotype caused by irradiation can be effectively relieved by inhibiting the MAPK/ERK pathway in a short time after irradiation. The method avoids side effects possibly brought by long-term inhibition of MAPK/ERK pathway, and provides a feasible means for clinically preventing and treating symptoms such as skin senilism and long-term callus ability disorder caused by ionizing radiation.

Previous studies have shown that DNA damage is a direct cause of aging and premature aging of skin epidermis and hair follicle stem cells, while ionizing radiation is one of the common causes of DNA damage in human skin. Thus, the present study suggests that irradiation stimulation leads to exhaustion of epithelial stem cells of the skin by activating MAPK/ERK signaling pathways, which in turn leads to subsequent skin aging. Therefore, the stem cell exhaustion can be effectively prevented and the subsequent skin aging can be prevented only by inhibiting the activation of MAPK/ERK pathway in the early stage after irradiation stimulation, and long-term continuous medication is not needed.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. Use of a MAPK/ERK pathway inhibitor for the preparation of a composition or formulation for the prevention or treatment of skin aging (or anti-skin aging).

2. The use of claim 1, wherein said composition comprises a pharmaceutical composition, a daily chemical composition.

3. The use according to claim 1, wherein the composition or formulation is for one or more applications selected from the group consisting of:

(b) improving the skin healing ability or promoting wound healing;

(c) promoting migration of keratinocytes;

(e) improving hair regeneration ability.

4. The use according to claim 3, wherein the epithelial stem cells are selected from the group consisting of: hair follicle stem cells, epidermal stem cells, stem cells of skin epithelial tissue, or a combination thereof.

5. The use of claim 1, wherein said skin aging comprises: skin aging caused by DNA damage, radiation-induced skin senilism, natural skin aging, and skin photoaging.

6. The use of claim 1, wherein said skin aging is induced by an inducer selected from the group consisting of: ionizing radiation, gamma irradiation, ultraviolet light, radiation therapy, excessive exposure to light, chemical mutagens, natural skin aging caused by natural proliferation of skin cells, or combinations thereof.

7. The use of claim 1, wherein said MAPK/ERK pathway inhibitor is selected from the group consisting of: trametinib (Trametinib), U0126, MEK162, SCH772984, lixertinib, AZD0364, KO-947, HH2710, PD0325901, PD184352, Rifamtinib (Refametinib), Carbotinib, AS-701255, AS-701173, pimasetib (Pimasertib), RDEA436, RO4987655, RG7420, or a combination thereof.

8. The use of claim 1, wherein said inhibition of the MAPK/ERK pathway inhibitor comprises: trametinib, SCH772984, or a pharmaceutically acceptable salt thereof.

9. A composition or formulation useful for preventing or treating skin aging, the composition or formulation comprising: (a) a physiologically acceptable carrier; and (b) an effective amount of an active ingredient which is a MAPK/ERK pathway inhibitor.

10. An in vitro method for screening potential substances for preventing or treating skin aging, comprising the steps of:

(1) adding a substance to be detected or a negative control substance to the test system, respectively; and

(2) respectively detecting the levels of MAPK/ERK pathways in the sample added with the substance to be detected and the sample added with the negative control substance; a substance potentially useful for preventing or treating a skin aging disorder if the sample to which the substance to be detected is added has a reduced level of the MAPK/ERK pathway compared to the sample to which the negative control is added.