JP7621765B2 - Screening method for agents for improving skin photoaging - Google Patents

Description

Article 30, paragraph 2 of the Patent Act applies. (1) Publication date: September 10, 2020. (2) Publication address: http://ifscc2020. com/scientific_program. html

The present invention relates to a photoaged cell model that mimics photoaged skin, and a screening method for photoaging prevention and improvement agents using the same.

As we age, the skin becomes harder and loses elasticity, resulting in changes such as loss of firmness, wrinkles, and sagging. Wrinkles and sagging are particularly noticeable in areas that are susceptible to chronic exposure to ultraviolet rays, such as the face. Skin aging that occurs in areas exposed to ultraviolet rays is called photoaging, and preventing and improving this is one of the biggest challenges for researchers of topical skin products.

The dermis, which is the supporting tissue of the skin, is mainly composed of extracellular matrix such as collagen fibers (collagen fibers), elastic fibers (elastin fibers and microfibrils), glycoproteins (laminin, fibronectin, etc.), proteoglycans (versican, etc.), and glycosaminoglycans (hyaluronic acid). Collagen fibers provide the strength of the skin, elastic fibers provide the elasticity of the skin, glycoproteins bind cells and the extracellular matrix, and proteoglycans and glycosaminoglycans contribute to the moisture retention and flexibility of the skin. In addition to the extracellular matrix, the dermis also contains fibroblasts as a cellular component, which produce the extracellular matrix and its decomposition enzymes (collagenase, elastase, matrix metalloproteinase, etc.). Fibroblasts survive by adhering to the extracellular matrix that serves as a scaffold via adhesion molecules such as integrins present in the cell membrane, while signals that control cell proliferation, differentiation, and phenotypic expression are transmitted from the extracellular matrix. In this way, the extracellular matrix not only serves as a physical support for skin tissues and cells, but also acts as a regulator that directly controls cell function.

In the dermis of photoaged skin, abnormalities in the composition of the extracellular matrix, such as a decrease in collagen fibers, abnormal deposition of elastin fibers (such as fiber thickening and amorphous mass accumulation), and accumulation of proteoglycans, are known to occur. In addition, it is known that the activity of fibroblasts is also changed, for example, there is a decrease in cell proliferation, a decrease in the contractile ability of collagen fibers, and a change in the production function of extracellular matrix components. Changes in the production function of the extracellular matrix include, in particular, an increase in the production of tropoelastin, the precursor of elastin fibers, and the production of enzymes that degrade the extracellular matrix (Non-Patent Document 1). Such changes in fibroblast function cause abnormalities in the composition of the main extracellular matrix of the dermis, and further reduce the repair ability of the tissue. As a result, an increase in the hardness and a decrease in elasticity of the skin are caused, which ultimately leads to a decrease in firmness, wrinkles, sagging, etc. (Non-Patent Documents 2 and 3).

In the past, when studying the functions of cells present in photoaged skin or when searching for materials that prevent and improve various symptoms of photoaging, a method has been widely used in which external stimuli are applied directly to cells to examine changes in their behavior, such as irradiating cultured cells with ultraviolet light or adding reagents that induce oxidative damage. Alternatively, cells have been isolated from animals or clinical specimens with the relevant symptoms and their behavior has been examined directly. However, although the former test is simple, it is very difficult to set the type, concentration, and frequency of external stimuli and reagents to be applied, and as a result, the behavior of cells often differs between cultured cells and actual biological reactions. In addition, the latter test requires living tissue from humans or animals with the relevant symptoms, so it is difficult to carry out. Therefore, a method that can easily and conveniently mimic the behavior of cells in photoaged skin is needed to evaluate the cell functions of photoaged skin and to search for agents that prevent and improve photoaging.

On the other hand, protein nitration is one of the post-translational modifications of proteins caused by reactive nitrogen species generated in the body, in which a nitro group is added to the benzene ring in the residues of the aromatic amino acids tyrosine and tryptophan that constitute proteins. Nitration occurs when the benzene ring in an amino acid undergoes an electrophilic substitution reaction with nitronium ions (NO2+) or nitrogen dioxide radicals formed by reactive nitrogen species (Non-Patent Document 4). The content of tryptophan in many proteins present in the body is much lower than that of tyrosine, and it is thought that protein nitration reactions mainly occur in tyrosine residues (Non-Patent Document 5). It is known that protein nitration affects cell function by causing a decrease in the function of enzymes and tyrosine kinase receptors (Non-Patent Document 6). It is also known that nitrated proteins accumulate in a number of diseases associated with aging (such as arteriosclerosis and cerebral ischemic disease), and it has been reported that they are involved in these diseases (Non-Patent Document 7). Nitrated proteins are also known to exist in the skin, and the amount of nitrated proteins present in the stratum corneum in particular is associated with skin color (Patent Document 1). However, the effects of nitrated proteins in the dermis on dermal cells and their involvement in photoaging skin symptoms remain unclear.

JP 2017-181423 A

Matrix Biology Plus, June 2020 Exp. Dermatol. , 2019, 28(8):914-921 Exp. Dermatol. , 2019, 28(8):981-984 Chem. Res. Toxicol. , 2009, 22(5):894-898 Front. Chem. , 2016, 3:70 Diabetes, 2008, 57(4):889-98 Science, 2000, 290(5493):985-9

The objective of the present invention is to provide a dermal photoaged cell model that mimics the behavior of fibroblasts in photoaged skin, and a method for producing the same. Furthermore, the objective of the present invention is to provide a screening method for materials that can prevent and/or improve various symptoms of photoaged skin by suppressing changes in fibroblast function associated with photoaging.

After extensive research into ways to easily and simply mimic the behavior of fibroblasts in photoaged skin, the inventors came up with the idea of focusing on the signals transmitted to fibroblasts from the extracellular matrix that exists around cells and serves as a scaffold for cell survival, rather than on the direct effects of external stimuli such as ultraviolet light on fibroblasts. In other words, the inventors were inspired by the idea that abnormalities in the extracellular matrix of the dermis caused by photoaging may also transmit abnormal signals to fibroblasts.

The inventors therefore investigated abnormalities occurring in the extracellular matrix in photoaged skin and found that nitration modification occurs in the extracellular matrix proteins in photoaged skin. Further investigation revealed that nitration modification occurs particularly in elastin fibers.

Furthermore, the inventors found that when extracellular matrix proteins are nitrated, the cellular functions of fibroblasts that use them as a scaffold change, resulting in, for example, decreased cell proliferation, decreased collagen contractile force, and changes in the production of extracellular matrix, in particular increased production of tropoelastin, a precursor of elastin. These changes in cellular functions are similar to the characteristics of fibroblasts in photoaged skin, and based on this finding, the inventors found that when the support matrix for cells is modified by nitration, a signal is transmitted from the nitrated support matrix to the dermal fibroblasts that use it as a scaffold, resulting in a change in cellular function, just as in photoaged skin. It was believed that this change in cellular function leads to abnormal deposition of elastin in photoaged skin and changes in the physical properties of the dermis, leading to the progression of photoaging.

Based on these new findings, we determined that by using nitrated extracellular matrix proteins as a culture support matrix, it would be possible to construct a photoaged cell model that mimics the behavior of fibroblasts in photoaged skin. We also believe that by using this photoaged cell model, it would be possible to evaluate and/or select materials that can prevent or improve skin hardening and loss of skin elasticity, as well as the associated formation of wrinkles, sagging, and loss of firmness, and have thus arrived at the present invention.

In the present invention, the above problem was solved by preparing a photoaged cell model in which a culture support matrix serving as a scaffold for cultured cells was subjected to a nitration treatment and fibroblasts were cultured together with the culture support matrix. Furthermore, the above problem was solved by using this photoaged cell model as an indicator of fibroblast proliferation, collagen gel contraction, and gene or protein expression levels that change in response to signals transmitted from the nitrated culture support matrix.

The present invention provides the following photoaged cell model and a method for producing and screening the same.
[1] Nitrated culture support substrate and photoaged cell model containing fibroblasts.
[2]
A) any one of the following steps: A)-1: a step of seeding fibroblasts on a nitrated culture support substrate; and A)-2: a step of mixing the nitrated culture support substrate with the fibroblasts. B) A method for producing a photoaged cell model, comprising the step of culturing A).
[3]
A method for screening an agent for preventing and/or improving photoaging, comprising:
A) culturing fibroblasts on a nitrated culture support substrate;
B) adding a test substance or a control substance and culturing; C) measuring the cell activity of the fibroblasts and/or the gene expression level or protein expression level of the fibroblasts;
D) A step of selecting a test substance that changes the cell activity and/or gene expression level or protein expression level in the test substance addition group compared to the test substance non-addition group or the control substance addition group;
The method comprising:
[4] The screening method according to [3], wherein the photoaging symptoms are at least one of wrinkles, sagging skin, loss of firmness, hardening of the skin, and loss of elasticity.
[5] The screening method according to [3] or [4], wherein the culture support substrate is an extracellular matrix protein.
[6] The screening method according to any one of [3] to [5], wherein the culture support substrate contains elastin.
[7] The screening method according to any one of [3] to [6], wherein the cellular activity is cell proliferation.
[8] The screening method according to any one of [3] to [7], wherein the gene expression level or protein expression level is the gene expression level or protein expression level of an extracellular matrix.
[9] The screening method according to any one of [3] to [8], wherein the culture support substrate contains collagen and the cell activity is collagen contraction.

The present invention provides a photoaged cell model that mimics the behavior of fibroblasts in photoaged skin and a method for producing the same. Furthermore, the present invention provides a method for screening new materials that increase skin hardness, inhibit loss of skin elasticity, and prevent and/or improve the associated symptoms of photoaging such as wrinkle formation, sagging, and loss of firmness. By using the present invention, it is possible to easily evaluate the cellular functions in photoaged skin and conduct research to elucidate the mechanism of photoaging without the need for human or animal testing, and further to develop materials that prevent and improve various symptoms of photoaging.

FIG. 1 shows cell nuclei, nitrotyrosine, elastin fibers, and superimposed images of these in sections of photoaged (exposed) and naturally aged (unexposed) skin.

The culture support substrate used in the present invention is not particularly limited. Although a substrate generally used as a culture support substrate can be used, it is preferable to use an extracellular matrix protein of the dermis. For example, collagen can be used as a collagen fiber component, elastin can be used as an elastic fiber component, and microfibril components such as fibrillin and fibrin can be used as glycoproteins such as laminin and fibronectin, and versican and aggrecan can be used as proteoglycans. These can be natural or artificially produced. Natural products include those extracted from vertebrates such as humans, monkeys, mice, rats, cows, horses, rabbits, goats, sharks, and salmon, as well as those extracted from invertebrates such as jellyfish, octopus, and abalone. Artificially produced products include extracellular matrix proteins extracted from isolated and cultured cells of the above-mentioned organisms, recombinant proteins produced by transfecting the genes of the extracellular matrix proteins of the above-mentioned organisms into microbial cells such as E. coli or yeast, or proteins produced in a cell-free system using enzymes in extracts of E. coli, wheat germ, cultured cells, etc. The method for extracting the extracellular matrix protein is not particularly limited, and known methods can be used. The extracted extracellular matrix protein may be used as is, or may be hydrolyzed, denatured, or crosslinked, or may be artificially shaped into a solid, fibrous, or gel-like composition. The culture support substrate may contain one or more of the above-mentioned extracellular matrix proteins, and may be used as a single protein or a mixture of multiple proteins. Examples of commercially available products that can be used include, but are not limited to, Cellmatrix Type I-A (Nitta Gelatin), Elastin Fiber Sheet (Extracellular Matrix Research Institute), and Matrigel (Corning).

The elastin used in the present invention is not particularly limited, and examples thereof include tropoelastin protein and elastin protein. These may be natural products or artificially produced products. Natural products include those extracted from vertebrates including humans, rats, cows, rabbits, salmon, etc. Artificial products include those produced by transfecting tropoelastin genes derived from the above-mentioned organisms into microbial cells such as E. coli or yeast, or those produced in a cell-free system using enzymes in extracts from E. coli, wheat germ, and cultured cells. Elastin may exist as a protein, or may be a fibrous or gel-like structure formed by crosslinking multiple elastins. For example, elastic fibers such as elastic fibers and elaunin fibers recovered from the above-mentioned organisms, or may be artificially crosslinked to form a fibrous structure or a gel-like structure. Furthermore, these proteins or fibrous or gel-like elastins can be used as they are, or may be fragmented by hydrolysis, enzymatic decomposition, or denatured.

The collagen used in the present invention is not particularly limited, but examples thereof include type I collagen and type III collagen. These may be natural or artificially produced. Natural collagens can be extracted from vertebrates including humans, rats, cows, rabbits, salmon, etc. Artificial collagens can be produced by transfecting collagen synthesis genes derived from the above-mentioned organisms, such as the COL1A1 gene or the COL3A1 gene, into microbial cells such as E. coli or yeast, or produced in a cell-free system using enzymes in extracts from E. coli, wheat germ, and cultured cells. Collagen may be present as a protein, or may be a fibrous or gel-like structure formed by crosslinking multiple collagens. For example, collagen fibers recovered from the above-mentioned organisms may be artificially crosslinked to form a fibrous structure or a gel-like structure. Furthermore, these protein or fibrous collagens can be used as they are, or may be fragmented by hydrolysis, enzymatic decomposition, or the like, or may be denatured.

The nitrated culture support substrate of the present invention can be prepared by mixing and reacting a culture support substrate with a nitration reagent, or a culture support substrate that has already been nitrated can be used. For example, a culture support substrate containing nitrotyrosine, dinitrotyrosine, nitrotryptophan, nitrophenylalanine, dinitrophenylalanine, nitrocysteine, etc. can be used as a culture support substrate that has already been nitrated. In addition, based on the above-mentioned new findings, it is highly likely that the extracellular matrix, especially elastin, present in photoaged skin is modified by nitration. Therefore, a culture support substrate extracted from photoaged skin of humans or animals exposed to ultraviolet rays for a long period of time, or a culture support substrate extracted after irradiating and culturing human or animal cells with ultraviolet rays, can be used as a culture support substrate that has already been nitrated.

As the nitration reagent used for nitrating the culture support substrate, a known one can be used. For example, active nitrogen species such as peroxynitrite can be used directly, or a group of compounds capable of generating active nitrogen species in the test system can be mixed and used. For example, nitric oxide and superoxide anion can be mixed, or myeloperoxidase (MPO) can be mixed with NaNO ₂ , hydrogen peroxide, etc. In addition, reagents such as tetranitromethane (TNM) and nitric acid that generate nitronium ions can be used. In addition, reagents such as nitroyl chloride, nitrosoperoxycarboxylate, sodium azide, and catalase can be used. The nitration reaction itself can be performed by a known method. By using the above reagent, a nitro group is introduced into tyrosine, tryptophan, phenylalanine, and cysteine residues contained in the extracellular matrix protein, which is the culture support substrate, to form a culture support substrate containing nitrotyrosine, dinitrotyrosine, nitrotryptophan, nitrophenylalanine, dinitrophenylalanine, nitrocysteine, etc. Furthermore, in the present invention, the order and timing of mixing the culture support substrate and the nitrating reagent are not important as long as they are both present in the test system at the same time.

The fibroblasts used in the present invention are not particularly limited as long as they are derived from humans or animals, and for example, fibroblasts derived from mammals such as humans, monkeys, and mice can be preferably used. Fibroblasts may be isolated from human or animal tissue, or already isolated commercially available products may be used. For the cell culture medium, it is advisable to select a medium appropriate for the fibroblasts used, such as Dulbecco's MEM (D-MEM) supplemented with 10% fetal bovine serum (FBS).

The photoaged cell model of the present invention may be any model that contains a nitrated culture support substrate and fibroblasts within its structure, and examples of such models include a structure in which fibroblasts are provided on the surface of the matrix layer of a nitrated culture support substrate, or a structure in which fibroblasts are encapsulated within the matrix layer of a nitrated culture support substrate.

The photoaged cell model of the present invention can be prepared by the following method. For example, a substrate layer of a nitrated culture support substrate is prepared in advance in a culture vessel, and then fibroblasts are seeded on the substrate layer of the nitrated culture support substrate and cultured. Alternatively, the nitrated culture support substrate and fibroblasts are mixed in advance and then added to the culture vessel to prepare a nitrated culture support substrate containing fibroblasts, which is then cultured. There are no particular limitations on the culture vessel, but in addition to cell culture plates and petri dishes, those with a support membrane such as chamber slides and transwells can be used.

In one embodiment of the method for producing a photoaged cell model of the present invention,
A) any one of the following steps: A)-1: a step of seeding fibroblasts on a nitrated culture support substrate; A)-2: a step of mixing the nitrated culture support substrate with the fibroblasts; B) a step of culturing A).

The substrate layer of the nitrated culture support substrate can be prepared by adding the nitrated culture support substrate to the culture vessel. For example, it can be prepared by adding a mixture of the culture support substrate and the nitration reagent to the culture vessel, or by mixing and reacting the culture support substrate and the nitration reagent in the culture vessel. Alternatively, it can be prepared by adding an already nitrated culture support substrate to the culture vessel.

In the step of seeding fibroblasts onto the substrate layer of the nitrated culture support substrate, fibroblasts diluted to an arbitrary density may be dropped onto the substrate layer of the nitrated culture support substrate. There are no particular limitations on the density of the fibroblasts, and for example, the seeding density used in general culture may be used.

In the process of mixing the nitrated culture support substrate with the fibroblasts, the fibroblasts diluted to any density may be mixed with the nitrated culture support substrate. There are no particular limitations on the density of the fibroblasts, and for example, the seeding density used in general culture may be used.

The culture may be generally performed in the same manner as when fibroblasts are cultured, by incubating a structure containing nitrated culture support substrate and fibroblasts, and allowing the fibroblasts to settle on the nitrated culture support substrate. The culture conditions may also be generally performed in the same manner as when fibroblasts are cultured, for example, under humidified conditions of 37°C and 5% _CO2 , for several hours to several days, or up to about 30 days. If necessary, fibroblasts can be passaged, and generally performed in the same manner as when fibroblasts are cultured.

There are no particular limitations on the storage and transportation of the photoaged cell model. It is sufficient that the storage and transportation are under conditions in which the fibroblasts can survive, and these may be general cell storage and transportation conditions or conditions suitable for the survival of the fibroblasts used in this model.

In one embodiment, the screening method of the present invention comprises:
Cultivating fibroblasts with the nitrated culture support substrate;
A step of adding a test substance or a control substance and culturing the fibroblasts; A step of measuring the cell activity and/or the gene or protein expression level of the fibroblasts;
A step of selecting a test substance that changes the cell activity and/or gene or protein expression level in a test substance addition group compared to a control substance addition group;
Includes.

In the process of culturing fibroblasts on a nitrated culture support substrate, fibroblasts are seeded and cultured on the substrate layer of the nitrated culture support substrate, or a mixture of the nitrated culture support substrate and fibroblasts is cultured. There are no particular limitations on the culture vessel, but in addition to cell culture plates and petri dishes, vessels with a support membrane such as chamber slides and transwells can be used.

The test substance used in the screening method of the present invention is not particularly limited. Extracts derived from animals and plants, fungal cultures, or products of these substances treated with enzymes, compounds or derivatives thereof, etc. can be used as the test substance, and they can be in liquid, gaseous, powdered, gel, etc.

The test substance can be added to the structure containing the nitrated culture support substrate and fibroblasts at any concentration and amount. The culture conditions after the addition of the test substance may be the same as those for culturing fibroblasts in general, for example, at 37°C under humidified conditions of 5% _CO2 for several hours to several days, or up to about 30 days.

In the screening method of the present invention, the cellular activity and/or gene expression level or protein expression level of fibroblasts are used as indicators.

When using fibroblast cell activity as an indicator, for example, cell proliferation of fibroblasts and/or contraction force of collagen gel can be used as an indicator of cell activity, but is not particularly limited thereto. When using fibroblast cell proliferation as an indicator of cell activity, for example, if the presence of the test substance can increase cell proliferation of fibroblasts cultured with a nitrated culture support substrate compared to a non-added group, it can be determined that there is an effect. A substance that increases fibroblast cell proliferation by preferably 5% or more, more preferably 10% or more compared to the absence of the test substance, may be selected.

When the contraction force of collagen gel by fibroblasts is used as an index of cell activity, for example, collagen gel is used as a culture support substrate, and if the presence of the test substance increases collagen gel contraction by fibroblasts cultured with nitrated collagen gel compared to the control group, and the collagen gel can be made to have a more contracted shape, it can be determined that the test substance is effective. A substance that increases collagen gel contraction by preferably 5% or more, and more preferably 10% or more, compared to the absence of the test substance can be selected.

When the gene expression level or protein expression level of fibroblasts is used as an index, it is not particularly limited, but for example, the gene expression level or protein expression level of an extracellular matrix protein, an extracellular matrix protein cross-linking enzyme, an extracellular matrix decomposition enzyme, or an inhibitor of an extracellular matrix decomposition enzyme produced by fibroblasts can be used. Examples of the extracellular matrix protein include collagen, tropoelastin, fibrillin, fibulin, EMILIN, versican, fibronectin, laminin, podocan, fibromodulin, etc. Examples of the extracellular matrix protein cross-linking enzyme include lysyl oxidase and transglutaminase. Examples of the extracellular matrix decomposition enzyme include matrix metalloproteinases (MMPs), elastase, serine protease, cysteine protease, etc. Examples of the inhibitor of the extracellular matrix decomposition enzyme include tissue inhibitor of metalloproteinase (TIMP) and elafin, etc.
When the presence of the test substance can increase or decrease the gene expression level or protein expression level of fibroblasts cultured with a nitrated culture support substrate compared to the non-addition group, it can be judged to be effective. A substance that increases or decreases the cell proliferation of fibroblasts by preferably 5% or more, more preferably 10% or more, compared to the non-addition of the test substance may be selected. For genes or proteins whose expression levels have been reported to increase in photoaging skin, a substance that decreases the expression level compared to the non-addition of the test substance may be selected, and for genes or proteins whose expression levels have been reported to decrease in photoaging skin, a substance that increases the expression level compared to the non-addition of the test substance may be selected. Examples of genes or proteins whose expression levels have been reported to increase in photoaging skin include tropoelastin, fibrillin, matrix metalloproteinases (MMPs), elastase, elafin, etc., and examples of genes whose expression levels have been reported to decrease in photoaging skin include collagen, podocan, fibromodulin, lysyl oxidase, etc.

The cell proliferation of fibroblasts can be measured by any method. For example, cell proliferation can be measured by known methods such as a method of directly counting the number of cells, a method of staining the cell nuclei and counting the number of cells, a method of measuring the DNA synthesis of cells using BrdU or the like, a method of measuring the metabolic activity of cells using metabolic reducing dyes such as WST and MTT, a method of measuring intracellular ATP by luminoassay, etc.

Collagen contraction by fibroblasts can be measured by known methods. For example, since gel-like collagen contracts due to the coexistence of fibroblasts and gel-like collagen, it is possible to evaluate collagen contraction by measuring the volume and surface area of the gel-like collagen before and after contraction directly or by photographing, or by visually scoring it.

The expression level of the gene in fibroblasts or the protein converted thereby can be measured by any method. For example, a DNA fragment having a sequence that specifically binds to the sequence of the gene can be used as a primer to perform quantitative detection by agarose electrophoresis or real-time PCR. The gene sequences encoding the various factors described above are each publicly available, and a person skilled in the art can design appropriate primers and subject them to PCR. In addition, the expression level of the protein produced in fibroblasts can be measured by known methods such as nucleic acid hybridization using solid-phase samples such as gene chips and arrays, subtraction, differential display, differential hybridization, and cross-hybridization. The expression level of the protein produced in fibroblasts can also be measured by quantitatively measuring the expression level by conventional methods such as Western blotting, ELISA, radioimmunoassay, immunostaining, and mass spectrometry. The amount of the protein can also be measured indirectly by measuring the metabolic products of the protein.

The present invention will be explained in more detail below with reference to examples, but the present invention is not limited to these examples.

<Experiment 1> Localization of elastin and nitrotyrosine in sections of photoaged and naturally aged skin Elastin and nitrotyrosine were detected in sections of skin by the following procedure.
Formalin-fixed, paraffin-embedded sections of photoaged skin (exposed area, cheek) and naturally aged skin (unexposed area, buttocks) were prepared from a woman in her 90s who gave informed consent, and immunostained using anti-nitrotyrosine antibody (Stressmarq) and elastin antibody (Abeam) as primary antibodies, and AlexaFluor(R) 488-labeled anti-mouse antibody (Abeam) and AlexaFluor(R) 594-labeled anti-rabbit antibody (Abeam) as secondary antibodies, and the fluorescent images were observed under a fluorescence microscope (Keyence) (magnification 10x).

As shown in Figure 1, more nitrotyrosine was detected in the dermis of sun-exposed skin than in the dermis of non-sun-exposed skin, indicating that nitrotyrosine is present in the extracellular matrix of the dermis in photoaged skin. Furthermore, nitrotyrosine in sun-exposed skin was localized in roughly the same location as elastin, suggesting the presence of nitrated elastin in photoaged skin.

<Experiment 2> Change in cell proliferation of fibroblasts cultured on nitrated elastin fibers The cell proliferation rate of fibroblasts cultured on elastin fibers that had been nitrated according to the following procedure was measured.
A silicon support sheet (Extracellular Matrix Research Institute, Inc.) was laid on a 30φ glass petri dish (As One Co., Ltd.), and an elastin fiber sheet (Extracellular Matrix Research Institute, Inc.) was laid on top of it. A silicon support sheet with a 1.2 mm circular hole in the center was then laid on top of it to press the periphery of the elastin fiber sheet. 0.2 M phosphate buffer (pH 7.5) was added to the glass petri dish, and a 10% tetranitromethane (TNM) solution dissolved in EtOH was added to a concentration of 0.1%. After incubation at room temperature for 20 minutes, an aqueous urea solution was added to a final concentration of 2 M to stop the reaction. The solution was discarded, and the sheet was washed several times with PBS (-) while stirring the glass petri dish. Human adult dermal fibroblasts were suspended in D-MEM supplemented with 10% FBS, seeded in 2 mL portions on glass petri dishes, and cultured at 37°C under humidified conditions of 5% CO ₂ /95% air for 5 days. After the culture was completed, the medium was discarded, and 2 mL of D-MEM containing 10% WST-8 (Dojindo) and 10% FBS was dispensed into glass dishes and incubated at 37°C under humidified conditions of 5% CO ₂ /95% air until the medium color changed. 200 μL of the medium was dispensed into 96-well plates (TrueLine), and the absorbance at 450 nm was measured using a microplate reader (TECAN), and cell proliferation was calculated according to formula 1.

As shown in Table 1, fibroblasts cultured on nitrated elastin fibers showed a significant decrease in cell proliferation compared to those cultured on untreated elastin fibers, confirming that fibroblasts using nitrated elastin as a culture support substrate showed decreased cell proliferation.

<Experiment 3> Collagen Contraction Effect of Fibroblasts Cultured on Nitrated Collagen Gel The collagen contraction effect of fibroblasts cultured on a nitrated collagen gel was evaluated according to the following procedure.
CellMatrixI-A (Nitta Gelatin), 10x PBS(-), and 0.05N NaOH were ice-cooled and mixed at a ratio of 8:1:1. This was dispensed into 6-well plates (Corning) at 1.5 mL each, and left to stand for 30 minutes at 37°C under humidified conditions of 5% CO ₂ /95% air to prepare collagen gels. A 10% tetranitromethane (TNM) solution dissolved in EtOH was added to PBS(-) to a concentration of 0.1%, and 2 mL each was dispensed into 6-well plates. After incubation at room temperature for 15 minutes, 2 mL each of 4M urea aqueous solution was added and mixed to stop the reaction. The solution was discarded and PBS(-) was added, and the plate was stirred for several hours. This washing operation with PBS(-) was repeated several times. Human adult dermal fibroblasts were suspended in D-MEM supplemented with 10% FBS, seeded in 3.5 mL portions in 6-well plates, and cultured for 1 day at 37°C under humidified conditions of 5% CO ₂ /95% air. After replacing the medium, the outer circumference of the collagen gel in the 6-well plate was traced with a syringe needle to release the adhesion between the collagen gel and the container. After further culturing for 5 days under humidified conditions of 37°C and 5% CO ₂ /95% air to cause the collagen gel to contract, an image of the collagen gel was taken with a camera. The area of the collagen gel was calculated from the image using Image J (National Institutes of Health), and collagen contraction by fibroblasts was calculated according to Equation 2.

As shown in Table 2, fibroblasts cultured on nitrated collagen gel showed reduced collagen gel contraction compared to those cultured on untreated collagen gel, confirming that fibroblasts using nitrated collagen gel as a culture support substrate showed reduced collagen contraction.

<Experiment 4> Changes in gene expression levels in fibroblasts cultured on nitrated elastin fibers The gene expression levels in fibroblasts cultured on nitrated elastin fibers were measured by the following procedure.
A silicon support sheet was laid on a 30φ glass petri dish, and an elastin fiber sheet was placed on top of it. A silicon support sheet with a 1.2 mm circular hole in the center was placed on top of the elastin fiber sheet to press the periphery of the elastin fiber sheet. 0.2 M phosphate buffer (pH 7.5) was added, and Peroxynitrite (Dojindo) was dropped to 300 μM. After incubation at 37°C for 2 days, the elastin fiber sheet was washed several times with PBS (-). Human adult dermal fibroblasts were suspended in D-MEM supplemented with 10% FBS, seeded on a glass petri dish, and cultured for 5 days at 37°C under humidified conditions of 5% CO ₂ /95% air. Total RNA was extracted using a Total RNA Purification Kit (Jena Bioscience). Then, reverse transcription was performed using PrimeScript RT Reagent Kit (TaKaRa) to synthesize cDNA. Using the obtained cDNA as a template, the expression levels of proteins constituting the extracellular matrix, type I collagen, tropoelastin, fibrillin-1, fibulin 2, fibulin 4, fibulin 5, EMILIN, versican, and GAPDH (glyceraldehyde 3-phosphate dehydrogenase; used as a housekeeping gene), were measured by real-time PCR (7500 Real Time PCR System, Applied Biosystems) using gene-specific primers and Power SYBR Green Master Mix (Applied Biosystems), and gene expression was analyzed by the comparative Ct method. In other words, the change in gene expression level of fibroblasts cultured on a nitrated elastin fiber sheet was calculated as a relative amount to the tropoelastin Ct value of fibroblasts cultured on an untreated elastin fiber sheet, which was set to 1, corrected by the Ct value of GAPDH.

As shown in Table 3, in fibroblasts cultured on nitrated elastin fibers, there was almost no change in the gene expression levels of type I collagen (COL1A1), fibrillin-1, fibulin-2, fibulin-4, and fibulin-5 compared to the control, but the gene expression level of tropoelastin increased and the gene expression levels of EMILIN and versican decreased.

The above results revealed that the dermis of photoaged skin contains many nitrated proteins, and that nitration occurs in extracellular matrix proteins, particularly elastin fibers. In addition, it was found that when the extracellular matrix proteins that serve as a scaffold for cells are nitrated, dermal fibroblasts experience reduced cell proliferation and reduced collagen gel contraction, as well as changes in the gene expression levels of extracellular matrix proteins. Since such changes in cell function are similar to the characteristics of fibroblasts in photoaged skin, it was believed that the behavior of fibroblasts in photoaged skin could be mimicked by culturing fibroblasts using a nitrated culture support matrix. The photoaged cell model of the present invention and the method for evaluating and/or selecting a substance that can prevent or improve signs of photoaging, such as hardening and reduced elasticity of the skin, and the associated formation of wrinkles, sagging, and reduced firmness, are based on the above findings.

Example 1: Preparation of a photoaged cell model by culturing fibroblasts on nitrated fibronectin A human plasma-derived fibronectin solution (FUJIFILM) was dissolved in PBS(-) to prepare a concentration of 10 μg/mL. 1 mL of the solution was dispensed into a 6-well plate and incubated at room temperature for 3 hours. The solution was removed and washed with PBS(-). 2 mL of PBS(-) was dispensed into a 6-well plate, and a 10% tetranitromethane (TNM) solution dissolved in EtOH was added to PBS(-) to a concentration of 0.1%. After incubation at room temperature for 10 minutes, 2 mL of 4M urea aqueous solution was added and mixed to stop the reaction. The solution was discarded, PBS(-) was added, and the plate was washed several times with PBS(-) while stirring. Human dermal fibroblasts isolated from neonatal foreskin were suspended in D-MEM supplemented with 10% FBS to a density of 2.0 x ¹⁰⁵ cells/mL. 2 mL of fibroblasts were seeded into 6-well plates and cultured overnight at 37°C under humidified conditions of 5% _CO2 /95% air. The medium was discarded, and 1 mL of D-MEM supplemented with 10% FBS was dispensed, followed by culture for 7 days at 37°C under humidified conditions of 5% _CO2 /95% air.

Example 2: Preparation of a photoaged cell model containing fibroblasts in nitrated collagen CellMatrix I-A (Nitta Gelatin Co., Ltd.), 10x PBS (-), and 0.05N NaOH were mixed in a ratio of 8:1:1 on ice. Peroxynitrite was added dropwise to this to make it 1 mM, and mixed well on ice. Mouse fetal fibroblasts were suspended in D-MEM supplemented with 10% FBS to a density of 2.0 x 10 ⁵ cells/mL, and 1/5 of the fibroblast suspension was added to the collagen solution and mixed well by inversion. 2 mL of this cell suspension collagen was seeded on a 12-well plate and cultured overnight at 37°C under humidified conditions of 5% CO ₂ /95% air to prepare a gel-like structure.

Example 3 Screening method for cell proliferation promoters Screening for cell proliferation promoters for fibroblasts cultured on a nitrated elastin sheet was carried out according to the following procedure.
Preparation of test substances
A 50% (v/v) ethanol solution was added to each of a number of dried plant substances in an amount ten times the weight of the original plant substances, and the mixture was extracted at room temperature for one week. The dried residue of the extract was diluted with ethanol and water in a weight ratio of 1:50:49 to prepare a test substance. A 50% ethanol solution, which was the solvent, was used as a control substance.
<Cell proliferation test>
A porcine-derived water-soluble elastin type-A solution (FUJIFILM) was dissolved in PBS(-) to prepare a concentration of 100 μg/mL. 50 μL was dispensed into a 96-well plate and incubated at room temperature for 3 hours. The solution was removed and washed with PBS(-). 100 μL of 20 mM sodium acetate solution was dispensed into a 96-well plate (TrueLine), 10 μL of a reaction solution containing 1 M NaNO ₂ , 1 mM FeCl ₃ , and 3% hydrogen peroxide was added, and the plate was incubated at 37° C. for 1 day, after which the solution was discarded and the plate was washed several times with PBS(-). Human adult dermal fibroblasts were suspended in D-MEM supplemented with 10% FBS to a density of 5.0×10 ⁴ cells/mL. Fibroblasts were seeded in 96-well plates at 100 μL each and cultured for 1 day at 37° C. under humidified conditions of 5% CO ₂ /95% air. The medium was replaced with a new one, and each test substance or control substance was added to a final concentration of 100 ppm, and cultured for 3 days at 37° C. under humidified conditions of 5% CO ₂ /95% air. After the culture was completed, the medium was discarded, and 200 μL each of D-MEM containing 10% WST-8 (Dojindo) and 10% FBS was dispensed into 96-well plates and incubated at 37° C. under humidified conditions of 5% CO ₂ /95% air until the medium color changed. The medium was dispensed in 200 μL portions into 96-well plates, and the absorbance at 450 nm was measured using a microplate reader (TECAN). The cell proliferation (%) of each test substance was calculated according to Equation 3, assuming that the addition of the control substance was 100%, and test substances that showed cell proliferation of 105% or more were determined to be effective ingredients.

If the cell proliferation of fibroblasts when the test substance is added is 105% or more compared to when the control substance is added, it can be determined that the test substance has a sufficient effect of promoting the proliferation of fibroblasts cultured on nitrated elastin. This method can be used to select a cell proliferation promoter for fibroblasts, and this screening method can be used to select substances that prevent or improve photoaging, such as skin hardening inhibitors, inhibitors of loss of skin elasticity, and agents for preventing and improving wrinkle formation, sagging formation, and loss of firmness.

Example 4 Screening method for tropoelastin gene expression inhibitors Screening for tropoelastin gene expression inhibitors in fibroblasts cultured on nitrated elastin sheets was carried out according to the following procedure.
Preparation of test substances
A ten-fold weight of water was added to each of the dried plant raw materials, and the mixture was heated and extracted at 60° C. for 5 hours. The dried residue of the extract was diluted with water at a weight ratio of 1:99 to prepare the test substance. Water, the solvent, was used as the control substance.
<Measurement of gene expression level>
A silicon support sheet was laid on a 24-well plate, and an elastin fiber sheet was attached to the silicon support sheet. 0.2 M phosphate buffer (pH 7.5) was added, and Peroxynitrite donor SIN-1 (Dojindo) was diluted with 20 mM HCl and dropped into the 24-well plate to a concentration of 100 μM. After incubation at 37°C for 3 days, the elastin fiber sheet was washed several times with PBS (-). Human dermal fibroblasts were suspended in D-MEM containing 10% FBS and seeded on the 24-well plate, and cultured at 37°C under humidified conditions of 5% CO ₂ /95% air for 5 days. The medium was replaced with a new one, and each test substance or control substance was added to a final concentration of 100 ppm, and cultured at 37°C under humidified conditions of 5% CO ₂ /95% air for 1 day. Total RNA was extracted using Total RNA Purification Kit (Jena Bioscience). Then, reverse transcription was performed using PrimeScript RT Reagent Kit (TaKaRa) to synthesize cDNA. Using the obtained cDNA as a template, the expression levels of tropoelastin, a precursor protein of elastin, and GAPDH (glyceraldehyde 3-phosphate dehydrogenase; used as a housekeeping gene) were measured by real-time PCR (7500 Real Time PCR System, Applied Biosystems) using gene-specific primers and Power SYBR Green Master Mix (Applied Biosystems), and gene expression was analyzed by the comparative Ct method. That is, the change in tropoelastin gene expression level due to the addition of a test substance was calculated as a relative amount to the Ct value of the control substance-added group, which was corrected with the Ct value of GAPDH and set as 1. Test substances that reduced the tropoelastin gene expression level in each test substance-added group by 10% or more compared to the control substance-added group were determined to be effective ingredients.

It is known that the expression level of tropoelastin increases in photoaged skin, and the inventors have confirmed that the expression level of the tropoelastin gene increases in fibroblasts cultured on nitrated elastin based on their findings. Therefore, an active ingredient was selected that reduces the expression level of the tropoelastin gene when a test substance is added. If the expression level of the tropoelastin gene in fibroblasts when a test substance is added is reduced by 10% or more compared to when a control substance is added, it can be determined that the test substance has a sufficient inhibitory effect on the expression level of tropoelastin in fibroblasts cultured on nitrated elastin. This method can be used to select an inhibitor of tropoelastin gene expression, and this screening method can be used to select substances that prevent or improve photoaging, such as skin hardening inhibitors, inhibitors of reduced skin elasticity, and agents for preventing and improving wrinkle formation, sagging formation, and reduced firmness.

Example 5 Screening method for collagen contraction promoters Screening for collagen contraction promoters for fibroblasts cultured in nitrated collagen was carried out according to the following procedure.
Preparation of test substances
A ten-fold weight of 50% 1,3-butylene glycol was added to each of the dried plant materials, and extraction was performed for three days at 40° C. The filtrate was used as the test substance, and the solvent, 50% 1,3-butylene glycol, was used as the control substance.
Measurement of collagen contraction
CellMatrix I-A (Nitta Gelatin), 10x PBS (-), and 0.05N NaOH were ice-cooled and mixed in a ratio of 8:1:1. Peroxynitrite was added dropwise to this to make it 1 mM, and mixed well under ice-cooling. Human adult fibroblasts were suspended in D-MEM supplemented with 10% FBS to a density of 2.0 x 10 ⁵ cells/mL, and 1/5 of the fibroblast suspension was added to the collagen solution and mixed well by inversion. Further, each test substance or control substance was added to a final concentration of 1%, and further each test substance or control substance was added to a final concentration of 1%, and cultured at 37°C under humidified conditions of 5% CO ₂ /95% air for 1 day to prepare a gel-like structure. The collagen cell suspension was seeded in 12-well plates at 2 mL each and cultured overnight at 37° C. under humidified conditions of 5% CO ₂ /95% air to produce a gel-like structure. The outer circumference of the collagen gel in the 12-well plate was traced with a syringe needle to release the adhesion between the collagen gel and the container. After further culturing for 3 days under humidified conditions of 37° C. and 5% CO ₂ /95% air to cause the collagen gel to contract, an image of the collagen gel was taken with a camera. The area of the collagen gel was calculated from the image using Image J (National Institutes of Health), and the collagen contraction (%) of each test substance was calculated according to Formula 4 when the addition of the control substance was taken as 100%, and the test substance with collagen contraction of 105% or more was determined to be an effective ingredient.

If the collagen contraction when the test substance is added is 105% or more compared to when the control substance is added, it can be determined that the test substance has a sufficient effect of promoting the contraction of nitrated collagen by fibroblasts. This method can be used to select a collagen contraction promoter, and this screening method can be used to select a substance that prevents or improves photoaging, such as a skin hardening inhibitor, an inhibitor of reduced skin elasticity, or an agent for preventing or improving wrinkle formation, sagging formation, and reduced firmness.

Claims (5)

A method for screening an agent for preventing and/or improving photoaging, comprising:
A) culturing fibroblasts on a nitrated culture support substrate;
B) adding a test substance or a control substance and culturing ;
C) measuring cell proliferation of fibroblasts and/or the amount of tropoelastin gene expression or the amount of tropoelastin protein expression of fibroblasts;
D) selecting a test substance that changes cell proliferation and/or tropoelastin gene expression level or tropoelastin protein expression level in a test substance-added group compared to a test substance-free group or a control substance-added group;
The method comprising: A method for screening an agent for preventing and/or improving photoaging, comprising:
A) culturing fibroblasts and a nitrated culture support substrate containing collagen in a culture support base ;
B) adding a test substance or a control substance and culturing ;
C) measuring the collagen contractile activity of fibroblasts and/or the amount of tropoelastin gene expression or tropoelastin protein expression in fibroblasts;
D) selecting a test substance that exhibits changes in collagen contraction activity and/or tropoelastin gene expression level or tropoelastin protein expression level in a test substance-added group compared to a test substance-free group or a control substance-added group;
The method comprising: 3. The method according to claim 1, wherein the photoaging symptom is at least one of wrinkles, sagging skin, loss of firmness, hardening of the skin, and loss of elasticity. The screening method according to any one of claims 1 to 3 , wherein the culture support substrate is an extracellular matrix protein. 5. The screening method according to claim 1, wherein the culture support matrix contains elastin.