Methods for evaluating skin dryness
Measuring NFAT5 gene expression addresses the limitations of existing methods by providing rapid and accurate skin dryness assessment, facilitating early detection and treatment.
Patent Information
- Authority / Receiving Office
- JP · JP
- Patent Type
- Applications
- Current Assignee / Owner
- TOA CORP
- Filing Date
- 2024-11-12
- Publication Date
- 2026-05-22
Smart Images
Figure 2026084932000001_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a method for evaluating the dry state of the skin for objectively measuring and evaluating the dry state of the skin.
Background Art
[0002] Generally, dryness of human skin is a problem that many cosmetic users have. It is known that dryness itself causes various inflammatory reactions even without external stimuli, and finally, itching, rough skin, and fine wrinkles are considered to appear.
[0003] [[ID=十六]]Moreover, skin dryness is greatly affected by environmental factors even in people who feel they have healthy skin. That is, the skin of modern people who cannot avoid being exposed to overheating or air conditioners for a long time at home, in the office, and on the road often shows symptoms such as itching, rough skin, and fine wrinkles.
[0004] As a conventional method for evaluating the dry state of the skin, in order to evaluate the degree of dryness of skin cells with symptoms such as itching, rough skin, and fine wrinkles, a method of measuring the expression levels of AGR2 and AGR3, which are protein expression genes such as mucin, is known (Patent Document 1). <>
[0005] In such an evaluation method, by examining the expression levels of genes such as AGR2 and AGR3 related to the secretion of mucus such as mucin produced, the dry state of the skin according to the amount of mucus is evaluated, and the degree of rough skin is evaluated.
[0006] <00000> Also, detection kits that detect the concentration of substances such as taurine as the osmotic pressure regulator to know the degree of dehydration are known (Patent Document 2).
Prior Art Documents
Patent Documents
[0007]
Patent Document 1
[0008] However, the evaluation method described in Patent Document 1 evaluates the degree of symptoms that appear in the final stage of skin dryness, making it difficult to determine the state of skin dryness in the initial or preceding stages before such symptoms appear. In other words, to prevent skin dryness, it is necessary to assess the dryness level quickly and accurately using the simplest method possible, before symptoms such as itching, skin irritation, and fine wrinkles appear.
[0009] Furthermore, in the method for detecting osmotic pressure-regulating substances and measuring their concentration described in Patent Document 2 mentioned above, an osmotic pressure difference is created between the body's overall fluids and the water content cells. After this difference is established, osmotic pressure-regulating substances such as taurine secreted into the interstitial fluid of the skin are separated using an ion exchange membrane and their concentration is measured. Therefore, while it is possible to determine the degree of dehydration that has manifested, it has been difficult to evaluate the dryness of the skin, a specific organ, before the onset of dehydration symptoms.
[0010] Furthermore, the above method for detecting osmotic pressure-regulating substances requires the collection of interstitial fluid from the skin surface by adsorption onto a fixative, followed by separation using an ion-exchange membrane. Therefore, it is difficult to evaluate the dryness of the skin from a small amount of sample cells.
[0011] Therefore, the objective of this invention is to solve the above-mentioned problems and to create a novel method for evaluating the dryness of the skin before symptoms such as itching, roughness, and fine wrinkles appear, which can evaluate the dryness of the skin rapidly and with high accuracy from a small amount of skin cells. [Means for solving the problem]
[0012] In this invention, we focused on the osmotic stress response to elucidate the mechanism of skin dryness sensing. We discovered that the Transient Receptor Potential Vanilloid (TRPV) channel is involved in osmotic stress sensing in dry environments. Based on this, we focused on the expression level of the NFAT5 gene, which is involved in osmotic stress, and completed this invention by experimentally confirming that its expression level responds to the dryness state of the skin.
[0013] In other words, in order to solve the above problems, this invention provides a method for evaluating the dryness of the skin by measuring the expression level of NFAT5 in a subject using human skin cells and evaluating the expression level as an indicator that responds to osmotic stress on the skin.
[0014] NFAT5 (Nuclear Factor of Activated T-cell 5) is a transcription factor that regulates the expression of genes induced by osmotic stress in mammalian cells such as human cells. It is a protein belonging to the NFAT family, and its structure is registered in the Protein Data Bank (PDB).
[0015] As described above, the invention, which is configured to measure the gene expression level of a predetermined protein, uses NFAT5 expression, which is expressed when skin cells sense osmotic stress, as an indicator. This is because the protein expressed by NFAT5 in human skin cells is a transcription factor that regulates the expression of genes induced by osmotic stress. In other words, it uses NFAT5 expression, which is expressed when skin cells sense osmotic stress, as an indicator. Compared to conventional methods that use proteins produced at the time when symptoms of skin dryness or roughness appear as indicators, this method allows for the rapid and highly accurate assessment of skin dryness at an early stage, before the onset of symptoms.
[0016] The subjects used to measure the expression level of NFAT5 in human skin cells can be skin cells collected and cultured from humans, or a three-dimensional cultured skin model can be used.
[0017] When evaluating the dry state of a subject's skin, the effect of such a drug can also be examined by administering a drug such as a suppressant for atopic dermatitis to the subject's skin in advance.
[0018] For example, as a suppressant for atopic dermatitis, it is possible to examine the effect of a TRPV4 inhibitor that inhibits the expression of TRPV4, which is a sensor molecule involved in a mechanism that senses various physicochemical stimuli applied to the body due to changes in osmotic pressure and transmits them to cells, and the said mechanism.
[0019] The above-described method for evaluating the dry state of the skin can rapidly evaluate the early dry state of the skin before the onset, and is also useful for prevention to prevent the symptoms of the dry state of the skin from appearing, selection of pharmaceutically active ingredients for alleviating the developed symptoms, and as a method for discriminating the quality of the efficacy of drugs.
[0020] In addition, since dry skin cells are also undergoing an inflammatory reaction under osmotic stress, When the above-described dry state of the skin is an inflammatory state due to skin dryness, the present invention can also be used as a method for evaluating the inflammatory state due to skin dryness.
Effect of the Invention
[0021] This invention measures the expression level of NFAT5 in a subject using human skin cells, and evaluates the dry state of the skin by using the expression level as an index responsive to skin osmotic stress. Thus, it becomes a method for evaluating the dry state of the skin before the symptoms of skin itching, rough skin, and fine wrinkles appear, and has the advantage that the dry state of the skin can be evaluated quickly and with high accuracy from a small amount of sample cells.
Brief Description of the Drawings
[0022] [Figure 1] Chart showing the relationship between the NFAT5 expression level in three-dimensional cultured epidermis and environmental humidity [Figure 2] Chart showing the relationship between the NFAT5 expression level in three-dimensional cultured epidermis and environmental humidity or a TRPV4 antagonist (inhibitor) [Figure 3] Chart showing the relationship between the IL-1a expression level in the three-dimensional cultured epidermis and environmental humidity
Mode for Carrying Out the Invention
[0023] In an embodiment of this invention, when evaluating the dry state of the skin, skin cells collected from a subject are used as a subject using human skin cells, preferably the skin cells are used as a subject in a three-dimensional cultured skin model, and the expression level of the NFAT5 gene according to the dry state of this subject is measured. At the same time, the change in the expression level according to the dry state is examined, and the expression level is evaluated as an index responding to the osmotic stress of the skin.
[0024] The "dry state of the skin" in this invention includes not only a state where some symptoms, that is, "itching of the skin, rough skin, and fine wrinkle symptoms appear" due to skin dryness, but also the stage where osmotic stress occurred before that.
[0025] The skin cells collected from a subject who is a human used in this invention are preferably skin cells from the perspective of evaluating the dry state of the skin. Among the skin cells, it is more preferable to use epidermal keratinocytes in the epidermal layer rather than fibroblasts in the dermis. For example, if necessary in an evaluation test, commercially available products such as normal human epidermal keratinocytes isolated from human skin (NHEK, manufactured by Kurabo Industries, Ltd., etc.) can be used.
[0026] Such skin cells can be cultured in a liquid medium with the collected skin cells as a subject, and preferably a three-dimensional cultured skin model (LabCyte EPI-MODEL) cultured in a commercially available assay medium so that the skin cells are layered into a three-dimensional structure close to human epidermis can be used as a subject.
[0027] The NFAT5 gene used in this invention is a well-known transcription factor known as Nuclear factor of activated T-cell 5, which regulates the expression of genes that respond to osmotic stress, such as organic osmotic pressure, aggrecan, pro-inflammatory cytokines, IL-1b, IL-8, and TNF-α.
[0028] As described later, the expression level of the NFAT5 gene can be evaluated as a relative value by synthesizing cDNA from RNA isolated from cultured cells using PCR, normalizing it to the RPL13A gene, comparing the relative mRNA level with a control sample, and calculating it using the ΔΔCt method. [Examples]
[0029] Three-dimensional cultured human epidermis (Reconstructed human epidermis; RHE, LabCyte EPI-MODEL, Japan Tissue Engineering Co., Ltd.) was cultured in assay medium (Japan Tissue Engineering Co., Ltd.) using a 24-well plate.
[0030] One group was incubated in a high-humidity environment (over 90% humidity) as the control group, while the other group was incubated in a dry environment using a desiccant (less than 15% humidity) as the treatment group. Both groups were incubated at 37°C and 5% CO2 for 6 hours.
[0031] Both the control group and the treatment group were divided into two groups: one receiving 10 nM of the TRPV4 antagonist (inhibitor) GSK2193874 (manufactured by Cayman Chemical), and the other not receiving it. The expression levels of NFAT5 in these four groups were then examined as follows.
[0032] Total RNA from three-dimensional cultured human epidermis (RHE) was extracted using the Quick Gene RNA Tissue Kit SII (Kurabo Industries Ltd.). Specifically, cDNA was synthesized from 1 μg of total RNA using the QuantiTect Reverse Transcription Kit (QIAGEN).
[0033] The real-time polyclonal chain reaction (RT-PCR) experiment in this case was performed using the ABI KAPASYBR FAST qPCR Kit (manufactured by Bio-Rad Laboratories). The expression of each gene was normalized relative to the RPL13A gene, and relative mRNA levels were calculated using the ΔΔCt method compared to an untreated, time-adjusted control sample.
[0034] As is clear from the experimental results shown in Figure 1, we confirmed that NFAT5 expression increases in experiments simulating dry skin conditions using a three-dimensional cultured human epidermis model incubated at low humidity.
[0035] These results suggest that when the skin senses dryness, it responds to osmotic stress caused by an increase in solute concentration. This provides new insights into solving the problem of dry skin, as measuring the expression level of NFAT5 can detect skin dryness.
[0036] Furthermore, to elucidate the molecular mechanism more precisely, we investigated the involvement of the TRPV4 channel, and the results are shown in Figure 2.
[0037] The TRPV family is a subfamily of TRP (Transient receptor potential) channels, and its function as an osmotic sensor has been demonstrated in experiments using the nematode *C. elegans*.
[0038] Furthermore, it is well known that TRPV4 is involved in osmotic pressure sensing and vasopressin secretion, as demonstrated using TRPV4-deficient mice. By comprehensively utilizing these findings, we inhibited the TRPV4 channel and evaluated NFAT5 expression in keratinocytes.
[0039] As is clear from the results shown in Figures 1 and 2, in the RHE model, applying a TRPV4 channel inhibitor under low humidity conditions suppressed the increase in NFAT5 expression induced by dryness. This result indicated that the TRPV4 channel is involved in dryness sensing in human skin.
[0040] Figure 3 shows the changes in IL-1a expression levels in RHE incubated at high humidity (High RH) and low humidity (Low RH) for 6 hours, with P<0.001.
[0041] As shown in Figure 3, IL-1a expression was upregulated, suggesting that drying stimulation may have induced IL-1a expression in RHE.
[0042] From the results of the above embodiments, the following was found, and the effectiveness of this invention was confirmed. We confirmed that NHEK cells, which are keratinocytes of the human epidermis, can respond to osmotic stimuli. Localized hyperosmotic stress is a contributing factor to skin diseases, and keratinocytes responded to this stress by producing NFAT5 or pro-inflammatory cytokines.
[0043] Since NFAT5 is a major transcription factor involved in body hydration, upregulation of NFAT5 expression has been shown to be involved in sensing osmotic pressure and detecting skin dryness.
[0044] In other words, based on the correlation that NFAT5 expression levels increase as the humidity environment of skin cells decreases, it was possible to use the expression level as an indicator of response to osmotic stress in the skin, and to evaluate the effect of adding a dry skin dermatitis inhibitor to the skin cells, etc., as a method for evaluating the dryness of the skin.
[0045] This finding represents a new aspect of how the skin detects dryness, suggesting that the TRPV4 channel may also be involved in a more precise molecular mechanism for sensing and evaluating skin dryness.
[0046] This invention relates to a method for objectively and accurately measuring and evaluating the dryness of the skin. This evaluation method can be used not only in the field of cosmetics to evaluate efficacy, but also in the fields of pharmaceuticals and cosmetics manufacturing and development, such as for developing and selecting medicinal ingredients that help prevent the appearance of symptoms of dry skin or alleviate symptoms that have occurred, and for determining the effectiveness of drugs.
Claims
1. A method for evaluating the dryness of the skin, comprising measuring the expression level of NFAT5 in a subject using human skin cells and evaluating the expression level as an indicator of the skin's response to osmotic stress.
2. The method for evaluating the dryness of the skin according to claim 1, wherein the subject is cultured human skin cells.
3. The method for evaluating the dryness of the skin according to claim 2, wherein the subject is a three-dimensional cultured skin model using cultured human skin cells.
4. A method for evaluating the dryness of the skin according to any one of claims 1 to 3, wherein the dryness of the skin described above is an inflammatory state caused by dryness of the skin.