How to assess dry skin

Abstract

To create a novel method for evaluating the dry state of skin before symptoms such as itching, rough skin and fine wrinkles appear, which can evaluate the dry state of skin quickly and with high accuracy using a small amount of sample cells. [Solution] The expression level of NFAT5 in a subject, such as a three-dimensional cultured skin model using skin cells, is measured, and based on the correlation that the lower the humidity environment of the skin cells, the higher the NFAT5 expression level, the expression level is used as an indicator of response to osmotic stress in the skin, and a method for evaluating the effect of adding an inhibitor of dry dermatitis to the skin cells is provided.

Description

[Technical Field]

[0001] The present invention relates to a method for evaluating the dryness of skin for objectively measuring and evaluating the dryness of skin. [Background technology]

[0002] In general, dry skin is a concern for many cosmetic users, and it is known that dry skin itself can trigger various inflammatory reactions even without any external stimuli, ultimately leading to symptoms such as itching, rough skin, and fine wrinkles.

[0003] Furthermore, even in people who consider themselves to have healthy skin, dry skin is greatly affected by environmental factors: modern people's skin is inevitably exposed to overheating and air conditioning for long periods at home, in the office, and on the road, and this often leads to itching, rough skin, and fine wrinkles.

[0004] A conventional method for evaluating the state of dry skin involves measuring the expression levels of AGR2 and AGR3, which are genes that express proteins such as mucin, to assess the degree of dryness of skin cells that manifest as symptoms such as itching, rough skin, and fine wrinkles (Patent Document 1).

[0005] This evaluation method evaluates the degree of rough skin by examining the expression levels of genes AGR2 and AGR3, which are involved in the secretion of mucus such as mucin, and evaluates the state of dryness of the skin according to the amount of mucus.

[0006] Also known is a detection kit that detects the concentration of taurine or the like as an osmotic pressure adjusting substance to determine the degree of dehydration (Patent Document 2). [Prior art documents] [Patent documents]

[0007] [Patent Document 1] Japanese Patent Application Laid-Open No. 2015-227865 [Patent Document 2] Japanese Patent Application Publication No. 2019-070577 Summary of the Invention [Problem to be solved by the invention]

[0008] However, the evaluation method described in Patent Document 1 evaluates the degree of symptoms that appear in the final stage of dry skin, making it difficult to determine the state of dry skin in the early or pre-stage before such symptoms appear. In other words, in order to prevent dry skin, it is necessary to evaluate the state of dryness quickly and accurately using as simple a method as possible before symptoms such as itching, rough skin, and fine wrinkles appear.

[0009] Furthermore, in the method of detecting osmotic pressure adjusting substances and the method of measuring their concentrations described in Patent Document 2, after an osmotic pressure difference occurs between the body fluids throughout the body and the water cells, an ion exchange membrane is used to separate osmotic pressure adjusting substances such as taurine secreted into the interstitial fluid of the skin, and the concentration is measured. This makes it possible to know the degree of dehydration that has occurred, but makes it difficult to evaluate the dryness state of a specific organ, namely the skin, before symptoms of dehydration appear.

[0010] Furthermore, the above-mentioned method for detecting osmotic pressure adjusting substances requires that skin interstitial fluid be collected from the skin surface by adsorption onto a fixing agent, and then separated using an ion exchange membrane, making it difficult to evaluate the dryness state of the skin from a small amount of sample cells.

[0011] Therefore, the object of the present invention is to solve the above-mentioned problems and to create a novel method for evaluating the state of dry skin before symptoms such as itchy skin, rough skin, and fine wrinkles appear, which can evaluate the state of dry skin quickly and with high accuracy using a small amount of skin cells. [Means for solving the problem]

[0012] In this invention, we focused on the osmotic stress response in order to elucidate the mechanism of skin dryness sensing, and discovered that Transient Receptor Potential Vanilloid (TRPV) channels are involved in osmotic stress sensing in dry environments.We then focused on the expression level of the NFAT5 gene, a gene involved in osmotic stress, and experimentally confirmed that its expression level responds to the dry state of the skin, thereby completing this invention.

[0013] In other words, in order to solve the above problem, this invention provides a method for evaluating the state of dry skin by measuring the expression level of NFAT5 in a subject using human skin cells and evaluating the expression level as an indicator of response to osmotic stress of 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, including humans. 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 expression level of a gene that expresses a specific protein, uses the protein expressed by NFAT5 in human skin cells as an indicator, which is a transcription factor that regulates the expression of genes induced by osmotic stress. In other words, the invention uses NFAT5 expression, which is expressed when skin cells sense osmotic stress, as an indicator. Therefore, compared to conventional methods that use as an indicator a protein produced when symptoms of dry or rough skin appear, the invention can evaluate the state of dry skin quickly and with high accuracy at an early stage before the onset of symptoms.

[0016] The subject used to measure the expression level of NFAT5 in human skin cells can be skin cells collected from a human and cultured, or a three-dimensional cultured skin model can also be used.

[0017] Furthermore, when evaluating the dryness state of the subject's skin, it is also possible to examine the effect of a drug such as a drug for suppressing dry dermatitis by administering the drug to the subject's skin in advance.

[0018] For example, as a suppressant of dry dermatitis, it is possible to investigate the effect of TRPV4 inhibitors that inhibit the expression of TRPV4, a sensor molecule involved in the mechanism that senses various physicochemical stimuli imposed on the body by changes in osmotic pressure and transmits them to cells, or that inhibit this mechanism.

[0019] The above-mentioned method for evaluating dry skin conditions can quickly evaluate the state of dry skin at an early stage before the onset of symptoms, and can also be used as a method for preventing the symptoms of dry skin from appearing, for selecting medicinal ingredients that will help alleviate symptoms that have already occurred, and for determining the effectiveness of drugs.

[0020] In addition, dry skin cells are subject to osmotic stress and cause an inflammatory response, When the dry skin condition is an inflammatory condition caused by dry skin, the present invention can also be used as a method for evaluating an inflammatory condition caused by dry skin. [Effects of the Invention]

[0021] This invention measures the expression level of NFAT5 in a subject using human skin cells, and evaluates the state of dry skin by using the expression level as an indicator of response to osmotic stress in the skin.This provides a method for evaluating the state of dry skin before symptoms such as itchy skin, rough skin, and fine wrinkles appear, and has the advantage of being able to evaluate the state of dry skin quickly and with high accuracy using a small amount of sample cells. [Brief explanation of the drawings]

[0022] [Figure 1] Graph showing the relationship between NFAT5 expression levels in three-dimensionally cultured epidermis and environmental humidity [Figure 2] Graph showing the relationship between NFAT5 expression levels in three-dimensionally cultured epidermis and environmental humidity or TRPV4 antagonists (inhibitors). [Figure 3] Graph showing the relationship between IL-1a expression levels in three-dimensionally cultured epidermis and environmental humidity DETAILED DESCRIPTION OF THE INVENTION

[0023] An embodiment of the present invention is a method for evaluating the dryness state of skin, in which skin cells collected from a subject are cultured as a subject using human skin cells, preferably as a three-dimensional cultured skin model, and the expression level of the NFAT5 gene in response to the dryness state of the subject is measured, and changes in the expression level in response to the dryness state are examined, and the expression level is evaluated as an indicator of response to osmotic stress of the skin.

[0024] In this invention, "dry skin condition" refers not only to a condition in which some symptoms are caused by dry skin, i.e., "skin itching, rough skin, fine wrinkles," but also to the stage prior to this in which osmotic stress occurs.

[0025] In order to evaluate the state of dry skin, it is preferable to use skin cells collected from a human subject in this invention, and among skin cells, it is preferable to use epidermal keratinocytes located in the epidermis layer rather than fibroblasts located in the dermis layer. For example, if necessary for an evaluation test, commercially available products such as normal human epidermal keratinocytes isolated from human skin (e.g., NHEK, manufactured by Kurabo Industries, Ltd.) can be used.

[0026] Such skin cells can be collected and cultured in a liquid medium to serve as a subject, preferably a three-dimensional cultured skin model (LabCyte EPI-MODEL) cultured in a commercially available assay medium so that the skin cells are stratified into a three-dimensional structure similar to that of human epidermis.

[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, proinflammatory cytokines, IL-1b, IL-8, and TNF-α.

[0028] The expression level of the NFAT5 gene can be evaluated as a relative value by synthesizing cDNA by PCR from RNA isolated from cultured cells, normalizing it to the RPL13A gene, comparing the relative mRNA level with that of a control sample, and calculating it using the ΔΔCt method, as described below. [Example]

[0029] Three-dimensionally cultured human epidermis (Reconstructed human epidermis; RHE, LabCyte EPI-MODEL, Japan Tissue Engineering) was cultured in assay medium (Japan Tissue Engineering) using a 24-well plate.

[0030] One group was incubated in a high humidity environment (over 90%) as a high humidity (control group), and the other group was incubated in a dry environment (under 15%) using a desiccant as a low humidity (treatment group), both at 37°C and 5% CO2 for 6 hours.

[0031] Both the control and treatment groups were divided into two groups: one with 10 nM GSK2193874 (Cayman Chemical), a TRPV4 antagonist (inhibitor), and one without. The expression levels of NFAT5 in the four groups were examined as follows.

[0032] Total RNA from three-dimensionally cultured human epidermis (RHE) was extracted using the Quick Gene RNA Tissue Kit SII (Kurabo). 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) experiments were carried out using the ABI KAPASYBR FAST qPCR Kit (Bio-Rad Laboratories). The expression of each gene was normalized to the RPL13A gene, and relative mRNA levels were calculated by the ΔΔCt method compared with untreated time-matched control samples.

[0034] As is clear from the experimental results shown in Figure 1, we confirmed that NFAT5 expression increased in experiments simulating dry skin conditions using a three-dimensional cultured human epidermal 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, providing new insight into solving the problem of dry skin, suggesting that dry skin can be detected by measuring the expression level of NFAT5.

[0036] Furthermore, to clarify the precise molecular mechanism, we investigated the involvement of TRPV4 channels, 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 pressure sensor has been demonstrated in experiments using the nematode C. elegans.

[0038] Furthermore, using TRPV4-deficient mice, it is well known that TRPV4 is involved in osmosensing and vasopressin secretion. Taking advantage of these findings, we inhibited the TRPV4 channel and evaluated the expression of NFAT5 in keratinocytes.

[0039] As shown in Figures 1 and 2, in the RHE model, application of a TRPV4 channel inhibitor under low humidity conditions suppressed the dry-stimulated increase in NFAT5 expression. These results indicate that the TRPV4 channel is involved in the perception of dryness in human skin.

[0040] FIG. 3 shows the changes in IL-1a expression levels in RHE incubated for 6 hours at high humidity (High RH) and low humidity (Low RH), with ***P<0.001.

[0041] As shown in Figure 3, IL-1a expression was upregulated, suggesting that dryness stimulation may induce IL-1a expression in RHE.

[0042] From the results of the above examples, the following was found, and the effects of the present invention were confirmed. We confirmed that human epidermal keratinocytes, NHEK cells, can respond to osmotic stress. Local hyperosmotic stress contributes to skin diseases, and keratinocytes respond to this stress by producing NFAT5 or proinflammatory cytokines.

[0043] Because NFAT5 is a major transcription factor involved in water management in the body, upregulation of NFAT5 expression indicated that it is involved in sensing osmotic pressure and detecting skin dryness.

[0044] In other words, based on the correlation that the lower the humidity environment of skin cells, the higher the NFAT5 expression level, the expression level can be used as an indicator of response to osmotic stress in the skin, and can be used as a method for evaluating the dry state of the skin to evaluate the effect, for example, of adding an inhibitor of dry dermatitis to the skin cells.

[0045] This finding represents a new aspect of how the skin detects dryness and suggests that TRPV4 channels may also be involved as a more sophisticated molecular mechanism for sensing and assessing skin dryness.

[0046] This invention relates to a method for evaluating the state of dry skin for objectively and accurately measuring and evaluating the state of dry skin. This evaluation method is not only useful in the field of cosmetics where efficacy is evaluated using this evaluation method, but can also be used in the fields of production and development of pharmaceuticals and cosmetics, such as medicines, as a method for developing and selecting medicinal ingredients that are useful for preventing the symptoms of dry skin from appearing or for alleviating symptoms that have already occurred, and for determining the efficacy of drugs.

Claims

1. A method for evaluating the dryness of skin, which involves measuring the expression level of NFAT5 in a subject using human skin cells by incubating the subject in a humidity environment of 90% or more and less than 15%, and using the expression level as an indicator for evaluation.

2. The method for evaluating the dryness state of skin according to claim 1, wherein the subject is cultured human skin cells.

3. The method for evaluating the dry state of skin according to claim 2, wherein the subject is a three-dimensional cultured skin model using cultured human skin cells.

4. The method for evaluating a dry skin condition according to any one of claims 1 to 3, wherein the dry skin condition is an inflammatory condition caused by dry skin.

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