JP7084214B2 - How to evaluate or search for a pleasant emotion enhancer - Google Patents

Description

The present invention relates to a method for evaluating or searching for a pleasant emotion enhancer, a dry skin or skin barrier function improving agent, or a skin texture improving agent that enhances pleasant emotions evoked by human tactile sensation.

The skin is susceptible to damage from external factors such as dryness and ultraviolet rays, and damage from internal factors such as stress, and such damage accumulates, reducing the amount of water inside the skin and the skin barrier function, resulting in firmness. Appearance of skin condition (skin texture) such as gloss, texture, moisture, and transparency is reduced. On the other hand, when using daily necessities such as cosmetics, humans experience various emotions. In particular, the feeling of using cosmetics arouses feelings of happiness / satisfaction, luxury, relaxation, refreshment, dissatisfaction, and disappointment.
Therefore, cosmetics development requires not only functional values such as improvement of dry skin or skin barrier function and improvement of skin texture, but also emotional values that exert pleasant feelings such as comfort, happiness, and satisfaction. , The development of materials that can add such value is underway.

In recent years, it has been reported that pleasant emotions evoked by tactile sensation are transmitted to the brain by C fibers called C tactile fibers, which are different from Aβ fibers and Aδ fibers. The intensity and speed of the stimulus are important for the excitement of such C tactile fibers, and it is known that the excitement is most intense when the skin is patted at a speed of 1 to 10 cm per second and 20 to 40 g with a brush member such as a brush. (Non-Patent Document 1). In addition, it has been reported that massage improves clinical indicators of atopic dermatitis such as lichen, scales, and abrasions (Non-Patent Document 4).

Recently, a G protein-coupled receptor, MrgprB4 (Mas-related G-protein coupled receptor member B4), is expressed in a nerve fiber corresponding to a C tactile fiber in mice, and by activating the nerve fiber expressing MrgprB4, It has been reported that pleasant emotions caused by tactile sensation are aroused (Non-Patent Document 2). Nerve fibers expressing MrgprB4 are considered to be activated during tactile stimulation such as affinity behavior and maternal behavior, and during self-stimulation such as self-grooming. It is also known that the response of cells expressing MrgprB4 can be measured by osmotic stimulation that expands the cells using primary cultured cells derived from the dorsal root ganglion of mice (Non-Patent Document 3).

Conventionally, it is known that activation of nerves expressed by MrgprB4 arouses pleasant emotions caused by tactile sensation. It was not revealed that pleasant feelings were aroused. Furthermore, since the human ortholog of MrgprB4 has not been identified, there is no known means for scientifically and quantitatively grasping the pleasant emotions evoked by the sense of touch in humans, and a material for improving the pleasant emotions evoked by the sense of touch. No effective means of searching for the disease has been known so far. Moreover, the relationship between MrgprB4 and the function of the skin and the texture of the skin is not known at all.

Loken et al., NATURE NEUROSCIENCE, Vol. 12, p547-548, 2009 Vrontou et al., NATURE, Vol. 493, p669-673, 2013 Yamaguchi and Otsuguro, NEUROSCIENCE LETTERS, Vol. 653, p139-145, 2017 Schachner et al., Pharmacology and Therapeutics, 1998

The present invention relates to a method for efficiently searching for a pleasant emotion improving agent, a dry skin or skin barrier function improving agent, or a skin texture improving agent caused by human tactile sensation.

The present inventor has clarified that an agent for improving pleasant emotions caused by human tactile sensation, a dry skin or skin barrier function improving agent, or a skin texture improving agent can be evaluated and searched using the activity of MrgprB4 as an index.

That is, the present invention relates to the following.
A human tactile-induced pleasant emotion enhancer, dry skin or A method for evaluating or searching for a skin barrier function improving agent or a skin texture improving agent.

According to the present invention, it is possible to easily and efficiently evaluate or search for a pleasant emotion improver, a dry skin or a skin barrier function improver, or a skin texture improver that improves the pleasant emotions evoked by human tactile sensation. Pleasure enhancers are useful as materials that enhance the emotional value of external skin preparations such as cosmetics.

Response activity of MrgprB4 by contraction stimulation after cell expansion. Effect of polyoxyethylene methylglucoside on relative MrgprB4 activity. Effect of polyoxyethylene methylglucoside on pleasant feelings due to tactile stimulation after application. Outline of the schedule of continuous tactile stimulation tests. Changes in the amount of inflammatory cytokines in the stratum corneum due to continuous tactile stimulation. Changes in skin texture due to continuous tactile stimulation.

In the present invention, the "pleasant emotion caused by the human tactile sensation" means the pleasant emotion evoked by the tactile sensation in humans. Pleasure includes comfort, happiness / satisfaction, luxury, and exhilaration (Kadoji et al., JOURNAL OF SOCIETY OF COSMETIC CHEMICALS OF JAPAN, Vol. 43, p10-18, 2009).
Examples of tactile stimuli that evoke pleasant emotions in humans include stimuli that excite C tactile fibers. Examples include the act of applying cream or lotion to the skin, and the act of gently wiping (stroking) the skin with a fabric product such as a towel.
It is known that the C tactile fibers are most strongly excited when the skin is patted with a brush member such as a brush at a speed of 1 to 10 cm per second and 20 to 40 g (Non-Patent Document 1).

In the present invention, "improvement of dry skin or skin barrier function" refers to a decrease in water content inside the skin or an increase in scale or roughness on the skin surface (dry skin), or a skin barrier function (evaporation of water from the inside of the skin). It means to suppress or improve the deterioration of the function of preventing and the function of preventing the invasion of substances from the outside into the skin).

In the present invention, the "skin texture" means the texture of the skin selected from the firmness, gloss, transparency, color unevenness, texture and smoothness of the skin surface, and is preferably firm, glossy, and transparent. And means the texture of the skin selected from uneven color.

The present inventors have found that when a cell expressing MrgprB4 is prepared and a stimulus is applied to the cell, the response of MrgprB4 changes accordingly.
Specifically, a material that reduces the response of MrgprB4 when a stimulus that causes the MrgprB4 expressing cells to expand and then contract is applied improves the pleasant emotions evoked by the excitement of human C tactile fibers. Was clarified (example below).
These results indicate that the response of MrgprB4 can be measured easily and efficiently, and that the response of MrgprB4 can be used as an index to evaluate and search for a pleasant emotion-improving agent caused by human tactile sensation.
Furthermore, the tactile stimulus that evokes pleasant feelings, that is, the stimulus that excites the C tactile fibers, suppresses the decrease in the water content of the stratum corneum of the skin, improves the roughness of scales and the surface of the skin, and relieves the inflammation of the skin. It was confirmed that the effect of improving the dry skin or skin barrier function was brought about (Example 2), the smoothness of the skin surface of the facial skin was improved, and the effect of improving the texture of the skin was brought about (Example 3).
Therefore, it can be said that it is possible to evaluate and search for a dry skin or skin barrier function improving agent and a skin texture improving agent using the response of MrgprB4 as an index.

The method for evaluating or searching for a pleasant emotion improving agent, a dry skin or a skin barrier function improving agent or a skin texture improving agent caused by the human tactile sensation of the present invention (I) stimulates MrgprB4 expressing cells in the presence of the test substance. It comprises a step of imparting and (II) a step of measuring the response of MrgprB4 to the stimulus.
Specifically, it is performed by the following steps (A) to (E).
(A) Step of expanding MrgprB4 expressing cells (B) Step of contracting expanded cells in the presence of a test substance (C) Step of measuring the response of MrgprB4 to cell contraction (D) Step of measuring MrgprB4 based on the measured response The process of identifying test substances that reduce the response of
(E) The identified test substance is 1) evaluated or selected as a tactile-induced pleasant emotion improving agent, 2) evaluated or selected as a dry skin or skin barrier function improving agent, or 3) skin texture. Process to evaluate or select as an improver

MrgprB4 (Mas-related G-protein coupled receptor member B4) is registered in NCBI as ID: 233230. Mouse MrgprB4 is a protein consisting of the amino acid sequence set forth in SEQ ID NO: 2, encoded by the gene having the nucleotide sequence set forth in SEQ ID NO: 1.

In the method of the present invention, as the MrgprB4 expressing cell, a tissue or cell that naturally expresses MrgprB4 such as an MrgprB4 positive posterior root ganglion nerve isolated from a living body, or a culture thereof; inherited to express MrgprB4. Examples thereof include recombinant cells or cultures thereof that have been manipulated.

Recombinant cells genetically engineered to express MrgprB4 are cells in which the nucleic acid encoding MrgprB4 has been introduced into the host cell so that the nucleic acid encoding MrgprB4 can be expressed, and the nucleic acid encoding MrgprB4 is the nucleic acid as an extrachromosomal element. Examples thereof include cells into which the nucleic acid has been introduced so as to be replicable, or cells into which the nucleic acid has been introduced so that the nucleic acid can be replicated by chromosomal integration. Here, the nucleic acid may be any of DNA, RNA, mRNA, cDNA and cRNA.
The cell in which the nucleic acid encoding MrgprB4 is expressively introduced into the host cell is MrgprB4 obtained by ligating the nucleic acid encoding MrgprB4 with a vector suitable for the expression of MrgprB4 in the host cell used. It can be obtained by introducing an expression vector into a host cell. Introduction of the nucleic acid encoding MrgprB4 into host cells is described, for example, in Sambrook, J., Fritsch, EF, and Maniatis, T., "Molecular Cloning A Laboratory Manual, Second Edition", Cold Spring Harbor Laboratory Press, (1989). It can be carried out according to the methods such as transformation and transfection described in the above, electroporation method, calcium phosphate method, lipofection method, DEAE dextran method, particle gun method, method using a virus and the like.

The nucleic acid encoding MrgprB4 is based on information on a known base sequence corresponding to a nucleic acid encoding MrgprB4, for example, information on a known base sequence corresponding to a nucleic acid encoding mouse MrgprB4 (SEQ ID NO: 1). It can be obtained by a PCR method using an appropriate primer pair prepared from a base sequence and / or a hybridization method using an appropriate probe prepared from the base sequence and a cDNA library.

The nucleic acid encoding MrgprB4 may be one or several in its base sequence if the polypeptide encoded by the nucleic acid expresses a function as a G protein-conjugated receptor after introduction into a cell. It may have a nucleotide deletion, substitution, addition or insertion. The nucleic acid encoding MrgprB4 is, for example, 1) a nucleic acid consisting of the base sequence shown in SEQ ID NO: 1, 2) 80% or more, preferably 90% or more, more preferably the base sequence shown in SEQ ID NO: 1. Is a nucleic acid consisting of a base sequence having 95% or more sequence identity, and the polypeptide encoded by the sequence is a polypeptide having a function as a G protein-conjugated receptor, 3) the base shown in SEQ ID NO: 1. Examples thereof include nucleic acids that hybridize under stringent conditions to the complementary strand of a nucleic acid consisting of a sequence, and the polypeptide encoded by the sequence is a polypeptide having a function as a G protein-conjugated acceptor. .. The nucleic acid of 2) or 3) may be a nucleic acid derived from a mouse or a nucleic acid derived from another animal such as a rat.

Here, the identity of the base sequence is calculated by the Lipman-Pearson method (Lipman, DJ., Pearson.WR .: Science, 1985, 227: 1435-1441). Specifically, it is calculated by performing an analysis with Unit size to homology (ktup) as 2 using a homology analysis (Search homology) program of genetic information processing software Genetyx-Win Ver.11 (software development).
Further, the "several" of "1 or several" used for deletion, substitution, addition or insertion of an integer in a base sequence is, for example, a maximum of 20% or less of the total number of integers in the target nucleotide sequence. An integer, preferably a maximum integer of 10% or less, more preferably a maximum integer of 5% or less, still more preferably a maximum integer of 4% or less, still more preferably a maximum integer of 3% or less, still more preferably 2 as well. It means a maximum integer of% or less, and more preferably a maximum integer of 1% or less. Also herein, "addition" of a nucleotide includes the addition of one or several nucleotides to one end and both ends of the sequence.

Further, the "stringent condition" regarding hybridization is a condition that enables confirmation of a gene having a base sequence having a sequence identity of about 80% or more or about 90% or more. Examples of the "stringent condition" include the conditions described in Molecular Cloning-A LABORATORY MANUAL THIRD EDITION (Joseph Sambrook, David W. Russell, Cold Spring Harbor Laboratory Press, 2001). Those skilled in the art of hybridization can appropriately create stringent conditions by adjusting the salt concentration, temperature, etc. of the hybridization solution according to the nucleotide sequence, concentration, length, etc. of the probe. For example, the above-mentioned "stringent condition" means that the hybridization condition is preferably 5 × SSC, 70 ° C. or higher, more preferably 5 × SSC, 85 ° C. or higher, and the washing condition is 1 × SSC. , 60 ° C or higher, more preferably 1 × SSC, 73 ° C or higher. The above combinations of SSC and temperature conditions are exemplary, and one of ordinary skill in the art can achieve appropriate stringency by appropriately combining the above or other factors that determine the stringency of hybridization.

The MrgprB4 expression vector is obtained by ligating the nucleic acid encoding MrgprB4 with a vector suitable for expression of MrgprB4 in the host cell used. The vector may be any vector that is easy to prepare, can be efficiently introduced into the host cell used, and can express MrgprB4 in the host cell, and is preferably an E. coli plasmid, a yeast plasmid, or a retrovirus. Animal viral vectors such as vectors are preferred.

The host cell may be any cell as long as the nucleic acid encoding the MrgprB4 polypeptide is efficiently expressed and easily cultured, and is not particularly limited. For example, a human embryonic kidney (HEK) such as HEK293 cells. Examples include cells, Sf-9 insect cells, African Tumegael egg mother cells, Chinese hamster ovary cells (CHO) and the like.

The cell into which the nucleic acid encoding MrgprB4 used in the method of the present invention can be expressed in a host cell is, for example, expression of the function of MrgprB4 in the cell, expression at the protein level, introduction of a fluorescent substance, etc. Can be selected as an index. An appropriate selective medium or the like can be used for selection of cells in which the nucleic acid encoding MrgprB4 has been introduced into a host cell so that it can be expressed.

The medium used for culturing MrgprB4 expressing cells includes components suitable for the growth of cells expressing MrgprB4, such as glucose, amino acids, peptones, vitamins, and cell growth promoting factors (eg, cell growth factors, hormones, etc.). Any medium may be used as a medium containing binding protein, cell adhesion factor, lipid), serum (for example, FBS, FCS, etc.), calcium chloride, magnesium chloride and the like. The medium may be a commercially available medium. The medium used for culturing the cells expressing MrgprB4 may be any medium suitable for such cells, and examples thereof include, but are not limited to, MEM medium and DMEM medium.

The cell concentration at the time of seeding of MrgprB4 expressing cells is preferably 10,000 to 50,000 cells / well, and more preferably 20,000 to 40,000 cells / well when a 96 Well plate is used.

In step (I), the stimulus given to the MrgprB4 expressing cell includes a stimulus that expands and / or contracts the cell, a mechanical stimulus, and specifically, an osmotic pressure stimulus and a stimulus that pushes the cell with an extra-fine glass tube. , Stimulation such as movement, convection stimulation of the solution in which the cells are immersed, and the like, and osmotic pressure stimulation is preferable.

The expansion of MrgprB4-expressing cells in step (A) is performed, for example, by placing (incubating) the cells in hypotonic buffer.
Examples of the hypotonic buffer include hypotonic buffers having an osmotic concentration of 10 to 40 mOsm / L, preferably 20 to 30 mOsm / L.
Examples of such a hypotonic buffer include a buffer containing at least Ca ²⁺ and HEPES, for example, a 20 mOsm / L buffer; 5 mM CaCl ₂ , 0.5 mM MgCl ₂ , 10 mM HEPES, pH 7.4 and the like.
Cell swelling is performed by incubating MrgprB4 expressing cells with the above hypotonic buffer at room temperature (25 ° C.) to 37 ° C., usually for 5 to 60 minutes.

The swollen MrgprB4 expressing cells in step (B) are contracted in the presence of the test substance.
Cell contraction is performed by placing the cells in a buffer having a higher osmotic concentration than the hypotonic buffer used in step (A).
Examples of the buffer solution used here include buffer solutions having an osmotic pressure concentration of 20 to 90 mOsm / L, preferably 30 to 70 mOsm / L.
Such hypotonic buffers include at least Ca ²⁺ , HEPES, D-mannitol-containing buffers such as 35 mOsm / L buffers; 10 mM NaCl, 0.57 mM CsCl, 7.1 mM CaCl ₂ , 0.71 mM MgCl ₂ . , 14.3 mM HEPES, 8.6 mM D-mannitol, 1.0 mM glucose, pH 7.4 and the like.
Cell contraction is performed by incubating the expanded MrgprB4 expressing cells with the above hypotonic buffer at room temperature (25 ° C.) to 37 ° C., usually for 5 to 60 minutes.
Since MrgprB4-expressing cells increase the response of MrgprB4 by swelling stimulation, but show higher sensitivity to contractile stimulation after cell expansion by osmotic stimulation, the evaluation of the response activity is performed on the expanded cells. It is preferable to evaluate by the degree of decrease in the response derived from MrgprB4 when a contractile stimulus is given.

The test substance used in the method of the present invention is particularly long as it is a substance desired to be used as a pleasant emotion improver, a dry skin or a skin barrier function improver, or a skin texture improver caused by human tactile sensation. Not limited. The test substance may be a naturally occurring substance, a substance artificially synthesized by a chemical or biological method, or a compound, a composition or a mixture. good.
In the method of the present invention, the addition of the test substance may be performed simultaneously with or prior to the stimulation of cell contraction. Preferably, the test substance is added to the osmotic buffer solution to a predetermined concentration, and then the buffer solution is incubated with the cells.
The concentration of the test substance added is preferably 0.001 to 0.03% by mass (solid residue) and 0.003 to 0.01% by mass (solid residue) so as not to affect the osmotic pressure in the solution. Solid residue) is more preferable.

The contact between the test substance and the MrgprB4 expressing cells is preferably incubated at room temperature (25 ° C.) to 37 ° C. for usually about 0.1 to 4 hours, preferably about 0.5 to 2 hours.

The response of MrgprB4 to cell contraction is then measured (step (C)).
The measurement is performed by any method known in the art as a method for measuring the response of the G protein-conjugated receptor, for example, a calcium imaging method, a cAMP assay, a TGF-α cleavage assay (TGF-α sheared assay), or the like. Just do it. Of these, the TGF-α cleavage assay is a method developed in 2012 as a method for measuring receptor activity with high accuracy and sensitivity (Inoue et al., Nature Methods, 9, 1021-1029 (Inoue et al., Nature Methods, 9, 1021-1029). 2012)), suitable for measuring the response of MrgprB4. In the TGF-α cleavage assay, cells expressing TGF-α (AP-TGF-α) in which the extracellular domain is labeled with alkaline phosphatase (AP) together with the target receptor are prepared. When the receptor is activated by stimulation, the extracellular domain of AP-TGF-α is cleaved and released in the culture supernatant, and the AP activity of this culture supernatant is referred to as the substrate p-Nitrophenyl Phosphate (pNpp). Receptor response is measured by detection based on color development response.
When the TGF-α cleavage assay system is used in the method of the present invention, as the MrgprB4 expressing cell, a cell obtained by gene-introducing the MrgprB4 expression plasmid vector and the AP-TGF-α expression plasmid vector into HEK293 cells is used.

Then, based on the measured response of MrgprB4, the effect of the test substance on the response of the receptor is evaluated, and the test substance that reduces the response of the receptor is identified (step (D)).
The effect can be evaluated, for example, by comparing the response activity of MrgprB4 (the amount of decrease based on the response during cell expansion) to the cell contraction measured when different concentrations of the test substance were added. More specific examples include cell contraction between the test substance-added group with a higher concentration and the test substance-added group with a lower concentration; between the test substance-added group and the non-added group; or before and after the test substance addition. The response of MrgprB4 to is compared. If the response of MrgprB4 is further reduced by the addition of the test substance or by the addition of a higher concentration of the test substance, the test substance can be identified as a substance that reduces the response of the MrgprB4.

For example, if the response of MrgprB4 in the test substance-added group is reduced to 50% or less, preferably 80% or less as compared with the control group, the test substance can be identified as a substance that reduces the response of MrgprB4. can.

Thus, the identified test substance that reduces the response of MrgprB4 is a pleasant sensation evoked when human skin is stimulated with a cheek brush (3-5 cm / sec, 30-50 G), as shown in Examples below. Was improved. Therefore, a test substance that reduces the response of MrgprB4 can be evaluated or selected as a pleasant emotion enhancer caused by human tactile sensation.
In addition, the tactile stimulus that evokes pleasant emotions suppresses the decrease in the amount of water in the stratum corneum of the skin, improves the roughness of scales and the surface of the skin, and alleviates the inflammation of the skin. (Example 2). In addition, the tactile stimulus that evokes pleasant emotions improves the smoothness of the skin surface of the facial skin and brings about the effect of improving the texture of the skin (Example 3). Therefore, the test substance that reduces the response of MrgprB4 can be evaluated or selected as a dry skin or skin barrier function improving agent, or as a skin texture improving agent.

The human tactile-induced pleasant emotion enhancer, dry skin or skin barrier function improver or skin texture improver thus selected is used as an external skin agent or enhances human tactile pleasant emotion. Therefore, it can be used in combination with an external skin preparation as a material or preparation for improving dry skin or skin barrier function, or for improving the texture of the skin, and enhances its emotional value or functional value. It is possible. In addition, the pleasant feeling improving agent improves the pleasant feeling when the cosmetics (milky lotion, cream, etc.) are applied to the skin after ingestion of the food, for example, by using it as a food or by blending it with the food, and the emotion of the cosmetics. It is possible to increase the target value.

Regarding the above-described embodiments, the following aspects are further disclosed in the present invention.
<1> A pleasant emotion-enhancing agent caused by human tactile sensation, which comprises (I) a step of stimulating MrgprB4 expressing cells in the presence of a test substance, and (II) a step of measuring the response of MrgprB4 to the stimulus. A method for evaluating or searching for a dry skin or skin barrier function improving agent or a skin texture improving agent.
<2> The method of <1>, which comprises the following steps (A) to (E).
(A) Step of expanding MrgprB4 expressing cells (B) Step of contracting expanded cells in the presence of a test substance (C) Step of measuring the response of MrgprB4 to cell contraction (D) Step of measuring MrgprB4 based on the measured response The process of identifying test substances that reduce the response of
(E) The identified test substance is evaluated or selected as 1) a tactile-induced pleasant feeling improver, 2) a step of evaluating or selecting as a dry skin or skin barrier function improving agent, or 3) as a skin texture improving agent. Step <3> The method of <2>, wherein the expansion of the cells in the step (A) is carried out by incubating the cells with a buffer having an osmotic concentration of 20 to 30 mOsm / L.
<4> The method of <2> or <3>, wherein the contraction of the cells in the step (B) is performed by incubating the expanded cells with a buffer solution having an osmotic concentration of 30 to 70 mOsm / L.
<5> The method according to any one of <1> to <4>, wherein the cells are HEK293 cells.
<6> The method according to any one of <1> to <5>, in which the pleasant emotion caused by the sense of touch is the pleasant emotion generated when the external skin preparation is applied to the skin.
<7> Any method of <1> to <6> in which pleasant feelings are comfort, happiness / satisfaction, luxury or exhilaration <8> Stimulation applied to cells causes cells to expand or expand and The method of <1>, which is a stimulus to contract.

Test Example 1
(1) Preparation of MrgprB4 transiently expressing cells Human fetal kidney cells HEK293 purchased from ATCC were used as cells. The medium was D-MEM / F-12 (Life technologies) and was cultured in a medium containing 10% serum (Fetal Bovine Serum, FBS). The cells were cultured under 37 ° C. and 5% CO ₂ conditions. MrgprB4 was cloned from Dorsal root ganglions of mice, and MrgprX1 was cloned from SK-N-MC cells into pcDNA3.1 (Life technologies) to prepare a plasmid, and the plasmid was introduced into HEK293 using TransIT-293 (Mirus). Transiently expressed cells were prepared.

(2) Detection of MrgprB4-derived activity by TGF-α cleavage assay According to the previous report (Inoue et al., Nat Methods., 10, 1021-1029, 2012), measurements were carried out. The (TGF-α-AP) -pRc plasmid and MrgprB4- or MrgprX1-pcDNA3.1 plasmid were gene-introduced into HEK293 with TransIT-293. The cells were then seeded and cultured in a Poly-D-lysine coated Black-walled clear-base 96-well plate. Remove the culture solution from the plate and use an isotonic buffer (320 mOsm / L; 105 mM NaCl, 6 mM CsCl, 5 mM CaCl ₂ , 0.5 mM MgCl ₂ , 10 mM HEPES, 90 mM D-Mannitol, 10 mM Glucose, pH 7 during swelling stimulation). .4), or hypotonic buffer (20 mOsm / L; 5 mM NaCl ₂ , 0.5 mM MgCl ₂ , 10 mM HEPES, pH 7.4) at 100 μl / well after 5 minutes. Various osmotic buffers (35 mOsm / L; 5.8 mM NaCl, 0.3 mM CsCl, 5 mM CaCl ₂ , 0.5 mM MgCl ₂ , 10 mM HEPES, 5 mM D-Mannitol, 0.5 mM Glucose, pH 7.4, 50 mOsm / L: 11.7 mM NaCl, 0.7 mM CsCl, 5 mM CaCl ₂ , 0.5 mM MgCl ₂ , 10 mM HEPES, 10 mM D-Mannitol, 1.1 mM Glucose, pH 7.4, 245 mOsm / L; 79 mM NaCl, 4. 5 mM CsCl, 5 mM CaCl ₂ , 0.5 mM MgCl ₂ , 10 mM HEPES, 67.5 mM D-Mannitol, 7.5 mM Glucose, pH 7.4, 270 mOsm / L; 87.2 mM NaCl, 5 mM CsCl, 5 mM CaCl ₂ , 0. 5 mM MgCl ₂ , 10 mM HEPES, 74.7 mM D-Mannitol, 8.3 mM Glucose, pH 7.4) was added at 100 μl / well and incubated for 1 hour under 37 ° C. and 5% CO ₂ conditions. Centrifuge the plate at 1,000 rpm for 1 minute to separate the cells and culture supernatant, transfer the supernatant to a new 96-well plate and lyse the pNpp tablet (Cayman) in AP Buffer (40 mM Tris-HCl, 40 mM NaCl, 10 mM). MgCl _2, pH 9.5, 1 buffer / 20 mL) was added to the culture plate and the plate to which the supernatant was transferred at 100 μL / well, and the absorbance (405 nm, OD405) was measured after incubating at 37 ° C. for 5 to 30 minutes. ..
The OD405 measured 5 minutes after the addition of the AP Buffer in which pNPP was dissolved was subtracted for each well from the value measured 30 minutes later, and the values were defined as ΔOD405 (Cell) and ΔOD405 (Conditioned medium, CM), respectively. In order to calculate the amount released extracellularly from the total amount of AP-TGF-α expressed in the cells, the value of ΔOD405 (CM) / (ΔOD405 (Cell) + ΔOD405 (CM)) was obtained for each well, and AP- The TGF-α release ratio (%) was used.

(3) Results Detection of MrgprB4-derived activity by TGF-α shearing assay From 320 mOsm / L isotonic buffer, stimulated with 245 270 mOsm / L buffer to swell cells, compared to MOCK. The AP-TGF-α release rate tended to increase in hyperexpressing cells, but the reproducibility was poor. On the other hand, after swelling the cells with a hypotonic buffer of 20 mOsm / L, the response when an osmotic stimulus to contract the cells was given with a buffer of 35 or 50 mOsm / L was measured. The results of changes in the AP-TGF-α release ratio based on the response when stimulated with 20 mOsm / L hypotonic buffer are shown in FIGS. 1a and 1b. As a result, the AP-TGF-α release ratio was reduced with good reproducibility in MrgprB4 transiently expressing cells as compared with MOCK by the cell contraction stimulation after cell expansion. Furthermore, as shown in FIG. 1b, since the response was the same as that of MOCK in MrgprX1 transiently expressing cells belonging to the same family, the decrease in AP-TGF-α release ratio due to cell contraction was specific to MrgprB4 transiently expressing cells. It became clear that. From this, it was clarified that the cells expressing MrgprB4 were highly sensitive to the contractile stimulus after the cell expansion by the osmotic pressure stimulus, and further lowered the AP-TGF-α release ratio by the cell contraction stimulus. The material was thought to reduce the activity of MrgprB4.

Example 1 Search for a material for reducing the response activity of MrgprB4 and evaluation by tactile stimulation (1) Method MrgprB4 stable expression cells were prepared by HEK293. The (TGF-α-AP) -pRc plasmid was introduced into MrgprB4 stable expression cells or HEK293 with TransIT-293. The cells were then seeded and cultured in a Poly-D-lysine coated Black-walled clear-base 96-well plate. Remove the culture from the plate, add hypotonic buffer at 100 μl / well, remove the buffer after 5 minutes, and remove 50 mOsm / L osmotic buffer (10 mM NaCl, 0.57 mM CsCl, 7.1 mM CaCl ₂ , Add 100 μl / well of 0.71 mM MgCl ₂ , 14.3 mM HEPES, 8.6 mM D-Mannitol, 1.0 mM Glucose, pH 7.4) and incubate for 1 hour under 37 ° C. and 5% CO ₂ conditions. Shrinked the cells with. Moreover, as a control, the one incubated with a hypotonic buffer solution of 20 mOsm / L was used. For the subsequent detection of activity, the AP-TGF-α release ratio was calculated in the same manner as in Test Example 1 (2), and the AP-TGF-α release ratio when stimulated with the control 20 mOsm / L hypotonic buffer solution was calculated. Subtraction was performed as a reference, and the amount of change when the cells were contracted with a buffer solution of 50 mOsm / L was calculated.
In the screening of the material for reducing the response activity of MrgprB4, the material was added to a buffer solution of 50 mOsm / L to stimulate the cells, the relative MrgprB4 activity was calculated by the following formula, and the material for reducing the relative MrgprB4 activity was searched for.

(2) Evaluation of pleasant emotions by tactile stimulation Five healthy men in their 20s and 30s were evaluated. Thirty seconds after applying 100 μL of saline to the extensor side of the forearm, the forearm was stroked 10 times with a cheek brush at a speed of 3-5 cm / s and a load of 30-50 g, and the pleasant feelings were expressed on the Visual Analog Scale (VAS; subjective). The degree of emotion was evaluated linearly (the left side feels nothing in particular, the right side is very pleasant). Immediately after that, 100 μL of physiological saline or the material for reducing the response activity of MrgprB4 selected in (1) was applied to the forearm, and 30 seconds later, the forearm was similarly stroked 10 times with a cheek brush, and the pleasant feeling was evaluated by VAS. did. Then, the rate of change from the VAS value of the comfort when stimulating the cheek brush after the first application of the saline solution is obtained from the comfort VAS value when the cheek brush is stimulated after the application of the saline solution to the forearm or the material. Calculated. The evaluation of the pleasant feelings of the tactile sensation before and after the application of the saline solution and the material was performed on different days. Stimulation with a cheek brush was performed at a load of about 3-5 cm / sec and 30-50 g, which activates C fibers that receive tactile stimuli that evoke pleasant emotions (Non-Patent Document 1).

(3) Results i) As a result of searching for substances that reduce the MrgprB4 response from about 60 kinds of cosmetic raw materials, polyoxyethylene methylglucoside (ethylene oxide addition mole number 20, GLUCAM E-20; manufactured by Lubrizol) was found. .. As shown in FIG. 2, in polyoxyethylene methylglucoside, polyoxyethylene methylglucoside reduced the relative MrgprB4 activity in a concentration-dependent manner, and the effect was maximized at 0.01%.

ii) As shown in FIG. 3, it is clear that the rate of change in the pleasant emotional VAS value before and after application of 0.01% polyoxyethylene methylglucoside is significantly higher than the rate of change before and after application of physiological saline. became.

Example 2 Skin function improvement effect by continuous tactile stimulation (1) Method i) Test protocol Six healthy men in their twenties and thirties were tested according to the schedule shown in FIG. That is, from one week before stimulating the forearm with a cheek brush, the extensor sides of both forearms were washed twice a day with soap. Then, immediately after the same 2 times / day wash, he stroked the extensor side of one forearm by himself with a cheek brush twice / day for 3 minutes / time for 4 weeks (for right-handed people). I stroked my left forearm.) The opposite forearm was not irritating except for irrigation. Blush brush stimulation was performed at a rate of about 3-5 cm / sec and a load of about 30-50 g to activate C fibers that receive tactile stimuli that evoke pleasant emotions (Loken et al., NATURE NEUROSCIENCE, Vol. . 12, p547-548, 2009). Due to concerns about strong rough skin caused by long-term washing with soap, soap was changed to washing with face wash 3 weeks after the start of the test (2 weeks after the start of blush brush stimulation).
At 1 and 5 weeks after the start of the test, the water content of the stratum corneum was measured with Corneometer CM825 (Courage + Khazaka), and the skin surface was measured with Visioscan VC98 USB (Courage + Khazaka). The data are shown as the rate of change relative to the measured values at 1 week. At 5 weeks after the start of the test, the stratum corneum on the extensor side of both forearms was collected by tape stripping.

ii) Extraction of stratum corneum protein, quantification of cytokines in the stratum corneum Protein was extracted from three stratum corneum tapes. The tape was cut into small pieces with a scalpel, immersed in PBS / 0.005% Tween 20 solution (v / v) (one tape was immersed in 500 μl), and allowed to stand on ice for 30 minutes. Then, ultrasonic crushing was performed in cold water for 10 minutes, centrifugation (14,000 rpm, 2 min, 4 ° C.), and then the supernatant was collected. After quantifying the amount of protein in the supernatant with a Micro BCA Protein Assay Kit (Thermo Cytokine) based on the calibration curve prepared with bovine serum albumin, the final concentration is adjusted to 0.5% (v / v) in the supernatant. BSA was added, and the amount of inflammatory cytokines was quantified using IL-1Alpha Human ELISA Kit (RayBio), IL-1ra Human ELISA Kit (RayBio), and IL-1Beta Human ELISA Kit (RayBio).

(2) Results i) Analysis of changes in skin function / properties There is a tendency (p = 0.067) that the decrease in the water content (capacity value) of the stratum corneum on the extensor side of the forearm, which is thought to be the effect of washing, is alleviated by cheek brush stimulation. Shown. Furthermore, skin surface analysis showed that the decrease in SESC (scale index) and SER (roughness index) due to washing was significantly improved by cheek brush stimulation. From these facts, it was clarified that the cheek brush stimulation alleviates or improves the onset of dry skin and the deterioration of skin function (skin barrier function) due to washing.

ii) Changes in the amount of cytokines in the stratum corneum At 5 weeks after the start of the test, the stratum corneum on the extensor sides of both forearms was collected by tape stripping, and the amount of inflammatory cytokines (IL-1RA / IL-1α, IL-1β) was measured. As a result, IL-1RA / IL-1α on the extensor side of the forearm significantly decreased and IL-1β tended to decrease due to the cheek brush stimulation (FIG. 5). Therefore, it was revealed that cheek brush irritation relieves skin inflammation caused by washing. From these facts, it was clarified that cheek brush stimulation ameliorated the deterioration of skin function due to washing.

Example 3 Improvement of skin texture score by continuous tactile stimulation (1) Method i) This was performed on 22 healthy women in their 20s and 40s.
ii) Tactile stimulation I stroked the extensor side of my forearm by myself for 4 weeks with a cheek brush twice a day (morning and evening) for 3 minutes. Blush brush stimulation was performed at a rate of about 3-5 cm / sec to activate C fibers that receive tactile stimuli that evoke pleasant emotions, with a load of 30-50 g (Loken et al., NATURE NEUROSCIENCE, Vol. 12). , P547-548, 2009).
iii) Visual evaluation of skin texture Visual evaluation of skin texture is based on certain evaluation criteria according to the method described in the literature (Nakamura et al., JOURNAL OF SOCIETY OF COSMETIC CHEMICALS OF JAPAN, Vo. 45, p306-314, 2011). It was carried out by an expert who had been trained in objective visual evaluation. For the evaluation, a 7-step scale method of "not (-3)" and "yes (3)" was used.

(2) Results It was clarified that the score value of the smoothness of the skin surface of the facial skin tends to be improved by continuing the stimulation of stroking the forearm with the cheek brush for 4 weeks, as shown in FIG.

Claims (5)

A method for evaluating or searching for a pleasant emotion improving agent, a dry skin or a skin barrier function improving agent, or a skin texture improving agent caused by human tactile sensation, which comprises the following steps (A) to (E).
(A) Step of expanding MrgprB4 expressing cells
(B) Step of contracting expanded cells in the presence of a test substance
(C) Step of measuring the response of MrgprB4 to cell contraction
(D) A step of identifying a test substance that reduces the response of MrgprB4 based on the measured response,
(E) The identified test substance is 1) evaluated or selected as a tactile-induced pleasant emotion improving agent, 2) evaluated or selected as a dry skin or skin barrier function improving agent, or 3) skin texture. Process to evaluate or select as an improver The method according to claim 1 , wherein the expansion of the cells in the step (A) is performed by incubating the cells with a buffer solution having an osmotic concentration of 20 to 30 mOsm / L. The method according to claim 1 or 2 , wherein the contraction of the cells in the step (B) is performed by incubating the expanded cells with a buffer solution having an osmotic concentration of 30 to 70 mOsm / L. The method according to any one of claims 1 to 3 , wherein the cell is a HEK293 cell. The method according to any one of claims 1 to 4 , wherein the pleasant emotion caused by the sense of touch is the pleasant emotion generated when the external skin preparation is applied to the skin.

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