Method for regulating perspiration, perspiration regulator and screening method of perspiration regulator

Abstract

To provide a method for regulating perspiration, a perspiration regulator, and a screening method of the perspiration regulator.SOLUTION: An active regulator of at least one TRP channel selected from the group consisting of TRPV1, TRPV3, and TRPM4 is used.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for regulating perspiration, a perspiration regulator, and a method for screening perspiration regulators. [Background technology]

[0002] Sweat glands are divided into two types: apocrine sweat glands and eccrine sweat glands. Apocrine sweat glands are found in specific locations (e.g., underarms), whereas eccrine sweat glands are found throughout the body and function as thermoregulatory organs.

[0003] In the case of thermal sweating, the hypothalamus of the brain detects an increase in body temperature, which results in the secretion of acetylcholine from the terminals of the sympathetic nerves. When this acetylcholine binds to muscarinic receptors present in the secretory ducts that make up the sweat glands, sweat that is isotonic with body fluids is produced. The ducts that make up the sweat glands are the pathways through which sweat travels before being secreted onto the body surface (skin). In the ducts, NaCl contained in sweat is reabsorbed into the body to prevent excessive loss of salt from the body, and hypotonic sweat is excreted onto the body surface (skin) (see Non-Patent Document 1).

[0004] Sweat is produced by store-operated Ca2+ secretion in the clear cells of the sweat glands. 2+ It is known that store-operated Ca in the clear cells, which are the secretory cells of the sweat glands, is secreted by activation of store-operated Ca 2+ Inactivation of store-operated Ca influx (SOCE) inhibits sweat secretion. 2+ Inactivation of SOCE influx is caused by Na + It is known that this occurs due to (see Non-Patent Document 2).

[0005] Research on human sweat glands has lagged behind because mice only have sweat glands on the palms of their hands, and because the functions of human and mouse sweat glands are different. Therefore, the mechanisms of sweat secretion and reabsorption in humans remain unclear. Furthermore, although sweating in humans is regulated by the central nervous system, it is unclear whether a mechanism for sweating regulation exists in the peripheral nervous system. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Concepcion AR and Feske S. Cell Calcium. 2017 May; 63: 53‐59 [Non-Patent Document 2] Monteith GR, et al., Nat Rev Cancer. 2007 Jul; 7(7): 519-530 Summary of the Invention [Problem to be solved by the invention]

[0007] As described above, in the current situation where research into human sweat glands is lagging behind, there is a demand for the development of a method for regulating sweating, a sweat regulating agent, and a method for screening for sweat regulating agents.

[0008] One aspect of the present invention aims to provide a method for regulating sweating, a sweating regulator, and a method for screening sweating regulators. [Means for solving the problem]

[0009] It is known that many organs, including skin epithelial keratinocytes, have a system in which the cells themselves sense the extracellular environment without the intervention of nerves. One such system is the transient receptor potential ion channels (TRP channels). TRP channels sense changes in the intracellular and extracellular environment by being activated by various stimuli (e.g., temperature, osmotic pressure, chemicals), and convert the changes into intracellular signals.

[0010] The inventors focused particularly on thermosensitive TRP channels and hypothesized that TRP channels may be responsible for regulating the secretion and reabsorption of sweat not mediated by sympathetic nerves by sensing changes in temperature. They then conducted research with the aim of elucidating the mechanism by which TRP channels regulate sweating.

[0011] The present inventors have discovered that TRP channels TRPV1, TRPV3, and TRPM4 are expressed in the secretory ducts and / or ducts that constitute sweat glands, and that the TRP channels mediate sweating by ions (e.g., Ca 2+ , Na + The present invention encompasses the following:

[0012] [1] A method for regulating sweating, comprising a step of contacting, in vitro, sweat gland cells with an activity regulator of at least one TRP channel selected from the group consisting of TRPV1, TRPV3, and TRPM4.

[0013] [2] A method for screening a sweat regulating agent, comprising the steps of contacting a test substance with a cell having at least one type of TRP channel activity selected from the group consisting of TRPV1, TRPV3, and TRPM4, and measuring a change in the activity of the TRP channel in the cell having TRP channel activity that has been contacted with the test substance.

[0014] [3] A sweat regulating agent comprising, as an active ingredient, an activity regulator of at least one TRP channel selected from the group consisting of TRPV1, TRPV3 and TRPM4. Effect of the Invention

[0015] According to one aspect of the present invention, a method for regulating sweating, a sweating regulator, and a method for screening sweating regulators can be provided. [Brief description of the drawings]

[0016] [Figure 1] 1 is a graph showing the expression of TRPV1, TRPV3 and TRPM4 in eccrine sweat glands in an example of the present invention. [Diagram 2] 1 is a graph showing the channel activity of TRPV1 and TRPV3 in an example of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] An example of an embodiment of the present invention will be described in detail below, but the present invention is not limited thereto. Various modifications of the present invention are possible within the scope of the claims. In addition, embodiments or examples obtained by appropriately combining the technical means disclosed in different embodiments or examples are also included in the technical scope of the present invention. Furthermore, new technical features can be formed by combining the technical means disclosed in each embodiment. All academic documents and patent documents described in this specification are incorporated herein by reference. Unless otherwise specified in this specification, "X to Y" representing a numerical range is intended to mean "X or more and Y or less."

[0018] [1. Sweat Regulator] A sweat regulator according to one embodiment of the present invention contains, as an active ingredient, an activity regulator of at least one TRP channel selected from the group consisting of TRPV1 (transient receptor potential vanilloid 1), TRPV3 (transient receptor potential vanilloid 3), and TRPM4 (transient receptor potential melastatin 4).

[0019] In this specification, "sweating" includes "secretion of sweat" and "reabsorption of sweat". The "reabsorption of sweat" includes "the release of ions related to sweating (e.g., Ca 2+ , Na + ) resorption.

[0020] The sweat regulator according to one embodiment of the present invention may also be an agent for adjusting the amount of ions inside and / or outside the cells that constitute sweat glands. Examples of the sweat glands include apocrine sweat glands and eccrine sweat glands. More specifically, examples of the sweat glands include secretory ducts and ducts that are parts that constitute sweat glands. Examples of the cells that constitute the sweat glands include luminal cells and myoepithelial cells that constitute the secretory ducts, and luminal cells and basal layer cells that constitute the ducts. Examples of the ions include positive ions. More specifically, examples of the ions include Ca 2+ and Na + Examples include:

[0021] The secretory ducts that make up the sweat glands are involved in the secretion of sweat, and the ducts that make up the sweat glands are involved in the reabsorption of sweat. Sweating also requires the release of ions (e.g., Ca 2+ , Na + ) is involved.

[0022] More specifically, sweat is produced by store-operated Ca2+ release in clear cells, the secretory cells of the sweat glands. 2+ It is known that Ca is secreted by activation of the SOCE influx (see Non-Patent Document 1). In addition, the secretory capacity of secretory glands, including sweat glands, can be evaluated by measuring the Ca 2+Inflow analysis is used (see non-patent document 1).

[0023] On the other hand, store-operated Ca in clear cells, which are the secretory cells of sweat glands, 2+ It is known that inactivation of store-operated Ca influx (SOCE) inhibits sweat secretion. 2+ Inactivation of SOCE influx is caused by Na + It is known that this occurs due to (see Non-Patent Document 2).

[0024] Therefore, regulators of the amount of ions within and / or outside the cells that make up sweat glands may also be sweat regulators.

[0025] The activity regulator may be an agonist or an inhibitor (antagonist) of at least one TRP channel selected from the group consisting of TRPV1, TRPV3, and TRPM4.

[0026] When the activity regulator is a TRPV1 or TRPV3 agonist, the sweat regulator containing the agonist functions as a sweat regulator for promoting sweat secretion, whereas when the activity regulator is a TRPV1 or TRPV3 inhibitor, the sweat regulator containing the inhibitor functions as a sweat regulator for suppressing sweat secretion.

[0027] Agonists of TRPV1 include, for example, capsaicin, nonivamide, and N-oleoyldopamine.

[0028] Examples of TRPV1 inhibitors include capsazepine, SB-366791 manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd., and AMG9810 manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.

[0029] Agonists of TRPV3 include, for example, menthol, camphor, dihydrocarveol, and 1,8-cineole.

[0030] Examples of TRPV3 inhibitors include Trpvicin, TRPV3 74a manufactured by Tocris Bioscience, and KM-001 (CAS number: 2682864-35-1).

[0031] When the activity regulator is a TRPM4 agonist, the sweat regulator containing the agonist functions as a sweat regulator for suppressing sweat secretion, whereas when the activity regulator is a TRPM4 inhibitor, the sweat regulator containing the agonist functions as a sweat regulator for promoting sweat secretion.

[0032] Agonists of TRPM4 include, for example, 3,5-bis(trifluoromethyl)pyrazole (BTP2), U73122 manufactured by Cayman Chemical, and potassium aluminum sulfate.

[0033] Examples of TRPM4 inhibitors include 9-phenanthrol and CBA (CAS number: 351424-20-9).

[0034] The amount of the active ingredient contained in the perspiration regulator according to one embodiment of the present invention is not particularly limited, and for example, when the perspiration regulator is taken as 100% by mass, the amount may be 0.00001% by mass to 100% by mass, 0.0001% by mass to 100% by mass, 0.001% by mass to 100% by mass, 0.01% by mass to 100% by mass, 0.1% by mass to 100% by mass, 0.1% by mass to 95% by mass, 0.1% by mass to 90% by mass, 0.1% by mass to 80% by mass, 0.1% by mass to 70% by mass, 0.1% by mass to 60% by mass, 0.1% by mass to 50% by mass, 0.1% by mass to 40% by mass, 0.1% by mass to 30% by mass, 0.1% by mass to 20% by mass, or 0.1% by mass to 10% by mass.

[0035] The perspiration regulating agent according to one embodiment of the present invention may contain ingredients other than the above-mentioned active ingredients.

[0036] The ingredients other than the active ingredient are not particularly limited, and may be, for example, a buffering agent, a pH adjusting agent, an isotonicity agent, a preservative, an antioxidant, a high molecular weight polymer, an excipient, a solvent, an antibacterial agent, and the like.

[0037] Examples of the buffer include phosphoric acid or a phosphate, boric acid or a borate, citric acid or a citrate, acetic acid or a acetate, carbonic acid or a carbonate, tartaric acid or a tartrate, ε-aminocaproic acid, and trometamol. Examples of the phosphate include sodium phosphate, sodium dihydrogen phosphate, disodium hydrogen phosphate, potassium phosphate, potassium dihydrogen phosphate, and dipotassium hydrogen phosphate. Examples of the borate include borax, sodium borate, and potassium borate. Examples of the citrate include sodium citrate, disodium citrate, and trisodium citrate. Examples of the acetate include sodium acetate and potassium acetate. Examples of the carbonate include sodium carbonate and sodium bicarbonate. Examples of the tartrate include sodium tartrate and potassium tartrate.

[0038] Examples of the pH adjuster include hydrochloric acid, phosphoric acid, citric acid, acetic acid, sodium hydroxide, and potassium hydroxide.

[0039] Examples of the isotonicity agent include ionic isotonicity agents (eg, sodium chloride, potassium chloride, calcium chloride, magnesium chloride) and non-ionic isotonicity agents (eg, glycerin, propylene glycol, sorbitol, mannitol).

[0040] Examples of the preservative include benzalkonium chloride, benzalkonium bromide, benzethonium chloride, sorbic acid, potassium sorbate, methyl parahydroxybenzoate, propyl parahydroxybenzoate, and chlorobutanol.

[0041] Examples of the antioxidant include ascorbic acid, tocopherol, dibutylhydroxytoluene, butylhydroxyanisole, sodium erythorbate, propyl gallate, and sodium sulfite.

[0042] Examples of the high molecular weight polymer include methyl cellulose, ethyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxyethyl methyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose, sodium carboxymethyl cellulose, hydroxypropyl methyl cellulose acetate succinate, hydroxypropyl methyl cellulose phthalate, carboxymethyl ethyl cellulose, cellulose acetate phthalate, polyvinylpyrrolidone, polyvinyl alcohol, carboxyvinyl polymer, polyethylene glycol, and atelocollagen.

[0043] Examples of the excipient include lactose, sucrose, D-mannitol, xylitol, sorbitol, erythritol, starch, and crystalline cellulose.

[0044] Examples of the solvent include water, physiological saline, and alcohol.

[0045] Examples of the antibacterial agent include β-lactam, aminoglycoside, tetracycline, lincomycin, chloramphenicol, macrolide, ketolide, polypeptide, and glycopeptide antibiotics; and pyridone carboxylic acid (quinolone), new quinolone, oxazolidinone, and sulfa drug synthetic antibacterial agents.

[0046] The amount of ingredients other than the active ingredient contained in the perspiration regulating agent according to one embodiment of the present invention is not particularly limited, and may be, for example, 0% by mass to 99.99999% by mass, 0% by mass to 99.9999% by mass, 0% by mass to 99.9999% by mass, 0% by mass to 99.999% by mass, 0% by mass to 99.999% by mass, 0% by mass to 99.99% by mass, 5% by mass to 99.9% by mass, or 5% by mass to 99.9% by mass. The content may be 10% by mass to 99.9% by mass, 20% by mass to 99.9% by mass, 30% by mass to 99.9% by mass, 40% by mass to 99.9% by mass, 50% by mass to 99.9% by mass, 60% by mass to 99.9% by mass, 70% by mass to 99.9% by mass, 80% by mass to 99.9% by mass, or 90% by mass to 99.9% by mass.

[0047] The dosage form of the sweat regulating agent according to one embodiment of the present invention is not particularly limited, and examples thereof include external preparations (e.g., liquids, gels, creams, sticks, sheets), tablets, powders, and granules.

[0048] The perspiration regulating agent according to one embodiment of the present invention may be in the form of a body lotion, a deodorant cosmetic, a lotion, a milky lotion, a skin care cream, a tonic, a stick cosmetic, a lip, a facial cleanser, a cleanser, or a sheet-like cosmetic.

[0049] The subjects to which the perspiration regulating agent according to one embodiment of the present invention is administered are not particularly limited, and examples thereof include humans, non-human animals (e.g., livestock, pets, and laboratory animals), tissues collected therefrom, cells collected therefrom, and established cell lines. The non-human animals are not particularly limited, and examples thereof include monkeys, chimpanzees, cows, pigs, sheep, goats, horses, dogs, cats, rabbits, mice, and rats.

[0050] The administration route of the perspiration regulating agent according to one embodiment of the present invention is not particularly limited, and examples thereof include parenteral administration (eg, transdermal administration) and oral administration.

[0051] The interval at which the sweat regulating agent according to one embodiment of the present invention is administered to a subject is not particularly limited, and examples include once every hour, once every 1 to 6 hours, once every 6 to 12 hours, once every 12 hours to once a day, once every 1 to 3 days, once every 1 to 5 days, and once every 1 to 7 days.

[0052] 2. How to regulate sweating A method for regulating sweating according to one embodiment of the present invention includes a step of contacting, in vitro, sweat gland cells with an activity regulator of at least one TRP channel selected from the group consisting of TRPV1, TRPV3, and TRPM4.

[0053] A method for regulating sweating according to one embodiment of the present invention may be a method comprising the step of contacting sweat gland cells collected from a living body with an activity regulator of at least one TRP channel selected from the group consisting of TRPV1, TRPV3 and TRPM4.

[0054] A method for regulating sweating according to one embodiment of the present invention can be used, for example, (i) as a new method for analyzing the mechanisms of sweat secretion and reabsorption, and (ii) as a new method for screening sweat regulating agents.

[0055] The sweat gland cells are not limited. For example, the sweat gland cells may be sweat gland cells contained in sweat gland tissue separated from a living body, or may be uncultured sweat gland cells (e.g., primary culture cells) separated from sweat gland tissue, or may be cultured sweat gland cells separated from sweat gland tissue, or may be immortalized sweat gland cells separated from sweat gland tissue. Examples of immortalized sweat gland cells include the immortalized sweat gland cells described in International Publication WO2019 / 031500, Japanese Patent Application No. 2020-157576, or Japanese Patent Application No. 2020-157577.

[0056] As the activity regulator of at least one TRP channel selected from the group consisting of TRPV1, TRPV3 and TRPM4, those described in the above section [1. Sweat Regulator] can be used. Here, the description of the activity regulator is omitted.

[0057] In the contacting step, the sweat gland cells may be contacted with at least one TRP channel activity regulator selected from the group consisting of TRPV1, TRPV3, and TRPM4, for example, in a culture medium, and then the sweat gland cells may be cultured for a desired time. During the culture, the activity regulator may or may not be in contact with the cells. The culture time is not limited and may be 1 hour to 1 day, 1 day to 3 days, 1 day to 7 days, or 7 days or more.

[0058] 3. Screening method for sweat regulators A method for screening for sweat regulating agents according to one embodiment of the present invention includes the steps of contacting a test substance with cells having at least one type of TRP channel activity selected from the group consisting of TRPV1, TRPV3 and TRPM4, and measuring a change in the activity of the TRP channel in the cells having TRP channel activity that have been contacted with the test substance.

[0059] As described above, a method for screening a sweat regulating agent according to one embodiment of the present invention includes step A of contacting a test substance with a cell having at least one TRP channel activity selected from the group consisting of TRPV1, TRPV3 and TRPM4.

[0060] The test substance is not limited, and examples thereof include inorganic compounds, organic compounds, plant extracts, microbial extracts, and culture supernatants of various cells. The test substance may be used as it is, or may be used in a state dissolved in a solvent. Examples of the solvent include physiological saline, phosphate buffered saline, and water.

[0061] The cells having at least one TRP channel activity selected from the group consisting of TRPV1, TRPV3 and TRPM4 are not limited. The cells may be, for example, sweat gland cells contained in sweat gland tissue separated from a living body, uncultured sweat gland cells separated from sweat gland tissue (e.g., primary culture cells), cultured sweat gland cells separated from sweat gland tissue, or immortalized sweat gland cells separated from sweat gland tissue. Examples of immortalized sweat gland cells include the immortalized sweat gland cells described in International Publication WO2019 / 031500, Japanese Patent Application No. 2020-157576, or Japanese Patent Application No. 2020-157577.

[0062] In the step A, the test substance may be contacted with cells having at least one TRP channel activity selected from the group consisting of TRPV1, TRPV3 and TRPM4, for example, in a medium, and then the cells may be cultured for a desired time. During the culture, the test substance may or may not be in contact with the cells. The culture time is not limited, and may be 1 hour to 1 day, 1 day to 3 days, 1 day to 7 days, or 7 days or more.

[0063] As described above, the screening method for a sweat regulating agent according to one embodiment of the present invention includes a step B of measuring a change in the activity of the TRP channel in a cell having TRP channel activity that has been contacted with the test substance.

[0064] The method for measuring the change in activity of the TRP channel is not limited and can be any known method for measuring the activity of a channel (e.g., an ion channel) (see, e.g., the Examples).

[0065] For example, the activity of the TRPV1 channel measured in step B when the test substance is contacted with the cells having TRPV1 channel activity in step A is set as a value α1 (screening test), and the activity of the TRPV1 channel measured in step B when the known agonist of the TRPV1 channel is contacted with the cells having TRPV1 channel activity in step A is set as a value β1 (control test). If the value α1 and the value β1 are close to each other, the test substance can be determined to be an agonist of the TRPV1 channel (in other words, an active ingredient of a sweat regulator for promoting sweat secretion and reabsorption).

[0066] For example, the activity of the TRPV1 channel measured in step B when the test substance is contacted with the cell having TRPV1 channel activity in step A is set as a value α2 (screening test), and the activity of the TRPV1 channel measured in step B when the known TRPV1 channel antagonist is contacted with the cell having TRPV1 channel activity in step A is set as a value β2 (control test). If the values ​​α2 and β2 are close to each other, the test substance can be determined to be an antagonist of the TRPV1 channel (in other words, an active ingredient of a sweat regulator for suppressing sweat secretion and reabsorption).

[0067] For example, the activity of the TRPV3 channel measured in step B when the test substance is contacted with the cell having TRPV3 channel activity in step A is set as a value α3 (screening test), and the activity of the TRPV3 channel measured in step B when the known agonist of the TRPV3 channel is contacted with the cell having TRPV3 channel activity in step A is set as a value β3 (control test). If the values ​​α3 and β3 are close to each other, the test substance can be determined to be an agonist of the TRPV3 channel (in other words, an active ingredient of a sweat regulator for promoting sweat secretion and reabsorption).

[0068] For example, the activity of the TRPV3 channel measured in step B when the test substance is contacted with the cell having TRPV3 channel activity in step A is set as a value α4 (screening test), and the activity of the TRPV3 channel measured in step B when the known TRPV3 channel antagonist is contacted with the cell having TRPV3 channel activity in step A is set as a value β4 (control test). If the value α4 and the value β4 are close to each other, the test substance can be determined to be an antagonist of the TRPV3 channel (in other words, an active ingredient of a sweat regulator for suppressing sweat secretion and reabsorption).

[0069] For example, the activity of the TRPM4 channel measured in step B when a test substance is contacted with cells having TRPM4 channel activity in step A is set to a value α5 (screening test), and the activity of the TRPM4 channel measured in step B when a known agonist of the TRPM4 channel is contacted with cells having TRPM4 channel activity in step A is set to a value β5 (control test). If the values ​​α5 and β5 are close to each other, the test substance can be determined to be an agonist of the TRPM4 channel (in other words, an active ingredient of a sweat regulator for suppressing the secretion and reabsorption of sweat).

[0070] For example, the activity of the TRPM4 channel measured in step B when a test substance is contacted with a cell having TRPM4 channel activity in step A is set to a value α6 (screening test), and the activity of the TRPM4 channel measured in step B when a known TRPM4 channel antagonist is contacted with a cell having TRPM4 channel activity in step A is set to a value β6 (control test). If the values ​​α6 and β6 are close to each other, the test substance can be determined to be an antagonist of the TRPM4 channel (in other words, an active ingredient of a sweat regulator for promoting sweat secretion and reabsorption).

[0071] [4. Other] An embodiment of the present invention can also be configured as follows.

[0072] <1> A method for regulating sweating, comprising the step of contacting a subject (e.g., a human or a non-human animal (e.g., a monkey, chimpanzee, cow, pig, sheep, goat, horse, dog, cat, rabbit, mouse, and rat)) with an activity regulator of at least one TRP channel selected from the group consisting of TRPV1, TRPV3, and TRPM4.

[0073] <2> 2. Use of an activity regulator of at least one TRP channel selected from the group consisting of TRPV1, TRPV3 and TRPM4 in the manufacture of a sweat regulator. EXAMPLES

[0074] <1. Sample preparation> <1-1. Human skin tissue> Human skin tissue was obtained from the donor after surgery after obtaining informed consent. The experiment using human skin tissue was approved by the ethical review committee of Osaka University. Table 1 shows the uses of human skin tissue in the examples described below.

[0075] [Table 1]

[0076] <1-2. Isolation of eccrine sweat glands from human skin tissue> Human skin tissue was immersed in Neutral Red (Sigma Aldrich, N4638) diluted to 300 μM to stain the eccrine sweat glands. Then, while observing the human skin tissue under a stereomicroscope (Leica, 10450167), the stained eccrine sweat glands were isolated using dissecting tweezers. The isolated eccrine sweat glands were collected in Phosphate Buffered Saline (Fujifilm, 049-29793).

[0077] <1-3. Extraction of RNA from eccrine sweat glands> The isolated eccrine sweat glands and 500 μL of TRI Reagent (registered trademark) (Molecular Research Center, Inc. TR118) were placed in a BioMasher II (Nippi, 320102) to crush the eccrine sweat glands. The crushed material was then left to stand at room temperature for 5 minutes.

[0078] 100 μL of chloroform (Fujifilm, 038-02606) was added to the disrupted material, and the disrupted material was mixed by inversion about 20 times and then allowed to stand at room temperature for 3 minutes.

[0079] The disrupted material was centrifuged at 12000 rpm and 4° C. for 15 minutes. Of the three liquid phases that separated, only the top liquid phase was collected. 500 μL of isopropanol (i-PrOH) (Fujifilm, 166-004836) was added to the collected liquid phase, which was stirred and then allowed to stand at room temperature for 10 minutes.

[0080] The liquid phase was centrifuged at 12000 rpm at 4° C. for 10 minutes, and then the supernatant was removed to collect the precipitate. 1 mL of 75% ethanol (EtOH) was gently added to the collected precipitate, and the mixture was centrifuged at 12000 rpm at 4° C. for 5 minutes.

[0081] The supernatant was removed to collect the precipitate, which was then naturally dried at room temperature for 15 minutes. 30 μL of RNase-free water was added to the precipitate, and the RNA concentration was measured using NanoDrop 2000 (Thermo Fisher Scientific, ND-2000).

[0082] <2. Real-time PCR> cDNA was synthesized from the RNA extracted in <1-3> above using SuperScript 3 First-Strand Synthesis System for RT-PCR (Thermo Fisher Scientific, 18080051).

[0083] A solution for PCR reaction was prepared using THUNDERBIRD SYBER qPCR Mix (TOYOBO, QPS-201). The PCR reaction consisted of 40 cycles of thermal denaturation at 95°C for 15 seconds, annealing at 55°C for 10 seconds, and extension at 72°C for 30 seconds. Gene expression analysis was performed using a ViiATM 7 Real Time PCR System (Thermo Fisher Scientific, ViiA 7). When detecting RNA expression levels, 18s RNA was used as an endogenous control to calculate relative expression levels. The primers used for gene analysis are listed in Table 2 below.

[0084] [Table 2]

[0085] The test results are shown in Figure 1. In Figure 1, the vertical axis is shown as "Means ± SD (n = 8, P = NS)." As is clear from Figure 1, among the TRP channels, TRPM4, TRPV1, and TRPV3 in particular were expressed in eccrine sweat glands.

[0086] <3. Expression analysis of TRP channels in sweat glands> <3-1.Whole mount immunohistochemical staining (WM:Whole mount staining)> Eccrine sweat glands were isolated anatomically from human skin tissue and fixed by incubating them overnight at 4°C in a tissue fixation solution (4% paraformaldehyde-phosphate buffer (4% PFA) (Fujifilm, 163-20145)).

[0087] The next day, the eccrine sweat glands were washed with PBS, and then the eccrine sweat glands were placed in a tissue blocking solution (5% bovine serum albumin (BSA) (Sigma, A7030) / PBS / 0.1% Tween 20 (Sigma, SLCJ0231), 1% Triton-X (Sigma, T8787)) and incubated overnight at 4°C for blocking.

[0088] The primary antibody was diluted to an appropriate dilution ratio using dilution solution (2% BSA / PBS / 0.1% Tween 20) to prepare a primary antibody solution. The eccrine sweat glands after blocking were placed in the primary antibody solution and incubated overnight at 4°C to carry out the primary antibody reaction. Information on the primary antibodies used is shown in Table 3.

[0089] [Table 3]

[0090] The next day, the eccrine sweat glands after the primary antibody reaction were washed with PBS. The secondary antibody was diluted to an appropriate dilution ratio using a dilution solution (2% BSA / PBS / 0.1% Tween 20), and then a secondary antibody solution was prepared by adding 1 / 1000 volume of Hoechst 33342 (Thermo Fisher Scientific, H3570) to the dilution solution. The washed eccrine sweat glands were placed in the secondary antibody solution and incubated overnight at 4°C in the dark to perform the secondary antibody reaction. Information on the secondary antibodies used is shown in Table 4.

[0091] [Table 4]

[0092] The next day, the eccrine sweat glands after the secondary antibody reaction were washed with PBS. After that, the eccrine sweat glands were permeabilized with 80% glycerol (Sigma, G9012), and then sandwiched between cover glasses and observed using a confocal laser scanning microscope (OLYMPUS, FV3000). The observation results are shown in <3-4. Observation Results> below.

[0093] <3-2. Immunohistochemical staining (IHC)> The human skin tissue was fixed by incubating it in 4% paraformaldehyde overnight at 4° C. The human skin tissue was dehydrated, replaced with an organic solvent, and replaced with paraffin to prepare a paraffin block. Paraffin sections having a thickness of 5 μm were cut from the paraffin block using a sliding microtome (Leica, SM2010).

[0094] The paraffin sections were deparaffinized, and the deparaffinized paraffin sections were then incubated in a citrate buffer at 100° C. for 20 minutes or in a Tris-EDTA buffer at 100° C. for 20 minutes to perform activation.

[0095] The paraffin sections were washed with PBS, and then the paraffin sections were placed in a section blocking solution (5% BSA / PBS / 0.1% Triton-X) and incubated at room temperature for 1 hour for blocking treatment.

[0096] The primary antibody solution was prepared by diluting the primary antibody to an appropriate dilution ratio using dilution solution (2% BSA / PBS / 0.1% Tween 20). The paraffin sections after blocking were placed in the primary antibody solution and incubated overnight at 4°C to carry out the primary antibody reaction. Information on the primary antibodies used is shown in Table 3.

[0097] The next day, the paraffin sections after the primary antibody reaction were washed with PBS. The secondary antibody was diluted to an appropriate dilution ratio using a dilution solution (2% BSA / PBS / 0.1% Tween 20), and then 1 / 1000 volume of Hoechst 33342 (Thermo Fisher Scientific, H3570) was added to the dilution solution to prepare a secondary antibody solution. The washed paraffin sections were placed in the secondary antibody solution and incubated overnight at 4°C in the dark to perform the secondary antibody reaction. Information on the secondary antibodies used is shown in Table 4.

[0098] The next day, the paraffin sections after the secondary antibody reaction were washed with PBS. Then, the paraffin sections were mounted between cover glasses using mounting medium Prolong Gold (Thermo Fisher Scientific, P36980) and observed using a confocal laser scanning microscope (OLYMPUS, FV3000). The observation results are shown in <3-4. Observation Results> below.

[0099] <3-3. Fluorescence in situ hybridization (FISH)> Fluorescence in situ hybridization was performed using the paraffin sections after deparaffinization as described above in <3-2> according to the protocol for RNAscope Multiplex Fluorescent Reagent Kit v2 (ACD, 323100).

[0100] RNAscope Target Probe-Hs-TRPV1 (ACD, 415381), RNAscope Target Probe-Hs-TRPV3 (ACD, 573471), and RNAscope Target Probe-Hs-TRPM4-C1 (ACD, 1181941-C1) were labeled with Opal 520 (Akoya Bioscience, FP1487001KT) (1:1500).

[0101] Paraffin sections were deparaffinized and stained with the labeled probes, followed by DAPI staining. The positive control probe was RNAscope Positive control Probe-Hs-PPIB (ACD, 313901), and the negative control probe was RNAscope Negative control Probe-DapB (ACD, 310043).

[0102] In addition, in order to perform subsequent immunostaining, the paraffin sections were washed with PBS and then placed in a section blocking solution (5% BSA / PBS / 0.1% Triton-X) and incubated at room temperature for 1 hour to perform a blocking treatment.

[0103] The primary antibody solution was prepared by diluting the primary antibody to an appropriate dilution ratio using dilution solution (2% BSA / PBS / 0.1% Tween 20). The paraffin sections after blocking were placed in the primary antibody solution and incubated overnight at 4°C to carry out the primary antibody reaction. Information on the primary antibodies used is shown in Table 3.

[0104] The next day, the paraffin sections after the primary antibody reaction were washed with PBS. The secondary antibody was diluted to an appropriate dilution ratio using a dilution solution (2% BSA / PBS / 0.1% Tween 20), and then 1 / 1000 volume of Hoechst 33342 (Thermo Fisher Scientific, H3570) was added to the dilution solution to prepare a secondary antibody solution. The washed paraffin sections were placed in the secondary antibody solution and incubated overnight at 4°C in the dark to perform the secondary antibody reaction. Information on the secondary antibodies used is shown in Table 4.

[0105] The next day, the paraffin sections after the secondary antibody reaction were washed with PBS. Then, the paraffin sections were mounted between cover glasses using mounting medium Prolong Gold (Thermo Fisher Scientific, P36980) and observed using a confocal laser scanning microscope (OLYMPUS, FV3000). The observation results are shown in <3-4. Observation Results> below.

[0106] <3-4. Observation results> The test results of whole mount immunohistochemical staining (WM), immunohistochemical staining (IHC), and fluorescent in situ hybridization (FISH) are shown in Table 5. In Table 5, "++" indicates that a large amount of expression was observed, "+" indicates that expression was observed, and "-" indicates that no expression was observed.

[0107] As shown in Table 5, TRPM4, TRPV1, and TRPV3 were found to be expressed in both the secretory ducts, which play a major role in sweat secretion, and the ducts, which play a major role in sweat reabsorption.

[0108] In particular, it was revealed that TRPM4, TRPV1, and TRPV3 are expressed in large amounts in luminal cells that compose secretory ducts, and in luminal cells and basal layer cells that compose ducts.

[0109] [Table 5]

[0110] <4. Functional analysis of TRP channels in sweat glands> 2.0 × 10 cells were placed in the wells of an ultra-low attachment surface 6-well plate (CORNING, 3471) filled with basal medium. 4 ~1.0×10 5 cell / cm 2Immortalized sweat gland cells (Hayakawa, T., Fujita, F., Okada, F., & Sekiguchi, K. (2022). Establishment and characterization of immortalized sweat gland myoepithelial cells. Scientific Reports, 12(1). https: / / doi.org / 10.1038 / s41598-021-03991-5) were seeded at a density of 100 μg / ml. The immortalized sweat gland cells were incubated at 37°C and 5% CO 2 The composition of the basal medium is shown in Table 6.

[0111] [Table 6]

[0112] On the 7th day of culture, the immortalized cells were collected. The immortalized sweat gland cells that had been cultured as spheroids for 7 days were collected into a 15 mL centrifuge tube (As One, 4-3632-01), and the tube was centrifuged at 300 g for 5 minutes, after which the supernatant was removed.

[0113] 2 mL of Accutase (BD Biosciences, 561527) was added to the collected immortalized sweat gland cells and incubated at 37° C. for 10 minutes. The mixture of immortalized sweat gland cells and Accutase was then pipetted for 1 minute to break the immortalized sweat gland cells into single cells.

[0114] The immortalized sweat gland cells were washed with PBS, and then 2.0 × 10 cells were added to wells of an ultra-low attachment surface 6-well plate filled with basal medium (see Table 6). 4 ~1.0×10 5 cell / cm 2 The immortalized sweat gland cells were seeded at a density of 100 μg / ml. The immortalized sweat gland cells were incubated at 37° C., 5% CO 2 The cells were cultured in an incubator.

[0115] 10 μM ICG-001 (Selleck Chemicals, S2662) was added to the basal medium, and the basal medium was replaced with fresh basal medium containing 10 μM ICG-001 every 7 days. On the 14th day of culture, the differentiation-induced immortalized sweat gland cells were collected.

[0116] The immortalized sweat gland cells were differentiated into single cells. 4 cells) were suspended in 30 μL of basal medium (see Table 6).

[0117] To the suspension, 0.5 μL of iMatrix-332 (MATRIXOME, 892031) and 0.5 μL of iMatrix-511 (MATRIXOME, 892011) were added, and the suspension was allowed to adhere onto a sterilized cover glass.

[0118] The cover glass was placed on an open chamber and incubated with standard bath solution (140 mM NaCl, 5 mM KCl, 2 mM MgCl 2 , 2 mM CaCl 2 The reagents were perfused with diluted 10 mM HEPES, 10 mM glucose, pH 7.4 and standard bath solution. The reagents and their concentrations are shown in Table 7.

[0119] [Table 7]

[0120] Free Ca in immortalized sweat gland cells 2+ Concentrations were measured by dual wavelength fura-2 (Invitrogen, Molecular Probes) microfluorometry with excitation at 340 / 380 nm and emission at 510 nm. Fura-2 Ratio images were calculated and acquired using an IP-Lab image processing system (Scanalytics Inc., Fairfax, VA USA).

[0121] The test results are shown in Figure 2. As is clear from Figure 2, the TRPV1 agonist and the TRPV4 agonist, like the positive control TRPV4 agonist, suppressed the free Ca2+ concentration in the immortalized sweat gland cells. 2+ The concentration was increased.

[0122] Sweat is produced by store-operated Ca2+ receptors in the clear cells, which are the secretory cells of the sweat glands. 2+ It is known that Ca is secreted by activation of the SOCE influx. In addition, Ca influx into cells is used to evaluate the secretory activity of secretory glands, including sweat glands. 2+ Inflow analysis has been used (see, for example, Non-Patent Document 1). The results in Figure 2 show that TRPV1 agonists and TRPV4 agonists promote sweat secretion. [Industrial Applicability]

[0123] The present invention can be widely used in the fields of cosmetics, medical products, and the like.

Claims

1. A method for regulating sweating, comprising a step of contacting, in vitro, sweat gland cells with an activity regulator of at least one TRP channel selected from the group consisting of TRPV1, TRPV3 and TRPM4.

2. A step of contacting a test substance with a cell having at least one type of TRP channel activity selected from the group consisting of TRPV1, TRPV3, and TRPM4; and measuring a change in activity of the TRP channel in a cell having TRP channel activity that has been contacted with the test substance.

3. A sweat regulating agent comprising, as an active ingredient, an activity regulator of at least one TRP channel selected from the group consisting of TRPV1, TRPV3 and TRPM4.

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