JP7199891B2 - Test sample evaluation method - Google Patents

Description

The present invention relates to methods for evaluating test samples. More particularly, the present invention relates to methods for evaluating test samples useful for the development of anti-inflammatory cosmetics, anti-inflammatory cosmetics, anti-inflammatory agents, anti-inflammatory agents, and the like.

Inflammation is one of biological reactions caused by chemical stimuli, physical stimuli, and the like. Inflammation may damage tissues and the like, so anti-inflammatory agents are sometimes applied to the inflammation. However, since inflammation has various onset mechanisms, new anti-inflammatory agents that suppress inflammation are eagerly awaited.

By the way, TRPV4 is a channel involved in sensory reception (see, for example, Non-Patent Document 1). However, at present, the present inventors have not found any literature that specifically describes the relationship between activation of TRPV4 and inhibition of inflammation.

Tomohiro Numata, et al., "Structure and Diverse Functions of TRP Channels," Japanese Biochemical Society of Japan, Seikagaku, Japan Biochemical Society, 2009, Vol. 81, No. 11, pp.962-983

The present invention has been made in view of the prior art described above, and an object of the present invention is to provide a method for evaluating a test sample that enables easy evaluation of the anti-inflammatory effect of the test sample.

The gist of the present invention is a test sample evaluation method for evaluating the anti-inflammatory action of a test sample, wherein the test sample is brought into contact with monocytes, and a physiological event caused by TRPV4 activation of the monocytes is measured. and a method for evaluating a test sample, which comprises evaluating the anti-inflammatory effect of the test sample based on the physiological event.

ADVANTAGE OF THE INVENTION According to this invention, the evaluation method of the test sample which can evaluate the anti-inflammatory effect which a test sample has easily is provided.

1 is a graph showing the results of examining the relationship between sample types and IL-1β relative levels in Example 1. FIG. 2 is a graph showing the results of examining the relationship between sample types and IL-1β relative levels in Example 2. FIG. 10 is a graph showing the results of examining the relationship between the type of RNA sample and the degree of increase in the amount of IL-1β mRNA in Example 3. FIG. 10 is a graph showing the results of examining the relationship between the type of RNA sample and the degree of increase in the amount of IL-1β mRNA in Example 4. FIG. 10 is a graph showing the results of examining the relationship between sample type and IL-1β mass concentration in Example 5. FIG.

The test sample evaluation method of the present invention is a test sample evaluation method for evaluating the anti-inflammatory effect of the test sample, wherein the test sample is brought into contact with monocytes, and the physiology caused by the activation of TRPV4 in the monocytes. measuring a physiological event, and evaluating the anti-inflammatory action of the test sample based on the physiological event.

According to the method of the present invention, the physiological event caused by TRPV4 activation of monocytes by the test sample is measured, and the anti-inflammatory effect of the test sample is evaluated based on the physiological event. Therefore, it is possible to easily evaluate the anti-inflammatory effect of the test sample, preferably the anti-inflammatory effect mediated by TRPV4 activation of monocytes.

As a specific example of the method of the present invention,
(A) contacting monocytes with a test sample and measuring physiological events triggered by activation of TRPV4;
(B) contacting the monocytes with a test sample under conditions that inhibit the function of TRPV4 on the monocytes, and (C) measuring physiological events caused by TRPV4 activation; and (C) steps (A) and (B) Based on the physiological events measured in (B), evaluate the activity-promoting effect of the test sample on TRPV4 of monocytes, and based on the evaluation result of the activity-promoting effect, the step of evaluating the anti-inflammatory effect of the test sample. However, the present invention is not limited only to such examples.

In step (A), monocytes are contacted with a test sample and physiological events triggered by TRPV4 activation are measured.

Monocytes may be isolated non-established monocytes (hereinafter referred to as “isolated monocytes”) or monocyte-established cells. Sources of isolated monocytes include, for example, human peripheral blood, human bone marrow, mouse peripheral blood, and mouse bone marrow, but the present invention is not limited to these exemplifications. Examples of isolated monocytes include isolated human monocytes, isolated mouse monocytes, and the like, but the present invention is not limited to these exemplifications. Human isolated monocytes include, for example, classical monocytes, intermediate monocytes, non-classical monocytes, etc., but the present invention is not limited only to such exemplifications. Human classical monocytes are CD14 positive and CD16 negative (CD14 ⁺ CD16 ⁻ ). Human intermediate monocytes are CD14-positive and CD16-positive (CD14 ⁺ CD16 ⁺ ). Human non-classical monocytes exhibit CD14 low or negative expression and CD16 positivity (CD14 ^lo/− CD16 ⁺ ). Mouse monocytes include, for example, classical monocytes, non-classical monocytes, etc., but the present invention is not limited to such exemplification. Mouse classical monocytes show high Ly6c expression (Ly6c ^hi ). Mouse non-classical monocytes show low Ly6c expression (Ly6c ^lo ). Isolated monocytes can be prepared, for example, using collected peripheral blood or collected bone marrow according to the method described in Preparation Example 1 below. Examples of monocyte cell lines include human monocyte cell lines such as THP-1, U937, and KG1, but the present invention is not limited to these exemplifications.

TRPV4 is one of the non-selective cation channels activated by, for example, a decrease in extracellular fluid osmotic pressure. TRPV4 has the amino acid sequence shown in GenBank Accession No: NM_021625.

Examples of test samples include inorganic compounds, organic compounds, plant extracts, cell culture supernatants, and cell extracts, but the present invention is not limited to these examples. When the test sample is liquid, it may be used as it is, or may be diluted with a solvent if necessary. Moreover, when the test sample is solid, it can be used by dissolving it in a solvent.

Since the solvent varies depending on the type of test sample, the type of physiological event to be measured, etc., it cannot be decided unconditionally. preferably. Solvents include, for example, physiological saline, phosphate buffered saline, purified water, ethanol, ethanol aqueous solution, calcium-containing solution [composition: 140 mM sodium chloride, 5 mM potassium chloride, 2 mM magnesium chloride, 2 mM calcium chloride, 10 mM glucose and 10 mM 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid (HEPES) hydrochloric acid buffer (pH 7.4)], calcium-free solution [composition: 140 mM sodium chloride, 5 mM potassium chloride, 2 mM magnesium chloride, 5 mM glycol ether diamine tetraacetic acid, 10 mM glucose and 10 mM HEPES hydrochloric acid buffer (pH 7.4)], dimethyl sulfoxide (DMSO), etc., but the present invention is limited only to such examples. is not.

Contacting monocytes with a test sample can be carried out, for example, by culturing monocytes in a monocyte medium containing the test sample under culture conditions suitable for maintaining the physiological functions of monocytes. .

A monocyte medium can be prepared, for example, by adding medium supplements to a basal medium. Examples of medium additives include serum and antibiotics, but the present invention is not limited to such examples. Basic media include, for example, RPMI1640 medium, MEM medium, IMDM medium, Ham's F12 medium, etc., but the present invention is not limited to these examples. The monocyte culture medium may be a serum starvation medium or a nonserum starvation medium. Serum starvation media usually have a serum concentration of preferably 0-1% (v/v), more preferably 0-0.5% (v/v). Serum-free starvation media usually have a serum concentration of preferably 2-10% (v/v), more preferably 5-10% (v/v). Among these media for monocytes, serum starvation media are preferred because they contain serum containing various physiologically active substances, and thus the effects of test samples can be evaluated more specifically.

The amount of the test sample in the monocyte culture medium varies depending on the type of test sample, the number of monocytes, etc., and cannot be determined unconditionally. is preferred. The monocyte culture medium used for contacting the monocytes with the test sample is the same as the monocyte culture medium used as the solvent for the test sample. The contact time between the monocytes and the test sample varies depending on the type of test sample, culture temperature, etc., and cannot be determined unconditionally. The contact time between the monocytes and the test sample is preferably 0.15 hours or more, more preferably 0, from the viewpoint of accurately evaluating anti-inflammatory effects, preferably anti-inflammatory effects mediated by TRPV4 activation of monocytes. .5 hours or more, more preferably 1 hour or more, still more preferably 3 hours or more, still more preferably 6 hours or more, and the same anti-inflammatory effect as described above, preferably via TRPV4 activation of monocytes. It is preferably 72 hours or less, more preferably 12 hours or less, still more preferably 8 hours or less, from the viewpoint of accurately evaluating the anti-inflammatory action.

The culture conditions include culture temperature, carbon dioxide concentration in the culture atmosphere, and the like. The culture temperature is preferably 35° C. or higher, more preferably 36.5° C. or higher, from the viewpoint of accurately evaluating the anti-inflammatory effect, preferably the anti-inflammatory effect mediated by TRPV4 activation of monocytes. Similarly, the temperature is preferably 38° C. or lower, more preferably 37.5° C. or lower, from the viewpoint of accurately evaluating anti-inflammatory effects, preferably anti-inflammatory effects mediated by TRPV4 activation of monocytes. Specifically, the culture temperature is usually preferably 35° C. to 38° C., more preferably 36° C., from the viewpoint of accurately evaluating the anti-inflammatory effect, preferably the anti-inflammatory effect mediated by TRPV4 activation of monocytes. 5 to 37.5°C. The carbon dioxide concentration in the culture atmosphere is preferably 4% (v/v) or more, more preferably 5% (v/v) or more, from the viewpoint of accurately evaluating the anti-inflammatory effect, preferably the anti-inflammatory effect mediated by TRPV4 activation of monocytes. % (v / v) or more, and from the viewpoint of accurately evaluating the anti-inflammatory effect as described above, preferably the anti-inflammatory effect mediated by TRPV4 activation of monocytes, preferably 10% (v / v) 7% (v/v) or less, more preferably 7% (v/v) or less. Specifically, the carbon dioxide concentration is usually preferably 4 to 10% (v / v) from the viewpoint of accurately evaluating the anti-inflammatory effect, preferably the anti-inflammatory effect mediated by the activation of TRPV4 of monocytes. , more preferably 5-7% (v/v).

Physiological events caused via activation of TRPV4 include, for example, (i) a decrease in the expression level of inflammation-related factors or their mRNA due to the activation of TRPV4 in monocytes, (ii) TRPV4 in monocytes. Suppression of differentiation from monocytes to M1-type macrophages due to activation, (iii) increase in intracellular calcium ion concentration of monocytes due to activation of TRPV4 in monocytes, (iv) activity of TRPV4 in monocytes. Examples include an increase in the membrane potential of monocytes due to quenching, but the present invention is not limited to such examples.

Methods for measuring physiological events include the following measurement methods 1 to 4, etc., but the present invention is not limited to these examples.

<Measurement method 1>
In Assay Method 1, the above-mentioned “(i) decrease in the expression level of an inflammation-related factor or its mRNA due to activation of TRPV4 in monocytes” is used as the physiological event. Measurement method 1 is
(1a) contacting monocytes with an inflammatory substance; and (1b) measuring the expression level of an inflammation-related factor or its mRNA.

Contacting monocytes with an inflammatory substance can be carried out, for example, by culturing the monocytes in a monocyte medium containing the inflammatory substance under culture conditions suitable for maintaining the physiological function of monocytes. can be done.

Examples of proinflammatory substances include lipopolysaccharide; CpG oligonucleotides such as K3CpG; lipopeptides such as Pam3CSK4;

The amount of inflammation-causing substance in the monocyte culture medium varies depending on the type of inflammation-causing substance, the number of monocytes, etc., and cannot be determined unconditionally. It is preferable to determine as appropriate. The culture conditions are similar to those used when monocytes are contacted with the test sample.

When measurement method 1 is used in step (A), in step (1a) of measurement method 1, the test sample and the proinflammatory substance may be brought into contact with monocytes simultaneously or separately. .

When the test sample and the inflammation-causing substance are brought into contact with the monocytes at the same time, for example, a monocyte culture medium containing the test sample and the inflammation-causing substance can be used. The culture temperature at which the test sample and the inflammatory substance are brought into contact with the monocytes at the same time is the same as the culture temperature under the culture conditions described above. The contact time between monocytes, the test sample, and the inflammation-causing substance varies depending on the type of test sample, the type of inflammation-causing substance, the culture temperature, etc., and cannot be determined indiscriminately. It is preferable to appropriately determine the amount depending on the type of culture, culture temperature, and the like.

When the test sample and the inflammatory substance are brought into contact with the monocytes separately, the inflammatory substance may be brought into contact with the monocytes after the test sample is brought into contact with the monocytes, or the inflammatory substance is brought into contact with the monocytes. After that, the test sample may be brought into contact with the monocytes. The culture temperature at which the inflammatory substance and monocytes are brought into contact is the same as the culture temperature under the culture conditions described above. The contact time between monocytes and inflammation-causing substances varies depending on the type of inflammation-causing substance, culture temperature, etc., and cannot be determined unconditionally. is preferred. The contact time between monocytes and inflammation-causing substances is preferably 0.15 hours or more, more preferably 0.15 hours or more, more preferably from the viewpoint of accurately evaluating anti-inflammatory effects, preferably anti-inflammatory effects mediated by TRPV4 activation of monocytes. 0.5 hours or more, more preferably 1 hour or more, still more preferably 3 hours or more, still more preferably 6 hours or more. It is preferably 72 hours or less, more preferably 12 hours or less, and still more preferably 8 hours or less, from the viewpoint of accurately evaluating the anti-inflammatory effect obtained.

Inflammation-related factors are factors present at sites of inflammation. Examples of inflammation-related factors include IFN-α, IFN-γ, IL-1α, IL-1β, IL-12p70, IL-13, IL-17α, IL-4, IL-6, TNF-α, GM- inflammatory cytokines such as CSF; chemokines such as IP-10, MCP-1, MIP-1α and MIP-1β; cell adhesion factors such as E-selectin, P-selectin and sICAM-1; is not limited only to such examples. Among these inflammation-related factors, IFN-α, IFN-γ, IL-1α, IL- 1β, IL-12p70, IL-13, IL-17α, IL-4, IL-6, TNF-α, GM-CSF, IP-10, MCP-1, MIP-1α, MIP-1β, E-selectin, P-selectin and sICAM-1 are preferred.

Examples of methods for measuring the expression level of inflammation-related factors include enzyme-labeled immunoassay (hereinafter referred to as "ELISA"), Western blotting, immunofluorescence staining, and fluorescence-activated cell sorting (hereinafter referred to as "FACS"). , multiplex assays using fluorescence-labeled beads, etc., but the present invention is not limited only to such exemplifications. Examples of methods for measuring the expression level of inflammation-related factor mRNA include real-time RT-PCR, northern blotting, and the like, but the present invention is not limited to these exemplifications.

Antibodies used in ELISA and Western blotting include, for example, antibodies against inflammation-related substances or antibody fragments thereof, but the present invention is not limited to such examples. Antibodies against inflammation-related substances may be monoclonal antibodies or polyclonal antibodies. As antibodies and antibody fragments thereof against inflammation-related substances, commercially available antibodies and antibody fragments thereof can be used.

Primer pairs used in the real-time RT-PCR method include, for example, a primer consisting of part of the base sequence of a nucleic acid encoding an inflammation-related factor and a primer consisting of part of the base sequence of the antisense strand of the nucleic acid. Examples include primers and the like, but the present invention is not limited to such examples. A commercially available primer pair can be used as the primer pair.

Probes used in Northern blotting include, for example, nucleic acids consisting of all or part of nucleic acids encoding inflammation-related factors, nucleic acids consisting of all or part of antisense strands of nucleic acids encoding inflammation-related factors, and the like. However, the invention is not limited to only such exemplifications. When the probe is a nucleic acid consisting of a part of a nucleic acid encoding an inflammation-related factor or a nucleic acid consisting of a part of the antisense strand of the nucleic acid, the length of the probe varies depending on the type of inflammation-related factor, etc. Since it cannot be determined, it is preferably determined as appropriate according to the type of inflammation-related factor. The length of the probe is usually preferably 20 to 500 nucleotides from the viewpoint of improving the measurement accuracy of the expression level of inflammation-related factors.

<Measurement method 2>
In Assay Method 2, the aforementioned “(ii) Suppression of differentiation of monocytes into M1-type macrophages caused by activation of TRPV4 in monocytes” is used as the physiological event. Differentiation of monocytes into M1-type macrophages can be performed by contacting the monocytes with GM-CSF. Measurement method 2 is
(2a) contacting monocytes with granulocyte-macrophage colony-stimulating factor (hereinafter referred to as "GM-CSF"); and (2b) measuring the number of M1-type macrophages.

Contacting the monocytes with GM-CSF is performed, for example, by culturing the monocytes in a monocyte medium containing GM-CSF under culture conditions suitable for maintaining the physiological functions of the monocytes. can be done.

Since the amount of GM-CSF in the monocyte culture medium varies depending on the number of monocytes and the like, it cannot be determined unconditionally. The amount of GM-CSF in the monocyte medium is usually preferably 5 ng/mL or more, more preferably 5 ng/mL or more, from the viewpoint of accurately evaluating the anti-inflammatory effect, preferably the anti-inflammatory effect mediated by TRPV4 activation of monocytes. is 10 ng/mL or more, and is preferably 200 ng/mL or less, more preferably 100 ng, from the viewpoint of accurately evaluating the anti-inflammatory effect, preferably the anti-inflammatory effect mediated by TRPV4 activation of monocytes. /mL or less. The monocyte culture medium is the same as the monocyte culture medium used as the solvent for the test sample. The culture conditions are similar to those used when monocytes are contacted with the test sample.

When measurement method 2 is used in step (A), in step (2a) of measurement method 2, the test sample and GM-CSF may be brought into contact with monocytes simultaneously or separately. .

When the test sample and GM-CSF are brought into contact with monocytes at the same time, for example, a monocyte culture medium containing the test sample and GM-CSF can be used. In this case, the contact time between the monocytes, the test sample, and GM-CSF varies depending on the type of the test sample, etc., and cannot be determined unconditionally. .

When the test sample and GM-CSF are brought into contact with monocytes separately, the order in which the test sample and GM-CSF are brought into contact with monocytes is not particularly limited. The contact time between monocytes and GM-CSF is preferably 3 days or more, more preferably 5 days or more, from the viewpoint of suppressing the differentiation of monocytes into M1-type macrophages due to TRPV4 activation of monocytes. From the viewpoint of accurately evaluating the anti-inflammatory effect, preferably the anti-inflammatory effect mediated by TRPV4 activation of monocytes, the period is preferably 14 days or less, more preferably 10 days or less.

Methods for measuring the number of M1-type macrophages include, for example, flow cytometry, but the present invention is not limited to such examples. When measuring the number of M1-type macrophages by flow cytometry, markers of M1-type macrophages include, for example, CD14, CD11b, CD68, CD80, CD163, etc., but the present invention is limited only to such exemplifications. not a thing Among these markers for M1-type macrophages, CD14, CD11b and CD68 are preferred from the viewpoint of easy differentiation between monocytes and M1-type macrophages. M1-type macrophages can be detected and counted as an indicator of "CD14-negative, CD11b-positive and CD68-positive".

<Measurement method 3>
In measurement method 3, the aforementioned “(iii) increase in intracellular calcium ion concentration in monocytes caused by activation of TRPV4 in monocytes” is used as the physiological event. The intracellular calcium ion concentration of monocytes can be measured by introducing a calcium indicator into the monocytes and using the amount of the calcium indicator bound to the calcium ions in the monocytes as an indicator.

Since the amount of the calcium indicator bound to calcium ions can be measured with a simple operation, the calcium indicator should be a reagent that can detect changes before and after binding to calcium ions by changes in optical properties, etc. is preferred. Changes in optical properties include, for example, changes in fluorescence intensity and changes in absorbance, but the present invention is not limited to these examples. Calcium indicators include, for example, fluorescent calcium indicators whose fluorescence intensity changes before and after binding with calcium ions, but the present invention is not limited to such examples. A specific example of a calcium indicator is 1-[6-amino-2-(5-carboxy-2-oxazolyl)-5-benzofuranyloxy]-2-(2-amino-5-methylphenoxy)ethane-N ,N,N′,N′-tetraacetic acid pentaacetoxymethyl ester (hereinafter referred to as “Fura 2-AM”), 1-[2-amino-5-(2,7-dichloro-6-hydroxy-3-oxo -9-xanthenyl)phenoxy]-2-(2-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (hereinafter referred to as "Fluo 3-AM"), 1-[2-amino-5-(2,7-difluoro-6-acetoxymethoxy-3-oxo-9-xanthenyl)phenoxy]-2-(2-amino-5-methylphenoxy)ethane-N,N, Examples thereof include fluorescent calcium indicators such as N',N'-tetraacetic acid tetraacetoxymethyl ester (hereinafter referred to as "Fluo 4-AM"), but the present invention is not limited to such examples. Among the calcium indicators, from the viewpoint of distinguishing between the dynamics of calcium ions and the dynamics of contaminants and accurately evaluating anti-inflammatory effects, preferably anti-inflammatory effects mediated by activation of TRPV4 in monocytes, calcium ions and Fluorescent calcium indicators that change fluorescence intensity before and after binding are preferred, with Fura 2-AM, Fluo 3-AM and Fluo 4-AM being more preferred.

The fluorescent calcium indicator may have one excitation wavelength, or two or more excitation wavelengths. When the calcium indicator is a fluorescent calcium indicator, it is preferable to use a fluorescent calcium indicator having two excitation wavelengths because the fluorescence intensity can be easily measured and the detection intensity is high. When measuring changes in intracellular calcium concentration, when a fluorescent calcium indicator having one excitation wavelength is used, changes in intracellular calcium concentration can be measured based on fluorescence intensity at the excitation wavelength. When measuring changes in intracellular calcium concentration, when using fluorescent calcium indicators having two or more excitation wavelengths, the viewpoint of accurately evaluating anti-inflammatory effects, preferably anti-inflammatory effects mediated by activation of TRPV4 in monocytes. From, two excitation wavelengths (first excitation wavelength and second excitation wavelength) selected from the two or more excitation wavelengths are selected, and the fluorescence intensity at each of the first excitation wavelength and the second excitation wavelength is calculated. Changes in intracellular calcium concentration can be measured based on the fluorescence intensity ratio obtained. When measuring the intracellular calcium concentration, for example, when using FURA 2-AM, which is a fluorescent calcium indicator having two excitation wavelengths, the fluorescence intensity at the first excitation wavelength (hereinafter also referred to as "first fluorescence intensity") The fluorescence intensity at the excitation wavelength of 380 nm can be used as the fluorescence intensity at the excitation wavelength of 340 nm and the fluorescence intensity at the second excitation wavelength (hereinafter also referred to as "second fluorescence intensity"). The fluorescence intensity ratio is, for example, the formula (I):
[Fluorescence intensity ratio]=[First fluorescence intensity]/[Second fluorescence intensity] (I)
can be determined based on

Methods for introducing a fluorescent calcium indicator into monocytes include, for example, a method of circulating a buffer solution containing the fluorescent calcium indicator in a perfusion chamber containing monocytes, but the present invention is limited to such examples. It is not limited. Examples of the method of contacting the monocytes after introducing the indicator with the test sample include a method of circulating a buffer solution containing the fluorescent calcium indicator in a perfusion chamber containing the monocytes introduced with the fluorescent calcium indicator. , the invention is not limited to such examples only. Examples of the method of contacting the cells with calcium ions after contact with the test sample include a method of circulating a buffer solution containing calcium ions and not containing the test sample, but the present invention is limited to such examples only. It is not limited. Examples of buffers include HEPES buffers, but the present invention is not limited to such examples. The temperature at which the fluorescent calcium indicator is introduced into the monocytes, the contact temperature between the monocytes and the test sample, and the contact temperature between the monocytes and the calcium ions have an anti-inflammatory effect, preferably via TRPV4 activation of the monocytes. From the viewpoint of accurately evaluating the anti-inflammatory action, the temperature is preferably 35° C. or higher, more preferably 36.5° C. or higher. From the viewpoint of accurately evaluating the action, the temperature is preferably 38° C. or lower, more preferably 37.5° C. or lower. The contact time between the monocytes and the test sample is preferably 0.5 minutes or more, more preferably 1 minute, from the viewpoint of accurately evaluating anti-inflammatory effects, preferably anti-inflammatory effects mediated by TRPV4 activation of monocytes. Minutes or more, preferably 1 hour or less, more preferably 0.5 hours or less, from the viewpoint of accurately evaluating changes in intracellular calcium. The contact time between monocytes and calcium ions is preferably 1 minute or more, more preferably 5 minutes or more, from the viewpoint of accurately evaluating anti-inflammatory effects, preferably anti-inflammatory effects mediated by TRPV4 activation of monocytes. From the viewpoint of accurately evaluating the anti-inflammatory effect, preferably the anti-inflammatory effect mediated by TRPV4 activation of monocytes, the time is preferably 2 hours or less, more preferably 1 hour or less.

<Measurement method 4>
In measurement method 4, the aforementioned “(iv) increase in monocyte membrane potential caused by activation of TRPV4 in monocytes” is used as the physiological event. Methods for measuring the membrane potential of monocytes include, for example, patch clamp methods such as the whole-cell method and the cell-attach method, but the present invention is not limited to these examples.

In step (B), the monocytes are brought into contact with a test sample under conditions that inhibit the function of TRPV4 on the monocytes, and the physiological events caused by the activation of TRPV4 are measured. Measurement of physiological events in step (B) can be performed under the same conditions and methods as in step (A), except that the measurement is performed under conditions that inhibit the function of TRPV4 on monocytes. Also, the physiological event measured in step (B) is the same type of physiological event as the physiological event measured in step (A).

Methods for suppressing the function of monocyte TRPV4 include, for example, a method of contacting a TRPV4 activity inhibitor with monocyte TRPV4, and a method of knocking down or knocking out monocyte TRPV4. , is not limited to such examples.

Examples of TRPV4 activity inhibitors include 2-methyl-1-[3-(4-morpholinyl)propyl]-5-phenyl-N-[3-(trifluoromethyl)phenyl]-1H-pyrrole-3-carboxamide (hereinafter referred to as “HC-067147”), 3-([1,4′-bipiperidin]-1′-ylmethyl)-7-bromo-N-(1-phenylcyclopropyl)-2-[3-(tri fluoromethyl)phenyl]-4-quinolinecarboxamide (hereinafter referred to as "GSK2193874"), N-[4-[[4-(1-methylethyl)-1-piperazinyl]sulfonyl]phenyl]-2-nitro-4- (Trifluoromethyl)benzamide hydrochloride (hereinafter referred to as "RN9893 hydrochloride"), 2,4-dichloro-N-(1-methylethyl)-N-{2-[(1-methylethyl)amino]ethyl} Examples include benzenesulfonamide (hereinafter referred to as “RN-1734”) and TRPV4 antagonists such as ruthenium red, but the present invention is not limited to such examples. Among these TRPV4 activity inhibitors, TRPV4 antagonists are preferable from the viewpoint of accurately measuring the physiological events caused via the activation of TRPV4 in monocytes. 1734 and ruthenium red are more preferred, and HC067147 and GGSK2193874 are even more preferred.

Since the amount of TRPV4 activity inhibitor to be brought into contact with TRPV4 of monocytes varies depending on the type of TRPV4 activity inhibitor, the number of monocytes, etc., it cannot be determined unconditionally. It is preferable to determine appropriately according to the number of .

The contact between the TRPV4 activity inhibitor and the monocyte TRPV4 is performed before or after the contact between the monocyte and the test sample, from the viewpoint of accurately evaluating the effect of the test sample under the condition of suppressing the TRPV4 function of the monocyte. Simultaneous operation is preferred.

When the TRPV4 activity inhibitor and monocytes are contacted with TRPV4 before the monocytes are contacted with the test sample, the contact time between the TRPV4 activity inhibitor and monocytes with TRPV4 is determined to have an anti-inflammatory effect, preferably on monocytes. From the viewpoint of accurately evaluating the anti-inflammatory effect mediated by the activation of TRPV4, it is preferably 5 minutes or more, more preferably 15 minutes or more, and the same anti-inflammatory effect as described above, preferably monocyte TRPV4 activation. From the viewpoint of accurately evaluating the anti-inflammatory effect via

When the contact between the TRPV4 activity inhibitor and the monocyte TRPV4 is performed simultaneously with the contact between the monocyte and the test sample, the contact time between the TRPV4 activity inhibitor and the monocyte TRPV4 is the time between the monocyte TRPV4 and the test substance. Similar to contact time.

Methods of knocking down TRPV4 in monocytes include, for example, a method of inhibiting TRPV4 gene expression by RNA silencing, a method of inhibiting normal TRPV4 function by expressing a dominant-negative mutant, and the like. However, the invention is not limited to only such exemplifications. Methods for knocking out TRPV4 in monocytes include, for example, a method of disrupting the TRPV4 gene by homologous recombination, a method of disrupting the TRPV4 gene by genome editing technology, etc., but the present invention is limited only to such examples. not to be

In step (C), based on the physiological events measured in steps (A) and (B), the activity-promoting effect of monocytes on TRPV4 by the test sample is evaluated, and based on the evaluation result of the activity-promoting effect, The anti-inflammatory action of the test sample is evaluated.

When the physiological event measured in steps (A) and (B) is "(i) a decrease in the expression level of an inflammation-related factor or its mRNA due to activation of TRPV4 in monocytes", the test sample is Based on at least one of the following indices (1a) and (1b), it is evaluated as having an activity-promoting effect on TRPV4 on monocytes.
<Indicator (1a)>
The expression level of the inflammation-related factor measured in step (A) is lower than the expression level of the inflammation-related factor measured in step (B) <Indicator (1b)>
The expression level of inflammation-related factor mRNA measured in step (A) is lower than the expression level of inflammation-related factor mRNA measured in step (B).

If the physiological event measured in steps (A) and (B) is "(ii) inhibition of differentiation of monocytes into M1-type macrophages due to TRPV4 activation of monocytes", the test sample is Based on the following index (2a), it is evaluated as having an activity-promoting effect on TRPV4 on monocytes.
<Indicator (2a)>
The number of M1-type macrophages measured in step (A) is less than the number of M1-type macrophages measured in step (B)

If the physiological event measured in steps (A) and (B) is "(iii) an increase in intracellular calcium ion concentration of monocytes due to activation of TRPV4 in monocytes", the test sample is: It is evaluated to have an activity-promoting effect on TRPV4 of monocytes based on the index (3a) of .
<Indicator (3a)>
The intracellular calcium ion concentration of monocytes measured in step (A) is higher than the intracellular calcium ion concentration of monocytes measured in step (B)

If the physiological event measured in steps (A) and (B) is "(iv) an increase in monocyte membrane potential due to activation of TRPV4 in monocytes", the test sample will have the following indicators ( Based on 4a), it is evaluated to have an activity-promoting effect on TRPV4 on monocytes.
<Indicator (4a)>
The monocyte membrane potential measured in step (A) is greater than the monocyte membrane potential measured in step (B)

A test sample can be evaluated as having an anti-inflammatory effect, preferably an anti-inflammatory effect mediated by activation of TRPV4, when it has an activity-promoting effect on TRPV4 of monocytes. If the test sample does not have an activity promoting effect on TRPV4 on monocytes, it can be evaluated as having no anti-inflammatory effect, preferably an anti-inflammatory effect mediated by TRPV4 activation.

As described above, according to the test sample evaluation method of the present invention, the anti-inflammatory effect of the test sample, preferably the anti-inflammatory effect mediated by TRPV4 activation, can be easily evaluated. Therefore, the test sample evaluation method of the present invention is expected to be suitably used for the development of anti-inflammatory cosmetics, anti-inflammatory cosmetics, anti-inflammatory agents, anti-inflammatory agents, and the like.

EXAMPLES The present invention will be described in more detail below with reference to examples, but the present invention is not limited only to these examples. In the following, "% (m/v)" indicates mass/volume percent, and "% (v/v)" indicates volume/volume percent (volume %). In the following, the capture antibody solution, quantification sample, biotin detection antibody solution, streptavidin HRP antibody solution, and substrate solution [A solution/B solution (volume ratio) = 1/1] were prepared using an ELISA kit [R · R&D Systems, trade name: Human IL-1beta/IL-1F2 DuoSet (registered trademark) ELISA Development]. The meaning of each abbreviation is as follows.

<Explanation of abbreviations>
BSA: bovine serum albumin EDTA: ethylenediaminetetraacetic acid FBS: fetal bovine serum GAPDH: glyceraldehyde-3-phosphate dehydrogenase LPS: lipopolysaccharide PBS: phosphate buffered saline PFA: paraformaldehyde

Preparation example 1
Peripheral blood mononuclear cells were separated from peripheral blood of healthy volunteers according to density gradient centrifugation using a medium for human lymphocyte separation [manufactured by GE Healthcare, trade name: Ficoll-Paque PLUS]. 4 mL of ACK lysis buffer (composition: 150 mM ammonium chloride, 10 mM potassium bicarbonate and 0.1 mM EDTA) was added to 2.0×10 ⁸ peripheral blood mononuclear cells. The resulting mixture was allowed to stand at room temperature for 15 minutes to hemolyze red blood cells in the mixture.

A dead cell removal kit [manufactured by Miltenyi Biotec, trade name: MACS (registered trademark) Dead Cell Removal Kit] was used to magnetically label dead cells contained in the mixture after hemolysis. The resulting mixture was applied to a cell separation column [manufactured by Militeny Biotech, trade name: LS column] to remove labeled dead cells from the mixture.

Peripheral blood mononuclear cells contained in the resulting mixture were magnetically labeled with a cell separation reagent [manufactured by Militeny Biotech, trade name: CD14 Micro Beads]. A CD14 positive (CD14 ⁺ ) cell population was obtained by magnetic separation of the labeled peripheral blood mononuclear cells from the resulting mixture. The resulting CD14-positive cell population was used as isolated monocytes below.

Preparation example 2
A human monocyte cell line THP-1 was cultured in medium A [RPMI1640 medium containing 0.5% (v/v) FBS] at 37° C. in a 5% (v/v) carbon dioxide atmosphere. LPS [manufactured by Sigma-Aldrich, trade name: Lipopolysaccharides from Escherichia coli O111: B4] and TRPV4 agonist [manufactured by Wako Pure Chemical Industries, Ltd., trade name: GSK1016790A] at concentrations shown in Table 1 was added to medium A so that THP-1 was then cultured for an additional 6 hours. LPS induces the expression of the inflammatory cytokine IL-1β.

Polyethylene glycol tert-octylphenyl ether (Triton X-100) was added to the resulting culture to a concentration of 0.1% (v/v). A THP-1 lysate was prepared by placing the resulting mixture on ice for 10 minutes. Experiment numbers 1-1 through 1-5 were obtained by subjecting the THP-1 lysate to centrifugation at 17800×g and 4° C. for 5 minutes and collecting the supernatant.

Example 1
100 μL of capture antibody solution was added to each well of the ELISA plate. The ELISA plate was incubated overnight (approximately 8 hours) at 4°C. After removing the liquid component in each well of the ELISA plate, each well was washed four times with 300 μL of washing solution [PBS solution containing 0.05% (v/v) polyoxyethylene sorbitan monoutarate (Tween-20)]. bottom. The remaining wash solution was removed from each well of the ELISA plate after washing.

After adding 300 μL of blocking buffer [0.1% (m/v) BSA-containing PBS solution] to each well of the ELISA plate, the ELISA plate was incubated at room temperature for 1 hour. After removing liquid components in each well of the ELISA plate, each well was washed four times with the washing solution.

100 μL of samples of Experiment Nos. 1-1 to 1-5 or 100 μL of quantification preparations were added to each well of the ELISA plate. The ELISA plate was then incubated at room temperature. After removing the liquid component in each well of the ELISA plate two hours after the start of incubation, the reaction product in each well was washed.

After washing, 100 μL of biotin detection antibody solution was added to each well of the ELISA plate. The ELISA plate was then incubated for 2 hours at room temperature. After removing the liquid component in each well of the ELISA plate two hours after the start of incubation, the reaction product in each well was washed.

After washing, 100 μL of streptavidin-HRP antibody solution was added to each well of the ELISA plate. The ELISA plate was then incubated at room temperature. After removing the liquid component in each well of the ELISA plate 20 minutes after the start of incubation, the reaction product in each well was washed.

After adding 100 μL of the substrate solution [A solution/B solution (volume ratio)=1/1] to each well of the ELISA plate, the ELISA plate was incubated at room temperature (25° C.) under light-shielding conditions to obtain a reaction product. was dyed. After 20 minutes of incubation, 50 μL of stop solution was added to each well of the ELISA plate.

The ELISA plate was subjected to a plate reader (manufactured by TECAN, trade name: Infinite F200 PRO) to measure the absorbance. Further, by measuring the absorbance of the standard for quantitative determination, a relational expression between the absorbance and the IL-1β mass concentration was obtained. Using the relational expression between absorbance and IL-1β mass concentration, the IL-1β mass concentration of each sample was calculated. Next, using the IL-1β mass concentration of each sample, formula (II):
[IL-1β relative value (fold)]
= [IL-1β mass concentration of sample]/[IL-1β mass concentration of sample of Experiment No. 1-1] (II)
IL-1β relative values were determined accordingly.

FIG. 1 shows the results of examining the relationship between sample types and IL-1β relative levels. In the figure, lane 1 is IL-1β relative value of sample of experiment number 1-1, lane 2 is IL-1β relative value of sample of experiment number 1-2, lane 3 is IL-1β of sample of experiment number 1-3. Lane 4 shows the IL-1β relative values of the samples of Experiment Nos. 1-4, and Lane 5 shows the IL-1β relative values of the samples of Experiment Nos. 1-5.

From the results shown in FIG. 1, the IL-1β relative values (lanes 3 to 5) of the samples of experiment numbers 1-3 to 1-5 are the relative IL-1β values of the samples of experiment number 1-2 (lane 2). ), the IL-1β expression level of the monocyte cell line when TRPV4 is activated is smaller than the IL-1β expression level of the monocyte cell line when TRPV4 is not activated. It can be seen that it is less than In addition, since the relative IL-1β level tends to decrease as the amount of TRPV4 agonist in medium A increases, it can be seen that the IL-1β expression level decreases as the TRPV4 activity is enhanced.

These results show that activation of TRPV4 in monocyte cell lines reduces IL-1β expression in monocyte cell lines under inflammation-inducing conditions with LPS, thereby suppressing inflammation. Thus, since the activation of TRPV4 in monocyte cell lines is associated with suppression of inflammation, the test sample is contacted with the monocyte cell line, and the physiological events caused by the activation of TRPV4 are detected. can be used to evaluate the anti-inflammatory effect of the test sample. It is also found that substances that activate TRPV4 in monocytic cell lines activate TRPV4 and suppress inflammation.

Preparation example 3
The isolated monocytes obtained in Preparation Example 1 were cultured in medium A [RPMI1640 medium containing 0.5% (v/v) FBS] at 37°C in a 5% (v/v) carbon dioxide atmosphere. The concentration of the TRPV4 antagonist [manufactured by TOCRIS, trade name: HC067047] in the medium A was adjusted to 0 μM or 30 μM, and the isolated monocytes were further cultured for 30 minutes.

LPS [manufactured by Sigma-Aldrich, trade name: Lipopolysaccharides from Escherichia coli O111: B4, product number: L2630] and TRPV4 agonist [manufactured by Wako Pure Chemical Industries, Ltd., trade name: GSK1016790A] at a concentration of LPS was added to the medium A at the concentration shown in Table 2. Isolated monocytes were then cultured for an additional 6 hours.

Polyethylene glycol tert-octylphenyl ether (Triton X-100) was added to the resulting culture to a concentration of 0.1% (v/v). A cell lysate was prepared by placing the resulting mixture on ice for 10 minutes. Samples for experiment numbers 2-1 to 2-8 were obtained by subjecting the cell lysate to centrifugation at 17800×g and 4° C. for 5 minutes and collecting the supernatant.

Example 2
In Example 1, the same operation as in Example 1 was performed except that the samples of Experiment Nos. 2-1 to 2-8 were used instead of the samples of Experiment Nos. 1-1 to 1-5, and the absorbance was measured. It was measured. A relational expression between the absorbance and the IL-1β mass concentration was obtained by measuring the absorbance of the standard for quantification. Using the relational expression between absorbance and IL-1β mass concentration, the IL-1β mass concentration of each sample was calculated. Using the IL-1β mass concentration of each sample, formula (III)
[IL-1β relative value (fold)]
= [IL-1β mass concentration of sample]/[IL-1β mass concentration of sample of Experiment No. 2-1] (III)
IL-1β relative values were determined accordingly.

FIG. 2 shows the results of examining the relationship between sample types and IL-1β relative levels. In the figure, lane 1 is IL-1β relative value of sample of experiment number 2-1, lane 2 is IL-1β relative value of sample of experiment number 2-2, lane 3 is IL-1β of sample of experiment number 2-3. 1β relative values, lane 4 IL-1β relative values of samples from experiment numbers 2-4, lane 5 relative IL-1β values from samples of experiment numbers 2-5, lane 6 IL from samples of experiment numbers 2-6 -1β relative values, lane 7 shows the IL-1β relative values of the samples of Experiment No. 2-7, and lane 8 shows the IL-1β relative values of the samples of Experiment No. 2-8.

From the results shown in FIG. 2, the IL-1β relative values (lanes 3-5) of the samples of experiment numbers 2-3 to 2-5 are the IL-1β relative values of the samples of experiment numbers 2-2 and 2-6 IL-1β expression in isolated monocytes when TRPV4 is activated is lower than that of IL-1β in isolated monocytes when TRPV4 is not activated (lanes 2 and 6). It can be seen that the expression level of -1β is smaller than that of -1β. In addition, since the relative IL-1β level tends to decrease as the amount of TRPV4 agonist in medium A increases, it can be seen that the IL-1β expression level decreases as the TRPV4 activity is enhanced.

The IL-1β relative values (lanes 7 and 8) of the samples of Experiment Nos. 2-7 and 2-8 are comparable to the IL-1β relative values (lane 1) of the sample of Experiment No. 2-1. , it can be seen that TRPV4 agonists and TRPV4 antagonists do not induce IL-1β.

These results show that activation of TRPV4 in isolated monocytes reduces IL-1β expression in isolated monocytes under inflammation-inducing conditions with LPS, thereby suppressing inflammation. Thus, since the activation of TRPV4 in isolated monocytes is associated with suppression of inflammation, the test sample is contacted with the isolated monocytes, and the physiological events caused by the activation of TRPV4 are used. Therefore, it is possible to evaluate the anti-inflammatory effect of the test sample. It is also found that a substance that activates TRPV4 in isolated monocytes activates TRPV4 and suppresses inflammation.

Preparation example 4
LPS [manufactured by SIGMA-Aldrich, trade name: Lipopolysaccharides from Escherichia coli O111: B4] and TRPV4 agonist [manufactured by Wako Pure Chemical Industries, Ltd., trade name: GSK1016790A] at concentrations shown in Table 3 was added to the medium A as above to obtain the medium BD.

Example 3
THP-1 in medium A (experiment number 3-1), medium B (experiment number 3-2), medium C (experiment number 3-3) or medium D (experiment number 3-4) at 37 ° C. 5% (v/v) cultured in a carbon dioxide atmosphere. After 6 hours from the start of culture, THP-1 was recovered and washed with PBS.

Using the obtained THP-1 and a total RNA extraction reagent [manufactured by Molecular Research, trade name: Trireagent], a total RNA-containing solution was obtained. Chloroform was added to the total RNA-containing solution and the aqueous layer containing RNA was collected. Isopropanol was added to the aqueous layer to precipitate RNA. After washing the precipitated RNA with a 70% (v/v) aqueous ethanol solution, it was dissolved in RNase-free water to obtain RNA samples of experiment numbers 3-1 to 3-4.

Using the RNA samples of experiment numbers 3-1 to 3-4 and a reverse transcription kit [manufactured by QIAGEN, trade name: QuantiTect reverse transcription kit], cDNA was synthesized. Real-time PCR was performed using the cDNA, the primer pairs shown in Table 4, and SYBR Green mix [manufactured by Toyobo Co., Ltd., trade name: THUNDERBIRD SYBR qPCR Mix] to determine the IL-1β Ct value and GAPDH Ct value of each sample. An average value was obtained.

Using the average IL-1β Ct value and GAPDH Ct value for each sample, formula (IV):
[IL-1β ΔCt]
= [IL-1β Ct value of sample] - [average GAPDH Ct value] (IV)
IL-1β ΔCt was determined accordingly. Next, the relative value of IL-1β ΔCt for each experiment number was calculated when the IL-1β ΔCt value for experiment number 3-1 was set to 1. In the following, the relative value of IL-1β ΔCt was used as the degree of increase in mRNA amount.

FIG. 3 shows the results of examining the relationship between the type of RNA sample and the degree of increase in the amount of IL-1β mRNA. In the figure, lane 1 is the degree of increase in the amount of IL-1β mRNA in the RNA sample of Experiment No. 3-1, lane 2 is the degree of increase in the amount of IL-1β mRNA in the RNA sample of Experiment No. 3-2, and lane 3 is the experiment number. Lane 4 shows the degree of increase in the amount of IL-1β mRNA in the RNA samples of experiment numbers 3-3, and the degree of increase in the amount of IL-1β mRNA in the RNA samples of Experiment Nos. 3-4.

From the results shown in FIG. 3, it can be seen that the increase in IL-1β mRNA level in the RNA sample of Experiment No. 3-3 is smaller than the increase in IL-1β mRNA level in the RNA sample of Experiment No. 3-2. Understand. These results indicate that IL-1β mRNA levels in monocyte cell lines when TRPV4 is activated are higher than IL-1β mRNA levels in monocyte cell lines when TRPV4 is not activated. I know it's less. Therefore, it can be seen that activation of TRPV4 suppresses the expression of IL-1β in monocyte cell lines at the mRNA level. The degree of increase in the amount of IL-1β mRNA in the RNA sample of Experiment No. 3-4 was less than or equal to the degree of increase in the amount of IL-1β mRNA in the RNA sample of Experiment No. 3-1. , alone has no effect on IL-1β mRNA expression in monocyte cell lines.

Preparation example 5
LPS [manufactured by Sigma-Aldrich, trade name: Lipopolysaccharides from Escherichia coli O111: B4] and TRPV4 agonist [manufactured by Wako Pure Chemical Industries, Ltd., trade name: GSK1016790A] at concentrations shown in Table 5 was added to the above medium A to obtain mediums EG.

Example 4
The isolated monocytes obtained in Preparation Example 1 were used in medium A (experiment number 4-1), medium E (experiment number 4-2), medium F (experiment number 4-3), or medium G (experiment number 4-4). ) in a 5% (v/v) carbon dioxide atmosphere at 37°C. After 6 hours from the start of culture, the isolated monocytes were collected and washed with PBS.

Using the obtained isolated monocytes and a total RNA extraction reagent (manufactured by Molecular Research, trade name: Trireagent), a total RNA-containing solution was obtained. Chloroform was added to the total RNA-containing solution and the aqueous layer containing RNA was collected. Isopropanol was added to the aqueous layer to precipitate RNA. After washing the precipitated RNA with a 70% (v/v) aqueous ethanol solution, it was dissolved in RNase-free water to obtain RNA samples of experiment numbers 4-1 to 4-4.

Using the RNA samples of experiment numbers 4-1 to 4-4 and a reverse transcription kit [manufactured by QIAGEN, trade name: QuantiTect reverse transcription kit], cDNA was synthesized. Real-time PCR was performed using the cDNA, the primer pairs shown in Table 3, and SYBR Green mix [manufactured by Toyobo Co., Ltd., trade name: THUNDERBIRD SYBR qPCR Mix] to determine the IL-1β Ct value and GAPDH Ct value of each sample. An average value was obtained.

Using the average IL-1β Ct value and GAPDH Ct value of each sample, IL-1β ΔCt was determined according to the above formula (IV). Next, the relative value of IL-1β ΔCt for each experiment number was calculated when the IL-1β ΔCt value for experiment number 4-1 was set to 1. In the following, the relative value of IL-1β ΔCt was used as the degree of increase in mRNA amount.

FIG. 4 shows the results of examining the relationship between the type of RNA sample and the degree of increase in the amount of IL-1β mRNA in Example 4. In the figure, lane 1 is the degree of increase in the amount of IL-1β mRNA in the RNA sample of Experiment No. 4-1, lane 2 is the degree of increase in the amount of IL-1β mRNA in the RNA sample of Experiment No. 4-2, and lane 3 is the experiment number. Lane 4 shows the degree of increase in the amount of IL-1β mRNA in the RNA sample of Experiment No. 4-3, and the degree of increase in the amount of IL-1β mRNA in the RNA sample of Experiment No. 4-4.

From the results shown in FIG. 4, the degree of increase in IL-1β mRNA level in the RNA sample of Experiment No. 4-3 was smaller than that in the RNA sample of Experiment No. 4-2. Understand. These results indicate that the amount of IL-1β mRNA in isolated monocytes when TRPV4 is activated is lower than that in isolated monocytes when TRPV4 is not activated. I understand. Thus, it can be seen that activation of TRPV4 in isolated monocytes suppresses IL-1β expression at the mRNA level, as in monocyte cell lines. The degree of increase in the amount of IL-1β mRNA in the RNA sample of Experiment No. 4-4 was less than or equal to the degree of increase in the amount of IL-1β mRNA in the RNA sample of Experiment No. 4-1. , alone has no effect on IL-1β mRNA expression in isolated monocytes.

Preparation example 6
The monocyte cell line THP-1 was cultured in medium A at 37° C. in a 5% (v/v) carbon dioxide atmosphere. A calcium chelating agent [manufactured by R&D Systems Co., trade name: BAPTA-AM] was added at concentrations shown in Table 6. After 30 minutes from the addition of the calcium chelating agent, LPS [manufactured by Sigma-Aldrich, trade name: Lipopolysaccharides from Escherichia coli O111: B4] and TRPV4 agonist [manufactured by Wako Pure Chemical Industries, Ltd., Trade name: GSK1016790A] was added to the concentration shown in Table 6, and THP-1 was further cultured for 6 hours.

Polyethylene glycol tert-octylphenyl ether (Triton X-100) was added to the resulting culture to a concentration of 0.1% (v/v). A THP-1 lysate was prepared by placing the resulting mixture on ice for 10 minutes. Samples for Experiment Nos. 5-1 to 5-4 were obtained by subjecting the THP-1 lysate to centrifugation at 17800×g and 4° C. for 5 minutes and collecting the supernatant.

Example 5
In Example 1, the same operation as in Example 1 was performed except that the samples of Experiment Nos. 5-1 to 5-4 were used instead of the samples of Experiment Nos. 1-1 to 1-5, and the absorbance was measured. It was measured. The mass concentration of IL-1β contained in the sample was calculated using the relational expression between the absorbance obtained by measuring the absorbance of the standard for quantification and the IL-1β mass concentration.

FIG. 5 shows the results of examining the relationship between the type of sample and the IL-1β mass concentration. In the figure, lane 1 is the IL-1β mass concentration of the sample of experiment number 5-1, lane 2 is the IL-1β mass concentration of the sample of experiment number 5-2, and lane 3 is the IL-1β concentration of the sample of experiment number 5-3. 1β mass concentration, Lane 4 shows the IL-1β mass concentration of the samples of Experiment Nos. 5-4.

From the results shown in FIG. 5, the IL-1β mass concentration of the sample of Experiment No. 5-3 (lane 3) is lower than the IL-1β mass concentration of the sample of Experiment No. 5-2 (lane 2). I understand. From these results, the IL-1β expression level of the monocyte cell line when TRPV4 of the monocyte cell line is activated by a TRPV4 agonist is higher than that of the monocyte cell line when TRPV4 is not activated. It can be seen that the IL-1β expression level is lower than that of Also, from the results shown in FIG. 5, the IL-1β mass concentration of the sample of Experiment No. 5-4 (lane 4) is higher than the IL-1β mass concentration of the sample of Experiment No. 5-2 (lane 2). I know it's expensive. In Experiment No. 5-4, since a calcium chelating agent was used, a chelate complex was formed between the calcium chelating agent and intracellular calcium ions. It is considered that the 1β expression inhibitory action is reduced.

Preparation example 7
FBS and an antibiotic [manufactured by Gibco, trade name: Antibiotic-Antimycotic] were added to RPMI1630 medium so that the FBS concentration was 20% (v/v) and the antibiotic concentration was 1% (v/v). , medium H was obtained.

GM-CSF [manufactured by SHENANDOAH, product number: 100-08], TRPV4 agonist [manufactured by Wako Pure Chemical Industries, Ltd., trade name: GSK1016790A] and TRPV4 antagonist [manufactured by Sigma-Aldrich, trade name: GSK2193874] was added to medium H at concentrations shown in Table 7 to obtain medium IK.

Preparation Example 8
M-CSF [manufactured by Shenandoah, product number: 100-03], TRPV4 agonist [manufactured by Wako Pure Chemical Industries, Ltd., trade name: GSK1016790A] and TRPV4 antagonist [manufactured by Sigma-Aldrich, trade name: GSK2193874] are shown in Table 8. was added to medium H at the concentration shown in , to obtain medium L to N.

Example 6
The isolated monocytes obtained in Preparation Example 1 were used in medium H (experiment number 6-1), medium I (experiment number 6-2), medium J (experiment number 6-3), medium K (experiment number 6- 4) 5% (v/v) dioxide at 37° C. in culture vessels containing medium L (experiment number 6-5), medium M (experiment number 6-6) or medium N (experiment number 6-7) Cultured in a carbon atmosphere. Fresh medium was added to the isolated monocyte culture system after 2 days and 5 days from the start of the culture.

Cell cultures were harvested 7 days after the initiation of culture. Adherent cells were collected by adding a cell dissociation reagent [Gibco, trade name: TrypLE ^™ express] to the culture vessel, standing at 37°C for 15 minutes, and then pipetting. The resulting cell culture was washed with PBS.

A dead cell staining dye [manufactured by Invitrogen, trade name: LIVE/DEAD Fixable Aqua Dead Cell Stain kit] was used to stain dead cells contained in the cell culture. Thereafter, the cell culture was incubated with an anti-CD14 antibody [manufactured by BioLegend, trade name: BV421 anti-human CD14] and an anti-CD11b antibody [manufactured by BioLegend, trade name: APC anti-human CD11b] at room temperature for 10 minutes. Cells contained in the cell culture were stained for CD14 and CD11b on the plasma membrane by incubating for 1 min. Cell cultures after staining were washed with PBS.

The washed cell culture was immersed in a 0.5% (v/v) PFA-containing PBS solution and fixed at room temperature for 10 minutes. Cell membrane permeabilization was performed by incubating the fixed cell culture in a permeabilizing agent [PBS solution containing 0.1% (v/v) polyethylene glycol tert-octylphenyl ether (Triton X-100)]. Cell cultures after treatment were washed with PBS.

The washed cell culture was incubated with an anti-CD68 antibody [manufactured by Biolegend, trade name: FITC anti-human CD68] for 10 minutes at room temperature to stain CD68 inside the cells contained in the cell culture. After the stained cell culture was washed with PBS, it was resuspended in PBS to obtain a sample.

The obtained sample was subjected to a flow cytometer [manufactured by BD, trade name: BD FACSAria ^™ II]. M1-type macrophages and M2-type macrophages in a sample by sorting cells showing weak CD14-positive, CD11b-positive and CD68-positive as M1-type macrophages and sorting cells showing CD14-positive, CD11b-positive and CD68-positive as M2-type macrophages. Each content rate was calculated. Table 9 shows the results.

The results shown in Table 9 show that the content of M1-type macrophages in Experiment No. 6-3 is lower than the content of M1-type macrophages in Experiment Nos. 6-2 and 6-4. These results show that activation of TRPV4 in isolated monocytes suppresses the differentiation of isolated monocytes into M1-type macrophages. Since M1-type macrophages induce inflammation, substances that activate TRPV4 in isolated monocytes are found to suppress inflammation.

Further, from the results shown in Table 9, it can be seen that the content of M2-type macrophages in Experiment No. 6-6 is at least the same as the content of M2-type macrophages in Experiment Nos. 6-5 and 6-7. . These results indicate that activation of TRPV4 in isolated monocytes does not suppress the differentiation of isolated monocytes into M2-type macrophages. Since M2-type macrophages suppress inflammation, it can be seen that a substance that activates TRPV4 in isolated monocytes suppresses inflammation.

Based on the above, in Examples 1 to 6, a test sample was used instead of using a TRPV4 agonist, a physiological event caused by TRPV4 activation of monocytes was measured, and based on the physiological event, the test sample was By adopting the operation of evaluating the anti-inflammatory action possessed, the anti-inflammatory action possessed by the test sample, preferably the anti-inflammatory action mediated by TRPV4 activation of monocytes, can be easily evaluated.

Preparation Example 9
LPS [manufactured by Sigma-Aldrich, product number: L2630], K3CpG [manufactured by Gene Design, product number: CN65003), Pam3CSK4 [manufactured by Invitrogen, product number: tlrl-pms], poly I:C [manufactured by Sigma-Aldrich, product number: p1530] and a TRPV4 agonist [manufactured by Wako Pure Chemical Industries, Ltd., trade name: GSK1016790A] were added to medium A at concentrations shown in Table 10, and serum starvation medium 1 was prepared. ~9 was obtained.

Preparation Example 10
LPS [manufactured by Sigma-Aldrich, product number: L2630], K3CpG [manufactured by Gene Design, product number: CN65003), Pam3CSK4 [manufactured by Invitrogen, product number: tlrl-pms], poly I:C [manufactured by Sigma-Aldrich, product number: p1530] and TRPV4 agonist [manufactured by Wako Pure Chemical Industries, Ltd., trade name: GSK1016790A] were added to medium A at concentrations shown in Table 11, and normal medium 1 to got 9.

Example 7
(1) Serum starvation culture The isolated monocytes obtained in Preparation Example 1 were subjected to serum starvation medium 1 (experiment number 7-1), serum starvation medium 2 (experiment number 7-2), serum starvation medium 3 (experiment number 7). -3), serum starvation medium 4 (experiment number 7-4), serum starvation medium 5 (experiment number 7-5), serum starvation medium 6 (experiment number 7-6), serum starvation medium 7 (experiment number 7-7 ), serum starvation medium 8 (experiment number 7-8) or serum starvation medium 9 (control; experiment number 7-9), cultured in 5% (v/v) carbon dioxide atmosphere at 37° C. for 6 hours.

Polyethylene glycol tert-octylphenyl ether (Triton X-100) was added to the resulting culture to a concentration of 0.1% (v/v). A monocyte lysate was prepared by placing the resulting mixture on ice for 10 minutes. Samples for Experiment Nos. 7-1 through 7-9 were obtained by subjecting the monocyte lysate to centrifugation at 17800×g and 4° C. for 5 minutes and collecting the supernatant.

Using the obtained samples and measurement reagents [manufactured by eBioscience, trade name: Procarta Plex Human Inflammation Panel (20plex)], the expression levels of inflammation-related factors were measured. By comparing the expression level of inflammation-related factors when using a medium containing no TRPV4 agonist and the expression level of inflammation-related factors when using a medium containing a TRPV4 agonist, activation of TRPV4 Inflammation-related factors whose expression levels were decreased were investigated.

(2) Normal culture In Example 7 (1), serum starvation medium 1, serum starvation medium 2, serum starvation medium 3, serum starvation medium 4, serum starvation medium 5, serum starvation medium 6, serum starvation medium 7, serum starvation Instead of using medium 8 or serum starvation medium 9, normal medium 1 (experiment number 8-1), normal medium 2 (experiment number 8-2), normal medium 3 (experiment number 8-3), normal medium 4 (experiment number 8-4), normal medium 5 (experiment number 8-5), normal medium 6 (experiment number 8-6), normal medium 7 (experiment number 8-7), normal medium 8 (experiment number 8-8) or normal The same procedure as in Example 7(1) was performed except that medium 9 (control; Experiment No. 8-9) was used, and inflammation-related factors whose expression level was decreased by activation of TRPV4 were examined.

Tables 12 to 14 show inflammation-related factors whose expression levels were decreased by activation of TRPV4.

From the above results, it can be seen that the inflammation-related factors listed in Tables 12 to 14 or a decrease in expression of their mRNA can be used as a physiological event caused by TRPV4 activation.

As explained above, activation of TRPV4 in monocytes suppresses the expression of IL-1β mRNA in monocytes under inflammation-inducing conditions, thus suppressing inflammation. Thus, since the activation of monocyte TRPV4 and the suppression of inflammation are related, the physiological events caused by the activation of monocyte TRPV4 by the test sample are measured, and the test sample is evaluated. It can be seen that the anti-inflammatory effect of the test sample can be evaluated by using physiological events. It is also found that a substance that activates TRPV4 on monocytes activates TRPV4 and suppresses inflammation. Therefore, the present invention is expected to be suitably used for the development of anti-inflammatory cosmetics, anti-inflammatory agents, and the like.

SEQ ID NO: 1 is the sequence of the IL-1β forward primer.
SEQ ID NO:2 is the sequence of the IL-1β reverse primer.
SEQ ID NO:3 is the sequence of the GAPDH forward primer.
SEQ ID NO: 4 is the sequence of the GAPDH reverse primer.

Claims (1)

A test sample evaluation method for evaluating the anti-inflammatory effect of a test sample, comprising contacting the test sample with monocytes, measuring a physiological event caused by TRPV4 activation of monocytes, and measuring the physiological event A method for evaluating a test sample, which comprises evaluating the anti-inflammatory effect of the test sample based on.

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