JP2011115152A - Screening method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a simple and secured screening method for a substance suitable as a cosmetic material (wherein, including a quasi-drug) and having the effect of improving skin-barrier function. <P>SOLUTION: The method for screening simply, efficiently and in high accuracy an ingredient having the effect of improving skin-barrier function is provided. This screening method includes the following: paying attention to the action mechanism of a substance having the effect of improving skin-barrier function, the respective levels of extent on the TRPV(transient receptor potential vanilloid)-activating effect and the effect of improving the formation of TJ(tight-junction) and AJ(adherents-junction) in cells are adopted as indicators. <P>COPYRIGHT: (C)2011,JPO&INPIT

Description

  The present invention relates to a method for screening a component for improving the skin barrier function, which is suitable as a cosmetic raw material, and a cosmetic comprising the cosmetic raw material having an action of improving the skin barrier function.

  In addition to the function of transmitting external stimuli such as heat and pain to the living body, the skin has a barrier function that prevents the evaporation of moisture and protects the body from external and physical stimuli, which is important for maintaining the functions of the living body. Plays an important role. It is known that such skin function is reduced by temperature, humidity, excessive friction, modulation of life rhythm, stress, etc., and due to the above causes, the skin becomes dry and becomes hypersensitive to irritation. Skin symptoms such as being susceptible to exposure occur. Furthermore, if this state progresses, direct damage due to invasion of bacteria and harmful substances from outside the body, allergic reactions, etc. occur, leading to the development of skin disorders such as atopic dermatitis and sebum-deficient eczema. Skin is also a major concern for many women because it touches the human eye and directly affects its visual beauty. For the purpose of keeping the skin condition healthy, a skin barrier function improving agent (see, for example, Patent Document 1), a rough skin improving agent (for example, see Patent Document 2), and a ceramide synthesis accelerator (for example, Patent Document 3) Various attempts have been made, for example, for external preparations for skin containing the reference). All of these are effective as they are, but it is difficult to say that the effects are sufficiently satisfactory, and only when a plurality of factors are treated, a large rough skin improvement effect is exhibited. For this reason, it can be said that development of a material that acts on a plurality of factors with a single material has been sought in order to more reliably improve rough skin.

  The non-selective cation channel TRP (Transient receptor potential) channel, which was identified in 1989 as the causative gene of a Drosophila photoreceptor mutant, is classified into at least nine subfamilies based on amino acid sequence homology. . Since the molecular entity of TRPV1 (Transient receptor potential vaniloid 1) has been elucidated for the first time, the temperature-sensitive receptor belonging to the TRP channel has been opened in mammals by each temperature stimulus from low to high temperatures. Types of temperature sensitive receptors (TRPV1, TRPV2, TRPV3, TRPV4, TRPM2, TRPM4, TRPM5, TRPM8, TRPA1) have been reported. TRPV4 was originally discovered as an osmotic pressure-sensitive receptor activated at low osmotic pressure, and was confirmed to be expressed in various tissues such as sensory nerve, hypothalamus, skin, kidney, lung, and inner ear. It is known to play an important role in the transmission of pain and inflammatory pain caused by stimulation and mechanical stimulation. TRPV4, which functions as a transmembrane calcium influx channel, is added by compounds such as endogenous cannabinoids, arachidonic acid metabolites, and 4α-PDD (4alpha-phorbol 12,13-didecanoate) in addition to the above physical stimulation. Is also activated. From another viewpoint, it can be said that TRPV4 controls various physiological actions by controlling the calcium ion concentration. As described above, TRPV4 expressed in various tissues and involved in a physical or chemical stimulus response may be related to many biological activities. In fact, stimulation and pain recognition, autism It is known to be deeply involved in neurological disease symptoms such as. Furthermore, energy metabolism-related activities such as weight gain in TRPV4-deficient mice, a significant inhibitory effect on total fat mass and visceral fat mass (see, for example, Patent Document 1) have been reported. In addition, studies on TRPV present in epidermal cells have reported that TRPV has a function as a moisture transpiration sensor, and activation by 4α-PDD suppresses moisture transpiration and enhances the skin barrier function. (For example, see Non-Patent Document 1 and Non-Patent Document 2).

Tight-junction (TJ) and adherence junction (A
J: Adherens-junction) is an intercellular adhesion structure that exists in the form of a belt around cells, closes gaps by adhering adjacent cells together, and continuously connects cells. In skin tissue, TJ is present in the epidermal cells of the granule layer and plays an important role in preventing water and substances from permeating through the cell gap and maintaining the skin barrier function (for example, non-patent literature). 3). In addition to the physical functions described above, TJ also regulates biological activity related to the transport of substances via TJ, such as calcium ion permeability and maintains the balance of ion distribution to differentiate epidermal cells. It is known that it affects maturation and also the skin condition (for example, see Non-Patent Document 4). In other words, it can be said that intracellular distribution of calcium ions plays an important role in improving the skin barrier function (see, for example, Patent Document 4). It is also known that normal formation of AJ plays an important role in the formation of TJ. However, the interaction between the cell-cell adhesion structures such as TJ and AJ and TRPV has never been known so far, although both exist on the epidermal cell membrane. Therefore, a component having an action of activating TRPV4 that is present in the epidermis and exhibits an action of improving the skin barrier function through an interaction, and a component having an action of improving the skin barrier function by the action of promoting the formation of TJ and / or AJ. The method for screening a component having a TJ and / or AJ formation promoting effect through TRPV4 activation is very useful for easily screening a substance expected as a skin barrier function improving agent having a new action mechanism. Useful.

JP 2007-001914 A JP 2008-044857 A JP 2006-232769 A JP 2006-250786 A

Denda M. et al., J. Invest. Dermatol, 127 (3), 654-659 (2007) Denda M., International Journal of Cosmetic Science, 31, 79-86 (2009) Kurasawa M. et al., Biochemical and Biophysical Reseach Communications, 381, 171-175 (2009) Maeda T. et al., 36th International Congress of Physiological Sciences, P1PM-16-8 (2009)

  The present invention has been made under such circumstances, and found an ingredient having an action of improving the skin barrier function suitable as a raw material for cosmetics (including quasi-drugs). It is an object of the present invention to provide a simple and reliable screening method for a substance having an action of improving the skin barrier function.

In view of such a situation, the present inventors have focused on the mechanism of action of a substance having an action of improving skin barrier function, and sought a method for screening a substance having such action in a simple, efficient and accurate manner. As a result of intensive efforts, the present inventors have found that there are materials having a TRPV4 activation action and a TJ and / or AJ formation promoting action in cosmetic materials. Based on this, a component having an effect of improving the skin barrier function with a simple, efficient and accurate index using the degree of activation of TRPV in cells and the degree of TJ and / or AJ formation promotion as an index. A screening method has been found and the present invention has been completed. The present invention is as follows.
<1> Measure the degree of TRPV receptor activation action of the test substance, measure the degree of tight junction and / or adherence junction formation promoting action, and use the magnitude of these actions as an index, A screening method for a skin barrier function-improving component, characterized in that those having a degree of action larger than that of each control are selected.
<2> The measurement of the degree of activation of the TRPV receptor and the degree of promoting the formation of tight junctions and / or adherence junctions is measured using human-derived cells. 1> The screening method for a skin barrier function improving component according to 1>.
<3> The formation promotion action of the tight junction and / or adherence junction is a formation promotion action of the tight junction and / or adherence junction of the epidermis, <1> or <2> The screening method of the skin barrier function improvement component of description.
<4> The method for screening a skin barrier function improving component according to any one of <1> to <3>, wherein the TRPV receptor is a TRPV receptor present in epidermal cells.
<5> The screening method for a skin barrier function improving component according to any one of <1> to <4>, wherein the TRPV receptor is TRPV4.
<6> The screening method for a skin barrier function improving component according to <5>, wherein the measurement of the degree of activation of TRPV4 measures the degree of increase in intracellular calcium ion concentration.
<7> The skin barrier function according to <6>, wherein the measurement of the degree of increase in intracellular calcium ion concentration is to measure the degree of increase in intracellular calcium ion concentration in TRPV4 overexpressing cells. Screening method for improving components.
<8> A skin barrier containing a skin barrier function improving component screened as a substance having a skin barrier function improving action by the method for screening a skin barrier function improving component according to any one of <1> to <7> A composition for improving functions.
<9> The composition according to <8>, which is a cosmetic (including a quasi-drug).
<10> The composition according to <8> or <9>, which is a skin external preparation.

  ADVANTAGE OF THE INVENTION According to this invention, the method of screening the component which has a skin barrier function improvement effect simply, efficiently, and accurately can be provided.

It is a figure which shows the TJ and / or AJ formation promotion effect by TRPV4 activator (4 (alpha) -PDD) by TER value measurement. It is a figure which shows TJ and / or AJ formation promotion effect by TRPV4 activator (4 (alpha) -PDD) by FITC-Dextran permeation | transmission amount measurement. It is a figure which shows TRPV4 knockdown efficiency by si-RNA transfection. It is a figure which shows the expression level of the cell adhesion related gene of a normal human newborn foreskin epidermal keratinocyte (NHEKn) by TRPV4 knockdown. It is a figure which shows the time-dependent change of the TER value in a TRPV4 knockdown cell. It is a figure which shows the result of the FITC-Dextran permeation | transmission amount measurement in a TRPV4 knockdown cell. It is a figure (photograph) which shows the result of the immunoprecipitation of TRPV4 and AJ related protein, and an immunoblot. It is a figure (photograph) which shows the change of the amount of active Rho by the temperature change in NHEK, and activation of TRPV4. It is a figure (photograph) which shows the influence on the localization of AJ related protein by the temperature change in NHEK and activation of TRPV4. It is a figure (photograph) which shows the influence on the localization of TJ related protein by the temperature change in NHEK and activation of TRPV4. It is a figure which shows the influence on the time-dependent change of the TER value by the temperature change in NHEK and activation of TRPV4. (A) It is a figure which shows the influence on the time-dependent change of the barrier function recovery rate by the temperature change in human skin tissue, and activation of TRPV4. (B) It is a figure (photograph) which shows the biotin permeability change by the temperature change in the human skin tissue after barrier destruction, and activation of TRPV4. It is a figure which shows the result of the TER value measurement (TJ and / or AJ formation promotion effect | action) of the plant extract of this invention. It is a figure which shows the result of the FITC-Dextran permeation | transmission amount measurement (TJ and / or AJ function improvement effect) of the plant extract of this invention. It is a figure which shows the increase effect | action of the intracellular calcium ion concentration of 4 (alpha) -PDD in a TRPV4 expression cell. It is a figure which shows the increase effect | action of the intracellular calcium ion concentration of the Asenya extract of this invention in a TRPV4 expression cell. It is a figure which shows the increase effect | action of the intracellular calcium ion density | concentration of the Crape myrtle extract of this invention in a TRPV4 expression cell. It is a figure which shows the increase effect | action of the intracellular calcium ion concentration of the Asenya extract of this invention in a Mock cell. It is a figure which shows the increase effect | action of the intracellular calcium ion density | concentration of the Crape myrtle extract of this invention in a Mock cell. It is a figure which shows the increase effect | action of the intracellular calcium ion density | concentration of the asenyaku extract of this invention in the extracellular calcium ion free environment in a TRPV4 expression cell. It is a figure which shows the increase effect | action of the intracellular calcium ion density | concentration of the giant crape myrtle extract of this invention in the extracellular calcium ion free environment of a TRPV4 expression cell. It is a figure which shows the increase effect | action of the intracellular calcium concentration of the Asenya extract of this invention in the extracellular calcium ion free environment of a Mock cell. It is a figure which shows the increase effect | action of the intracellular calcium ion density | concentration of an Acacia yak extract in an extracellular calcium ion free environment and calcium presence environment with respect to a mouse | mouth TRPV4 expression cell.

<Screening method of component having skin barrier function improving action of the present invention>
The method for screening a component having an action of improving skin barrier function according to the present invention measures the degree of TRPV activation in a test substance cell, measures the degree of TJ and / or AJ formation promoting action, and these actions. The skin should be included in a skin external preparation for improving rough skin, characterized by selecting those whose degree of action is greater than their respective controls, using the magnitude of the degree of the action as an index. This is a screening method for a barrier function improving component. In order to carry out such a screening method and to screen for components that improve the skin barrier function, the TRPV activation action in cells is measured, and the one having TRPV activation action is screened, and then the TRPV activation action The component having TJ and / or AJ formation promoting action is measured, and only those having both actions are selected and used as the cosmetic material of the present invention, or vice versa. And / or measuring the formation promoting action of AJ, screening for a material exhibiting such an effect, and then measuring the activation action of TRPV for the material selected in this screening, and selecting the one having this action Or, with one material, it can activate TRPV and promote TJ and / or AJ formation. Sometimes measured, to implement any of selecting a material having both effects can be preferably exemplified. Details will be described below.
First, TJ and / or AJ will be described. The skin barrier function is roughly classified into two types, one derived from the stratum corneum and one derived from the epidermal granule layer. The stratum corneum barrier function is a function to keep the body from being moisturized and various external stimuli from the outside world. On the other hand, the barrier function of the epidermal granule layer is a protein complex that exists at the cell-cell adhesion site and controls cell adhesion, such as tight junction (TJ: Tight-junction) and adhesive junction (AJ: Adherens-junction). A special intercellular adhesion structure is a function for regulating the permeability of substances such as water, calcium ions, and drugs. In particular, TJ and AJ play an important role in improving the skin barrier function through intracellular distribution of calcium ions. For this reason, the substance which can promote TJ and / or AJ formation is expected to improve the skin barrier function. On the other hand, nine types of temperature-sensitive receptors (TRPV1, TRPV2, TRPV3, TRPV4, TRPM2, TRPM4, TRPM5, TRPM8, and TRPA1) that are opened by each temperature stimulus from low temperature to high temperature are known for the TRP channel. In particular, TRPV3 and TRPV4 are temperature-stimulated receptors expressed in skin and immune system cells (TRPV3 is 32 ° C. or higher, TRPV4 is 27 ° C. or higher), and in skin, it is mainly present in epidermal cells. . TRPV is a molecular entity of a temperature-sensitive calcium ion channel, and is considered to be deeply involved in the skin barrier function by adjusting the intracellular calcium concentration. Furthermore, it has been clarified by the present inventor that the formation of TJ and AJ is promoted through the activation of TRPV4. For this reason, an intercellular adhesion structure such as TJ or AJ and TRPV (particularly TRPV4) are expected to be additively or synergistically improved skin barrier function by interaction.

The method for screening for the TJ and / or AJ formation promoting action in the screening method for the component having the skin barrier function improving action of the present invention may be an evaluation method capable of evaluating the TJ and / or AJ formation promoting action. For example, the method can be applied without any particular limitation, but in particular, a screening method for a component having an action of promoting the formation of TJ and / or AJ in the epidermis is preferable.
The component having TJ and / or AJ formation promoting action in the present invention is a component that directly or indirectly acts on an intercellular adhesion structure such as TJ and / or AJ to promote TJ and / or AJ formation. If there is, it can be applied without any particular limitation. As a method for evaluating the TJ and AJ formation promoting action, for example, a three-dimensional epidermis model described in Non-Patent Document 3 or a method for confirming intercellular mass transfer using a biotin-labeled diffusion tracer using living skin, a cultured cell sheet is used. FITC-dextran intercellular substance permeation test method, method for analogizing the function of strands by TJ strand observation by freeze fracture method, and transepithelial cell electrical resistance value described in JP-A-2007-174931 ( Examples include a method of measuring TER (transepitherial electrical resistance), but from the viewpoint of ease of operation and measurement accuracy, a FITC-Dextran permeability test method, a method of measuring transepithelial cell electrical resistance (TER value) Can be suitably exemplified.

  According to the method for evaluating the TJ and / or AJ formation promoting action, the degree of intercellular mass transfer in the epidermis keratinocyte layer constructed on the support is determined based on the intercellular substance permeability or transepithelial cell electrical resistance value ( TER value) is measured, and the degree of intercellular mass transfer is suppressed in the presence of the test substance compared to the absence of the test substance (control), or the TER is in the absence of the test substance ( When it is increased in the presence of the test substance compared to the control), it is judged that the TJ and / or AJ of the epidermal keratinocyte layer is densely constructed, and when this is applied to the skin in vivo. It is determined that the substance permeation suppressing action of the stratum corneum or the epidermal granule layer is improved, and thus the skin barrier function is improved.

  The epidermal keratinocyte layer in the present invention is cultured in a liquid medium containing about 0.04 to 0.2 mM of calcium ions until the epidermal keratinocytes become confluent on the support. It is characterized by being able to be cultured (calcium ion switch) in a liquid medium containing about 2 mM.

  As the epidermal keratinocytes used for the formation of the epidermal keratinocyte layer, normal epidermal keratinocytes having self-proliferation can be used, and cells of different animals such as mice, pigs and rats can also be used. However, since it is intended to be applied to human skin, it is preferable to use human cells. In the case of using animal epidermal keratinocytes, epidermal keratinocytes can be collected from the animals and processed according to a conventional method, or can be purchased and used. As human cells, normal human epidermal keratinocytes (NHEK) are commercially available from many manufacturers and can be purchased and used, which is preferable.

As a liquid medium containing about 0.04 mM to 0.2 mM of calcium ions for culturing such epidermal keratinocytes, a commercially available medium for epidermal keratinocyte culture can be used, for example, 0.15 mM calcium. “Humdia-KG2” (manufactured by Kurashiki Boseki Co., Ltd.), which is a liquid medium containing ions, can be suitably exemplified. Using such a medium, inoculate about 2.5 × 10 ⁵ cells / cm ² of epidermal keratinocytes in the presence of the support, and culture at 35 to 40 ° C. in a 5% carbon dioxide stream for about 24 to 96 hours. Thus, the cell layer is formed in a confluent state on the support. Here, the confluent state means a state in which at least one cell exists on the entire surface of the support, and partially remains in a stack of at most about three layers. The support is not particularly limited as long as it is a support used for constructing such a cell layer, and a membrane having micropores of about 0.1 to 12 μm can be preferably exemplified. There are already commercially available supports for constructing such a cell layer, and such commercially available products can be purchased and used. For example, since films made of polyethylene telesterate, polycarbonate, and collagen-treated polytetrafluoroethylene are commercially available from Corning and Millipore, these can be purchased and used, which is preferable. Among these, “Transwell” manufactured by Corning (polyethylene terephthalate film having a 0.4 μm pore) can be preferably exemplified. In addition, a liquid medium containing about 1 to 2 mM of calcium ions can be prepared and used by appropriately adding a calcium salt such as calcium chloride to the “Humdia-KG2”. Cell differentiation is promoted by culturing the epidermis keratinocyte layer constructed confluently on the support in such a liquid medium at 35-40 ° C. under a 5% carbon dioxide stream for about 24 to 96 hours, The cell layer can have a TJ structure. The test substance is added to the cells and further cultured for about 24 to 96 hours until a sufficient difference occurs under the same conditions, and the difference in permeability or TER value of the cell layer is observed with the case of continuing the culture without adding the test substance. Thus, the influence of the test substance on the epidermal keratinocyte layer can be known. The difference in permeability can be screened as described in the following screening method.

  The TJ and / or AJ formation promoting action in the screening method for the skin barrier function improving component of the present invention is, for example, in one chamber in two chambers separated by an epidermis keratinocyte layer constructed on the support. After the treatment with the test substance or simultaneously with the test substance, the labeling substance is filled together with the culture medium and the like, and the degree to which the movement of the labeling substance to the other chamber is suppressed in the presence of the test substance is evaluated as an index. Here, as a labeling substance, any substance can be used as long as its movement can be clearly detected, regardless of its type, such as an ionic substance whose absorbance can be grasped by a change in potential difference, electrical conductivity, electrical resistance, etc., absorbance or fluorescence intensity. Illustrative examples include fluorescent labeling substances and colorants whose movement can be grasped. Particularly preferred are ionic substances. Such an ionic substance may be contained in a culture medium. In this case, the test substance and the labeling substance are simultaneously administered, and the degree of movement of the ionic substance can be screened by measuring the electric resistance value of the cell layer. . Examples of the fluorescent labeling substance include fluorescent labeling dextran, and it is preferable to further change the fluorescence intensity by further incubation after the addition.

In such screening, when the cell-to-cell movement of the labeling substance is suppressed in the formation of the epidermal keratinocyte layer in the presence of the test substance, the TJ and / or AJ construction of the epidermal keratinocyte layer is made dense. Thus, when applied to the skin in vivo, it is determined that the substance permeation suppressing effect of the stratum corneum or epidermal granule layer is improved, thereby improving the skin barrier function. Therefore, such a test substance is determined to be a material that improves the skin barrier function when applied to the skin. This effectiveness is determined to be effective when movement suppression of 10% or more is observed in the presence of the test substance in the presence of the test substance in the intercellular movement of the labeling substance. The Further, it is determined that the effect of improving the skin barrier function is remarkable as the degree of the suppression is remarkable.

As a method for evaluating the activation effect of TRPV in the screening method for a component having an action of improving the skin barrier function of the present invention, any method capable of evaluating the activation effect of TRPV can be applied without particular limitation. In particular, a method for evaluating the activation effect of TRPV using the intracellular calcium ion concentration as an index is preferable. Of the methods for evaluating the activation effect of TRPV, a method for evaluating the activation effect of TRPV4 is preferred. This is because TRPV4 has been confirmed to exist in epidermal cells and is expected to be involved in the skin barrier function. Among the methods for evaluating the activation effect of TRPV, it is more preferable that the activity of TRPV4 is measured using the intracellular calcium ion concentration in cells in which TRPV4 expression is confirmed, more preferably in cells overexpressing TRPV4 as an index. A method for discriminating the chemical action is preferred. As a method for measuring intracellular calcium ion concentration, which serves as an index for discriminating the activation action of TRPV4, any method can be used without particular limitation as long as it is a method for measuring intracellular calcium ion concentration. For example, Sokabe T, Tsujiuchi S, Kadowaki T, Tominaga M .: Drosophila painless is a Ca ²⁺ -requiring channel activated by noxious heat .: J Neurosci., 2008 Oct 1:28 (40): 9929-38 etc., and the calcium imaging method or the patch clamp method is preferable.
According to such a method for evaluating the activation effect of TRPV, the intracellular calcium ion concentration in cells in which TRPV4 expression has been confirmed or cells overexpressing TRPV4 is confirmed by the calcium imaging method or patch clamp method, and the like. When the degree of calcium ion concentration is increased in the presence of the test substance compared to the absence of the test substance (control), it is judged that the function of TRPV is activated, and this is applied to the skin in vivo. In this case, it is determined that the calcium ion permeation effect of TRPV in the stratum corneum or epidermal granule layer is promoted, and thus the skin barrier function is improved. This effectiveness is preferably higher in the presence of the intracellular calcium ion concentration in the presence of the test substance than in the positive control such as ionomycin in the presence of the test substance. It is determined that the concentration is effective when an increase in concentration of at least 30%, more preferably an increase in concentration of at least 30% is observed. Further, it is determined that the effect of improving the skin barrier function is remarkable as the degree of the suppression is remarkable.
The rate of increase in intracellular calcium ion concentration by the test substance relative to the positive control such as ionomycin was selected by randomly selecting 3 or 5 cells whose intracellular calcium ion concentration increased by the addition of the test substance. The initial value from the calcium ion concentration after addition, that is, the calcium ion concentration before addition is subtracted to calculate the amount of increase in the calcium ion concentration by the test substance, and then the initial value from the calcium ion concentration after addition of positive control such as ionomycin, ie, addition Calculate the increase in calcium ion concentration by the positive control by subtracting the previous calcium ion concentration, divide the increase in calcium ion concentration by the test substance by the increase in calcium ion concentration by the positive control, and then multiply by 100. Calculated as a value.

  The cells used for evaluating the component having TRPV activation activity of the present invention can be applied without particular limitation as long as TRPV expression is confirmed, but from the viewpoint of measurement sensitivity and accuracy. More preferably, TRPV overexpressing cells are used. In particular, for the production of TRPV1 overexpressing cells, for example, according to the method described in JP-A-2009-082053, and for the production of TRPV4 overexpressing cells, TRPV4 overexpression is carried out by the method described below based on the above method. Cells can be made.

  Regarding the cells in which the expression of TRPV4 used for the evaluation of the TRPV4 activation action in the present invention has been confirmed, any cell expressing TRPV4 can be applied without particular limitation, and more preferable is a TRPV4 expression vector. A cell in which TRPV4 is overexpressed by introducing A into the host cell is preferred. Examples of host cells overexpressing TRPV4 include bacterial cells, plant cells, animal cells, insect cells, etc. More preferably, HEK293 cells, CHO cells, COS-7 cells, NIH3T3 cells and the like are suitable. It can be illustrated. The host cell preferably takes in a TRPV4 expression vector, efficiently expresses TRPV4, and is easy to culture. Further, in the preparation of a TRPV4 expression vector, any cDNA encoding TRPV4 can be used without any particular limitation. More preferably, TRPV4 is added to pcDNA3 (manufactured by Invitrogen), which is a vector for mammalian cells. A preferred example is a ligated encoded nucleic acid. As the nucleic acid encoding TRPV4, a nucleic acid encoding all nucleotide sequences can be used, for example, one obtained by extracting mRNA and allowing reverse transcriptase to act on this, and using the TRPV4 main function. An appropriate primer can be selected only for the encoded portion, amplified by PCR, and ligated using this. Particularly preferred is the oligonucleotide shown in SEQ ID NO: 1 (SEQ ID NO: 1), and the oligonucleotides of such sequences use the primers of SEQ ID NO: 2 (SEQ ID NO: 2) and SEQ ID NO: 3 (SEQ ID NO: 3). It can be obtained by subjecting the extracted DNA or cDNA to PCR reaction.

  The cells overexpressing TRPV4 that can be used for evaluation of the TRPV activation action of the present invention are selected based on the function expression of TRPV4 in the cells, expression at the protein level, co-introduction of a fluorescent substance, and the like as indices. I can do it. An appropriate selection medium can be used for selection of cells in which the nucleic acid encoding TRPV4 has been introduced into a host cell so that the nucleic acid can be expressed. For example, a material obtained by adding a growth factor such as FBS to DMEM or RPMI medium can be suitably exemplified.

  Examples of the medium used for culturing cells that overexpress TRPV4 include components suitable for growth of cells that overexpress TRPV4, such as glucose, amino acids, peptone, vitamins, cell growth promoting factors (for example, cell Any medium containing growth factors, hormones, binding proteins, cell adhesion factors, lipids), serum (eg, FBS, FCS, etc.), calcium chloride, magnesium chloride, etc. may be used. The medium may be a commercially available medium. The medium used for culturing the cells overexpressing TRPV4 is not particularly limited as long as it is a medium suitable for such cells, and examples thereof include MEM medium, DMEM medium, and RPMI 1640 medium. For example, when the host cell used is HEK293 cells, a DMEM medium containing high glucose and 10% by mass FBS is used.

  The substances having TRPV4 activation action and TJ and / or AJ formation promoting action in the present invention act additively or synergistically by acting together with each target molecule TRPV4 and TJ and / or AJ. Appears to improve skin barrier function. Furthermore, TPV and / AJ of the TRPV4 and the cell-cell adhesion structure interact, in other words, by acting on TRPV4, formation of TJ and / or AJ is promoted, and the skin barrier function improving action is exhibited. Therefore, further improvement of the skin barrier function is expected. The interaction regarding the improvement of the skin barrier function between TRPV (particularly TRPV4) and TJ and / or AJ of the intercellular adhesion structure in the present invention is confirmed by the following test results (Test Examples 1 to 6). That is, TJ and / or AJ formation promotion was confirmed by TRPV4 activation by 4α-PDD which is a TRPV4 ligand. That is, it can be said that the component having the TRPV4 activating effect exhibits the skin barrier function improving effect by promoting the formation of TJ and / or AJ.

<Ingredient for Promoting the Formation of TJ and / or AJ of the Present Invention, and Improvement of Skin Barrier Function Having TRPV Activation Action>
The screening method for a skin barrier function improving component, which is the subject of the present invention, determines the degree of TJ and / or AJ formation promoting action and TRPV activation action when selecting a component having a skin barrier function improving action. As an index, both of these actions are selected to be larger than their respective controls. The skin barrier function-improving component selected by the screening method means a generic name of mixed compositions such as simple chemical substances, extracts from plants and animals and plants, and fractionated purified products thereof. In addition, the skin barrier function improving component of the present invention exerts an effect of improving the skin barrier function by adjusting the intracellular calcium ion concentration by acting on TRPV4 existing in the epidermis and TJ and AJ of the intercellular adhesion structure. To do. Furthermore, by promoting the formation of TJ and AJ through the activation action of TRPV4, an additive or synergistic improvement of the skin barrier function is expected. The same component may have a TJ and AJ formation promoting action by another mechanism in addition to the TJ and AJ formation promoting action via the TRPV4 activation action.

  Here, the degree of TJ and / or AJ formation promoting action in the present invention is measured, and the component selected as having a high degree of this action is the TJ and / or AJ formation promoting action evaluation system ( Any component that exhibits TJ and / or AJ formation promoting action in TER value measurement and FITC-Dextran substance permeation test) can be used without particular limitation. Specifically, the substance having the TJ and / or AJ formation promoting action is preferably more preferable in the “FITC-Dextran substance permeation test” than in the presence of the test substance in the presence of the test substance. Means a substance having an intercellular substance permeation effect of 10% or more, or, in the “transepithelial cell electrical resistance (TER value) measurement test”, in the presence of the test substance, it is compared with the absence of the test substance. The TER value means a substance having an action of preferably increasing by 10% or more. If the above-mentioned action is recognized in any of these evaluation methods, it can be said that it is a component having a TJ and AJ formation promoting action. Moreover, the said evaluation method can also be used independently and can also be used together.

  In addition, as a component that measures the degree of TRPV activation in the present invention and is selected as having a high degree of this effect, any component that exhibits activity in the evaluation of TRPV activation can be applied without particular limitation. I can do it. As a TRPV activation action evaluation system, for example, a TRPV activation action evaluation system such as a calcium imaging method and a patch clamp method using TRPV4 overexpressing cells can be preferably exemplified. Among these, a preferable example is a calcium imaging method using the intracellular calcium ion concentration of the TRPV4 activation evaluation system described later as an index. As the component having TRPV activation action of the present invention, in particular, the component having TRPV activation action in the following “TRPV activation action evaluation 1: TRPV4 activation action evaluation by calcium imaging method of plant extract in the present invention” Is preferred. In the evaluation system, the substance having a TRPV activation action is a substance that specifically promotes calcium ion influx from outside the cell into cells expressing TRPV4. Preferably, the action is 4α- It means a substance having an action equivalent to or higher than that of PDD. The evaluation method concerning the TRPV activation action can be used alone or in combination.

  The skin barrier function-improving components that have been determined to have such a TJ and / or AJ formation-promoting action and a TRPV activation action and selected as having both of these actions are various. It is suitably used for the treatment or prevention of various symptoms and diseases. The route of administration is not limited, but a method of administration close to the lesion can be suitably exemplified, and transdermal administration is particularly preferred. Preferred examples of the disease include rough skin, inflammation, and atopic dermatitis.

  As a component having a skin barrier function improving action, which is determined to have such a TJ and / or AJ formation promoting action and a TRPV activating action degree, and these actions are both large, An extract obtained from a plant belonging to the genus Crape myrtle and Rubiaceae can be preferably exemplified, and more preferably, an extract obtained from the crape myrtle genus Cranalis and Rubi genus beer can be suitably exemplified. Preferred examples of the site used for preparing the plant extract include plants, flowers, leaves, branches, roots, fruits, bark, above-ground parts, tree trunks, and the like. The giant crape myrtle is a evergreen small Takagi native to tropical Asia. In Japan, it is cultivated in Okinawa and Kyushu. In addition to being used as a diabetic drug, the leaf of the giant clam is used to produce banaba tea by drying the leaves and used as a Chinese medicine, supplement, and the like. Rubiaceae genus beer is a plant native to the coast of Malacca Strait. An extract made from dried beer leaves is called asenyaku (Asen drug), and its anti-fibrinolytic activity, antithrombotic activity, antitumor activity, etc. are known. The extract obtained from the above-mentioned plant in the present invention can be prepared using a plant grown in Japan, or a plant made in Japan sold as a herbal medicine raw material, etc. Commercial extracts sold by companies that handle plant extracts such as companies can be purchased and used. In the extraction, the plant body, the above-ground part or the tree trunk part is preferably processed in advance so as to improve the extraction efficiency by crushing or chopping. The extract is immersed in 1 to 30 parts by mass of a solvent with respect to 1 part by mass of the plant body, the above-ground part or its dried product, and immersed for several days at room temperature and for several hours at a temperature near the boiling point. After the immersion, the solution can be cooled to room temperature, insoluble matter can be removed if desired, and then the solvent can be removed by concentration under reduced pressure. Thereafter, fractionation and purification can be performed by column chromatography packed with silica gel or ion exchange resin to obtain a desired extract. In the present invention, the extract means a generic name of the extract itself, a fraction of the extract, a purified fraction, an extract or a fraction, and a solvent-removed product of the purified product. The preferred content of such components in the composition is 0.000001-20% by mass, more preferably 0.00001% -10% by mass, and still more preferably 0.0001-5% by mass. . This is because if the concentration is too high, the effect may reach its peak, and if it is too low, the effective concentration may not be obtained.

  The extraction solvent is preferably a polar solvent, alcohols such as water, ethanol, isopropyl alcohol and butanol, polyhydric alcohols such as 1,3-butanediol and polypropylene glycol, ketones such as acetone and methyl ethyl ketone, and diethyl ether. 1 type or 2 types or more selected from ethers, such as tetrahydrofuran, can illustrate suitably.

<Composition containing the component which has the skin barrier function improvement effect | action of this invention>
The composition containing a component having an action of improving the skin barrier function of the present invention measures the degree of TRPV activation action in cells, measures the degree of TJ and / or AJ formation promoting action, and measures the degree of these actions. It is characterized by containing an ingredient for improving the skin barrier function, which is screened as having an action size as large as that of each control, as compared with the respective controls.

In formulating a component having an action of improving the skin barrier function, an optional component used in formulating normal foods, pharmaceuticals, cosmetics and the like can be contained. As such an optional component, if it is a composition for oral administration, for example, excipients such as lactose and sucrose, binders such as starch, cellulose, gum arabic, and hydroxypropyl cellulose, sodium carboxymethylcellulose, carboxymethylcellulose calcium, etc. Disintegrants, soy lecithin, surfactants such as sucrose fatty acid esters, sweeteners such as maltitol and sorbitol, sour agents such as citric acid, buffering agents such as phosphate, film forming agents such as shellac and zein, Lubricants such as talc and waxes, light anhydrous silicic acid, glidants such as dry aluminum hydroxide gel, diluents such as physiological saline and aqueous glucose solution, flavoring agents, coloring agents, bactericides, preservatives, A fragrance | flavor etc. can be illustrated suitably. For transdermal administration compositions, hydrocarbons such as squalane, petrolatum, microcrystalline wax, esters such as jojoba oil, carnauba wax, octyldodecyl oleate, triglycerides such as olive oil, beef tallow, coconut oil, stearin Acids, fatty acids such as oleic acid and retinoic acid, lower alcohols such as ethanol and isopropanol, higher alcohols such as oleyl alcohol, stearyl alcohol and octyldodecanol, anionic surfactants such as sulfosuccinic acid ester and sodium polyoxyethylene alkyl sulfate , Amphoteric surfactants such as alkylbetaine salts, cationic surfactants such as dialkylammonium salts, sorbitan fatty acid esters, fatty acid monoglycerides, their polyoxyethylene adducts, poly Nonionic surfactants such as xylethylene alkyl ether, polyoxyethylene fatty acid ester, polyoxyethylene castor oil, polyoxyethylene hydrogenated castor oil, dipropylene glycol, polyethylene glycol, glycerin, 1,3-butanediol, 1, Polyhydric alcohols such as 2-pentanediol, thickening / gelling agents, antioxidants, ultraviolet absorbers, colorants, preservatives, powders and the like can be contained. Manufacture can be done without difficulty by treating these components according to conventional methods.

  As the composition containing a component having an action of improving the skin barrier function of the present invention, pharmaceuticals, cosmetics, foods, beverages and the like can be suitably exemplified and can be taken on a daily basis, so that they can be applied to foods, cosmetics, etc. Is preferred. The route of administration can be either oral or transdermal. For the purpose of improving the skin barrier function, transdermal administration with high efficiency in reaching the relevant organs has been adopted, and cosmetics, external skin drugs, etc. It is preferable to adopt the form of a transdermal composition. Of course, the active ingredient can also reach the target organ skin by oral administration or the like, and the oral administration system such as food is not excluded.

  Hereinafter, the present invention will be described in more detail with reference to examples, but it goes without saying that the present invention is not limited to such examples.

<Test Example 1: TER value measurement test>
Frozen normal human epidermal keratinocytes (NHEK) (manufactured by Kurashiki Boseki Co., Ltd.) are thawed, and a 0.15 mM calcium ion-containing medium (Humdia-KG2: Kurashiki Boseki Co., Ltd.) at 37 ° C., 5% carbon dioxide stream Cultured under. Set Transwell (Corning, 3460-Clear) on a Millicell Tissue Culture Plate (Millipore), put 0.5 mL of the upper layer and 1.5 mL of the lower layer, and add 2 NHEKs to the upper Transwell well. The cells were seeded at 5 × 10 ⁵ cells / cm ² and cultured at 37 ° C. in a 5% carbon dioxide stream for 24 hours. After confirming that the NHEK had grown to confluence, the medium was replaced with a Humeria-KG2 medium containing 1.5 mM calcium chloride, and cultured at 33 ° C. under a 5% carbon dioxide stream for 24 to 72 hours to form keratinized skin. A cell layer was constructed. Thereafter, the medium was replaced with a 1.5 mM calcium chloride-containing Humedia-KG2 medium supplemented with 10 μM 4α-PDD (manufactured by Sigma), 3 mM Camphor (manufactured by Camphor, Sigma) or methanol (vehicle, manufactured by Wako Pure Chemical Industries, Ltd.). The cells were cultured under a TRPV4 / TRPV3 activation temperature threshold value of 24 ° C to 28 ° C under a 5% carbon dioxide stream. The TER value was measured after 0, 3, 6, and 9 hours after changing to the medium containing each substance. The TER value was measured using Millicell ERS (manufactured by Millipore) after the sample was acclimated for 30 minutes in a clean bench. The results are shown in FIG.

Specific expression of TRPV3 and TRPV4 has been confirmed in the epidermis deeply involved in the skin barrier function. From the results shown in FIG. 1, in the TRPV4 inactive temperature region, NHEK cultured in the presence of 4α-PDD, which is a TRPV4 ligand, has a statistically significant increase in TER value compared to NHEK cultured in the absence. confirmed. On the other hand, in NHEK cultured in the presence of camphor of the TRPV3 ligand, an increase in TER value was not observed as compared with the absence. It was shown that the increase in the TER value, that is, the action of improving the skin barrier function by promoting TJ and / or AJ formation is exhibited by specific activation of TRPV4.

<Test Example 2: FITC-Dextran substance permeation test>
In the TER value measurement test, the medium of the sample 6 hours after the medium exchange used for measurement was removed. 0.5 mL of Apical Buffer (1.45 mM CaCl ₂ , 10 mM glucose, PBS containing 1 mg / mL FITC-Dextran) in the upper layer of the Transwell, and 1.5 mL of Basolatal Buffer (1.45 mM CaCl ₂ , 10 mM glucose in the lower layer) PBS containing was added and incubated for 3 hours. After collecting the basal buffer, a standard curve group (100 mg / mL to 0 mg / mL) is prepared on an 96-well plate using an Apical Buffer. The recovered basal buffer was added to each 96 wells in 200 mL, and then measured with a spectrophotometer (excitation 485/535 nm). The results are shown in FIG.

  From the results shown in FIG. 2, NHEK cultured in the presence of TRPV4 ligand 4α-PDD below the TRPV4 activation temperature threshold is statistically significant compared to NHEK cultured in the absence. Suppression of the amount was confirmed. On the other hand, in the presence of camphor of the TRPV3 ligand, suppression of FITC-Dextran permeation amount was not observed. It was shown that the suppression of FITC-Dextran permeation, that is, the skin barrier function improving action by promoting the formation of TJ and / or AJ is exhibited by specific activation of TRPV4.

<Test Example 3: si-RNA transfection test>
Frozen normal human epidermal keratinocytes (NHEK) (manufactured by Kurashiki Boseki Co., Ltd.) are thawed and cultured until they become subconfluent, then detached and collected with trypsin EDTA, and 7.5 × 10 ⁵ cells / well in a 6-well plate. And cultured at 37 ° C. under a 5% carbon dioxide stream for 2 hours. Moreover, si-RNA transfection (volume is described for 1 well) was performed according to the following procedures. To 500 μL OPTI-MEM, 5 μL of 10 μM siRNA was added, then 15 μL of HiperFect was added and mixed by vortexing, and then allowed to stand at room temperature for 10 minutes. After dripping into the well and diffusing gently, culturing under a 5% carbon dioxide stream at 37 ° C. for 24 hours, and then replacing with 1.5 mM calcium chloride-containing (+)-Humdia-KG2 medium, and the TER value with time And FITC-Dextran material permeability was measured. Also, the TRPV4 knockdown efficiency by si-RNA was confirmed by performing RT-PCR using Hs TRPV4 1 SG QuantiTect Primer Assay (Qiagen: QT00077217, sequence not disclosed) as a primer to determine the expression level of TRPV4 mRNA. did. The results are shown in FIGS. For si-RNA, commercially available designed si-RNA ON-TARGET plus SMART pool Human TRPV4 (manufactured by Thermo scientific) was used.

  From the results of FIG. 3 and FIG. 4, the expression of TRPV4 mRNA was suppressed with extremely high knockdown efficiency by si-RNA transfection in NHEK, and the suppression action was specific to TRPV4. Further, according to the results of FIG. 5 and FIG. 6, NHEK (TRPV4 / KD) in which TRPV4 is knocked down has a statistically significant TER value compared to NHEK (Mock) transfected with control si-RNA. Inhibition was confirmed. Furthermore, TRPV4 / KD was confirmed to have a statistically significant increase in FITC-Dextran permeability.

<Test Example 4: Expression of TRPV4 in human normal epidermal keratinocytes>
In order to confirm the presence of TRPV4 protein and TRPV4 binding molecule in human normal epidermal keratinocytes (NHEK) (manufactured by Kurashiki Boseki Co., Ltd.), immunoprecipitation was performed. Poly-Ab anti-β-catenin (Chemicon), mAb anti-E-cadherin (Takara Bio) and polyAb anti-TRPV4 (prepared against the N-terminal peptide of TRPV4) were used as antibodies.
The results are shown in FIG. In the left lane, NHEK membrane fraction was immunoprecipitated (IP) using an anti-β-catenin antibody, and a sample of the obtained complex was separated and immunoblotted (IB) using an anti-N-terminal TRPV4 antibody. Is. Black triangles indicate the position of TRPV4 co-precipitated with β-catenin. The right lane is replotted with anti-E-cadherin antibody. Black triangles indicate E-cadherin. That is, TRPV4 was coimmunoprecipitated with β-catenin and E-cadherin present in the cell membrane. β-catenin and E-cadherin are the main constituent proteins of AJ, suggesting that TRPV4 is present as part of the AJ complex in human keratinocytes.

<Test Example 5: Effect of temperature change and TRPV4 activation on differentiation process in human normal epidermal keratinocytes>
The Rho family of GTPases has been reported to play an important role in the differentiation of epidermal keratinocytes. The Rho family is activated by increasing intracellular calcium ion concentration and mediates keratinocyte differentiation including actin formation, intercellular adhesion formation and superficial actin formation. When activated, the TRP ion channel causes calcium ions to flow from outside the cell into the cell and promotes an increase in the intracellular calcium ion concentration. Thus, the expression level of active Rho was evaluated in NHEK cultured at 33 ° C. where TRPV3 and TRPV4 are activated and 28 ° C. below the activation temperature threshold.
After induction of differentiation by the calcium ion switch, NHEK was cultured at 33 ° C. or 28 ° C. for 24 hours. Thereafter, NHEK cultured at 28 ° C. was treated with 10 μM 4α-PDD or 3 mM camphor and cultured for 8 hours. For each of these NHEKs, the expression level of active Rho was evaluated using an immunoprecipitation method and a Western blotting method.
The results are shown in FIG. The expression level of active Rho is shown in the upper panel. 15 μg of total protein was loaded as input (lower panel).
The expression of the active form of NHEK cultured at 28 ° C. was lower than the expression of the active form of NHEK cultured at 33 ° C. The expression level of active Rho at 28 ° C. was remarkably increased by the addition of 4α-PDD which is a TRPV4 activator, but was not increased by camphor which was a TRPV3 activator.
This indicates that below the activation temperature threshold of TRPV3 and TRPV4 is insufficient to enhance Rho activity, but is specifically compensated by chemical activation of TRPV4, and TRPV4 mediated intracellular calcium It was suggested that ion influx is involved in Rho activation.

Next, compare the localization of AJ and TJ related proteins (E-cadherin, β-catenin, actin and occludin) in NHEK cultured at 33 ° C or 28 ° C or chemically activated TRPV4 and TRPV3 at 28 ° C In order to do this, immunofluorescence staining was performed.
The results are shown in FIG. After induction of differentiation by the calcium ion switch, NHEK was cultured at 33 ° C. or 28 ° C. for 24 hours. Thereafter, NHEK cultured at 28 ° C. was treated with 10 μM 4α-PDD or 3 mM camphor. These NHEK intercellular adhesion formation processes were confirmed over a time course (0-48 hours). Actin (green in the original image) surrounding the periphery of the cell membrane, β-catenin (purple in the original image) and E-cadherin (red in the original image) present at the intercellular adhesion are shown in an enlarged image. The white arrow indicates the “zipper structure” of AJ consisting of β-catenin and E-cadherin. )
In NHEK cultured at 33 ° C. and 28 ° C. 4 hours after differentiation induction by the calcium ion switch, both AJ-related proteins β-catenin and E-cadherin are localized along the cell membrane margin. “Zipper structure” was shown, suggesting that the initial cell-cell adhesion was formed regardless of the culture temperature. After 24 and 48 hours at 33 ° C., the cells layered together, and the surrounding surface actin clearly showed a honeycomb-like network. In contrast, cell-cell adhesion at 28 ° C. remained at the initial formation stage even after 24 and 48 hours. This immature differentiation was restored by the addition of 4α-PDD but not with camphor.

Furthermore, 48 hours after differentiation induction by the calcium ion switch, immunofluorescence staining of occludin, a TJ-related protein, was performed to confirm its localization. As the antibody, mAb anti-Occludin (manufactured by Invitrogen) was used.
The results are shown in FIG. Occludin, one of the TJ-related proteins, was localized along the cell-cell junctions 48 hours after differentiation induction in NHEK cultured at 33 ° C (red in the original figure), while 28 ° C In, the localization of occludin at the cell-cell junction was intermittent. Further, as in the case of the AJ-related protein, occludin was continuously localized by 4α-PDD at 28 ° C., but not by camphor.
When the expression levels of AJ and TJ-related mRNAs were confirmed by the RT-PCR method, changes in the expression levels of these mRNAs due to the addition of 4α-PDD were not confirmed.
From these results, TRPV4 activation does not contribute to the expression level of AJ and TJ-related molecules, but promotes the formation of AJ structure during the differentiation process of human normal epidermal keratinocytes and consequently promotes TJ formation. It was suggested.

<Test Example 6: Effect on barrier function in human keratinocytes and human skin tissue by temperature change and TRPV4 activation>
The barrier function in the skin is roughly divided into a stratum corneum barrier function and an epidermal cell (TJ) barrier function. In this study, in order to evaluate whether the TEK barrier function of NHEK is affected by temperature and TRPV4 activation, the transepithelial electrical resistance (TER) value used as an index when evaluating the TJ barrier function is expressed by Millicell- Measurements were made using ERS (Millipore).
NHEK subjected to differentiation induction by a calcium ion switch was cultured at 33 ° C. (◇) or 28 ° C. (□), and 24 hours after the start of the culture, NHEK cultured at 28 ° C. was added to 4α-PDD (Δ) or camphor (○ ) Was added to perform chemical enhancement of TRPV4 and TRPV3 activation.
The results are shown in FIG. Arrows indicate the timing of chemical enhancement. Data are the mean ± SEM of 5 independent experiments. D. It shows with. (**: P <0.01, N.S .: not significant.) Between 33 ° C. and 28 ° C., the presence of TRPV activator at 28 ° C. is not determined by t-test. Significant difference test was performed by Dunnett method using the value of as a control. A value of P <0.05 was considered significant.
The TER value of NHEK cultured at 28 ° C. was significantly lower than that cultured at 33 ° C., supporting the result of the occludin fragmented immunofluorescence signal at 28 ° C. (FIG. 10). As shown in Test Example 1 above, the TER value of NHEK chemically enhanced at 28 ° C. was significantly enhanced by 4α-PDD compared to the control without TRPV activator. It was not enhanced.
These facts suggested that the TJ barrier function of human keratinocytes decreases below the TRPV4 activation temperature threshold, but is overcome by specifically activating TRPV4 through chemical enhancement.
Furthermore, in order to confirm that the change in the TJ barrier function as described above is a phenomenon specifically caused by the involvement of TRPV4, TREK4 knockdown of NHEK using si-RNA was performed. The TER value of NHEK knocked down with TRPV4 was significantly reduced compared to NHEK (Mock) transfected with control si-RNA. (Fig. 5)
These facts suggested that the TJ barrier function of human keratinocytes is reduced by suppressing the expression of TRPV4.

Furthermore, the effect of temperature on the recovery of skin barrier function after stratum corneum barrier destruction was evaluated by ex vivo evaluation using fresh human skin tissue. The human skin tissue used at this time was the skin tissue (Biopredic and Juntendo Urayasu Hospital) excised by surgical operation after obtaining the consent of the patient through informed consent. These human skin tissues are pre-cultured using Skin Long Term Culture Medium (manufactured by Biopredic), and then stripped with C40SH02 adhesive tape (manufactured by Seikagaku Corporation) to completely remove the stratum corneum. As a result, the barrier was destroyed. Immediately after the disruption of the barrier due to removal of the stratum corneum, an aqueous solution containing 10 μM 4α-PDD, 3 mM camphor, or 50 μl of water alone is directly administered to the horny layer removal site of human skin tissue and cultured at 33 ° C. or 28 ° C. did. To restore skin barrier function, measure transepidermal water transpiration (TEWL) with VAPO SCAN AS-VT100RS (Asahi Techno Lab) and apply it to the following formula to calculate skin barrier function recovery rate (%). ,evaluated:
(TEWL immediately after stratum corneum barrier breakdown−TEWL at each measurement point) / (TEWL immediately after stratum corneum barrier breakdown−TEWL before stratum corneum barrier breakdown) × 100
Further, 2% EZ-link sulfo-NHS-LC-biotin (Pierce) was injected into the skin as an intercellular substance permeation marker, and the intercellular permeability of the marker was evaluated by immunofluorescence staining.
The results are shown in FIG. (A) shows the barrier recovery rate of the skin tissue at 1.5, 4 and 24 hours after barrier destruction. Each label indicates the following. ◇: administered at 33 ° C. water, □: administered at 28 ° C. water, Δ: administered at 28 ° C. 4α-PDD, ○: administered at 28 ° C. camphor. Data are the mean ± SEM of 3 independent experiments. D. It shows with. (*: P <0.05, **: P <0.01.) Between 33 ° C. and 28 ° C., the presence or absence of TRPV activator at 28 ° C. Significance test was performed by Dunnett method with values set as controls. A value of P <0.05 was considered significant.
The recovery rate of the barrier function of the skin tissue when cultured at 33 ° C. and 28 ° C. for 24 hours after the barrier destruction is significantly reduced in the skin tissue cultured at 28 ° C. compared to the skin tissue cultured at 33 ° C. It was. Regarding the barrier recovery rate of skin tissue treated with 4α-PDD or camphor, skin tissue cultured at 28 ° C. was compared as a control, and skin tissue treated with 4α-PDD showed a significantly higher recovery rate compared to control. As shown, the skin tissue treated with camphor showed a significantly lower recovery rate compared to the control.
These results are also supported by the evaluation of intercellular substance permeation described below.

The results are shown in FIG. (B) shows an immunofluorescent image of a cross section of skin tissue 24 hours after destruction of the stratum corneum barrier. The arrow indicates occludin (green in the original figure), which is one of the major TJ-related proteins localized in the epidermal granule layer. A white triangle indicates a substance permeation marker (red in the original drawing) that has passed through the position of occludin.
In the skin tissue cultured at 33 ° C., permeation of the substance permeation marker was blocked at the outermost layer of the epidermal granular layer where occludin was localized. On the other hand, in the skin tissue cultured at 28 ° C., the substance permeation marker leaked upward from the outermost layer of the epidermal granule layer where occludin was localized. Leakage of the substance permeation marker at 28 ° C. was suppressed by 4α-PDD, but was not suppressed by camphor, indicating that intercellular adhesion, particularly TJ strength, depends on activation of TRPV4.
Previous reports by the inventors have shown that epidermal TJ has an important role in stratum corneum barrier formation by imparting polarity to keratinocytes and promoting lamellar body secretion. Therefore, it is suggested that the activation of TRPV4 regulates not only the TJ barrier function but also the stratum corneum barrier function.
From the above results, that is, at a low temperature below the TRPV4 activation temperature threshold, it was shown that recovery of the skin barrier is delayed, but is specifically compensated by activation of TRPV4.

[Example 1]
<Method for producing plant extract>
A plant extract (including an extract obtained from the honey bee family Crape myrtle, Rubiaceae genus beer) can be produced according to the quasi-drug raw material standard 2006, and is commercially available from Maruzen Co., Ltd. You can also purchase and use plant extracts. In this example, a plant extract purchased from Maruzen Co., Ltd. was used.

[Example 2]
<Evaluation of TJ and AJ formation promoting action 1: Measurement of TER value of plant extract in the present invention>
According to the test method described in Test Example 1, the TER value of the plant extract was measured. The results are shown in FIG. In FIG. 13, the vertical axis represents the TER value (Ωcm ² ), and the horizontal axis represents the measurement time. From the results shown in FIG. 13, among the plant extracts, the extracts obtained from the beetle family Crape myrtle and the Rubiaceae gan beer show a marked increase in TER value, and the function promoting action of TJ and / or AJ. Was recognized.

[Example 3]
<TJ and AJ formation promoting action evaluation 2: FITC-Dextran substance permeation test of plant extract in the present invention>
According to the test method described in Test Example 2 above, the FITC-Dextran substance permeability of the plant extract was evaluated. The results are shown in FIG. From the results shown in FIG. 14, among the plant extracts, the extract obtained from the honey beetle genus Coleoptera and the Rubiaceae ganbei has a significant inhibition of FITC-Dextran substance permeability, and TJ and / or Or the formation promotion effect of AJ was recognized.

  From the results shown in FIG. 13 and FIG. 14, the extract of the present invention obtained from the honey bee family Crape myrtle, Rubiaceae ganbei is evaluated for the TJ and / or AJ formation promoting action (TER value measurement). And FITC-Dextran substance permeation test) both showed TJ and AJ formation promoting action.

[Example 4]
<Preparation of cells expressing human TRPV4>
Human TRPV4-expressing cells were prepared according to the following experimental procedure. CDNA encoding human TRPV4 [SEQ ID NO: 1: GenBank accession number: NM 021625, 90 bp to 2705 bp polynucleotide] is a vector for mammalian cells, the product name: pcDNA3 (manufactured by Invitrogen), MCS BamHI site and By inserting between XhoI sites, a human TRPV4 expression vector was obtained. Human TRPV4 expression vector obtained (corresponding to 0.5 μg) and DsRed (corresponding to 0.1 μg), plus Regent (trade name, catalog number: 11514-015, manufactured by Invitrogen), 6 μL, OPTI-MEM (registered trademark) 100 μL of I Reduced-Serum Medium (catalog number: 11058021, manufactured by Invitrogen) was mixed to obtain a mixture 1. Further, 4 μL of Lipofectamine (registered trademark, catalog number: 18324-012, manufactured by Invitrogen) and 100 μL of OPTI-MEM were mixed to obtain a mixture 2. On the other hand, HEK293 cells (5 × 10 ⁵ cells / 35 mm diameter petri dish) were cultured in a DMEM medium containing 10% by mass FBS to 37% confluence at 37 ° C. in a 5% carbon dioxide stream. Thereafter, the mixture of the mixture 1 and the mixture 2 was added to the obtained cells. Thereby, the human TRPV4 expression vector was introduced into HEK293 cells to obtain TRPV4-expressing cells. Further, a vector in which human TRPV4 was not inserted was prepared in the same manner, and this vector was introduced into HEK293 cells to prepare Mock cells.

[Example 5]
<TRPV activation effect evaluation 1: TRPV4 activation effect evaluation by the calcium imaging method of the plant extract in this invention>
The plant extract of the present invention was added to solution A [composition: 140 mM NaCl, 5 mM KCl, 2 mM MgCl ₂ , 2 mM CaCl ₂ , 10 mM glucose, 10 mM HEPES (pH 7.4)] to prepare a test solution. The concentration of the plant extract in each test solution was 0.1% by mass. The TRPV4-expressing cells or Mock cells prepared according to Example 4 above were incubated for 60 to 90 minutes in DMEM medium containing 10% by mass FBS containing 1 to 20 μg / mL FURA 2-AM (manufactured by Invitrogen) (37 FURA 2-AM was introduced into TRPV4-expressing cells or Mock cells. TRPV4-expressing cells or Mock cells after introduction of FURA 2-AM were set in a chamber (RC-26G, manufactured by Warner Instruments) of an inverted microscope of calcium imaging apparatus and washed with solution A. Subsequently, the fluorescence intensity at an excitation wavelength of 340 nm and the fluorescence intensity at an excitation wavelength of 380 nm were measured while circulating the test solution in a chamber. Thereafter, a fluorescence intensity ratio (fluorescence intensity at 340 nm / fluorescence intensity at 380 nm) between the fluorescence intensity at the excitation wavelength of 340 nm and the fluorescence intensity at the excitation wavelength of 380 nm when the test solution was used was calculated. The enhancement effect of the test substance on the change in intracellular calcium ion concentration (increased calcium ion concentration) was analyzed by IpLab software (manufactured by Scanalytics). Moreover, the same evaluation was carried out using 4alpha-phorbol 12,13-didecanoate (4α-PDD) (manufactured by Sigma Aldrich), which is a TRPV4 ligand, as a positive control. The results are shown in FIGS.

From the results of FIGS. 15 to 17, the positive control 4α-PDD showed a marked increase in intracellular calcium ion concentration, confirming the objectivity of this evaluation system. In addition, it was confirmed that the plant extract of the present invention significantly increased intracellular calcium ion concentration in TRPV4-expressing cells. On the other hand, it was not confirmed in Mock cells (FIGS. 18 and 19). Furthermore, the Acacia yak extract was added as a plant extract of the present invention to the calcium ion-free solution B [composition: 140 mM NaCl, 5 mM KCl, 2 mM MgCl ₂ , 5 mM EGTA, 10 mM glucose, 10 mM HEPES (pH 7.4)]. However, changes in intracellular calcium ion concentration in TRPV4-expressing cells were not confirmed (FIGS. 20 and 21), and were not confirmed in Mock cells (FIGS. 22 and 23). From this, it can be seen that the increase effect of intracellular calcium ion concentration by Acacia yak extract is influx of calcium ions from the outside via TRPV4.

[Example 6]
<Composition of the present invention>
<Manufacture of a composition (skin external preparation) containing a component having an action of improving the skin barrier function of the present invention>
According to the formulations shown in Tables 1 and 2, a lotion cosmetic, which is a composition (external preparation for skin) containing a component having an action of improving the skin barrier function of the present invention, was prepared. That is, the formulation components were stirred at 80 ° C. to solubilize, and then cooled with stirring to obtain lotion cosmetics [Cosmetics 1 and 2 (external preparations for skin 1 and 2)]. In addition, the same operation was performed to prepare Comparative Example 1 in which the “component having an action of improving skin barrier function” of the composition of the present invention (external preparation for skin) was replaced with “water”.

[Example 7]
<Skin roughness improvement test of the external preparation for skin of the present invention>
Using the paneler, cosmetics 1 and 2 and Comparative Example 1 which are external preparations of the present invention were evaluated for rough skin improvement effects in a rough skin model prepared by tape stripping. That is, three parts of 1.5 cm × 1.5 cm are defined on one forearm, tape stripping is carried out 15 times for each part, and transepidermal water transpiration (TEWL) is “Tevameter” manufactured by Integral. Measured with. After that, once a day, 50 μL of each sample was randomly selected for each subject (which of cosmetics 1 and 2 and Comparative Example 1 is applied to one site, and which sample is applied to which site). Next, TEWL was measured again on day 7. The TEWL value on day 7 was subtracted from the TEWL value on the first day, divided by the TEWL value on the first day, and multiplied by 100 to calculate the TEWL improvement rate (%). The n number was set to 15. The results are shown in Table 3. From this, it can be seen that the external preparation for skin of the present invention is excellent in the rough skin improving action.

  INDUSTRIAL APPLICABILITY The present invention can screen for a component having a skin barrier function improving action suitable as a cosmetic raw material (including quasi-drugs), and can be applied to a skin external preparation such as a cosmetic.

Claims (10)

  Measure the degree of TRPV receptor activation of the test substance, measure the degree of tight junction and / or adherence junction formation promotion, and use these levels as indicators A screening method for a skin barrier function-improving component, characterized in that those having a greater degree than the respective controls are selected.   The activation of the TRPV receptor and the measurement of the degree of tight junction and / or promotion of formation of adherence junction are measured using human-derived cells. The screening method of the skin barrier function improvement component of description.   The skin barrier function according to claim 1, wherein the formation promoting action of the tight junction and / or adherence junction is a promoting action of formation of the tight junction and / or adherence junction of the epidermis. Screening method for improving components.   The method for screening a skin barrier function improving component according to any one of claims 1 to 3, wherein the TRPV receptor is a TRPV receptor present in epidermal cells.   The method for screening a skin barrier function improving component according to any one of claims 1 to 4, wherein the TRPV receptor is TRPV4.   6. The method for screening a skin barrier function improving component according to claim 5, wherein the measurement of the degree of activation of TRPV4 measures the degree of increase in intracellular calcium ion concentration.   The measurement of the degree of increase in intracellular calcium ion concentration is to measure the degree of increase in intracellular calcium ion concentration in TRPV4 overexpressing cells. Screening method.   A skin barrier function improving component comprising a skin barrier function improving component screened as a substance having a skin barrier function improving action by the method for screening a skin barrier function improving component according to any one of claims 1 to 7. Composition.   The composition according to claim 8, wherein the composition is a cosmetic (however, including a quasi-drug).   The composition according to claim 8 or 9, which is an external preparation for skin.