Screening method for peroxynitrite activity inhibitors

Abstract

To provide a method for inhibiting peroxynitrite activity and a method for screening an activity inhibitor.SOLUTION: As a result of intensive examination in order to solve the above objective, it was made possible to inhibit peroxynitrite activity by promoting the nitration reaction of peroxynitrite by a method for maintaining the pH of skin to 6.5 or below, which has resulted in the present invention. Furthermore, a method for screening a peroxynitrite activity inhibitor is provided, with which it can be determined that the capability of inhibiting peroxynitrite activity is progressively higher as a nitrated protein content increases.EFFECT: The action of peroxynitrite, except the nitration reactions of protein such as cell differentiation and growth inhibition, can be suppressed by use of the method for inhibiting peroxynitrite activity or an activity inhibitor found out by use of the method for screening the activity inhibitor, according to the present invention.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for inhibiting peroxynitrite activity in skin and a method for screening for an inhibitor of peroxynitrite activity. [Background technology]

[0002] Peroxynitrite (ONOO-) is a compound produced by the reaction of nitric oxide and superoxide. Because of its extremely high reactivity, it is known to react with various biomolecules through oxidation and nitration reactions. For example, it is known to react with proteins, nucleic acids, lipids, glutathione, and other biomolecules (Non-Patent Document 1).

[0003] In the skin, peroxynitrite has been reported to be involved in skin aging such as wrinkles, sagging, and pigmentation (Patent Document 1). In addition, it is known that nitrated proteins generated by reacting with proteins cause yellowing of the skin (Patent Document 2), and therefore, inhibition of peroxynitrite activity is important for keeping the skin healthy.

[0004] Conventional methods for inhibiting peroxynitrite activity have been reported that use peroxynitrite activity inhibitors such as corn extract, coffee extract, grape extract (Patent Document 3), D-penicillamine, bucillamine, thioproline, and thiosalicylic acid (Patent Document 4). In addition, screening methods for these peroxynitrite activity inhibitors involve reacting tyrosine or protein with peroxynitrite to select components that can reduce the amount of nitrotyrosine or nitrated protein produced.

[0005] However, as mentioned above, peroxynitrite reacts with various substances in the body, including nucleic acids and lipids, in addition to proteins. In other words, even if the nitration reaction of proteins is inhibited, it cannot be determined whether the action of peroxynitrite on other biological molecules is inhibited. For example, even if a component that prevents the nitration of tyrosine is found by the conventional method, if the component does not react with peroxynitrite but only inhibits the nitration of tyrosine, it will not prevent the reaction of peroxynitrite with biological substances other than tyrosine. In this way, conventional screening methods can find inhibitors of protein nitration and inhibitors of cell damage induced by protein nitration, but they do not demonstrate the effect of peroxynitrite on the nitration and oxidation reaction of biological substances such as nucleic acids and lipids, and are insufficient as a screening method for peroxynitrite activity inhibitors. At the same time, protein nitration itself has been considered to be one of the cell damages caused by peroxynitrite, and the benefits of protein nitration on the skin have not been investigated until now.

[0006] On the other hand, as mentioned above, damage to the skin caused by peroxynitrite is known to cause wrinkles, sagging, pigmentation, etc., but its effects on epidermal cell differentiation and proliferation, the moisture retention ability of the stratum corneum, and the barrier function of the stratum corneum were not known. [Prior art documents] [Patent documents]

[0007] [Patent Document 1] JP 2002-265387 A [Patent Document 2] JP 2017-181423 A [Patent Document 3] JP 2002-265387 A [Patent Document 4] JP 2005-170849 A [Non-patent literature]

[0008] [Non-Patent Document 1] Szabo C et al., Peroxynitrite: biochemistry, pathophysiology and development of therapeutics., Nat Rev Drug Discov,Aug;6(8),662-80,2007. Summary of the Invention [Problem to be solved by the invention]

[0009] An object of the present invention is to provide a method for inhibiting peroxynitrite activity and a method for screening for an activity inhibitor. [Means for solving the problem]

[0010] In order to solve the above problems, the inventors conducted extensive research and found that pH contributes to the generation of nitrated proteins by peroxynitrite, and that the nitration reaction of proteins is unlikely to occur near neutral pH, but is likely to occur as the pH approaches acidity. Furthermore, the inventors clarified that under conditions where nitration is unlikely to occur, peroxynitrite reduces the differentiation and proliferation of epidermal cells and the barrier function of the stratum corneum, but under conditions where nitration is likely to occur, such as under acidic pH conditions, the differentiation and proliferation of epidermal cells and the reduction in the barrier function of the stratum corneum are suppressed. Based on these findings, the inventors have been able to promote the nitration reaction of peroxynitrite by, for example, maintaining the pH of the epidermis at 6.5 or less, thereby inhibiting the nitration and oxidation activity of peroxynitrite on biological substances other than proteins, thereby completing the present invention. Furthermore, the inventors can provide a method for screening peroxynitrite activity inhibitors, which can be determined to have a higher ability to inhibit peroxynitrite activity as the amount of nitrated proteins increases. Effect of the Invention

[0011] By using the peroxynitrite activity inhibition method of the present invention or an activity inhibitor discovered using the activity inhibitor screening method, it is possible to suppress the effects of peroxynitrite, such as inhibition of cell differentiation and proliferation, excluding protein nitration reactions. [Brief description of the drawings]

[0012] [Figure 1] FIG. 1 shows the amount of nitrated proteins produced by adding peroxynitrite to cultured epidermal cells. [Diagram 2] FIG. 2 shows changes in gene expression upon addition of peroxynitrite to cultured epidermal cells. [Diagram 3] FIG. 3 shows the amount of protein nitration reaction by peroxynitrite under different pH conditions. [Figure 4] FIG. 4 shows the amount of nitrated protein produced by adding peroxynitrite to cultured epidermal cells when the pH was brought closer to acidic. [Diagram 5] FIG. 5 shows a comparison of gene expression changes caused by the addition of peroxynitrite to cultured epidermal cells under different pH conditions. [Figure 6] FIG. 6 shows a correlation analysis between the moisture content of the stratum corneum of the cheek and the amount of nitrated protein. [Figure 7] FIG. 7 is a graph comparing changes in the stratum corneum barrier function caused by the addition of peroxynitrite to a three-dimensional skin model. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0013] In the present invention, inhibition of peroxynitrite activity means inhibition of the damaging activity of peroxynitrite on the skin other than the nitration of proteins, including inhibition of the oxidation and nitration of biological molecules other than proteins by peroxynitrite. In particular, in the present invention, the nitration of proteins by peroxynitrite is promoted, thereby preventing peroxynitrite from reacting with biological factors other than proteins, and as a result, inhibiting its activity.

[0014] In the present invention, the method for inhibiting peroxynitrite activity refers to a method for enhancing the nitration reactivity of peroxynitrite against proteins, and the peroxynitrite activity inhibitor refers to a substance that enhances the nitration reactivity of peroxynitrite against proteins.

[0015] Peroxynitrite treatment in screening for peroxynitrite activity inhibitors refers to allowing peroxynitrite to act on proteins in epidermal cells, etc., and the conditions are not particularly limited. For example, proteins may be dissolved in a buffer solution such as PBS and peroxynitrite may be added, or peroxynitrite may be added to a culture medium in which epidermal cells are cultured.

[0016] There is no particular limitation on the test material. Animal extracts, fungal cultures, or enzyme-treated products thereof, compounds or derivatives thereof, etc. can be used as test substances, and they can be in liquid, powder, gel, etc. The method for analyzing the amount of nitrated protein can be appropriately selected depending on the test material.

[0017] In the present invention, the protein used for screening is used in a concept including individual amino acids and peptides that constitute the protein, except when referring to the expression level of the protein. The type of protein used for screening is not particularly limited. Disintegrated cultured epidermal cells, proteins that constitute the skin such as keratin, collagen, elastin, etc. may be used, or commercially available proteins such as BSA (bovine serum albumin), which is a protein that does not constitute the skin, may be used as long as they undergo a nitration reaction. When it is desired to take into account the influence of biological reactions, it is preferable to use cultured epidermal cells as the protein.

[0018] The amount of nitrated protein may be measured directly, or indirectly, for example, by decomposing the nitrated protein by enzymatic treatment or the like, and measuring the amount of isolated nitro groups or the amount of isolated nitrated amino acid residues obtained, and using these as the amount of nitrated protein. In measuring the amount of nitrated protein, compounds that can be amino acid residues constituting nitrated protein, such as nitrotyrosine, 3,5-dinitrotyrosine, nitrotryptophan, nitrophenylalanine, 2,4-dinitrophenylalanine, and proteins containing these compounds, can be used as indicators of the amount of nitrated protein.

[0019] The amount of nitrated protein can be measured by a known method, for example, absorbance, immunostaining, Western blotting, radioimmunoassay, ELISA, liquid chromatography, gas chromatography, mass spectrometry, NMR, autofluorescence, etc.

[0020] In the screening method of the present invention, the greater the amount of nitrated protein produced by the action of peroxynitrite on a protein in the presence of a test material, the greater the ability to inhibit peroxynitrite activity, or to promote differentiation and proliferation of epidermal cells, improve the barrier function of the stratum corneum, and enhance the water-retaining ability of the stratum corneum. For example, screening is performed in the presence or absence of a test material, and if the amount of nitrated protein increases when the test material is added, it can be evaluated as being usable as a peroxynitrite activity inhibitor, an agent for promoting differentiation and proliferation of epidermal cells, an agent for improving the barrier function of the stratum corneum, or an agent for enhancing the water-retaining ability of the stratum corneum.

[0021] The stratum corneum moisture retention capacity refers to the ability of the stratum corneum to retain moisture. Since the moisture content of the stratum corneum depends on the moisture retention capacity of the stratum corneum as well as on the application of a cosmetic preparation and environmental conditions, materials discovered by the method for enhancing the moisture retention capacity of the stratum corneum and the screening method for enhancers of the stratum corneum moisture retention capacity of the present application contribute to increasing the moisture content of the stratum corneum.

[0022] The term "maintaining the pH of the epidermis at 6.5 or less" refers to maintaining the pH at 6.5 or less for a certain period of time or more. The longer the pH is maintained, the greater the effect can be expected. For example, the term refers to maintaining the pH at 6.5 or less for a total of 1 hour or more, preferably 3 hours or more, more preferably 6 hours or more, and even more preferably 12 hours or more per day. It is preferable to maintain the pH at 6.5 or less continuously, but it is acceptable to maintain the pH at 6.5 or less at appropriate intervals. It is preferable to maintain the pH at all layers of the epidermis from the basal layer to the stratum corneum, but it is also acceptable to maintain the pH at 6.5 or less in layers above the spinous layer or above the granular layer. As a method of maintaining the pH, it is possible to use a method such as promoting the production of acidic substances in the skin by cosmetics or the like.

[0023] The present invention will be specifically described with reference to the following examples, although the present invention is not limited thereto. EXAMPLES

[0024] <Example 1: Verification of the effect of peroxynitrite on epidermal cells> Normal human epidermal keratinocytes (KURABO) derived from newborns (5.0×104 cells / mL) were dispersed in antibacterial Humedia KG2 (KURABO), and 500μL was seeded in a 24-well plate. Incubated at 37℃ under 5% CO2 for 3 days. 25μL of peroxynitrite diluted with 0.1M sodium hydroxide to 2mM (final concentration 100μM), 1mM (final concentration 50μM: peroxynitrite+), and 0.5mM (final concentration 25μM) was added. 25μL of 0.1M sodium hydroxide was added as a control (peroxynitrite-). Incubated at 37℃ under 5% CO2 for 24 hr.

[0025] The medium was removed, and 50 μL of Extraction Buffer included with the Nitro tyrosine ELISA Kit was added. The mixture was left in the refrigerator for 30 minutes, and the supernatant was collected to obtain the extracted protein. The total protein amount (g / mL) of the extracted protein was determined using the BCA method in a standard manner. The extracted protein was then subjected to a Nitrotyrosine ELISA Kit to calculate the amount of nitrotyrosine (nmol / mL). The amount of nitrotyrosine (nmol / g) per fixed amount of extracted protein was determined. The amount of nitrotyrosine per fixed amount of protein for each condition was averaged, and the amount of nitrotyrosine per amount of protein for each condition was determined.

[0026] Total RNA was extracted from the cells in each well using a Total RNA Purification Kit (JenaBioscience). Reverse transcription was performed using the PrimeScriptTM RT Reagent Kit (TaKaRa) to synthesize cDNA. Real-time PCR was performed, and the gene expression of p21, keratin 5 (KRT5), keratin 14 (KRT14), keratin 1 (KRT1), keratin 10 (KRT10), involucrin (INV), and loricrin (LOR) was calculated by the comparative Ct method, with the gene expression level of peroxynitrite+ being calculated as "expression change due to peroxynitrite 1" when peroxynitrite- was set to 1. GAPDH was used as the housekeeping gene.

[0027] In this method, the addition of peroxynitrite did not increase nitrated proteins (Figure 1). On the other hand, changes were observed in gene expression, with the addition of peroxynitrite increasing the expression of p21 and decreasing the expression of keratin 1, keratin 10, and loricrin (Figure 2). These results demonstrated that peroxynitrite acts on biological substances other than proteins, suppressing cell proliferation and differentiation, under conditions that do not produce nitrated proteins.

[0028] <Example 2: Verification of the effect of pH on the production of nitrated proteins> Normal human epidermal keratinocytes (5.0×104 cells / mL) derived from newborns were dispersed in antibacterial Humedia KG2, and 500μL was seeded in a 24-well plate. Incubated at 37℃ under 5% CO2 for 3 days. The medium was replaced with 100μL of McIlvaine buffer adjusted to pH 4.5, 5.5, 6.5, or 7.5. 10μL of peroxynitrite diluted to 10mM (final concentration 1mM) was added. Incubated at 37℃ under 5% CO2 for 10min.

[0029] 50 μL of Extraction Buffer included with the Nitro tyrosine ELISA Kit was added. After leaving it in the refrigerator for 30 minutes, the supernatant was collected and used as the extracted protein. The total protein amount (g / mL) of the extracted protein was determined using the BCA method in the usual manner. Furthermore, the extracted protein was subjected to the Nitrotyrosine ELISA Kit to calculate the amount of nitrotyrosine (nmol / mL). The amount of nitrotyrosine (nmol / g) per fixed amount of extracted protein was determined. The amount of nitrotyrosine per fixed amount of protein for each condition was averaged, and the amount of nitrotyrosine per amount of protein for each condition was determined.

[0030] Compared to pH 7.5, more nitrotyrosine was produced at pH 4.5-6.5 (Figure 3). These results demonstrated that peroxynitrite produces more nitrated proteins under acidic pH conditions than under neutral pH conditions.

[0031] <Example 3: Verification of the effect of peroxynitrite on epidermal cells 2> Normal human epidermal keratinocytes derived from newborns (5.0×104 cells / mL) were dispersed in antibacterial Humedia KG2, and 500μL was seeded in a 24-well plate. The plate was incubated at 37℃ under 5% CO2 for 3 days. 25μL of 0.2M hydrochloric acid (final concentration 10mM) or distilled water was added. 25μL of peroxynitrite diluted to 1mM (final concentration 50μM) was added (peroxynitrite+). As a control, 25μL of 0.1M sodium hydroxide was added (peroxynitrite-). The plate was incubated at 37℃ under 5% CO2 for 24 hours.

[0032] 50 μL of Extraction Buffer included with the Nitro tyrosine ELISA Kit was added. After leaving it in the refrigerator for 30 minutes, the supernatant was collected and used as the extracted protein. The total protein amount (g / mL) of the extracted protein was determined using the BCA method in the usual manner. Furthermore, the extracted protein was subjected to the Nitrotyrosine ELISA Kit to calculate the amount of nitrotyrosine (nmol / mL). The amount of nitrotyrosine (nmol / g) per fixed amount of extracted protein was determined. The amount of nitrotyrosine per fixed amount of protein for each condition was averaged, and the amount of nitrotyrosine per amount of protein for each condition was determined.

[0033] Total RNA was extracted from the cells of each well using the Total RNA Purification Kit. Reverse transcription was performed using the PrimeScriptTM RT Reagent Kit to synthesize cDNA. Real-time PCR was performed, and the gene expression levels of p21, keratin 5 (KRT5), keratin 14 (KRT14), keratin 1 (KRT1), keratin 10 (KRT10), involucrin (INV), and loricrin (LOR) were calculated by the comparative Ct method, with the gene expression levels of peroxynitrite+ calculated as "expression change amount 2 due to peroxynitrite" when peroxynitrite- was set to 1. The expression increase rate of "expression change amount 2 due to peroxynitrite" in this example was calculated when the "expression change amount 1 due to peroxynitrite" shown in Example 2 was set to 1, and is shown in FIG. 6. GAPDH was used as the housekeeping gene.

[0034] By adding 0.2M hydrochloric acid to cultured epidermal cells and then peroxynitrite, nitrated proteins were produced at approximately 1.36 times the level in the control group (Fig. 4). Furthermore, gene expression was also altered, with p21, keratin 5, and keratin 14 expression decreased, while involucrin and loricrin expression increased, compared to conditions in which nitrated proteins were not produced (Fig. 5). These results indicate that under conditions in which nitrated proteins are easily produced, the inhibition of cell differentiation and proliferation by peroxynitrite is alleviated. This is believed to be because the reaction of peroxynitrite with proteins reduces the oxidation and nitration of other substances in the body. In addition, by increasing the amount of nitrated proteins, it is possible to promote epidermal cell differentiation and proliferation, and at the same time, it was determined that it is possible to screen for agents that promote epidermal cell differentiation and proliferation using the amount of nitrated proteins as an indicator.

[0035] Example 4: Relationship between the amount of nitrated protein in the stratum corneum and the moisture retention capacity of the stratum corneum The subjects were 16 healthy men and women in their 20s to 40s living in Japan. Each subject washed their face with cleanser and rested for 20 minutes in a room at 22°C and 50% humidity before the measurement to keep the moisture state of the cheek constant. The moisture content of the stratum corneum of each subject's cheek was measured five times using SKICON (Yayoi). The stratum corneum was collected from the test site using tape. The tape was placed on the cheek, gently rubbed with a finger five times over the tape, and then peeled off. The collection of the stratum corneum using tape was repeated three times from the same site. The tape with the stratum corneum attached was placed in a 1.5 mL tube. 1 mL of 6N hydrochloric acid was added and incubated at 37°C for 48 hours. The mixture was neutralized by mixing with sodium hydroxide to obtain a stratum corneum extract.

[0036] The total protein amount (g / mL) in the stratum corneum extract was determined using the BCA method according to standard methods. The stratum corneum extract was then subjected to a Nitrotyrosine ELISA Kit (Abcam) to calculate the amount of nitrotyrosine (nmol / mL). The amount of nitrotyrosine in each of the three tapes to which stratum corneum samples were attached, taken from the same subject, was divided by the total amount of protein to determine the amount of nitrotyrosine (nmol / g) per fixed amount of protein on each tape. The amount of nitrotyrosine per amount of protein on the three tapes for each subject was averaged to determine the amount of nitrotyrosine per amount of protein for each subject.

[0037] A Pearson correlation test was performed on the relationship between the average moisture content of the stratum corneum measured at five points and the amount of nitrotyrosine per protein amount calculated, and it was found that there was a positive correlation between them (Figure 6). The moisture content of the stratum corneum was measured after the skin condition was stabilized, so it can be considered that the moisture retention ability of the stratum corneum was evaluated. This result showed that the moisture retention ability of the stratum corneum increased with an increase in the amount of nitrated protein in the stratum corneum. Taking these results together with Examples 1 to 3, it was determined that by increasing the amount of nitrated protein, it is possible to improve the moisture retention ability of the stratum corneum, and at the same time, it is possible to screen for an agent for improving the moisture retention ability of the stratum corneum using the amount of nitrated protein as an index.

[0038] <Example 5: Verification of the effect of peroxynitrite on barrier function> The three-dimensional skin model LabCyte EPI-MODEL 6D (J-TEC) was incubated at 37°C under 5% CO2 for 7 days. During the 7-day culture period, the following treatments were performed three times. The medium was replaced with 25 μL of 0.2 M hydrochloric acid (HCl+) or distilled water. 25 μL of peroxynitrite / 0.1 M sodium hydroxide diluted to 1 mM (final concentration 50 μM) was added (peroxynitrite+). As a control, 25 μL of 0.1 M sodium hydroxide was added (control). The medium was incubated at 37°C under 5% CO2 for 1 hour and then replaced with normal medium.

[0039] After 7 days of incubation, 200 μL of 0.1% fluorescein sodium salt (FLUKA) solution was added and incubated at room temperature for 2 hours. The tissue was separated with a scalpel, embedded in Tissue-Tek OCT compound (Sakura Finetech Japan), and frozen. 5 μm-thick sections were prepared using a cryostat (Leica Microsystems) and photographed under a fluorescent microscope.

[0040] The addition of peroxynitrite promoted the penetration of fluorescent substances, whereas the addition of peroxynitrite to a culture medium made weakly acidic by the addition of hydrochloric acid inhibited the penetration (Figure 7). These results demonstrated that peroxynitrite reduces the barrier function of the stratum corneum and promotes the penetration of exogenous substances into the stratum corneum, but that making the medium weakly acidic increases the reactivity of peroxynitrite with proteins and inhibits reactions with other biological factors, thereby inhibiting the decrease in barrier function.

[0041] <Example 6: Screening method 1 for peroxynitrite activity inhibitors> Normal human epidermal keratinocytes (5.0×104 cells / mL) derived from newborns were dispersed in antibacterial Humedia KG2, and 500μL was seeded in a 24-well plate. The plate was incubated at 37℃ under 5% CO2 for 3 days. Wells to which the test material was added and wells without the test material were prepared, and 10μL of the test material was added to the wells to which the test material was added. 25μL of peroxynitrite diluted to 1mM was added. The medium was removed, and 50μL of Extraction Buffer included with the Nitro tyrosine ELISA Kit was added. The plate was left in the refrigerator for 30 minutes, and the supernatant was collected to obtain the extracted protein. The total protein amount (g / mL) of the extracted protein was calculated using the BCA method according to the usual method. The extracted protein was then subjected to the Nitrotyrosine ELISA Kit to calculate the amount of nitrotyrosine (nmol / mL). The amount of nitrotyrosine (nmol / g) per a given amount of extracted protein was calculated. For each condition, the compound with the highest amount of nitrotyrosine per a given amount of protein was selected as an inhibitor of peroxynitrite activity.

[0042] <Example 7: Screening method 2 for peroxynitrite activity inhibitors> Normal human epidermal keratinocytes (5.0×104 cells / mL) derived from newborns were dispersed in antibacterial Humedia KG2, and 500μL was seeded in a 24-well plate. The plate was incubated at 37℃ under 5% CO2 for 3 days. Wells to which the test material was added and wells without the test material were prepared, and 10μL of the test material was added to the wells to which the test material was added. 25μL of peroxynitrite diluted to 1mM was added. Total RNA was extracted from the cells in each well using the Total RNA Purification Kit. Reverse transcription was performed using the PrimeScriptTM RT Reagent Kit, and cDNA was synthesized. Real-time PCR was performed, and the test material that reduced the p21 expression level compared to the absence of the test material was selected as a peroxynitrite activity inhibitor by the comparative Ct method.

Claims

1. A method for screening for a peroxynitrite activity inhibitor, comprising the steps of: (1) treating a protein with peroxynitrite in the presence and absence of a test substance; (2) measuring an indicator of the amount of nitrated protein of the protein; and (3) determining that the test material is a peroxynitrite activity inhibitor when the amount of nitrated protein increases in the presence of the test material compared to when the test material is not present;

2. The method of claim 1, wherein the protein is a protein of a cultured epidermal cell.

3. The method for screening according to claim 1 or 2, wherein the amount of the nitrated protein is the amount of nitrated aromatic amino acids constituting the nitrated protein.

4. The method for screening according to claim 1 or 2, wherein the amount of nitrated protein is the amount of nitrotyrosine constituting the nitrated protein.

5. A method for screening for peroxynitrite activity inhibitors (excluding those that inhibit the damaging activity caused by protein nitration) using as an indicator the expression levels of differentiation / proliferation-related mRNA and / or protein in cultured epidermal cells treated with peroxynitrite.

6. A method for screening for peroxynitrite activity inhibitors (excluding those that inhibit the damaging activity caused by protein nitration) using as indicators the mRNA expression levels and / or protein expression levels of p21, involucrin, and loricrin in cultured epidermal cells treated with peroxynitrite.

7. A method for screening an agent for promoting differentiation and proliferation of epidermal cells, which comprises using the method for screening an inhibitor of peroxynitrite activity according to any one of claims 1 to 6.

8. A method for screening for a stratum corneum water-retaining ability enhancer, comprising using the method for screening for a peroxynitrite activity inhibitor according to any one of claims 1 to 6.

9. A method for screening for an agent for improving the barrier function of the stratum corneum, using the method for screening for a peroxynitrite activity inhibitor according to any one of claims 1 to 6.

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