JP7625681B2 - Screening method for peroxynitrite activity inhibitors - Google Patents
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Description
本発明は皮膚のペルオキシナイトライト活性阻害方法、およびペルオキシナイトライト活性阻害剤のスクリーニング方法に関する。 The present invention relates to a method for inhibiting peroxynitrite activity in the skin and a method for screening for inhibitors of peroxynitrite activity.
ペルオキシナイトライト（パーオキシナイトライト：ｐｅｒｏｘｙｎｉｔｒｉｔｅ：ＯＮＯＯ－）は一酸化窒素とスーパーオキシドが反応することで生成する化合物である。非常に高い反応性を持つことから、酸化反応やニトロ化反応を通して、様々な生体内分子と反応することが知られる。例えば、タンパク質、核酸、脂質、グルタチオン、その他生体内分子と反応することが知られる（非特許文献１）。 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 biological molecules through oxidation and nitration reactions. For example, it is known to react with proteins, nucleic acids, lipids, glutathione, and other biological molecules (Non-Patent Document 1).
皮膚では、ペルオキシナイトライトは、しわやたるみ、色素沈着などの皮膚の老化に関与すると報告されている（特許文献１）。また、タンパク質と反応することで生じるニトロ化タンパク質は、肌の黄ぐすみの原因になることが分かっており（特許文献２）、皮膚を健康に保つためには、ペルオキシナイトライトの活性阻害が重要である。 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 reaction with proteins cause yellowing of the skin (Patent Document 2), and therefore inhibiting the activity of peroxynitrite is important for keeping the skin healthy.
従来のペルオキシナイトライト活性阻害方法としては、とうもろこし抽出物、コーヒー抽出物、ブドウ抽出物（特許文献３）、Ｄ―ペニシラミン、ブシラミン、チオプロリン、チオサリチル酸（特許文献４）などのペルオキシナイトライト活性阻害剤を用いる方法が報告されている。また、これらペルオキシナイトライト活性阻害剤のスクリーニング方法として、チロシンやタンパク質をペルオキシナイトライトと反応させ、生じるニトロチロシンやニトロ化タンパク質量を減少させることができる成分を選択している。 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, a screening method for these peroxynitrite activity inhibitors involves reacting tyrosine or protein with peroxynitrite to select components that can reduce the amount of nitrotyrosine or nitrated protein produced.
しかしながら上述のように、ペルオキシナイトライトは生体内において、タンパク質以外にも核酸、脂質をはじめとして、様々な物質と反応を引き起こす。つまり、タンパク質のニトロ化反応を阻害できているからといって、その他生体内分子へのペルオキシナイトライトの作用を阻害できているかは判断できない。例えば、従来の方法でチロシンのニトロ化を防ぐ成分が見つかったとしても、その成分がペルオキシナイトライトと反応するのではなく、チロシンのニトロ化を阻害しているだけであったら、ペルオキシナイトライトのチロシン以外の生体内物質との反応は防げない。このように、従来のスクリーニング方法では、タンパク質のニトロ化抑制剤や、タンパク質のニトロ化により誘発される細胞傷害の抑制剤は見出すことができるが、ペルオキシナイトライトによる、核酸、脂質などの生体内物質へのニトロ化・酸化反応に対する効果を実証するものではなく、ペルオキシナイトライト活性阻害剤のスクリーニング方法としては不十分であった。同時に、このようにタンパク質のニトロ化自体がペルオキシナイトライトによる細胞傷害の一つだととらえられてきた側面もあり、これまでタンパク質がニトロ化することによる皮膚への恩恵については調べられてこなかった。 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 using conventional methods, if the component does not react with peroxynitrite but only inhibits the nitration of tyrosine, it will not be able to prevent peroxynitrite from reacting 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 to the skin have not been investigated until now.
一方で、上述のように、ペルオキシナイトライトによる皮膚への傷害については、しわやたるみ、色素沈着などが知られるが、表皮細胞の分化、増殖、角層水分保持能や角層のバリア機能に対する作用は知られていなかった。 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 unknown.
本発明の課題は、ペルオキシナイトライト活性阻害方法、および活性阻害剤のスクリーニング方法を提供することである。 The objective of the present invention is to provide a method for inhibiting peroxynitrite activity and a method for screening for activity inhibitors.
前記課題を解決すべく、鋭意検討を行った結果、ペルオキシナイトライトによるニトロ化タンパク質生成にはｐＨが寄与しており、中性付近ではタンパク質のニトロ化反応が起こりづらいが、酸性に近づくと、タンパク質のニトロ化が起こりやすくなることを見出した。さらに、ニトロ化が起こりづらい条件では、ペルオキシナイトライトにより表皮細胞の分化・増殖、角層のバリア機能が低下するが、ｐＨが酸性条件にあるなど、ニトロ化が起こりやすい条件では、表皮細胞の分化・増殖、角層のバリア機能の低下が抑えられることを明らかにした。これらの知見に基づいて、表皮のｐＨを６．５以下に維持するなどの方法で、ペルオキシナイトライトのニトロ化反応を促進することで、結果としてペルオキシナイトライトによる、タンパク質以外の生体内物質へのニトロ化・酸化活性を阻害することが可能となり、本発明に至った。さらに、ニトロ化タンパク質量が増加するほどペルオキシナイトライト活性阻害能が高いと判断できる、ペルオキシナイトライト活性阻害剤スクリーニング方法を提供できる。 As a result of intensive research to solve the above problem, it was found that pH contributes to the production 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, it was revealed 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 decline in the barrier function of the stratum corneum are suppressed. Based on these findings, it has become possible to inhibit the nitration and oxidation activity of peroxynitrite on biological substances other than proteins by promoting the nitration reaction of peroxynitrite by methods such as maintaining the pH of the epidermis at 6.5 or less, thereby arriving at the present invention. Furthermore, it is possible to provide a method for screening peroxynitrite activity inhibitors that can determine that the greater the amount of nitrated protein, the higher the ability to inhibit peroxynitrite activity.
本発明のペルオキシナイトライト活性阻害方法、または活性阻害剤スクリーニング方法を利用して見出される活性阻害剤を用いることで、細胞分化・増殖阻害などの、タンパク質のニトロ化反応を除いたペルオキシナイトライトの作用を抑制できる。 By using the peroxynitrite activity inhibition method of the present invention or an activity inhibitor found 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.
本発明におけるペルオキシナイトライト活性阻害とは、ペルオキシナイトライトの皮膚に対する、タンパク質のニトロ化以外の傷害活性を阻害することを意味し、ペルオキシナイトライトによるタンパク質以外の生体内分子への酸化やニトロ化反応の阻害を含む。本発明では特に、ペルオキシナイトライトによるタンパク質のニトロ化を促進することで、ペルオキシナイトライトがタンパク質以外の生体内因子と反応することを防ぎ、結果として活性を阻害する。 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, and includes inhibition of the oxidation and nitration reactions of biological molecules other than proteins by peroxynitrite. In particular, the present invention promotes the nitration of proteins by peroxynitrite, thereby preventing peroxynitrite from reacting with biological factors other than proteins, and as a result inhibiting its activity.
本発明におけるペルオキシナイトライト活性阻害方法とは、ペルオキシナイトライトのタンパク質に対するニトロ化反応性を高める方法を意味し、ペルオキシナイトライト活性阻害剤とは、ペルオキシナイトライトのタンパク質に対するニトロ化反応性を高める物質を意味する。 In the present invention, the method for inhibiting peroxynitrite activity refers to a method for increasing the nitration reactivity of peroxynitrite against proteins, and the peroxynitrite activity inhibitor refers to a substance that increases the nitration reactivity of peroxynitrite against proteins.
ペルオキシナイトライト活性阻害剤スクリーニング時の、ペルオキシナイトライト処理とは、ペルオキシナイトライトを表皮細胞などのタンパク質に作用させることであり、その条件は特に限定されない。例えば、ＰＢＳ等の緩衝液中にタンパク質を溶解し、ペルオキシナイトライトを添加しても良いし、表皮細胞を培養している培地中に添加しても良い。 When screening for peroxynitrite activity inhibitors, peroxynitrite treatment refers to allowing peroxynitrite to act on proteins in epidermal cells and the like, 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 the medium in which epidermal cells are cultured.
被験素材は、特に制限はない。動物由来エキス、菌類の培養物、又はこれらの酵素等処理物、化合物又はその誘導体等であっても被検物質として用いることが出来、液状の他、粉末状、ジェル状等であっても差し支えない。被験素材に応じてニトロ化タンパク質の量の分析方法を適宜選択すればよい。 There are no particular limitations on the test material. Animal extracts, fungal cultures, or enzyme-treated products of these, compounds or their derivatives, 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 selected appropriately depending on the test material.
本発明において、スクリーニングに使用するタンパク質は、タンパク質の発現量を指す場合を除き、タンパク質を構成する単体のアミノ酸やペプチドも含む概念で用いる。スクリーニングに使用するタンパク質の種類は特に限定されない。培養表皮細胞の破砕物や、皮膚を構成するタンパク質であるケラチン、コラーゲン、エラスチンなどを用いても良いし、ニトロ化反応が起こるタンパク質であれば、皮膚を構成しないタンパク質であるＢＳＡ（ウシ血清アルブミン）などの市販のタンパク質を用いても良い。生体反応の影響を加味したい場合は、タンパク質として培養表皮細胞を用いることが好ましい。 In the present invention, the protein used for screening is used to include the individual amino acids and peptides that make up the protein, except when referring to the expression level of the protein. There are no particular limitations on the type of protein used for screening. Crushed cultured epidermal cells, keratin, collagen, elastin, and other proteins that make up the skin may be used, or commercially available proteins such as BSA (bovine serum albumin), a protein that does not make up the skin, may be used as long as they undergo a nitration reaction. When it is desired to take into account the effects of biological reactions, it is preferable to use cultured epidermal cells as the protein.
ニトロ化タンパク質量は直接的に測定してもよいし、例えばニトロ化タンパク質を酵素処理等にて分解し、得られた単離ニトロ基の量や単離ニトロ化アミノ酸残基の量を測定したものをニトロ化タンパク質量とするなど間接的に測定することもできる。ニトロ化タンパク質量測定においては、ニトロチロシンや３，５－ジニトロチロシン、ニトロトリプトファン、ニトロフェニルアラニン、２,４-ジニトロフェニルアラニン等、ニトロ化タンパク質を構成するアミノ酸残基となりうる化合物、およびこれらを含むタンパク質を、ニトロ化タンパク質量の指標とすることができる。 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 this as the amount of nitrated protein. In measuring the amount of nitrated protein, compounds that can become amino acid residues that make up nitrated proteins, such as nitrotyrosine, 3,5-dinitrotyrosine, nitrotryptophan, nitrophenylalanine, and 2,4-dinitrophenylalanine, as well as proteins that contain these compounds, can be used as indicators of the amount of nitrated protein.
ニトロ化タンパク質の量の測定方法は、公知の方法で行うことができる。例えば、吸光度法、免疫染色法、ウエスタンブロッティング、放射免疫測定 （Ｒａｄｉｏｉｍｍｕｎｏａｓｓａｙ）、ＥＬＩＳＡ、液体クロマトグラフィー、ガスクロマトグラフィー、質量分析法、ＮＭＲ法、自家蛍光等を用いて測定することができる。 The amount of nitrated protein can be measured by known methods. For example, it can be measured using absorbance, immunostaining, Western blotting, radioimmunoassay, ELISA, liquid chromatography, gas chromatography, mass spectrometry, NMR, autofluorescence, etc.
本発明におけるスクリーニング方法では、被験素材存在下で、ペルオキシナイトライトをタンパク質に作用させて生成するニトロ化タンパク質量が多いほど、ペルオキシナイトライト活性阻害力、あるいは表皮細胞の分化・増殖促進力、角層のバリア機能向上力、角層水分保持能増進力が高いと判断する。例えば、被験素材の有無でスクリーニングを行い、被験素材添加時にニトロ化タンパク質量が増加していれば、ペルオキシナイトライト活性阻害剤、表皮細胞の分化・増殖促進剤、角層のバリア機能向上剤、角層水分保持能増進剤として利用可能であると評価できる。 In the screening method of the present invention, the greater the amount of nitrated protein produced by the action of peroxynitrite on 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 retention 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 retention ability of the stratum corneum.
角層水分保持能とは角層が水分を保持する能力を指す。角層水分量は化粧品製剤の塗布や環境条件などに加え、角層水分保持能に依存するため、本願の角層水分保持能増進方法や角層水分保持能増進剤スクリーニング方法により見出された素材は角層水分量の増加に寄与する。 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 the cosmetic preparation and environmental conditions, materials discovered by the method of enhancing stratum corneum moisture retention capacity and the screening method for stratum corneum moisture retention capacity enhancers of the present application contribute to increasing the moisture content of the stratum corneum.
表皮のｐＨを６．５以下に維持するとは、一定時間以上、ｐＨを６．５以下に維持されればよい。長時間維持されれば効果がより期待できる。例えば、１日合計で１時間以上、好ましくは３時間以上、より好ましくは６時間以上、更に好ましくは１２時間以上の維持を指す。連続で維持することが望ましいが、適宜間隔をあけてｐＨ６．５以下に維持しても差し支えない。表皮細胞の基底層から角層にかけての全層で維持することが望ましいが、有棘層より上層又は、顆粒層より上層でも問題ない。維持の方法としては、皮膚中の酸性物質の生成を、化粧品等により促進するなどの方法を用いることが可能である。 Maintaining the pH of the epidermis at 6.5 or less means maintaining the pH at 6.5 or less for a certain period of time or more. The longer it is maintained, the greater the effect can be expected. For example, it means maintaining the pH at 6.5 or less in total for a day 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. 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 in all layers of the epidermal cells from the basal layer to the stratum corneum, but it is also acceptable to maintain the pH in layers above the spinous layer or above the granular layer. Methods for maintaining the pH include promoting the production of acidic substances in the skin with cosmetics, etc.
以下の実施例により、本発明を具体的に説明する。なお、本発明はこれに限定されるものではない。 The present invention will be specifically explained by the following examples. Note that the present invention is not limited to these examples.
＜実施例１：ペルオキシナイトライトの表皮細胞に対する作用の検証＞
新生児由来正常ヒト表皮ケラチノサイト（ＫＵＲＡＢＯ）５．０×１０４ Ｃｅｌｌｓ／ｍＬを抗菌剤含有Ｈｕｍｅｄｉａ ＫＧ２（ＫＵＲＡＢＯ）に分散し、２４ ｗｅｌｌ ｐｌａｔｅに５００μＬを播種した。３７℃、５%ＣＯ２下で３日間インキュベートした。０．１Ｍ水酸化ナトリウムで２ｍＭ（終濃度１００μＭ）、１ｍＭ（終濃度５０μＭ：ｐｅｒｏｘｙｎｉｔｒｉｔｅ＋）、０．５ｍＭ（終濃度２５μＭ）に薄めたペルオキシナイトライトを２５μＬ添加した。コントロール（ｐｅｒｏｘｙｎｉｔｒｉｔｅ－）としては０．１Ｍ水酸化ナトリウムを２５μＬ添加した。３７℃、５%ＣＯ２下で２４ ｈｒインキュベートした。
Example 1: Verification of the effect of peroxynitrite on epidermal cells
5.0 x 104 cells/mL of normal human epidermal keratinocytes derived from newborns (KURABO) were dispersed in antibacterial agent-containing Humedia KG2 (KURABO), and 500 μL was seeded in a 24-well plate. Incubated at 37°C and 5% CO2 for 3 days. 25 μL of peroxynitrite diluted with 0.1 M sodium hydroxide to 2 mM (final concentration 100 μM), 1 mM (final concentration 50 μM: peroxynitrite+), and 0.5 mM (final concentration 25 μM) was added. 25 μL of 0.1 M sodium hydroxide was added as a control (peroxynitrite-). Incubated at 37°C and 5% CO2 for 24 hr.
培地を取り除き、Ｎｉｔｒｏ ｔｙｒｏｓｉｎｅ ＥＬＩＳＡ Ｋｉｔ付属のＥｘｔｒａｃｔｉｏｎ Ｂｕｆｆｅｒを５０μＬ加えた。冷蔵庫で３０分放置し、上清を回収し抽出タンパク質とした。常法に従い、ＢＣＡ法にて抽出タンパク質の総タンパク質量（ｇ／ｍＬ）を求めた。さらに抽出タンパク質をＮｉｔｒｏｔｙｒｏｓｉｎｅ ＥＬＩＳＡ Ｋｉｔに供し、ニトロチロシン量（ｎｍｏｌ／ｍＬ）を算出した。各抽出タンパク質一定量あたりのニトロチロシン量（ｎｍｏｌ／ｇ）を求めた。条件ごとに、タンパク質一定量あたりのニトロチロシン量を平均し、条件ごとのタンパク質量あたりのニトロチロシン量を求めた。 The medium was removed, and 50 μL of Extraction Buffer included with the Nitrotyrosine 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 the usual manner. 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 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.
Ｔｏｔａｌ ＲＮＡ Ｐｕｒｉｆｉｃａｔｉｏｎ Ｋｉｔ（ＪｅｎａＢｉｏｓｃｉｅｎｃｅ）を使用し、各ｗｅｌｌの細胞からＴｏｔａｌ ＲＮＡを抽出した。ＰｒｉｍｅＳｃｒｉｐｔＴＭ ＲＴ Ｒｅａｇｅｎｔ Ｋｉｔ（ＴａＫａＲａ) を用い、逆転写を行い、ｃＤＮＡを合成した。リアルタイムＰＣＲを行い、比較Ｃｔ法により、ｐ２１、ケラチン５（ＫＲＴ５）、ケラチン１４（ＫＲＴ１４）、ケラチン１（ＫＲＴ１）、ケラチン１０（ＫＲＴ１０）、インボルクリン（ＩＮＶ）、ロリクリン（ＬＯＲ）の遺伝子発現について、ｐｅｒｏｘｙｎｉｔｒｉｔｅ－を１とした場合の、ｐｅｒｏｘｙｎｉｔｒｉｔｅ＋の遺伝子発現量を、「ペルオキシナイトライトによる発現変化量１」として求めた。なお、ハウスキーピング遺伝子としては、ＧＡＰＤＨを用いた。 Total RNA was extracted from the cells in each well using Total RNA Purification Kit (JenaBioscience). Reverse transcription was performed using PrimeScriptTM RT Reagent Kit (TaKaRa) 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+ being calculated as "expression change due to peroxynitrite 1" when peroxynitrite- was set to 1. GAPDH was used as the housekeeping gene.
本方法において、ペルオキシナイトライトを添加しても、ニトロ化タンパク質は増加しなかった（図１）。一方で、遺伝子発現には変化が見られ、ペルオキシナイトライトを添加することで、ｐ２１の発現増加、ケラチン１、ケラチン１０、ロリクリンの発現低下が見られた（図２）。これらの結果より、ペルオキシナイトライトはニトロ化タンパク質を生成しない条件で、タンパク質以外の生体内物質に作用して、細胞増殖や細胞分化を抑制していることが分かった。 In this method, the addition of peroxynitrite did not increase nitrated proteins (Figure 1). However, 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 demonstrate that peroxynitrite acts on biological substances other than proteins, suppressing cell proliferation and differentiation, under conditions that do not produce nitrated proteins.
＜実施例２：ｐＨによるニトロ化タンパク質生成への影響検証＞
新生児由来正常ヒト表皮ケラチノサイト５．０×１０４ Ｃｅｌｌｓ／ｍＬを抗菌剤含有Ｈｕｍｅｄｉａ ＫＧ２に分散し、２４ ｗｅｌｌ ｐｌａｔｅに５００μＬを播種した。３７℃、５%ＣＯ２下で３日間インキュベートした。ｐＨ４．５、５．５、６．５、７．５に調製したマッキルベイン緩衝液１００μＬに置換した。１０ｍＭ（終濃度１ｍＭ）に薄めたペルオキシナイトライトを１０μＬ添加した。３７℃、５%ＣＯ２下で１０ｍｉｎインキュベートした。
Example 2: Verification of the effect of pH on the production of nitrated proteins
5.0 x 104 cells/mL of normal human epidermal keratinocytes derived from newborns were dispersed in antibacterial agent-containing Humedia KG2, and 500 μL was seeded in a 24-well plate. Incubated at 37°C and 5% CO2 for 3 days. Replaced with 100 μL of McIlvaine buffer adjusted to pH 4.5, 5.5, 6.5, and 7.5. 10 μL of peroxynitrite diluted to 10 mM (final concentration 1 mM) was added. Incubated at 37°C and 5% CO2 for 10 min.
Ｎｉｔｒｏ ｔｙｒｏｓｉｎｅ ＥＬＩＳＡ Ｋｉｔ付属のＥｘｔｒａｃｔｉｏｎ Ｂｕｆｆｅｒを５０μＬ加えた。冷蔵庫で３０分放置し、上清を回収し抽出タンパク質とした。常法に従い、ＢＣＡ法にて抽出タンパク質の総タンパク質量（ｇ／ｍＬ）を求めた。さらに抽出タンパク質をＮｉｔｒｏｔｙｒｏｓｉｎｅ ＥＬＩＳＡ Ｋｉｔに供し、ニトロチロシン量（ｎｍｏｌ／ｍＬ）を算出した。各抽出タンパク質一定量あたりのニトロチロシン量（ｎｍｏｌ／ｇ）を求めた。条件ごとに、タンパク質一定量あたりのニトロチロシン量を平均し、条件ごとのタンパク質量あたりのニトロチロシン量を求めた。 50 μL of Extraction Buffer included with the Nitrotyrosine 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 according to standard methods. 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 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.
ｐＨ７．５の条件と比較して、ｐＨ４．５～６．５ではニトロチロシンがより多く生成された（図３）この結果より、ペルオキシナイトライトはｐＨが中性条件よりも酸性条件で、よりニトロ化タンパク質を生成することが明らかとなった。 Compared to pH 7.5, more nitrotyrosine was produced at pH 4.5-6.5 (Figure 3). These results demonstrate that peroxynitrite produces more nitrated proteins under acidic pH conditions than under neutral pH conditions.
＜実施例３：ペルオキシナイトライトの表皮細胞に対する作用の検証２＞
新生児由来正常ヒト表皮ケラチノサイト５．０×１０４ Ｃｅｌｌｓ／ｍＬを抗菌剤含有Ｈｕｍｅｄｉａ ＫＧ２に分散し、２４ ｗｅｌｌ ｐｌａｔｅに５００μＬを播種した。３７℃、５%ＣＯ２下で３日間インキュベートした。０．２Ｍ塩酸（終濃度１０ｍＭ）または蒸留水を２５μＬ加えた。１ｍＭ（終濃度５０μＭ）に薄めたペルオキシナイトライトを２５μＬ添加した（ｐｅｒｏｘｙｎｉｔｒｉｔｅ＋）。コントロールとしては０．１Ｍ水酸化ナトリウムを２５μＬ添加した（ｐｅｒｏｘｙｎｉｔｒｉｔｅ－）。３７℃、５%ＣＯ２下で２４ ｈｒインキュベートした。
Example 3: Verification of the effect of peroxynitrite on epidermal cells 2
5.0 x 104 cells/mL of normal human epidermal keratinocytes derived from newborns were dispersed in antibacterial agent-containing Humedia KG2, and 500 μL was seeded in a 24-well plate. The mixture was incubated at 37°C and 5% CO2 for 3 days. 25 μL of 0.2 M hydrochloric acid (final concentration 10 mM) or distilled water was added. 25 μL of peroxynitrite diluted to 1 mM (final concentration 50 μM) was added (peroxynitrite+). As a control, 25 μL of 0.1 M sodium hydroxide was added (peroxynitrite-). The mixture was incubated at 37°C and 5% CO2 for 24 hours.
Ｎｉｔｒｏ ｔｙｒｏｓｉｎｅ ＥＬＩＳＡ Ｋｉｔ付属のＥｘｔｒａｃｔｉｏｎ Ｂｕｆｆｅｒを５０μＬ加えた。冷蔵庫で３０分放置し、上清を回収し抽出タンパク質とした。常法に従い、ＢＣＡ法にて抽出タンパク質の総タンパク質量（ｇ／ｍＬ）を求めた。さらに抽出タンパク質をＮｉｔｒｏｔｙｒｏｓｉｎｅ ＥＬＩＳＡ Ｋｉｔに供し、ニトロチロシン量（ｎｍｏｌ／ｍＬ）を算出した。各抽出タンパク質一定量あたりのニトロチロシン量（ｎｍｏｌ／ｇ）を求めた。条件ごとに、タンパク質一定量あたりのニトロチロシン量を平均し、条件ごとのタンパク質量あたりのニトロチロシン量を求めた。 50 μL of Extraction Buffer included with the Nitrotyrosine 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 according to standard methods. 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 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.
Ｔｏｔａｌ ＲＮＡ Ｐｕｒｉｆｉｃａｔｉｏｎ Ｋｉｔを使用し、各ｗｅｌｌの細胞からＴｏｔａｌ ＲＮＡを抽出した。ＰｒｉｍｅＳｃｒｉｐｔＴＭ ＲＴ Ｒｅａｇｅｎｔ Ｋｉｔを用い、逆転写を行い、ｃＤＮＡを合成した。リアルタイムＰＣＲを行い、比較Ｃｔ法により、ｐ２１、ケラチン５（ＫＲＴ５）、ケラチン１４（ＫＲＴ１４）、ケラチン１（ＫＲＴ１）、ケラチン１０（ＫＲＴ１０）、インボルクリン（ＩＮＶ）、ロリクリン（ＬＯＲ）の遺伝子発現について、ｐｅｒｏｘｙｎｉｔｒｉｔｅ－を１とした場合の、ｐｅｒｏｘｙｎｉｔｒｉｔｅ＋の遺伝子発現量を、「ペルオキシナイトライトによる発現変化量２」として求めた。実施例２で示した「ペルオキシナイトライトによる発現変化量１」を１とした場合の、本実施例における「ペルオキシナイトライトによる発現変化量２」の発現増加率を求め、図６に示した。なお、ハウスキーピング遺伝子としては、ＧＡＰＤＨを用いた。 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 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+ being calculated as "changes in expression due to peroxynitrite 2" when peroxynitrite- was set to 1. The expression increase rate of "expression change amount 2 due to peroxynitrite" in this example was calculated when "expression change amount 1 due to peroxynitrite" shown in Example 2 was set to 1, and is shown in Figure 6. GAPDH was used as the housekeeping gene.
培養表皮細胞に０．２Ｍ塩酸を加えた後にペルオキシナイトライトを添加することで、コントロールに比較して、ニトロ化タンパク質が約１．３６倍生成した（図４）。さらに、遺伝子発現の変化もあり、ニトロ化タンパク質が生成しなかった条件と比較して、ｐ２１、ケラチン５、ケラチン１４が発現低下、インボルクリン、ロリクリンが発現増加した（図５）。これらの結果より、ニトロ化タンパク質が生成しやすい条件では、ペルオキシナイトライトによる細胞分化・増殖の抑制が緩和されることが分かった。これは、ペルオキシナイトライトがタンパク質との反応を起こすことで、その他生体内物質への酸化・ニトロ化反応が減少するためであると考察される。また、ニトロ化タンパク質量を増加させることで、表皮細胞分化・増殖を促進することが可能となり、同時にニトロ化タンパク質量を指標として表皮細胞分化・増殖促進剤のスクリーニングが可能になると判断できた。 Adding 0.2 M hydrochloric acid to cultured epidermal cells followed by peroxynitrite produced 1.36 times more nitrated protein than in the control (Figure 4). Furthermore, gene expression was also altered, with expression of p21, keratin 5, and keratin 14 decreased, and expression of involucrin and loricrin increased, compared to conditions in which nitrated proteins were not produced (Figure 5). These results indicate that under conditions in which nitrated proteins are likely to be 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 reactions of other substances in the body. In addition, it was determined that by increasing the amount of nitrated proteins, it is possible to promote epidermal cell differentiation and proliferation, and at the same time, it is possible to screen for agents that promote epidermal cell differentiation and proliferation using the amount of nitrated proteins as an indicator.
＜実施例４：角層ニトロ化タンパク質量と角層水分保持能の関係＞
日本在住の２０～４０代の健康な男女１６名を被験者とした。各被験者は、測定前に洗顔料を用いた洗顔、および２２℃、湿度５０％の室内で２０分間安静にすることで、頬の水分状態を一定にした。各被験者の頬の角層水分量をＳＫＩＣＯＮ（ヤヨイ）で５回測定した。テープを用いて被験部位から角層を採取した。この際、テープを頬部にあて、テープの上から５回指で優しくこすり、テープをはがした。テープを用いた角層の採取は、同一部位を３度繰り返した。角層が付着したテープは１．５ｍＬチューブに入れた。６Ｎ塩酸を１ｍＬ加え、３７℃で４８時間 インキュベートした。水酸化ナトリウムを混合することで中和し、角層抽出物とした。
Example 4: Relationship between the amount of nitrated protein in the stratum corneum and the moisture retention ability of the stratum corneum
Sixteen healthy men and women in their 20s to 40s living in Japan were used as subjects. 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. At this time, the tape was placed on the cheek, and the tape was gently rubbed five times with a finger, and then the tape was 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.
常法に従い、ＢＣＡ法にて角層抽出物中の総タンパク質量（ｇ／ｍＬ）を求めた。さらに角層抽出物をＮｉｔｒｏｔｙｒｏｓｉｎｅ ＥＬＩＳＡ Ｋｉｔ（ａｂｃａｍ ）に供し、ニトロチロシン量（ｎｍｏｌ／ｍＬ）を算出した。同被験者から採取した角層が付着した、３枚のテープそれぞれのニトロチロシン量を総タンパク質量で割り、各テープのタンパク質一定量あたりのニトロチロシン量（ｎｍｏｌ／ｇ）を求めた。被験者ごとに、３枚のテープのタンパク質量あたりのニトロチロシン量を平均し、被験者ごとのタンパク質量あたりのニトロチロシン量を求めた。 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 the stratum corneum sampled from the same subject was attached was divided by the total amount of protein to determine the amount of nitrotyrosine (nmol/g) per fixed amount of protein for each tape. The amount of nitrotyrosine per amount of protein for the three tapes for each subject was averaged to determine the amount of nitrotyrosine per amount of protein for each subject.
測定した５点の角層水分量の平均と、求めたタンパク質量あたりのニトロチロシン量の関係について、ピアソンの相関関係の検定を行ったところ、これらに正の相関関係にあることが明らかとなった（図６）。角層水分量測定にあたっては、皮膚状態を一定にしてから測定しているため、角層水分保持能を評価できているとみなすことができる。この結果より、角層のニトロ化タンパク質量が多いほど、角層水分保持能が増加することが示された。この結果を実施例１～３と総合すると、ニトロ化タンパク質量を増加させることで、角層水分保持能を向上させることが可能となり、同時にニトロ化タンパク質量を指標として角層水分保持能向上剤のスクリーニングが可能になると判断できた。 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, revealing a positive correlation between the two (Figure 6). Because the moisture content of the stratum corneum was measured after the skin condition was stabilized, it can be considered that the moisture retention ability of the stratum corneum was evaluated. This result showed that the greater the amount of nitrated protein in the stratum corneum, the greater the moisture retention ability of the stratum corneum. Taking these results into account in conjunction 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 agents that improve the moisture retention ability of the stratum corneum using the amount of nitrated protein as an indicator.
＜実施例５：ペルオキシナイトライトのバリア機能に対する作用の検証＞
三次元皮膚モデルＬａｂＣｙｔｅ ＥＰＩ―ＭＯＤＥＬ ６Ｄ（Ｊ－ＴＥＣ）を３７℃、５％ＣＯ２下で７日間インキュベートした。７日間の培養期間中、次の処理を３回実施した。０．２Ｍ塩酸（ＨＣｌ＋）または蒸留水を２５μＬ加えた培地に交換した。１ｍＭ（終濃度５０μＭ）に薄めたペルオキシナイトライト／０．１Ｍ水酸化ナトリウムを２５μＬ添加した（ｐｅｒｏｘｙｎｉｔｒｉｔｅ＋）。コントロールとしては０．１Ｍ水酸化ナトリウムを２５μＬ添加した（ｃｏｎｔｒｏｌ）。３７℃、５％ＣＯ２下で１時間インキュベートし、通常の培地に置換した。
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 replaced with normal medium.
７日間のインキュベート終了後、０．１％Ｆｌｕｏｒｅｓｃｅｉｎ ｓｏｄｉｕｍ ｓａｌｔ（ＦＬＵＫＡ）水溶液を２００μＬ添加し、２時間常温でインキュベートした。メスで組織を分離し、ティシュー・テック Ｏ．Ｃ．Ｔ．コンパウンド（サクラファインテックジャパン) 中に包埋し凍結した。クリオスタット (Ｌｅｉｃａ Ｍｉｃｒｏｓｙｓｔｅｍｓ) を使用して５μｍ厚の切片を作成し、蛍光顕微鏡にて撮影した。 After 7 days of incubation, 200 μL of 0.1% fluorescein sodium salt (FLUKA) was added and incubated at room temperature for 2 hours. The tissue was separated with a scalpel, embedded in Tissue-Tec O.C.T. Compound (Sakura Finetech Japan), and frozen. 5 μm-thick sections were prepared using a cryostat (Leica Microsystems) and photographed under a fluorescent microscope.
ペルオキシナイトライトを添加すると蛍光物質の浸透が促進した一方、塩酸を添加して弱酸性条件にした培地にペルオキシナイトライトを添加すると浸透が抑制された（図７）。この結果より、ペルオキシナイトライトは角層のバリア機能を低下させ、角層への外因物質の浸透を促進させるが、弱酸性条件にすることで、ペルオキシナイトライトのタンパク質との反応性が向上し、その他生体内因子との反応を阻害することで、バリア機能の低下を抑制できることが明らかとなった。 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 decline in barrier function.
＜実施例６：ペルオキシナイトライト活性阻害剤のスクリーニング方法１＞
新生児由来正常ヒト表皮ケラチノサイト５．０×１０４ Ｃｅｌｌｓ／ｍＬを抗菌剤含有Ｈｕｍｅｄｉａ ＫＧ２に分散し、２４ ｗｅｌｌ ｐｌａｔｅに５００μＬを播種した。３７℃、５％ＣＯ２下で３日間インキュベートした。被験素材を添加するウェルと、被験素材無添加のウェルを用意し、被験素材を添加するウェルに被験素材を１０μＬ添加した。１ｍＭに薄めたペルオキシナイトライトを２５μＬ添加した。培地を取り除き、Ｎｉｔｒｏ ｔｙｒｏｓｉｎｅ ＥＬＩＳＡ Ｋｉｔ付属のＥｘｔｒａｃｔｉｏｎ Ｂｕｆｆｅｒを５０μＬ加えた。冷蔵庫で３０分放置し、上清を回収し抽出タンパク質とした。常法に従い、ＢＣＡ法にて抽出タンパク質の総タンパク質量（ｇ／ｍＬ）を求めた。さらに抽出タンパク質をＮｉｔｒｏｔｙｒｏｓｉｎｅ ＥＬＩＳＡ Ｋｉｔに供し、ニトロチロシン量（ｎｍｏｌ／ｍＬ）を算出した。各抽出タンパク質一定量あたりのニトロチロシン量（ｎｍｏｌ／ｇ）を求めた。条件ごとに、タンパク質一定量あたりのニトロチロシン量が多いものを、ペルオキシナイトライト活性阻害剤として選択した。
Example 6: Screening method 1 for peroxynitrite activity inhibitors
5.0 x 104 cells/mL of normal human epidermal keratinocytes derived from newborns were dispersed in antibacterial agent-containing Humedia KG2, and 500 μL was seeded in a 24-well plate. Incubated at 37°C and 5% CO2 for 3 days. Wells to which the test material was added and wells without the test material were prepared, and 10 μL of the test material was added to the wells to which the test material was added. 25 μL of peroxynitrite diluted to 1 mM was added. The medium was removed, and 50 μL of Extraction Buffer included with the Nitro tyrosine ELISA Kit was added. The mixture was left in a 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 by the BCA method according to a conventional method. The extracted protein was further subjected to Nitrotyrosine ELISA Kit to calculate the amount of nitrotyrosine (nmol/mL). The amount of nitrotyrosine (nmol/g) per a certain amount of each extracted protein was determined. For each condition, the one with the highest amount of nitrotyrosine per a certain amount of protein was selected as a peroxynitrite activity inhibitor.
＜実施例７：ペルオキシナイトライト活性阻害剤のスクリーニング方法２＞
新生児由来正常ヒト表皮ケラチノサイト５．０×１０４ Ｃｅｌｌｓ／ｍＬを抗菌剤含有Ｈｕｍｅｄｉａ ＫＧ２に分散し、２４ ｗｅｌｌ ｐｌａｔｅに５００μＬを播種した。３７℃、５％ＣＯ２下で３日間インキュベートした。被験素材を添加するウェルと、被験素材無添加のウェルを用意し、被験素材を添加するウェルに被験素材を１０μＬ添加した。１ｍＭに薄めたペルオキシナイトライトを２５μＬ添加した。Ｔｏｔａｌ ＲＮＡ Ｐｕｒｉｆｉｃａｔｉｏｎ Ｋｉｔを使用し、各ｗｅｌｌの細胞からＴｏｔａｌ ＲＮＡを抽出した。ＰｒｉｍｅＳｃｒｉｐｔＴＭ ＲＴ Ｒｅａｇｅｎｔ Ｋｉｔを用い、逆転写を行い、ｃＤＮＡを合成した。リアルタイムＰＣＲを行い、比較Ｃｔ法により、被験素材無添加に比較して、ｐ２１発現量を低下させた被験素材をペルオキシナイトライト活性阻害剤として選択した。
Example 7: Screening method 2 for peroxynitrite activity inhibitors
5.0 x 104 cells/mL of normal human epidermal keratinocytes derived from newborns were dispersed in antibacterial agent-containing Humedia KG2, and 500 μL was seeded in a 24-well plate. Incubated at 37°C and 5% CO2 for 3 days. Wells to which the test material was added and wells to which the test material was not added were prepared, and 10 μL of the test material was added to the wells to which the test material was added. 25 μL of peroxynitrite diluted to 1 mM was added. 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 carried out, and the test material that reduced the expression level of p21 compared to the case where the test material was not added was selected as an inhibitor of peroxynitrite activity by the comparative Ct method.
Claims (9)
（１）被験素材共存の条件と非共存の条件で、タンパク質をペルオキシナイトライト処理する工程、
（２）前記タンパク質の、ニトロ化タンパク質量の指標を測定する工程、
及び
（３）被験素材非共存時と比較して、被験素材共存時にニトロ化タンパク質量が増加する際に、被験素材はペルオキシナイトライト活性阻害剤であると判断する工程 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;
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| WO2016035342A1 (en) * | 2014-09-02 | 2016-03-10 | 北野 勝久 | Sterilization method, preparation for sterilization, and device for producing bactericidal liquid |
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