JP5199884B2 - Inflammatory bowel disease preventive and therapeutic agent - Google Patents
Inflammatory bowel disease preventive and therapeutic agent Download PDFInfo
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- JP5199884B2 JP5199884B2 JP2008548248A JP2008548248A JP5199884B2 JP 5199884 B2 JP5199884 B2 JP 5199884B2 JP 2008548248 A JP2008548248 A JP 2008548248A JP 2008548248 A JP2008548248 A JP 2008548248A JP 5199884 B2 JP5199884 B2 JP 5199884B2
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Description
【技術分野】
【０００１】
本発明は、新規な炎症性腸疾患及び/又は過敏性腸症候群の予防および治療剤、特に腸上皮細胞のTNF-α刺激による腸上皮細胞由来活性酸素生産抑制および腸上皮細胞由来IL-8産生抑制剤を提供する。本発明の炎症性腸疾患及び/又は過敏性腸症候群の予防および治療剤は、経口経路によって与えることができ、ヒト消化管における潰瘍性大腸炎およびクローン病などの炎症性腸疾患、ないしは炎症との関連が示唆される過敏性腸症候群などを予防し、これらの疾患に伴う血便、下血、腹痛、体重減少、および食欲不振などの諸症状の改善、ならびにこれらの疾患に伴う便秘、下痢、ガス、腹痛などの諸症状を予防、治療又は寛解することができる。
【背景技術】
【０００２】
炎症性腸疾患(IBD)の代表的な疾患には、潰瘍性大腸炎(UC)とクローン病(CD)があり、これらの疾患は、欧米において患者数が多く、近年、日本における症例数も増加傾向にある。これらの疾患は主に10〜20代の若者の発病が多く、腸管に広く炎症や潰瘍が生じ、腹痛や下痢を呈する。症状としては、学童期における成長遅延、直腸脱、鉄欠乏、ないし貧血、消耗、倦怠感、発熱、体重減少などが報告されている。また結腸癌への移行が頻繁に生じることが知られている。現在のところ病因は不明であり、再燃と緩解を繰り返す難治性の疾患である。臨床的知見やノックアウトマウスモデルによる解析から、これら疾患における組織障害では、腸内細菌をはじめ種々の腸管腔に存在する抗原に対する粘膜免疫寛容の破綻と免疫担当細胞の活性化が生じ、炎症が惹起されることが示唆されている。またマクロファージに由来する炎症性サイトカインの過剰産生が認められている。一方、臨床的には確立された有効な治療は無く、現在栄養療法およびサルファ剤やステロイド剤、5-ASA誘導体などを用いた薬物療法や免疫抑制剤などの使用、もしくはこれらの組み合わせ療法が症例ごとに検討されている。
【０００３】
一方、過敏性腸症候群(IBS)の有病率は一般人口の10〜15％で１年間の罹患率は1〜2％と概算される。主要文明国では過敏性腸症候群が医療費の増加に及ぼす影響が大きい。さらに過敏性腸症候群による生活の質（QOL）の低下による経済的損失も大きい。治療法については心理的なコントロールを行うとともに、抗コリン剤、緩下剤、整腸剤、粘膜麻痺剤、自律神経調節剤、抗不安剤、抗うつ剤、睡眠薬、抗精神病薬など、症状に応じた段階的な薬物療法が施されている。しかしこれら薬物による副作用が問題となっている。
【０００４】
炎症性腸疾患治療にも分子生物学的手法が応用され、炎症を断ち切るための抗サイトカイン療法が適用されるようになった。これらの療法は、有効率は高いものの、なお副作用には注意が必要とされている。
【０００５】
炎症性腸疾患の発症、再燃については遺伝的素因、ならびに食事や腸内細菌などの環境因子の影響との相乗効果が考えられ、これらによってもたされる免疫の調節異常が疾患に大きな影響を及ぼしていると考えられる。近年、腸内細菌、特にバクテロイデス属細菌と炎症性腸疾患との因果関係がクローズアップされ、疾患の治療に有効でありかつ副作用の無いプレバイオテクスやプロバイオテクスが注目されており、腸内細菌のバランスの是正や粘膜防御機構の制御が治療上における新しい戦略になると考えられている。また、乳酸菌ラクトバチルス・ガセリが潰瘍性大腸炎の病態に対して防御効果を有することが報告されている（特開２００３-９５９６３号）。
【０００６】
これらの炎症性腸疾患の発症原因は腸粘膜免疫系の調節の破錠に伴ったサイトカイン産生異常など免疫系の異常が考えられるが、そのメカニズムについてはほとんど解明されていない。しかしながら活動期腸管粘膜ではマクロファージより産生される腫瘍壊死因子（TNF-α）やインターロイキンIL-1、IL-6、IL-8、インターフェロンγなどの炎症性サイトカインが過剰に産生されていることが明らかにされている。TNF-αやIL-8等炎症性サイトカインの抑制剤は、免疫系を調節する作用があり、過敏性腸症候群を含め、上記したすべての腸疾患に関して、下痢や腹痛、出血など様々な症状を改善すると考えられる。
【０００７】
このような理由から、炎症性腸疾患の発症メカニズムの解明と、安全で有効な炎症性腸疾患の予防治療剤の開発が望まれている。
【０００８】
本明細書において引用される参考文献は以下のとおりである。これらの文献に記載される内容はすべて本明細書の一部としてここに引用する。これらの文献のいずれかが、本明細書に対する先行技術であると認めるものではない。
【特許文献１】
特開２００３-９５９６３号
【発明の開示】
【課題を解決するための手段】
【０００９】
本発明者らは、バチルス・ズブチリス種の細菌、とりわけC-3102の菌体およびまたはその発酵産物が、腸管上皮細胞においてTNF-α誘導性の活性酸素産生抑制およびIL-8産生抑制作用を有することを見いだして本発明を完成させた。
【００１０】
本発明は、バチルス属に属する細菌の発酵産物、培養上清、または培養物を有効成分とする、腸上皮細胞からの活性酸素の産生抑制剤ならびに腸上皮細胞からのＩＬ−８の産生抑制剤を提供する。別の観点においては、本発明は、バチルス属に属する細菌の発酵産物、培養上清、または培養物を有効成分とする、炎症性腸疾患及び/又は過敏性腸症候群の予防および治療剤を提供する。好ましくは、バチルス属に属する細菌はバチルス・ズブチリスであり、より好ましくは、バチルス・ズブチリスＣ−３１０２（FERM BP-1096）である。また好ましくは、炎症性腸疾患は潰瘍性大腸炎である。
【００１１】
本明細書においては、「培養物」とはバチルス属に属する細菌を適当な培地で培養して得られる、細菌菌体、培地および発酵生成物を含む混合物を表す。「発酵産物」とは培養物のうち菌体以外の部分を表し、培養物から菌体を除いて得られる調製物を含む。また、「発酵産物」には培養物から菌体以外の発酵生成物含有物を抽出したものも含まれる。特に液体培地を用いる場合の「発酵産物」を「培養上清」と称する。なお、「発酵産物」および「培養上清」は、培養物から菌体を除去するために当該技術分野において通常用いられる手段、例えば濾過や遠心分離の操作により得ることができる。なお、発酵産物、培養上清は、遠心分離などの操作により得た場合、菌体、または、その断片が全く存在しないことを意味するものではない。
【図面の簡単な説明】
【００１２】
【図１】図１は、ヒト大腸癌由来細胞株における、TNF-α刺激によるO2 -の産生量の増強に与えるC-3102生菌の前処理の効果を示す。
【図２】図２は、ヒト大腸癌由来細胞株における、TNF-α刺激によるO2 -の産生量増強に与えるC-3102生菌、死菌および培養上清の前処理の効果を示す。
【図３】図３は、ヒト大腸癌由来細胞株における、TNF-α刺激によるO2 -の産生量増強に与えるC-3102培養上清の前処理の影響を示す。
【図４】図４は、ヒト大腸癌由来細胞株における、TNF-刺激によるIL-8 mRNAの発現に与えるC-3102生菌、死菌、培養上清の効果を示す。
【図５】図５は、C-3102培養上清がTNF-αに及ぼす作用の検討結果を示す。
【図６】図６は、C-3102株大豆培養物を添加した飼料を与えたDSS誘発潰瘍性大腸炎モデルラットの体重の推移を示す。
【図７】図７は、C-3102株大豆培養物を添加した飼料を与えたDSS誘発潰瘍性大腸炎モデルラットの血便、下血など症状の発生率を示す。
【図８】図８は、C-3102株大豆培養物を添加した飼料を与えたDSS誘発潰瘍性大腸炎モデルの血漿中のIL-8発現率を示す。
【図９】図９は、C-3102株大豆培養物を添加した飼料を与えたDSS誘発潰瘍性大腸炎モデルラットの体重の推移を示す。
【図１０−１】図１０−１は、C-3102株大豆培養物を添加した飼料を与えたDSS誘発潰瘍性大腸炎モデルラットの血便、下血など症状の発生率を示す。
【図１０−２】図１０−２は、C-3102株大豆培養物を添加した飼料を与えたDSS誘発潰瘍性大腸炎モデルラットの血便、下血など症状の発生率を示す。
【発明を実施するための最良の形態】
【００１３】
バチルス属の菌（Bacillus subtilisを含む）は古くからヒトの食生活に深くかかわっており、その機能性についての情報は多いが、炎症性腸疾患の予防や治療の効果があることは未だ報告されていない。
【００１４】
本発明においては、下記の実施例に示されるように、バチルス属に属する細菌の培養物がDSS（Dextran Sulfate sodium）モデルにおいて潰瘍性大腸炎の治療・予防効果を示すことが実証された。さらに、一時的な大腸の炎症は臓器の感覚機能を変化させて過敏性腸症候群の症状発現に先行することが知られており、結腸直腸拡張による自律神経の応答を評価するラットでの試験系で炎症の誘発剤としてTNBS（Trinitrobenzene Sulfonic Acid）が使用されている（Adam B,et al.,2006,Pain 123(1-2):179-86）。この試薬はDSSと同様に潰瘍性大腸炎の試験系でも用いられているものであり（Charles O.Elson,et al.,1995,Gastroenterology 109:1344-1367）、本発明にかかるバチルス属に属する細菌の培養物は過敏性腸症候群に対しても治療・予防効果を示すと考えられる。
【００１５】
すなわち、本発明において、バチルス・ズブチリスC-3102が炎症性腸疾患及び過敏性腸症候群への予防ないし治療効果を有することが明らかになった。加えて、C-3102培養上清がヒトの大腸上皮細胞に対するTNF-α刺激に起因する活性酸素およびIL-8産生抑制作用を示すことが見出された。下記の実施例に示されるように、バチルス・ズブチリスC-3102株の培養上清画分で前処理したヒト大腸上皮細胞では、TNF-α刺激によるIL-8 mRNAの発現が抑制され、抗炎症作用が認められた。また、培養上清とTNF-αをあらかじめ37℃で反応させた場合、TNF-α刺激によるO2 -の産生の抑制およびIL-8 mRNAの発現が抑制された。このことから、C-3102によって細胞外に分泌される因子が炎症性サイトカインのTNF-αの作用の中和に関与していることが示唆された。
【００１６】
本発明の炎症性腸疾患及び/又は過敏性腸症候群の予防および治療剤は、バチルス属に属する細菌の培養物、好ましくはバチルス・ズブチリス（Ｂａｃｉｌｌｕｓ ｓｕｂｔｉｌｉｓ）の培養物を有効成分として含むことを特徴とする。バチルス・ズブチリスの菌学的性質はバージーズ・マニュアル・オブ・バクテリオロジー Ｖｏｌ．１１（１９８６）等に記載されており、具体的には例えば以下の特徴を有する。
（１）グラム陽性
（２）卵円形の芽胞を形成
（３）桿菌
（４）運動性：あり
（５）好気性
（６）カタラーゼ：陽性
（７）５０℃における発育：＋
（８）ｐＨ５．７における発育：＋
（９）クエン酸塩の利用：＋
（１０）糖類からの酸生成の有無：アラビノース、グルコース、キシロース、マンニット：＋
（１１）ＶＰ反応：＋
（１２）デンプンの加水分解：＋
（１３）硝酸塩の還元性：＋
（１４）インドールの生成：−
（１５）ゼラチンの加水分解：＋
（１６）カゼインの加水分解：＋
（１７）液体培地での被膜形成：＋
（１８）牛乳の凝固：−
（１９）牛乳のペプトン化：＋
【００１７】
本発明の炎症性腸疾患及び/又は過敏性腸症候群の予防および治療剤に用いるバチルス・ズブチリスとしては、例えば、バチルス・ズブチリスＣ−３１０２株（生命工学工業技術研究所寄託番号 ＦＥＲＭ ＢＰ−１０９６、寄託日１９８５年１２月２５日）を挙げることができる。
【００１８】
バチルス・ズブチリスは、例えば、特開昭63-209580に記載の方法により得ることができる。培地としては、微生物培養に通常使用される炭素源、窒素源、無機物等を含む液体培地又は固体培地を用いて培養することができる。炭素源としては、バチルス・ズブチリスが資化可能な炭素源であればよく、例えばグルコース、フルクトース、スクロース、スターチ、糖蜜等を、また窒素源としては、例えばペプトン、カゼイン加水分解物、肉エキス、硫安等を挙げることができる。更に、必要に応じて燐酸、カリウム、マグネシウム、カルシウム、ナトリウム、鉄およびマンガン等の塩類、ビタミン類、アミノ酸類、界面活性剤等を添加することもできる。また、これらの合成培地の他、大豆油かすなどの天然物由来物質を用いて培養してもよい。培養条件としては、好気的条件が好ましく、培養装置としては例えばジャーファーメンターによる通気撹拌液体培養、棚式固体培養、自動製麹培養装置等が好ましく、培養温度は２０〜５０℃、特に３０〜４５℃が好ましく、培養時間は１２時間〜７日間、培養初発ｐＨはｐＨ５〜９、特に好ましくはｐＨ６〜８である。
【００１９】
このようにして得られた培養物は、そのまま用いてもよく、培養物を濃縮して用いてもよい。あるいは、濾過、遠心分離、抽出などの方法により、培養物から菌体を分離して発酵産物または培養上清を調製して用いてもよい。さらに、これらの培養物、発酵産物または培養上清に賦形剤等を加えて乾燥粉末、顆粒、錠剤等の製剤として用いてもよい。特に好ましい態様においては、バチルス・ズブチリスは、大豆油かす、大豆煮豆、小豆煮豆、米飯、麦飯、小麦ふすま、煮とうもろこし、その他の穀類などの食用に適した天然物由来物質用いて培養し、培養物から菌体を分離することなく、そのまま食品に配合する。
【００２０】
本発明の炎症性腸疾患及び/又は過敏性腸症候群の予防および治療剤は、液体、粉末、造粒物、錠剤等の形で投与してもよく、食品添加物として飲食品中に配合して摂取してもよい。飲食品としては、例えば、飲料、製菓錠菓、ペースト、パン、魚肉加工製品、乳製品などが挙げられる。本発明の炎症性腸疾患及び/又は過敏性腸症候群の予防および治療剤をこれらの様々な食品素材に添加して、健康飲料、健康食品あるいは機能性食品として提供することができる。
【００２１】
本明細書において明示的に引用される全ての特許および参考文献の内容は全て本明細書の一部としてここに引用する。また，本出願が有する優先権主張の基礎となる出願である日本特許出願２００６−３２９５９６号の明細書および図面に記載の内容は全て本明細書の一部としてここに引用する。
【実施例】
【００２２】
以下に実施例により本発明をより詳細に説明するが、本発明はこれらの実施例により限定されるものではない。
【００２３】
実施例１．バチルス・ズブチリスC-3102株がヒト大腸上皮細胞に及ぼす作用の検証
以下の実施例においては、バチルス属に属する細菌の例として、バチルス・ズブチリスＣ−３１０２株（生命工学工業技術研究所寄託番号 ＦＥＲＭ ＢＰ−１０９６、寄託日１９８５年１２月２５日）を用いた。バチルス・ズブチリスC-3102株の大豆培養物は家畜に対して、腸内細菌叢改善、増体、感染防御、卵殻強化、肉質改善、便臭改善等の効果があり、添加物として利用されている（特公平４−２４０２２）。またこの株の保健効果として、整腸作用、腸内腐敗産物の減少などが知られている。（腸内細菌学会誌 第18巻 第二号 93-99（2004））。
【００２４】
バチルス・ズブチリスＣ−３１０２は、下記配列１及び配列２のＰＣＲプライマーを用いてＰＣＲ反応を行うと約７００ｂｐｓの断片が増幅するという特徴を持つ。他のバチルス・ズブチリスでは、このＰＣＲプライマーによっては増幅は起こらない。バチルス・ズブチリスＣ−３１０２で増殖された約７００ｂｐｓの断片は、アミラーゼの配列と相同性を有しないという特徴をもち、他のバチルス・ズブチリスと明確に識別される。
配列１：５’−ＧＣＣＣＣＧＣＡＣＡＴＡＣＧＡＡＡＡＧＡＣＴＧＧＣＴＧＡＡＡ−３’（配列番号１）
配列２：５’−ＧＧＡＴＣＣＣＡＣＧＴＴＧＴＧＡＴＴＡＡＡＡＧＣＡＧＣＧＡＴ−３’（配列番号２）
【００２５】
さらに、バチルス・ズブチリスＣ−３１０２株は以下の性質を有する：
（１）プラスミドＤＮＡを有しない。
（２）ゲノムＤＮＡを調製し、制限酵素ＮｏｔＩまたはＳｆｉＩで消化してアガロース電気泳動により分離したときの消化パターンは図１に示されるとおりである。
（３）B.cerous抗菌物質を産生する。
（４）アンピシリン、クロラムフェニコール、シプロフロキサシン、エリスロマイシン、ゲンタマイシン、カナマイシン、リネゾリド、キヌプリスチン／ダルフォプリスチン、リファムピン、ストレプトマイシン、テトラサイクリン、トリメトプリム、バンコマイシンに対して耐性を有しない（いずれも最小阻害濃度０．０３〜４μｇ／ｍｌ）。
【００２６】
バチルス・ズブチリスC-3102株の生菌、死菌および培養上清の画分につき、TNF-αによって誘導される大腸上皮細胞の炎症の抑制作用を、活性酸素ならびにサイトカインの誘導を指標として検討した。
【００２７】
材料と方法
（A）細胞
使用細胞はヒト大腸癌由来のT84細胞株を用いた。培養培地として5％牛胎児血清(FCS)、100 μg/mlストレプトマイシン、100 U/mlペニシリンを添加したDMEM/HAM-F12(1:1)混合培地（DF）を用いて培養する。T84細胞は24穴カルチャープレートまたは35 mmカルチャーディッシュにまき、37℃、5%CO2-雰囲気下で2日間培養を行った後の細胞を使用した。
【００２８】
（B）C-3102凍結乾燥菌末の調製
C-3102株はTS培地（Tripticase Soy Broth：BBL社+2％agar）に37℃一晩単離培養し、出現したコロニーを5mlのTSブロス（上述培地のagarを除いた培地）に接種し、37℃一晩培養したものをバルクスターターとした。300mlのTSブロスに3mlのバルクスターターを加え、37℃一晩振とう培養後、遠心分離（7000rpm、20min）にて集菌した。集菌ペレットは滅菌水で洗浄し、ふたたび遠心分離して得られたペレットは一晩凍結後凍結乾燥した。このようにして得られた菌末を、（１）C-3102生菌（L）：菌末に無血清のDFを加え、15分ボルテックスミキサーで攪拌し溶解した。（２）C-3102死菌（HK）：生菌を100℃で5分間処理したのち、1,000×gで15分遠心し、ペレットに再びDFを加えて ボルテックスミキサーで撹拌し溶解した。（３）C-3102培養上清（CM）：無血清のDFで24時間培養した菌の培養上清を0.2μmフィルターでろ過した。以上の三通りの方法で調製した。
【００２９】
（C）C-3102の処理方法と条件
37℃、5%CO2-雰囲気下で2日培養を行ったT84細胞をPBSにて5回洗浄し、無血清のDFに交換した。1時間後にC-3102の生菌（L）または死菌（HK）を、T84細胞1個あたり0〜2cfuの濃度に加えた。培養上清（CM）は、C-3102の生菌を24時間培養した上清を0〜2cfu/cellの濃度に相当する量で加えた。
【００３０】
（D）TNF-αの処理条件
ヒトリコンビナントTNF-α（R&D systems社）を24穴カルチャープレートまたは35mmカルチャーディッシュで培養したT84細胞に20ng/mlの濃度で添加した。
【００３１】
（E）スーパーオキシドアニオン（O2 -）の測定
細胞は24穴カルチャープレートで培養し、C-3102の生菌（L）、死菌（HK）または培養上清（CM）で2時間前処理をし、TNF-α刺激を行った。24時間後の細胞をHBSSで5回洗浄し、チトクロームc（1mg/ml）+PMA溶液を加えて37℃、5％CO2-雰囲気下に1時間インキュベートした。反応液の吸光度（550nm）を測定し、残った細胞はHBSSで3回洗浄し、Lowry変法を用いて蛋白質の定量を行った。1穴あたりのO2 -の産生量を算出しnmol/mg-タンパク質/hで示した。
【００３２】
（F）RT-PCR
35mmカルチャーディッシュで培養し、TNF-α処理を行った細胞をPBSで3回洗浄し、ISOGEN(ニッポンジーン、富山)を用いてRNAを抽出した。得られたRNA(1μg)はTAKARA RT-PCR kit(タカラ、東京)を用いて逆転写を行った。得られた逆転写産物は、IL-8のプライマーセットとして、センスプライマー5’-TTGGCAGCCTTCCTGATTTCT-3’（配列番号３）;アンチセンスプライマー, 5’-TTTCCTTGGGGTCCAGACAGA-3'（配列番号４）; グリセルアルデヒド3リン酸デヒドロゲナーゼ(GAPDH)のセンスプライマー, 5’-TCATGACCACAGTCCATGCCATCACT-3’（配列番号５）, アンチセンスプライマー5-GCCTGCTTCACCACCTTCTTGATGT-3’（配列番号６）を用いてPCRを行った。
【００３３】
結果
（A）TNF-α刺激後のO2 -の産生量の増強に与える生菌の前処理の効果
24穴カルチャープレートで培養したT84細胞を0〜2cfu/cellの生菌（L）で2時間前処理した後、20ng/mlのTNF-αを添加した。T84細胞からのO2 -産生は、TNF-α刺激によって増強されたが、生菌（L）の前処理による影響は見られなかった。2cfu/cellでは細胞が傷害されており、細胞数の減少に伴うO2 -産生の増強であると考えられた（図1）。以上の結果から、生菌（L）の前処理はTNF-α刺激によるO2 -産生の増強に影響を与えないことが示唆された。
【００３４】
（B）TNF-α刺激によるO2 -の産生量増強に与える生菌、死菌および培養上清の前処理の効果
次に死菌（HK）および培養上清（CM）について同様の検討を行った。24穴カルチャーディッシュで培養したT84細胞を0.5cfu/cellの生菌（L）、死菌（HK）および培養上清（CM）で2時間前処理した後、20ng/mlのTNF-αを添加した。O2 -は、無処理（N）と比較してTNF-α刺激（M）によって増強された。2時間の前処理による影響について検討した結果生菌ではO2 -の産生に影響を与えなかった。死菌（HK）は細胞の傷害が激しく細胞がはがれておりO2 -を測定することができなかった。しかしながら培養上清（CM）では、TNF-α刺激によるO2 -の産生を有意に抑制していた（図2）。
【００３５】
（C）TNF-α刺激によるO2 -の産生量増強に与える培養上清の前処理の影響
培養上清（CM）による抑制効果が見られる濃度について以下に検討した。24穴カルチャーディッシュで培養したT84細胞を0.05〜1cfu/cellの培養上清（CM）で2時間前処理した後、20ng/mlのTNF-αを添加した。O2 -は、無処理（N）と比較してTNF-α刺激（M）によって増強された。培養上清0.05cfu/cellの濃度から有意なO2 -産生抑制効果が見られた（図3）。
以上の結果より、C-3102の培養上清（CM）がT84細胞において、TNF-α刺激によるO2 -の産生を抑制することが示唆された。
【００３６】
(D)TNF-刺激によるIL-8 mRNAの発現に与える生菌、死菌、培養上清の効果
さらに、TNF-刺激による炎症性サイトカインIL-8 mRNAの発現に与える生菌、死菌および培養上清の前処理の効果について検討した。35mmカルチャーディッシュで培養したT84細胞に20ng/mlのTNF-αを添加して継時的にIL-8 mRNAの発現をRT-PCR法を用いて解析した。T84細胞にTNF-α刺激を加えると、1時間よりIL-8 mRNAの発現が見られ3時間でピークとなった。あらかじめ0.5cfu/cellの濃度で生菌（L）、死菌（HK）、培養上清（CM）を2時間前処理した細胞に、20ng/mlの濃度のTNF-をα添加して3時間後のIL-8 mRNAについて解析した。生菌（L）、培養上清（CM）単独で2時間前処理した細胞ではIL-8 mRNAの発現が見られなかったが、死菌（HK）においてはIL-8 mRNAの発現が見られた。これは、死菌（HK）では、抗炎症成分の産生が生じないためであると考えられた。さらに同様な実験の繰り返しを行い、培養上清（CM）で前処理したものでは、IL-8 mRNAの発現が抑制され、生産物が抗炎症作用を有することを強く示唆する結果をえた。以上の結果よりTNF-α刺激によるIL-8 mRNAの発現に対しても培養上清（CM）が抑制的に作用することが明らかになった。
【００３７】
(E)培養上清（CM）前処理のIL-8 mRNAの発現抑制効果
さらにこの培養上清（CM）による前処理がIL-8 mRNAの発現を抑制する効果について詳しく検討した。T84細胞を培養上清（CM）で2時間前処理後、細胞を洗浄し、TNF-αを添加したもの（CM-re）では、IL-8 mRNAの発現が抑制されなかった。このことより、培養上清（CM）はT84細胞に作用していないことが示唆された。また、培養上清（CM）を100℃で5分処理したもの（b-CM）でT84細胞を2時間前処理し、TNF-αを添加してIL-8 mRNAの発現について解析したが、抑制効果は見られなかった。このことより、培養上清（ＣＭ）中に分泌されている因子がＴＮＦ−α活性を中和する可能性が示唆された（図４）。これらの知見は、培養上清（ＣＭ）がＩＬ−８ ｍＲＮＡの発現を抑制する機構は、ＳＯＤやペプチド等の既存の抗炎症因子とは異なることを示唆している。
［００３８］
培養上清自体がＴＮＦ−αに直接的に作用しているかどうかについて検討を行った。あらかじめ培養上清とＴＮＦ−αを混ぜて３７℃で２時間インキュベートして反応させたもの（ＣＭ＋ＴＮＦ−α）をＴ８４細胞に添加し、ＩＬ−８ ｍＲＮＡの発現を解析した（Ｂ）。培養上清とＴＮＦ−αをあらかじめ混ぜ合わせてインキュベートしたものではＩＬ−８ ｍＲＮＡの発現が抑制された。ＤＦとＴＮＦ−αを同様に３７℃で２時間インキュベートしたもの（Ｍ＋ＴＮＦ−α）では抑制効果が見られなかった。また、培養上清とＴＮＦ−αを同時に添加してもＩＬ−８ ｍＲＮＡの発現は完全には抑制されなかった。以上の結果より、Ｃ−３１０２の培養上清中に分泌されている何らかの因子がＴＮＦ−αに対して作用して、炎症の拡大を抑制していることが示唆された（図５）。
［００３９］
実施例２．潰瘍性大腸炎モデルに対する効果
潰瘍性大腸炎モデル動物として、デキストラン硫酸ナトリウムを摂取させたラットを用いた。大豆油かす造粒品５Ｋｇに水道水５Ｋｇを加えて１２１℃、１２０分間殺菌して、予め前培養しておいたバチルス・ズブチリス Ｃ−３１０２（ＦＥＲＭ ＢＰ−１０９６）の培養液を摂取し、３７℃４０時間培養したものを乾燥粉砕して、Ｃ−３１０２株大豆培養物を得た。これをＣＥ−２（日本クレア）粉末飼料に混合（約１％）した（２．５×１０８ｃｆｕ／ｇ）。５週齢の雄性Ｓｐｒａｇｕｅ Ｄａｗｌｅｙ（ＩＧＳ）ラット（１群１０匹）に、飼料を約１週間自由摂取させた。対照群には、菌末のベースである大豆油かす造粒品を粉砕機にて粉砕し、試験群と同量でＣＥ−２粉末飼料に混合したものを摂取させた。対照食、試験食の摂食期間中、デキストラン硫酸ナトリウム（分子量５０００、硫黄含量１５．０〜２０．０％、和光純薬工業）を飲水投与（３％ｗ／ｖ）した。
［００４０］
試験期間中、体重、便性状、および血漿中のＩＬ−８（ＣＩＮＣ−１ ＩＬ−８：株パナファームラボラトリーズ）を測定し、試験食最終投与日の翌日に動物を犠牲死させ、解剖して、脾臓重量、結腸長さおよび盲腸重量を測定した。体重の推移を図６に、便性状を図７に、血漿中のＩＬ−８を図８に示す。また、解剖データを表１に示す。なお、図７の★は対照群の投与日数に対して試験食群ではｐ＜０．０１（ボンフェローニ調整済みχ２検定）で有意差ありを示す。また、図７の☆は対照群の投与日数に対して試験食群ではp<0.05（ボンフェローニ調整済みχ２検定)で有意差ありを示す。
【００４１】
【表１】
飲水量ならびに摂食量については両群間に差は認められなかったが、体重について試験食群では対照群と比較して重く推移し、最終日には有意差が認められた（p<0.05）（図6）。また便性状については対照群では血便が2日目から認められ、試験食群では4日目、5日目、6日目、7日目ならびに8日目において有意な便性状の差が認められた（図7）。結腸の長さは試験食群で有意に長く（p<0.01）。ラット体重100g当たりの洗浄後の盲腸重量も有意に軽かった（p<0.05）（表1）。また血漿中のIL-8については、試験食群で低下傾向が認められ（図8）、炎症反応の抑制が生じている可能性を示唆している。これらの結果より、C-3102がDSSで誘発される大腸炎の病態に対し、優れた防御効果を有することを確認した。
【００４３】
実施例３．潰瘍性大腸炎モデルに対す有効成分の確認試験
実施例２においてC-3102株大豆培養物に抗炎症効果が認められたため、有効成分が菌体にあるいは発酵産物、培養上清成分に存在するのかを調べた。また、C-3102株大豆培養物を水と混合しこれを遠心分離してた水抽出物を加熱処理することによりプロテアーゼ活性を有する成分を失活させ、このことが抗炎症効果に影響するかどうかを調べた。使用動物および試験方法は実施例２と同様である。ラットは1群４匹とし、試験食としては、CE-2粉末飼料に、
（１）C-3102株大豆培養物を１％添加したもの＜図9 C-3102、図10 C-3102＞、
（２）C-3102株大豆培養物の上記（１）と同量を水と混合しこれを遠心分離して水抽出物を得た。この水抽出物を凍結乾燥し、添加したもの＜図9 protease+、図10 extract＞、
（３）C-3102株大豆培養物の上記（１）と同量を水と混合しこれを遠心分離して水抽出物を得た。この水抽出物を100℃、5分間加熱後、凍結乾燥し、添加したもの＜図9 protease-、図10 Heated extract＞、
（４）C-3102株大豆培養物の上記（１）と同量を水と混合しこれを遠心分離して水抽出物を得た。この残りの残渣を凍結乾燥したもの＜図9 残渣、図10 residue＞、
（５）大豆カス増粒物を１％添加したもの＜図9 Cont、図10 Control＞
のいずれかの飼料を調製して与えた。対照群については、実施例２に用いた対照食と同じものを与えた。プロテアーゼ活性はProtease Assay Kit(PIERCE社)を用いて測定した。体重の推移を図9に、また便性状の変化を図10に示した。体重の推移には群間に有意な差は認められなかった。C-3102株大豆培養物の１％添加分量を水と混合し、遠心分離して取得した水抽出物を凍結乾燥したもの（上記（２）・プロテアーゼ活性あり）を加えた飼料を与えた群については、他群よりも炎症の程度が軽く、加熱処理（上記（３）・プロテアーゼ活性なし）群については対照群（上記（５）・Control）と同程度であったため、抗炎症効果を示す有効成分は加熱失活したものと思われた。
【００４４】
上記の実施例２および３に示されるように、潰瘍性大腸炎モデル試験において、飼料にC-3102株大豆培養物を1％添加することによって、体重の推移、血便および下血の発生率、大腸組織での抗炎症作用、ならびに血漿IL-8濃度の低下などで示されるように、大腸炎が抑制されていることがわかった。さらに、実施例３においては、バチルス・ズブチリスC-3102株大豆培養物の水抽出物において抗炎症作用が確認され、この作用は水抽出物を加熱することにより失われた。これらのことから、C-3102株の培養物、発酵産物、培養上清、はTNF-α刺激によって誘導される大腸上皮細胞の炎症応答を抑制する作用を持つことが示唆された。以上の結果より、バチルス・ズブチリスC-3102の菌体または、その培養物、発酵産物、培養上清を用いることにより、経口投与できる、有効性の高い炎症性腸疾患及び/又は過敏性腸症候群の予防・治療剤を提供することが可能となる。【Technical field】
[0001]
The present invention provides a novel preventive and therapeutic agent for inflammatory bowel disease and / or irritable bowel syndrome, in particular, suppression of intestinal epithelial cell-derived reactive oxygen production and intestinal epithelial cell-derived IL-8 production by TNF-α stimulation of intestinal epithelial cells Provide an inhibitor. The prophylactic and therapeutic agent for inflammatory bowel disease and / or irritable bowel syndrome of the present invention can be given by the oral route, and is an inflammatory bowel disease such as ulcerative colitis and Crohn's disease or inflammation in the human gastrointestinal tract. To prevent irritable bowel syndrome, which is suggested to be related to the above, and to improve symptoms such as bloody stool, melena, abdominal pain, weight loss and loss of appetite associated with these diseases, as well as constipation associated with these diseases, diarrhea, Various symptoms such as gas and abdominal pain can be prevented, treated or ameliorated.
[Background]
[0002]
Representative diseases of inflammatory bowel disease (IBD) include ulcerative colitis (UC) and Crohn's disease (CD), and these diseases have a large number of patients in Europe and the United States. It is increasing. These diseases are mainly caused by adolescents in their teens and twenties, causing widespread inflammation and ulcers in the intestine, and abdominal pain and diarrhea. Symptoms have been reported such as growth retardation, rectal prolapse, iron deficiency, anemia, wasting, malaise, fever and weight loss in schoolchildhood. Moreover, it is known that the transition to colon cancer occurs frequently. The etiology is unknown at present, and it is an intractable disease that repeats relapse and remission. Based on clinical findings and analysis using knockout mouse models, tissue damage in these diseases resulted in failure of mucosal immune tolerance and activation of immunocompetent cells against antigens present in various intestinal tract lumens including intestinal bacteria, leading to inflammation It has been suggested that In addition, overproduction of inflammatory cytokines derived from macrophages has been observed. On the other hand, there is no clinically established effective treatment. Currently, nutritional therapy, pharmacological treatment using sulfa drugs, steroids, 5-ASA derivatives, etc. Has been considered.
[0003]
On the other hand, the prevalence of irritable bowel syndrome (IBS) is estimated to be 10-15% of the general population and the annual morbidity is 1-2%. In major civilized countries, irritable bowel syndrome has a significant impact on medical expenses. In addition, there is a significant economic loss due to a decline in quality of life (QOL) due to irritable bowel syndrome. In addition to psychological control, therapies are graded according to symptoms, such as anticholinergic agents, laxatives, intestinal regulating agents, mucosal palsy agents, autonomic nerve regulators, anxiolytics, antidepressants, hypnotics, and antipsychotics. Pharmacotherapy is given. However, the side effects of these drugs are a problem.
[0004]
Molecular biological techniques have also been applied to treat inflammatory bowel disease, and anti-cytokine therapy has been applied to cut off inflammation. Although these therapies are highly effective, caution is still required for side effects.
[0005]
The onset and relapse of inflammatory bowel disease may have a genetic predisposition and a synergistic effect with the influence of environmental factors such as diet and intestinal bacteria. It is thought that it is exerting. In recent years, the causal relationship between enterobacteria, especially Bacteroides spp., And inflammatory bowel disease has been highlighted, and prebiotics and probiotics that are effective in treating diseases and have no side effects have attracted attention. Correcting the balance of bacteria and controlling the mucosal defense mechanism are considered to be new therapeutic strategies. Moreover, it has been reported that lactic acid bacteria Lactobacillus gasseri have a protective effect against the pathological condition of ulcerative colitis (Japanese Patent Laid-Open No. 2003-95963).
[0006]
The onset of these inflammatory bowel diseases may be due to abnormalities in the immune system such as cytokine production abnormalities accompanying the breakage of the regulation of the intestinal mucosal immune system, but the mechanism is hardly elucidated. However, in the active intestinal mucosa, tumor necrosis factor (TNF-α) produced by macrophages and inflammatory cytokines such as interleukins IL-1, IL-6, IL-8, and interferon γ are excessively produced. It has been revealed. Inhibitors of inflammatory cytokines such as TNF-α and IL-8 have the effect of regulating the immune system and have various symptoms such as diarrhea, abdominal pain, and bleeding for all of the above intestinal diseases including irritable bowel syndrome. It is thought to improve.
[0007]
For these reasons, it is desired to elucidate the onset mechanism of inflammatory bowel disease and to develop a safe and effective preventive and therapeutic agent for inflammatory bowel disease.
[0008]
References cited in this specification are as follows. All the contents described in these documents are cited here as a part of this specification. None of these documents is admitted to be prior art to this specification.
[Patent Document 1]
JP 2003-95963 A
DISCLOSURE OF THE INVENTION
[Means for Solving the Problems]
[0009]
The present inventors have demonstrated that Bacillus subtilis bacteria, particularly C-3102 cells and / or fermentation products thereof have TNF-α-induced suppression of active oxygen production and IL-8 production in intestinal epithelial cells. As a result, the present invention was completed.
[0010]
The present invention relates to an inhibitor of production of active oxygen from intestinal epithelial cells and an inhibitor of IL-8 production from intestinal epithelial cells, comprising as an active ingredient a fermentation product, culture supernatant or culture of a bacterium belonging to the genus Bacillus. I will provide a. In another aspect, the present invention provides a preventive and therapeutic agent for inflammatory bowel disease and / or irritable bowel syndrome, comprising as an active ingredient a fermentation product, culture supernatant, or culture of a bacterium belonging to the genus Bacillus. To do. Preferably, the bacterium belonging to the genus Bacillus is Bacillus subtilis, more preferably Bacillus subtilis C-3102 (FERM BP-1096). Also preferably, the inflammatory bowel disease is ulcerative colitis.
[0011]
In the present specification, the “culture” refers to a mixture containing bacterial cells, a medium, and a fermentation product obtained by culturing bacteria belonging to the genus Bacillus in an appropriate medium. “Fermentation product” refers to a portion of the culture other than the cells and includes a preparation obtained by removing the cells from the culture. In addition, “fermentation products” include those obtained by extracting fermentation product-containing materials other than bacterial cells from the culture. In particular, a “fermentation product” when a liquid medium is used is referred to as a “culture supernatant”. The “fermentation product” and the “culture supernatant” can be obtained by means usually used in the art for removing cells from the culture, for example, filtration or centrifugation. In addition, when a fermentation product and a culture supernatant are obtained by operations such as centrifugation, it does not mean that the cells or fragments thereof are not present at all.
[Brief description of the drawings]
[0012]
[Fig. 1] Fig. 1 shows TNF-α-stimulated O in human colon cancer-derived cell lines.2 -The effect of the pretreatment of C-3102 viable bacteria on the enhancement of the production amount is shown.
[Fig. 2] Fig. 2 shows TNF-α-stimulated O in human colon cancer-derived cell lines.2 -The effect of pretreatment of C-3102 live bacteria, dead bacteria, and culture supernatant on the increase in the production amount of.
[Fig. 3] Fig. 3 shows ONF induced by TNF-α in a human colon cancer-derived cell line.2 -The effect of the pretreatment of C-3102 culture supernatant on the increase in the production amount of is shown.
FIG. 4 shows the effects of C-3102 live cells, dead cells, and culture supernatant on TNF-stimulated IL-8 mRNA expression in human colon cancer-derived cell lines.
FIG. 5 shows the results of examining the effect of C-3102 culture supernatant on TNF-α.
FIG. 6 shows changes in body weight of DSS-induced ulcerative colitis model rats fed a diet supplemented with a C-3102 strain soybean culture.
FIG. 7 shows the incidence of symptoms such as bloody stool and melena in DSS-induced ulcerative colitis model rats fed a diet supplemented with C-3102 strain soybean culture.
FIG. 8 shows the IL-8 expression rate in plasma of a DSS-induced ulcerative colitis model fed with a diet supplemented with a C-3102 strain soybean culture.
FIG. 9 shows changes in body weight of DSS-induced ulcerative colitis model rats fed a diet supplemented with a C-3102 strain soybean culture.
FIG. 10-1 shows the incidence of symptoms such as bloody stool and melena in DSS-induced ulcerative colitis model rats fed a diet supplemented with C-3102 strain soybean culture.
FIG. 10-2 shows the incidence of symptoms such as bloody stool and melena in DSS-induced ulcerative colitis model rats fed a diet supplemented with C-3102 strain soybean culture.
BEST MODE FOR CARRYING OUT THE INVENTION
[0013]
Bacillus subtilis (including Bacillus subtilis) has long been deeply involved in the human diet, and there is much information on its functionality, but it has been reported that it is effective in preventing and treating inflammatory bowel disease. Not.
[0014]
In the present invention, as shown in the following examples, it was demonstrated that a culture of bacteria belonging to the genus Bacillus exhibits a therapeutic / preventive effect on ulcerative colitis in a DSS (Dextran Sulfate sodium) model. Furthermore, it is known that transient inflammation of the large intestine precedes the onset of symptoms of irritable bowel syndrome by altering the sensory function of the organ, and a test system in rats that evaluates the autonomic response due to colorectal dilatation TNBS (Trinitrobenzene Sulfonic Acid) is used as an inflammation inducer (Adam B, et al., 2006, Pain 123 (1-2): 179-86). This reagent is used in the ulcerative colitis test system as well as DSS (Charles O. Elson, et al., 1995, Gastroenterology 109: 1344-1367) and belongs to the genus Bacillus according to the present invention. Bacterial cultures are considered to have a therapeutic / preventive effect on irritable bowel syndrome.
[0015]
That is, in the present invention, it was revealed that Bacillus subtilis C-3102 has a preventive or therapeutic effect on inflammatory bowel disease and irritable bowel syndrome. In addition, it was found that the culture supernatant of C-3102 exhibits an active oxygen and IL-8 production inhibitory effect caused by TNF-α stimulation on human colonic epithelial cells. As shown in the examples below, in human colon epithelial cells pretreated with the culture supernatant fraction of Bacillus subtilis C-3102 strain, the expression of IL-8 mRNA by TNF-α stimulation was suppressed, and anti-inflammatory The effect was recognized. In addition, when the culture supernatant and TNF-α are reacted in advance at 37 ° C, ONF by stimulation with TNF-α2 -And the expression of IL-8 mRNA was suppressed. This suggests that a factor secreted extracellularly by C-3102 is involved in neutralizing the action of the inflammatory cytokine TNF-α.
[0016]
The preventive and therapeutic agent for inflammatory bowel disease and / or irritable bowel syndrome of the present invention comprises a culture of bacteria belonging to the genus Bacillus, preferably a culture of Bacillus subtilis as an active ingredient. And The bacteriological properties of Bacillus subtilis are described in Birge's Manual of Bacteriology Vol. 11 (1986), etc., and specifically has the following characteristics, for example.
(1) Gram positive
(2) Oval spore formation
(3) Neisseria gonorrhoeae
(4) Mobility: Yes
(5) Aerobic
(6) Catalase: positive
(7) Growth at 50 ° C: +
(8) Growth at pH 5.7: +
(9) Use of citrate: +
(10) Presence or absence of acid generation from sugars: arabinose, glucose, xylose, mannitol: +
(11) VP reaction: +
(12) Starch hydrolysis: +
(13) Reduction of nitrate: +
(14) Production of indole: −
(15) Gelatin hydrolysis: +
(16) Casein hydrolysis: +
(17) Film formation in liquid medium: +
(18) Milk coagulation:-
(19) Milk peptone: +
[0017]
Examples of Bacillus subtilis used for the preventive and therapeutic agent for inflammatory bowel disease and / or irritable bowel syndrome of the present invention include, for example, Bacillus subtilis C-3102 strain (Department of Biotechnology Industrial Technology Research Institute FERM BP-1096, (Deposit date: December 25, 1985).
[0018]
Bacillus subtilis can be obtained, for example, by the method described in JP-A-63-209580. The medium can be cultured using a liquid medium or a solid medium containing a carbon source, a nitrogen source, an inorganic substance and the like that are usually used for culturing microorganisms. The carbon source may be any carbon source that can assimilate Bacillus subtilis, for example, glucose, fructose, sucrose, starch, molasses, etc., and the nitrogen source, for example, peptone, casein hydrolyzate, meat extract, An ammonium sulfate etc. can be mentioned. Furthermore, salts such as phosphoric acid, potassium, magnesium, calcium, sodium, iron and manganese, vitamins, amino acids, surfactants and the like can be added as necessary. Moreover, you may culture | cultivate using natural product origin substances, such as soybean oil residue other than these synthetic culture media. As the culture conditions, aerobic conditions are preferable, and as the culture apparatus, for example, aeration and agitation liquid culture using a jar fermenter, shelf-type solid culture, an automatic koji culture apparatus, and the like are preferable. The culture time is preferably 12 hours to 7 days, and the initial culture pH is
[0019]
The culture thus obtained may be used as it is, or the culture may be concentrated and used. Or you may isolate | separate a microbial cell from a culture by methods, such as filtration, centrifugation, and extraction, and you may prepare and use a fermentation product or a culture supernatant. Furthermore, excipients and the like may be added to these cultures, fermentation products, or culture supernatants, and used as preparations such as dry powders, granules, and tablets. In a particularly preferred embodiment, Bacillus subtilis is cultivated using a natural product-derived substance suitable for edible use such as soybean oil cake, boiled soybeans, boiled soybeans, cooked rice, barley rice, wheat bran, boiled corn, and other grains. Without separating the cells from the food, add it to the food as it is.
[0020]
The preventive and therapeutic agent for inflammatory bowel disease and / or irritable bowel syndrome of the present invention may be administered in the form of a liquid, powder, granulated product, tablet, etc., and is incorporated in food and drink as a food additive. May be taken. Examples of the food and drink include beverages, confectionery tablets, pastes, breads, processed fish products, and dairy products. The preventive and therapeutic agents for inflammatory bowel disease and / or irritable bowel syndrome of the present invention can be added to these various food materials to provide health drinks, health foods or functional foods.
[0021]
The contents of all patents and references explicitly cited herein are hereby incorporated by reference as part of the present specification. In addition, all the contents described in the specification and drawings of Japanese Patent Application No. 2006-329596, which is the application on which the priority of the present application is based, are cited herein as a part of this specification.
【Example】
[0022]
EXAMPLES The present invention will be described below in more detail with reference to examples, but the present invention is not limited to these examples.
[0023]
Example 1. Verification of the effect of Bacillus subtilis strain C-3102 on human colonic epithelial cells
In the following examples, Bacillus subtilis C-3102 strain (Deposit number FERM BP-1096, deposit date December 25, 1985) was used as an example of bacteria belonging to the genus Bacillus. The soy culture of Bacillus subtilis strain C-3102 has effects such as improving intestinal microflora, increasing body weight, protecting against infection, strengthening eggshell, improving meat quality, and improving stool odor. (Japanese Patent Publication No. 4-24022). In addition, as a health effect of this strain, it is known that the intestinal regulating action, the reduction of intestinal rot products, etc. (Journal of Intestinal Bacteria Vol.18, No.2, 93-99 (2004)).
[0024]
Bacillus subtilis C-3102 is characterized in that a fragment of about 700 bps is amplified when PCR reaction is performed using PCR primers of the
Sequence 1: 5'-GCCCCGCACATACGAAAAGACTGGCTGAAA-3 '(SEQ ID NO: 1)
Sequence 2: 5'-GGATCCCACGTTGGTATTAAAAGCAGCGAT-3 '(SEQ ID NO: 2)
[0025]
Further, Bacillus subtilis C-3102 strain has the following properties:
(1) Does not have plasmid DNA.
(2) The digestion pattern when genomic DNA is prepared, digested with restriction enzymes NotI or SfiI and separated by agarose electrophoresis is as shown in FIG.
(3) Produces B.cerous antibacterial substances.
(4) Not resistant to ampicillin, chloramphenicol, ciprofloxacin, erythromycin, gentamicin, kanamycin, linezolid, quinupristin / dalfopristin, rifapampine, streptomycin, tetracycline, trimethoprim, vancomycin Inhibitory concentration 0.03-4 μg / ml).
[0026]
Inhibition of TNF-α-induced inflammation of colonic epithelial cells in the fractions of viable, dead, and culture supernatants of Bacillus subtilis strain C-3102 was examined using active oxygen and cytokine induction as indicators. .
[0027]
Materials and methods
(A) Cell
The cells used were T84 cell lines derived from human colon cancer. Culture is performed using DMEM / HAM-F12 (1: 1) mixed medium (DF) supplemented with 5% fetal calf serum (FCS), 100 μg / ml streptomycin, and 100 U / ml penicillin. T84 cells are seeded in 24-well culture plates or 35 mm culture dishes, 37 ° C, 5% CO2-Cells were used after 2 days of culture in an atmosphere.
[0028]
(B) Preparation of C-3102 freeze-dried bacterial powder
C-3102 strain is isolated and cultured overnight in TS medium (Tripticase Soy Broth: BBL + 2% agar) at 37 ° C, and the colonies that emerge are inoculated into 5 ml of TS broth (medium excluding the above-mentioned medium agar). What was cultured overnight at 37 ° C. was used as a bulk starter. 3 ml of bulk starter was added to 300 ml of TS broth, and the cells were collected by centrifugation (7000 rpm, 20 min) after shaking culture at 37 ° C. overnight. The collected pellet was washed with sterile water, and the pellet obtained by centrifugation again was frozen overnight and then lyophilized. The bacterial powder thus obtained was dissolved in (1) viable C-3102 (L): bacterial powder by adding serum-free DF and stirring for 15 minutes with a vortex mixer. (2) C-3102 killed bacteria (HK): The live bacteria were treated at 100 ° C. for 5 minutes, then centrifuged at 1,000 × g for 15 minutes, DF was added again to the pellet, and the mixture was stirred and dissolved with a vortex mixer. (3) C-3102 culture supernatant (CM): The culture supernatant of bacteria cultured in serum-free DF for 24 hours was filtered through a 0.2 μm filter. It was prepared by the above three methods.
[0029]
(C) C-3102 treatment method and conditions
37 ℃, 5% CO2-T84 cells cultured for 2 days in an atmosphere were washed 5 times with PBS and replaced with serum-free DF. One hour later, C-3102 viable bacteria (L) or dead bacteria (HK) were added to a concentration of 0-2 cfu per T84 cell. The supernatant obtained by culturing viable C-3102 for 24 hours was added to the culture supernatant (CM) in an amount corresponding to a concentration of 0 to 2 cfu / cell.
[0030]
(D) TNF-α processing conditions
Human recombinant TNF-α (R & D systems) was added at a concentration of 20 ng / ml to T84 cells cultured in a 24-well culture plate or 35 mm culture dish.
[0031]
(E) Superoxide anion (O2 -) Measurement
The cells were cultured in a 24-well culture plate, pretreated with C-3102 live bacteria (L), dead bacteria (HK), or culture supernatant (CM) for 2 hours, and stimulated with TNF-α. After 24 hours, the cells were washed 5 times with HBSS, and cytochrome c (1 mg / ml) + PMA solution was added.2-Incubated for 1 hour under atmosphere. The absorbance (550 nm) of the reaction solution was measured, the remaining cells were washed 3 times with HBSS, and the protein was quantified using the modified Lowry method. O per hole2 -Was calculated and expressed as nmol / mg-protein / h.
[0032]
(F) RT-PCR
Cells cultured in a 35 mm culture dish and treated with TNF-α were washed 3 times with PBS, and RNA was extracted using ISOGEN (Nippon Gene, Toyama). The obtained RNA (1 μg) was reverse transcribed using TAKARA RT-PCR kit (Takara, Tokyo). The obtained reverse transcription product was used as a primer set of IL-8 as sense primer 5'-TTGGCAGCCTTCCTGATTTCT-3 '(SEQ ID NO: 3); antisense primer, 5'-TTTCCTTGGGGTCCAGACAGA-3' (SEQ ID NO: 4); PCR was performed using a sense primer of aldehyde 3-phosphate dehydrogenase (GAPDH), 5′-TCATGACCACAGTCCATGCCATCACT-3 ′ (SEQ ID NO: 5), and antisense primer 5-GCCTGCTTCACCACCTTCTTGATGT-3 ′ (SEQ ID NO: 6).
[0033]
result
(A) O after TNF-α stimulation2 -Of pretreatment with viable bacteria on the enhancement of the production of rice
T84 cells cultured in a 24-well culture plate were pretreated with 0 to 2 cfu / cell viable bacteria (L) for 2 hours, and then 20 ng / ml TNF-α was added. O from T84 cells2 -Production was enhanced by TNF-α stimulation, but no effect of pretreatment with live bacteria (L) was observed. In 2cfu / cell, cells are damaged and O2 -This was thought to be an increase in production (Figure 1). Based on the above results, pretreatment of viable bacteria (L) was achieved by TNF-α stimulation.2 -It was suggested that it does not affect the production enhancement.
[0034]
(B) O by TNF-α stimulation2 -Of pretreatment of live bacteria, dead bacteria and culture supernatants on the production enhancement of rice
Next, the same examination was performed on dead bacteria (HK) and culture supernatant (CM). T84 cells cultured in a 24-well culture dish are pretreated with 0.5 cfu / cell live bacteria (L), dead bacteria (HK), and culture supernatant (CM) for 2 hours, and then added with 20 ng / ml TNF-α did. O2 -Was enhanced by TNF-α stimulation (M) compared to no treatment (N). As a result of examining the effect of pretreatment for 2 hours, O2 -Did not affect the production of. Dead bacteria (HK) are severely damaged by cells and are peeled off.2 -Could not be measured. However, in the culture supernatant (CM), O2 -Production was significantly suppressed (FIG. 2).
[0035]
(C) O by TNF-α stimulation2 -Of Pretreatment of Culture Supernatant on Enhancement of Yeast Production
The concentration at which the inhibitory effect by the culture supernatant (CM) was observed was examined below. T84 cells cultured in a 24-well culture dish were pretreated with 0.05 to 1 cfu / cell culture supernatant (CM) for 2 hours, and then 20 ng / ml TNF-α was added. O2 -Was enhanced by TNF-α stimulation (M) compared to no treatment (N). Significant O from concentration of culture supernatant 0.05cfu / cell2 -A production inhibitory effect was observed (Fig. 3).
Based on the above results, the culture supernatant (CM) of C-3102 was found to be O in the T84 cells upon stimulation with TNF-α.2 -It was suggested to suppress the production of.
[0036]
(D) Effects of live, dead, and culture supernatants on IL-8 mRNA expression induced by TNF-stimulation
Furthermore, the effect of pretreatment of live bacteria, dead bacteria and culture supernatant on the expression of inflammatory cytokine IL-8 mRNA by TNF-stimulation was examined. 20 ng / ml TNF-α was added to T84 cells cultured in a 35 mm culture dish, and IL-8 mRNA expression was analyzed using RT-PCR over time. When TNF-α stimulation was applied to T84 cells, IL-8 mRNA expression was observed from 1 hour and peaked at 3 hours. Add 20 ng / ml of TNF-α to the cells pre-treated with live bacteria (L), dead bacteria (HK), and culture supernatant (CM) for 2 hours at a concentration of 0.5 cfu / cell for 3 hours. Later IL-8 mRNA was analyzed. IL-8 mRNA expression was not observed in cells pretreated for 2 hours with live bacteria (L) or culture supernatant (CM) alone, but IL-8 mRNA expression was observed in dead bacteria (HK) It was. This was thought to be due to the absence of production of anti-inflammatory components in killed bacteria (HK). Furthermore, the same experiment was repeated, and pretreatment with the culture supernatant (CM) suppressed IL-8 mRNA expression, and strongly suggested that the product had an anti-inflammatory effect. From the above results, it was clarified that the culture supernatant (CM) also acts to suppress IL-8 mRNA expression by TNF-α stimulation.
[0037]
(E) IL-8 mRNA expression inhibitory effect of culture supernatant (CM) pretreatment
Furthermore, the effect of pretreatment with this culture supernatant (CM) to suppress the expression of IL-8 mRNA was examined in detail. When T84 cells were pretreated with culture supernatant (CM) for 2 hours, cells were washed, and TNF-α was added (CM-re), IL-8 mRNA expression was not suppressed. This suggested that the culture supernatant (CM) did not act on T84 cells. In addition, T84 cells were pretreated for 2 hours with culture supernatant (CM) treated at 100 ° C for 5 minutes (b-CM), and TNF-α was added to analyze IL-8 mRNA expression. No inhibitory effect was seen. This suggested that the factor secreted in the culture supernatant (CM) may neutralize TNF-α activity (FIG. 4). These findings suggest that the mechanism by which culture supernatant (CM) suppresses the expression of IL-8 mRNA is different from existing anti-inflammatory factors such as SOD and peptides.
[0038]
Whether the culture supernatant itself acts directly on TNF-α was examined. The culture supernatant and TNF-α mixed in advance and incubated at 37 ° C. for 2 hours and reacted (CM + TNF-α) were added to T84 cells, and the expression of IL-8 mRNA was analyzed (B). When the culture supernatant and TNF-α were mixed and incubated in advance, the expression of IL-8 mRNA was suppressed. In the same manner, DF and TNF-α incubated at 37 ° C. for 2 hours (M + TNF-α) showed no inhibitory effect. Further, even when the culture supernatant and TNF-α were added simultaneously, the expression of IL-8 mRNA was not completely suppressed. From the above results, it was suggested that some factor secreted in the culture supernatant of C-3102 acts on TNF-α to suppress the spread of inflammation (FIG. 5).
[0039]
Example 2 Effect on ulcerative colitis model
Rats ingested with dextran sulfate sodium were used as ulcerative colitis model animals. Add 5 kg of tap water to 5 kg of granulated soybean oil and sterilize at 121 ° C. for 120 minutes. What was cultured at 40 ° C. for 40 hours was dried and ground to obtain a soybean culture of C-3102 strain. This was mixed (about 1%) with CE-2 (Japan Claire) powdered feed (2.5 × 108cfu / g). Five-week-old male Sprague Dawley (IGS) rats (10 per group) were allowed free intake of food for about 1 week. In the control group, the soybean oil residue granulated product, which is the base of the fungus powder, was pulverized with a pulverizer and mixed with CE-2 powdered feed in the same amount as in the test group. During the intake period of the control diet and the test diet, dextran sodium sulfate (molecular weight 5000, sulfur content 15.0 to 20.0%, Wako Pure Chemical Industries, Ltd.) was administered with drinking water (3% w / v).
[0040]
During the study period, body weight, fecal properties, and plasma IL-8 (CINC-1 IL-8: strain Panafarm Laboratories) were measured, and the animals were sacrificed and dissected the day after the last dose of the test meal. Spleen weight, colon length and cecal weight were measured. FIG. 6 shows changes in body weight, FIG. 7 shows stool properties, and FIG. 8 shows IL-8 in plasma. The anatomical data is shown in Table 1. 7 indicates that there is a significant difference in p <0.01 (Bonferroni adjusted χ2 test) in the test food group with respect to the administration days of the control group. In FIG. 7, ☆ indicates that there is a significant difference in p <0.05 (Bonferroni adjusted χ2 test) in the test food group with respect to the administration days of the control group.
[0041]
[Table 1]
There was no difference between the two groups regarding water consumption and food intake, but the body weight of the test food group was greater than that of the control group, and a significant difference was observed on the last day (p <0.05). (Figure 6). As for fecal characteristics, blood stool was observed from the second day in the control group, and significant differences in fecal characteristics were observed in the test food group on the fourth, fifth, sixth, seventh, and eighth days. (Figure 7). Colon length is significantly longer in the test meal group (p <0.01). The weight of the cecum after washing per 100 g of rat body weight was also significantly light (p <0.05) (Table 1). In addition, a decrease in plasma IL-8 was observed in the test food group (Fig. 8), suggesting the possibility of suppression of the inflammatory response. From these results, it was confirmed that C-3102 has an excellent protective effect against the pathogenesis of colitis induced by DSS.
[0043]
Example 3 FIG. Confirmation test of active ingredients for ulcerative colitis model
In Example 2, since the anti-inflammatory effect was observed in the C-3102 strain soybean culture, it was examined whether the active ingredient was present in the cells, the fermentation product, or the culture supernatant component. In addition, by heating the water extract obtained by mixing the C-3102 soybean culture with water and centrifuging it, the components having protease activity are inactivated, and does this affect the anti-inflammatory effect? I checked. The animals used and the test method are the same as in Example 2. There are 4 rats per group, and the test meal is CE-2 powdered diet.
(1) 1% C-3102 strain soybean culture <Figure 9 C-3102, Figure 10 C-3102>
(2) The same amount of C-3102 strain soybean culture as above (1) was mixed with water and centrifuged to obtain a water extract. This water extract was freeze-dried and added <Fig. 9 protease +, Fig. 10 extract>,
(3) The same amount of the C-3102 strain soybean culture as above (1) was mixed with water and centrifuged to obtain a water extract. This water extract was heated at 100 ° C. for 5 minutes, freeze-dried and added <Fig. 9 protease-, Fig. 10 Heated extract>,
(4) The same amount of the C-3102 strain soybean culture as above (1) was mixed with water and centrifuged to obtain a water extract. This remaining residue was lyophilized <Fig. 9 residue, Fig. 10 residue>,
(5) 1% soy bean granule added <Fig. 9 Cont, Fig. 10 Control>
Either feed was prepared and given. For the control group, the same food as the control food used in Example 2 was given. Protease activity was measured using Protease Assay Kit (PIERCE). The change in body weight is shown in FIG. 9, and the change in fecal properties is shown in FIG. There was no significant difference between groups in the change in body weight. Group fed with 1% added amount of C-3102 soybean culture mixed with water and freeze-dried water extract obtained by centrifugation (above (2) with protease activity) Since the degree of inflammation was lighter than that of the other groups, and the heat treatment (above (3), no protease activity) group was similar to the control group (above (5), Control), the anti-inflammatory effect was exhibited. The active ingredient appeared to have been deactivated by heating.
[0044]
As shown in Examples 2 and 3 above, in the ulcerative colitis model test, by adding 1% of C-3102 strain soybean culture to the feed, the change in body weight, the incidence of bloody stool and melena, It was found that colitis was suppressed as shown by anti-inflammatory action in colon tissue and a decrease in plasma IL-8 concentration. Furthermore, in Example 3, the anti-inflammatory action was confirmed in the water extract of Bacillus subtilis C-3102 soybean culture, and this action was lost by heating the water extract. From these results, it was suggested that the culture, fermentation product, and culture supernatant of the C-3102 strain have an action of suppressing the inflammatory response of colonic epithelial cells induced by TNF-α stimulation. Based on the above results, highly effective inflammatory bowel disease and / or irritable bowel syndrome that can be administered orally by using the cells of Bacillus subtilis C-3102 or its culture, fermentation product, and culture supernatant It becomes possible to provide a preventive / therapeutic agent.
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