JP5941248B2 - Probiotic strain GM-263 (ADR-1) used for the treatment of renal fibrosis due to diabetes and use thereof - Google Patents
Probiotic strain GM-263 (ADR-1) used for the treatment of renal fibrosis due to diabetes and use thereof Download PDFInfo
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- JP5941248B2 JP5941248B2 JP2011019444A JP2011019444A JP5941248B2 JP 5941248 B2 JP5941248 B2 JP 5941248B2 JP 2011019444 A JP2011019444 A JP 2011019444A JP 2011019444 A JP2011019444 A JP 2011019444A JP 5941248 B2 JP5941248 B2 JP 5941248B2
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- lactobacillus
- probiotic strain
- diabetes
- renal fibrosis
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Description
本発明はプロバイオティクス菌株の用途に関し、特に、プロバイオティクス菌株GM−263(ADR−1)が糖尿病による腎線維化の治療に用いられる組成物及びその用途に関する。 The present invention relates to the use of probiotic strains, and in particular, to a composition in which probiotic strain GM-263 (ADR-1) is used for the treatment of renal fibrosis due to diabetes and the use thereof.
高血糖が糖尿病性腎症(diabetic nephropathy;DN)を誘発する主な原因であり、透析を受けている患者において、糖尿病による糖尿病性腎症(diabetic nephropathy;DN)患者が40%を占め、それに糖尿病性腎症が末期腎不全(end−stage renal disease;ESRD)を誘発する主な原因でもある。糖尿病性腎症は、通常、糖尿病発症後の15〜20年から臨床反応が発生し、高い割合で末期腎不全に発展してしまう。同時に、例えば、高血圧症、高血脂症、高尿酸症、心血管疾患などの他の疾病が伴う可能性もある。 Hyperglycemia is the main cause of diabetic nephropathy (DN), and among patients undergoing dialysis, 40% of patients with diabetic nephropathy (DN) are diabetic nephropathy (DN). Diabetic nephropathy is also a major cause of end-stage renal disease (ESRD). Diabetic nephropathy usually develops a clinical reaction from 15 to 20 years after the onset of diabetes, and develops into end stage renal failure at a high rate. At the same time, other diseases such as hypertension, hyperlipidemia, hyperuric acid, cardiovascular disease may be accompanied.
糖尿病性腎症は、おおよそ過剰濾過(hyperfiltration)期、静止期(silent phase)、微量アルブミン尿(microalbuminuria)期、タンパク尿(proteinuria)期及び末期腎不全である五つの時期に分けられる。一般的に、糖尿病性腎症の治療は血糖制御、血圧制御、飲食制御、薬物制御を多く採用し、それによって、血糖、血圧、タンパク質の摂食量を制御する。 Diabetic nephropathy is divided into five stages, which are roughly the hyperfiltration stage, the silent phase, the microalbuminuria stage, the proteinuria stage and the end stage renal failure. In general, the treatment of diabetic nephropathy employs many blood glucose controls, blood pressure controls, food and drink controls, and drug controls, thereby controlling blood sugar, blood pressure, and protein intake.
数多くの研究において、腸内菌叢が体の健康に影響を与えることが提示されている。菌叢のバランスを維持する方法としては、プロバイオティクス(probiotics又はprobiotic bacteria)を摂取する方法がある。プロバイオティクスは、一般的に人体内からの、腸内健康に有益な生菌を指し、外部から補充され、体に有益になる可能性がある微生物をも指しており、例えば、乳酸菌(lactic acid bacteria;LAB)と一部の酵母菌が挙げられ、その内、乳酸菌は、乳糖又はその他の糖類を乳酸に転換する微生物の総称である。乳酸菌はグラム陽性菌に属し、常に食品工業における発酵に用いられる。 Numerous studies have suggested that the gut microbiota affects body health. As a method of maintaining the balance of the flora, there is a method of ingesting probiotics (probiotics or probiotic bacteria). Probiotics generally refer to living bacteria beneficial to intestinal health from within the human body, and also to microorganisms that are supplemented from the outside and may be beneficial to the body. For example, lactic acid bacteria (lactic LAB) and some yeasts. Among them, lactic acid bacteria are a general term for microorganisms that convert lactose or other saccharides into lactic acid. Lactic acid bacteria belong to Gram-positive bacteria and are always used for fermentation in the food industry.
近年、流行病学に関する研究から、乳酸菌は、アレルギーに関する疾病及び胃腸病、例えば炎症性腸疾患(inflammatory bowel disease;IBD)などを改善できることが知られてきた。そして、更に乳酸菌が免疫反応を刺激することができ、無害のアレルゲン(innocent allergens)に対しての免疫寛容(tolerance)を発達させる。その他の研究において、乳酸菌が人間や家畜の抗生剤に関する下痢(antibiotic−associated diarrhea)、旅行者下痢(travellers’ diarrhea)、小児下痢(pediatric diarrhea)、炎症性腸疾患(IBD)、過敏性腸症候群(irritable bowel syndrome)、アトピー性疾患(atopic disease)などを有効に改善できることも示されている。 In recent years, it has been known from research on epidemiology that lactic acid bacteria can improve diseases related to allergies and gastrointestinal diseases such as inflammatory bowel disease (IBD). Furthermore, lactic acid bacteria can stimulate immune responses and develop immune tolerance against innocent allergens. In other studies, lactic acid bacteria are associated with antibiotic-associated diarrhea, traveler's diarrhea, pediatric diarrhea, inflammatory bowel disease (IBD), irritable bowel syndrome It has also been shown that it can effectively improve (irritable bowel syndrome), atopic disease, and the like.
しかし、前記研究において、プロバイオティクス菌株が糖尿病による腎線維化に応用できるかどうかについて殆ど検討せず、プロバイオティクス菌株の関与可能な制御機構も提示していない。 However, in the said research, it is hardly examined whether a probiotic strain can be applied to the renal fibrosis by diabetes, and the control mechanism in which a probiotic strain can participate is not shown.
上記事情に鑑みて、糖尿病による腎線維化の治療におけるプロバイオティクス菌株の新用途を提供し、プロバイオティクス菌株をその他に応用するよう開発する必要がある。 In view of the above circumstances, there is a need to develop new applications of probiotic strains in the treatment of renal fibrosis due to diabetes and to apply the probiotic strains to others.
そこで、本発明の一態様において、プロバイオティクス菌株GM−263(ADR−1)を含み、その含有量が糖化ヘモグロビン(HbA1c)と血糖濃度を有効に低下させることができ、体重と腎重を正常な範囲に回復させて、更に糖尿病による腎線維化を治療する、糖尿病による腎線維化の治療用組成物が提供される。 Therefore, in one embodiment of the present invention, the probiotic strain GM-263 (ADR-1) is contained, and the content thereof can effectively reduce glycated hemoglobin (HbA1c) and blood glucose concentration. A therapeutic composition for renal fibrosis due to diabetes is provided that restores to a normal range and further treats renal fibrosis due to diabetes.
本発明の他の一態様において、プロバイオティクス菌株GM−263(ADR−1)の用途が提供され、このプロバイオティクス菌株GM−263(ADR−1)は、ヤヌスキナーゼ2(Janus kinase 2;p−JAK2)/シグナル伝達性転写因子1(signal transducer and activator of transcription 1;p−STAT1)のシグナル伝導経路を特異的に抑制し、腎線維化に関するタンパク質の発現を抑制することによって、糖尿病による腎線維化を有効に治療することができる。 In another aspect of the present invention, the use of probiotic strain GM-263 (ADR-1) is provided, which probiotic strain GM-263 (ADR-1) is Janus kinase 2; p-JAK2) / signal transduction transcription factor 1 (signal-transducer of transcription 1; p-STAT1) specifically suppresses the signal conduction pathway and suppresses the expression of proteins related to renal fibrosis, thereby causing diabetes. Renal fibrosis can be effectively treated.
本発明の前記態様によれば、糖尿病による腎線維化の治療に用いられる組成物が提供される。一実施例において、この組成物は、プロバイオティクス菌株GM−263(ADR−1)を含み、このプロバイオティクス菌株GM−263(ADR−1)は、例えばラクトバチルス・ロイテリ(Lactobacillus reuteri) GM−263(ADR−1)(中国典型培養物保蔵センターに寄託されており、受託番号はCCTCC M 209263)であってもよい。 According to the said aspect of this invention, the composition used for the treatment of the renal fibrosis by diabetes is provided. In one example, the composition includes probiotic strain GM-263 (ADR-1), which is, for example, Lactobacillus reuteri GM. -263 (ADR-1) (deposited at the Chinese Traditional Culture Storage Center, the deposit number may be CCTCC M 209263).
本発明の一実施例によれば、前記プロバイオティクス菌株GM−263(ADR−1)が生菌又は不活化菌である。 According to one embodiment of the present invention, the probiotic strain GM-263 (ADR-1) is a live or inactivated bacterium.
本発明の一実施例によれば、前記プロバイオティクス菌株GM−263(ADR−1)は、JAK2/STAT1のリン酸化を特異的に抑制し、腎線維化に関するタンパク質の発現を特異的に抑制することに用いられる。腎線維化に関するタンパク質としては、例えばプラスミノーゲン・アクチベーター・インヒビタ−1(plasminogen activator inhibitor;PAI−1)、サイクリン依存性キナーゼ阻害因子(cyclin−dependent kinase inhibitor;CDKI)P21Waf1/Cip1、平滑筋α−アクチン(smooth muscle α−actin;α−SMA)又はフィブロネクチン(fibronectin)が挙げられる。 According to one embodiment of the present invention, the probiotic strain GM-263 (ADR-1) specifically suppresses phosphorylation of JAK2 / STAT1, and specifically suppresses the expression of proteins related to renal fibrosis. Used to do. Examples of proteins related to renal fibrosis include plasminogen activator inhibitor 1 (PAI-1), cyclin-dependent kinase inhibitor (CDKI) P21 WaflCl , smooth. Examples include muscle α-actin (α-SMA) or fibronectin.
本発明の一実施例によれば、前記組成物は、医薬組成物、補助飲食品、食品又はその組成分である。 According to one embodiment of the present invention, the composition is a pharmaceutical composition, an auxiliary food or drink, a food or a composition thereof.
本発明の他の一態様によれば、プロバイオティクス菌株GM−263(ADR−1)の用途が提供され、前記プロバイオティクス菌株GM−263(ADR−1)は、JAK2とSTAT1のリン酸化を特異的に抑制し、PAI−1、P21Waf1/Cip1、α−SMA、フィブロネクチンのタンパク質発現を抑制することができる。 According to another aspect of the present invention, the use of probiotic strain GM-263 (ADR-1) is provided, and the probiotic strain GM-263 (ADR-1) is phosphorylated between JAK2 and STAT1. Can be specifically suppressed, and protein expression of PAI-1, P21 Wafl / Cip1 , α-SMA, and fibronectin can be suppressed.
本発明の、糖尿病による腎線維化の治療におけるプロバイオティクス菌株GM−263(ADR−1)の用途を応用する場合、前記プロバイオティクス菌株GM−263(ADR−1)を用いて、糖化ヘモグロビンと血糖濃度を低下させ、体重と腎重を正常な範囲に回復させて、更にJAK2/STAT1のシグナル伝導経路を特異的に抑制し、腎線維化に関するタンパク質(例えばPAI−1、P21Waf1/Cip1、α−SMA、フィブロネクチン等)の発現を抑制することによって、糖尿病による腎線維化を有効に治療する。それに従い、プロバイオティクス菌株GM−263(ADR−1)をその他に応用するようも開発する。 When applying the use of probiotic strain GM-263 (ADR-1) in the treatment of renal fibrosis due to diabetes according to the present invention, glycated hemoglobin is used using the probiotic strain GM-263 (ADR-1). Decrease blood glucose concentration, restore body weight and kidney weight to normal ranges, and specifically inhibit the signal conduction pathway of JAK2 / STAT1, and proteins related to renal fibrosis (for example, PAI-1, P21 Wafl / Cip1) , Α-SMA, fibronectin and the like) are effectively treated for renal fibrosis due to diabetes. Accordingly, the probiotic strain GM-263 (ADR-1) will be developed for other applications.
前述によれば、本発明は、プロバイオティクス菌株GM−263(ADR−1)が糖尿病による腎線維化の治療に用いられる組成物及びその用途を提供し、プロバイオティクス菌株GM−263(ADR−1)を用いて組成物を製造し、このプロバイオティクス菌株GM−263(ADR−1)の含有量が糖尿病による腎線維化を有効に治療することができる。 According to the foregoing, the present invention provides a composition in which probiotic strain GM-263 (ADR-1) is used for the treatment of renal fibrosis due to diabetes and its use, and probiotic strain GM-263 (ADR) -1) can be used to produce a composition, and the content of this probiotic strain GM-263 (ADR-1) can effectively treat renal fibrosis due to diabetes.
下記の図面の詳細な説明は、本発明の前記又はその他の目的、特徴、利点及び実施例をより分りやすくするためのものである。 The detailed description of the drawings below is intended to make the foregoing and other objects, features, advantages and embodiments of the present invention more comprehensible.
ここで、本発明の「プロバイオティクス菌株GM−263(ADR−1)」とは、ラクトバチルス・ロイテリ(Lactobacillus reuteri)GM−263(ADR−1)であり、中国典型培養物保存センター(China Center for Type Culture Collection;CCTCC;中国湖北省武漢市武昌珞珈山)に寄託され、その受託番号はCCTCC M 209263である(なお、寄託時の微生物名は、ラクトバチルス・ロイテリGMNL−263とされている)。 Here, the “probiotic strain GM-263 (ADR-1)” of the present invention is Lactobacillus reuteri GM-263 (ADR-1), which is a Chinese typical culture preservation center (China). Center for Type Culture Collection; CCTCC; deposited in Wuchang, Wuhan City, Hubei Province, China. The deposit number is CCTCC M 209263 (the microorganism name at the time of deposit is Lactobacillus reuteri GMNL-263) ing).
前記プロバイオティクス菌株GM−263(ADR−1)は、従来の方法、例えば、健康な大人の消化管から候補菌株を選別し、従来の菌種同定方法、例えば、16S rDNA配列解析及び菌種の生理生化学特性を同定する市販品により、プロバイオティクス菌株がラクトバチルス・ロイテリGM−263(ADR−1)であると同定することができるが、ここで詳しく述べない。 The probiotic strain GM-263 (ADR-1) is a conventional method, for example, selecting a candidate strain from the digestive tract of a healthy adult, and a conventional bacterial species identification method, such as 16S rDNA sequence analysis and bacterial species. Although the probiotic strain can be identified as Lactobacillus reuteri GM-263 (ADR-1) by a commercial product that identifies the physiological and biochemical properties of, it is not described in detail here.
簡単に言えば、前記同定されたプロバイオティクス菌株GM−263(ADR−1)はMRSブロス培地(broth medium;DIFCO(登録商標)0881、最終pH 6.5±0.2)を用いて、37℃の温度で、嫌気又は好気で培養される。他の一例において、前記MRSブロス培地を用いる菌液を寒天培地に画線接種(streak plating)することができる。 Briefly, the identified probiotic strain GM-263 (ADR-1) uses MRS broth medium (DIFCO® 0881, final pH 6.5 ± 0.2), Incubate anaerobically or aerobically at a temperature of 37 ° C. In another example, the bacterial solution using the MRS broth medium can be streak plated on an agar medium.
一実施例において、生体内の(in vivo)動物実験によって、本発明のプロバイオティクス菌株GM−263(ADR−1)は、確かに糖化ヘモグロビン(glycated hemoglobin)と血糖(blood sugar)濃度を低下させ、体重と腎重を正常な範囲に回復させ、更にJAK2/STAT1のシグナル伝導経路を特異的に抑制し、腎線維化に関するタンパク質(PAI−1、P21Waf1/Cip1、α−SMA、フィブロネクチン等)の発現を特異的に抑制することによって、糖尿病による腎線維化を有効に治療できることが証明されている。 In one example, through in vivo animal experiments, the probiotic strain GM-263 (ADR-1) of the present invention indeed reduces glycated hemoglobin and blood sugar concentrations. The body weight and kidney weight are restored to the normal range, and the signal conduction pathway of JAK2 / STAT1 is specifically suppressed, and proteins relating to renal fibrosis (PAI-1, P21 Wafl / Cip1 , α-SMA, fibronectin, etc.) It has been proved that renal fibrosis due to diabetes can be effectively treated by specifically suppressing the expression of).
ここで特に説明すべきことは、本発明の「動物実験」には、例えばストレプトゾトシン(streptozotocin;STZ)のような薬物を用いることによって、糖尿病を人為的に誘発した実験動物を含み、これにより糖尿病による腎線維化に対するプロバイオティクス菌株GM−263(ADR−1)の治療効果を評価することができる。 It should be particularly noted that the “animal experiment” of the present invention includes an experimental animal in which diabetes is artificially induced by using a drug such as streptozotocin (STZ). The therapeutic effect of probiotic strain GM-263 (ADR-1) on renal fibrosis due to can be evaluated.
ここで補充するのは、一実施例において、前記プロバイオティクス菌株GM−263(ADR−1)(例えば、ラクトバチルス・ロイテリGM−263(ADR−1);受託番号はCCTCC M 209263)は、糖尿病による腎線維化の治療用組成物を製造することに用いられる場合、生(live)菌又は不活化(inactive)菌であってもよい。一例において、前記プロバイオティクス菌株GM−263(ADR−1)は、医薬組成物、補助飲食品、食品又はその組成分であってもよい。他の一例において、前記プロバイオティクス菌株GM−263(ADR−1)は凍結乾燥の形にしてもよく、また、このプロバイオティクス菌株GM−263(ADR−1)はその他の成分を更に含んでもよく、例えばグルコース、マルトデキストリン(maltodextrin)、乳児用調製粉乳、フラクトオリゴ糖(fructooligosaccharides)、ステアリン酸マグネシウム(magnesium stearate)、ヨーグルト香料(yogurt spices)、その他の分離しにくい成分、又は前記の任意の組み合わせを更に含んでもよい。 Here, in one embodiment, the probiotic strain GM-263 (ADR-1) (eg, Lactobacillus reuteri GM-263 (ADR-1); accession number CCTCC M 209263) is: When used for producing a composition for the treatment of renal fibrosis due to diabetes, it may be a live or inactive bacterium. In one example, the probiotic strain GM-263 (ADR-1) may be a pharmaceutical composition, an auxiliary food or drink, a food, or a composition thereof. In another example, the probiotic strain GM-263 (ADR-1) may be in a lyophilized form, and the probiotic strain GM-263 (ADR-1) further comprises other components. For example, glucose, maltodextrin, infant formula, fructooligosaccharides, magnesium stearate, yoghurt spices, other difficult-to-separate ingredients, or any of the above Further combinations may be included.
また、その他の実施例において、前記プロバイオティクス菌株GM−263(ADR−1)(例えば、ラクトバチルス・ロイテリGM−263(ADR−1);受託番号はCCTCC M 209263)は更にその他の混合菌株を選択的に含んで、糖尿病による腎線維化の治療用組成物を製造することに用いることもできる。一例において、前記その他の混合菌株は、アシドフィルス菌(Lactobacillus acidophilus)、ラクトバチルス・プランタルム(Lactobacillus plantarum)、ビフィドバクテリウム・ロンガム(Bifidobacterium longum)、発酵乳酸桿菌(Lactobacillus fermentum)、ラクトバチルス・ブルガリカス(Lactobacillus bulgaricus)、サーモフィルス菌(Streptococcus thermophilus)、ラクトバチルス・クレモリス(Lactobacillus cremoris)、ラクトバチルス・パラカゼイ・サブスピーシーズ・パラカゼイ(Lactobacillus paracasei subsp. paracasei)、ラクトバチルス・ラムノーサス・GG(Lactobacillus rhamnosus GG)又は前記の任意の組み合わせを含むが、これらに限られない。 In another embodiment, the probiotic strain GM-263 (ADR-1) (for example, Lactobacillus reuteri GM-263 (ADR-1); the accession number is CCTCC M 209263) is further mixed with other strains. Can also be used to produce a composition for the treatment of renal fibrosis due to diabetes. In one example, the other mixed strains include Lactobacillus acidophilus, Lactobacillus plantarum, Bifidobacterium longum, Lactobacillus lumbactyls (Lactobacillus bulgaricus), Thermophilus (Streptococcus thermophilus), Lactobacillus cremoris (Lactobacillus cremoris), Lactobacillus paracasei subspices paracasei (Lactobacillus paspis) ubsp. paracasei), Lactobacillus rhamnosus · GG (Lactobacillus rhamnosus GG) or any combination of the, not limited to these.
以下、実施例を用いて本発明の応用を説明したが、それは本発明を限定するものではなく、当業者であれば、本発明の精神と範囲から逸脱しない限り、種々の変更及び修正することができる。 In the following, the application of the present invention has been described using examples, but it is not intended to limit the present invention, and various changes and modifications can be made by those skilled in the art without departing from the spirit and scope of the present invention. Can do.
実 施 例 1:
動物評価モデルの作製
1.プロバイオティクス菌株GM−263(ADR−1)の同定と調製
この実施例では、プロバイオティクス菌株GM−263(ADR−1)(受託番号はCCTCC M 209263)を用いて、動物実験を行い、糖尿病による腎線維化を治療するプロバイオティクス菌株GM−263(ADR−1)の効果を評価した。
Example 1:
Preparation of animal evaluation model Identification and Preparation of Probiotic Strain GM-263 (ADR-1) In this example, animal experiments were performed using probiotic strain GM-263 (ADR-1) (accession number CCTCC M 209263), The effect of probiotic strain GM-263 (ADR-1) to treat renal fibrosis due to diabetes was evaluated.
このプロバイオティクス菌株GM−263(ADR−1;CCTCC M 209263)は、健康な大人の胃腸管から選別された分離株の種別を同定するために、16S rDNA配列解析及び菌種の生理生化学特性を同定する市販品(例えば、API同定システム)を用いた。前記16S rDNA配列解析及びAPI同定システムに関する技術は、当業者に熟知されているので、ここで詳しく述べない。 This probiotic strain GM-263 (ADR-1; CCTCC M 209263) is a 16S rDNA sequence analysis and bacterial biophysiology to identify the type of isolate selected from the gastrointestinal tract of healthy adults. A commercial product (for example, API identification system) for identifying characteristics was used. Techniques relating to the 16S rDNA sequence analysis and API identification system are well known to those skilled in the art and will not be described in detail here.
プロバイオティクス菌株GM−263(ADR−1;CCTCC M 209263)の16S rDNAの一部の配列解析の結果は、配列番号1に示されたヌクレオチド(nucleotide)配列のようであり、この16S rDNA配列解析は、財団法人食品工業発展研究所に委託して行った。配列番号1に示された16S rDNAには、560個のヌクレオチドがあり、配列を対比したところ、ラクトバチルス・ロイテリ(Lactobacillus reuteri)と99%の類似度(similarity)を有することが示された。 The result of sequence analysis of a part of 16S rDNA of probiotic strain GM-263 (ADR-1; CCTCC M 209263) seems to be the nucleotide sequence shown in SEQ ID NO: 1, and this 16S rDNA sequence The analysis was commissioned to the Food Industry Development Laboratory. The 16S rDNA shown in SEQ ID NO: 1 has 560 nucleotides and sequence comparison showed that it had 99% similarity to Lactobacillus reuteri.
次に、例えばAPI(登録商標) 50 CHL同定システム(API(登録商標) 50 CHL system;bioMerieux Inc., France)又は機能が相当する他の商品のような、菌種の生理生化学特性を同定する市販品によって、ラクトバチルス・ロイテリ(Lactobacillus reuteri;ATCC 23272)のような標準菌株と比べることで、プロバイオティクス菌株の「種(species)」別を同定した。 Next, identify the physiobiochemical properties of the fungus species, for example, API® 50 CHL identification system (API® 50 CHL system; bioMerieux Inc., France) or other products with equivalent functionality By comparison with a standard strain such as Lactobacillus reuteri (ATCC 23272), the “species” of probiotic strains were identified.
表1は、API(登録商標) 50CHL同定システムによってこのプロバイオティクス菌株GM−263(ADR−1;CCTCC M 209263)を分析した結果を示す。 Table 1 shows the results of analyzing this probiotic strain GM-263 (ADR-1; CCTCC M 209263) by the API® 50 CHL identification system.
前記16S rDNA配列解析と表1の結果によれば、このプロバイオティクス菌株GM−263(ADR−1;CCTCC M 209263)の配列類似度と生理代謝活性は、いずれもラクトバチルス・ロイテリ(Lactobacillus reuteri;ATCC 23272)に近い。よって、このプロバイオティクス菌株GM−263(ADR−1;CCTCC M 209263)はラクトバチルス・ロイテリ(Lactobacillus reuteri)であることが判明した。 According to the 16S rDNA sequence analysis and the results in Table 1, both the sequence similarity and the physiological metabolic activity of this probiotic strain GM-263 (ADR-1; CCTCC M 209263) were found to be Lactobacillus reuteri (Lactobacillus reuteri). Close to ATCC 23272). Thus, it was found that this probiotic strain GM-263 (ADR-1; CCTCC M 209263) is Lactobacillus reuteri.
前記同定されたラクトバチルス・ロイテリGM−263(ADR−1)は、MRSブロス培地(broth medium;DIFCO(登録商標)0881、最終pH 6.5±0.2)を用いて、37℃の温度で、嫌気又は好気で培養される。又は、前記MRSブロス培地を用いる菌液を寒天培地に画線接種(streak plating)することができる。動物実験に供するために、このラクトバチルスロイテリを大量に培養してもよい。 The identified Lactobacillus reuteri GM-263 (ADR-1) is obtained at a temperature of 37 ° C. using MRS broth medium (DIFCO® 0881, final pH 6.5 ± 0.2). In an anaerobic or aerobic culture. Alternatively, the bacterial solution using the MRS broth medium can be streaked on an agar medium. This Lactobacillus reuteri may be cultured in large quantities for use in animal experiments.
動物実験を行う場合に、ラクトバチルスロイテリGM−263(ADR−1)(受託番号CCTCC M 209263)の菌量は、1gあたり約1×106から約1×1011まで、または1×108までのコロニー形成単位(colony−forming unit;CFU)(CFU/g)であってもよい。このラクトバチルスロイテリGM−263(ADR−1)(受託番号CCTCC M 209263)は、凍結乾燥の形にしてもよく、また、その他の成分を含んでもよく、例えば賦形剤、グルコース、マルトデキストリン、脱脂粉乳、乳児用調製粉乳、フラクトオリゴ糖、ステアリン酸マグネシウム、ヨーグルト香料、その他の分離しにくい成分、又は前記の任意の組み合わせを含んでもよい。一例として、このラクトバチルスロイテリGM−263(ADR−1)(受託番号CCTCC M 209263)は凍結乾燥の形である場合に、前記賦形剤は、脱脂粉乳、トレハロース(trehalose)、フラクトオリゴ糖を含んでもよく、その割合(w/v)については、例えば、約2:1:1であってもよい。 When conducting animal experiments, the amount of Lactobacillus reuteri GM-263 (ADR-1) (Accession No. CCTCC M 209263) is about 1 × 10 6 to about 1 × 10 11 or 1 × 10 8 per gram. Colony-forming unit (CFU) (CFU / g). This Lactobacillus reuteri GM-263 (ADR-1) (Accession No. CCTCC M 209263) may be in lyophilized form and may contain other ingredients such as excipients, glucose, maltodextrin, Non-fat dry milk, infant formula, fructooligosaccharides, magnesium stearate, yogurt flavor, other hard-to-separate ingredients, or any combination of the above. As an example, when the Lactobacillus reuteri GM-263 (ADR-1) (Accession No. CCTCC M 209263) is in a freeze-dried form, the excipient includes skim milk powder, trehalose, fructooligosaccharides. The ratio (w / v) may be about 2: 1: 1, for example.
2.アレルゲン感作動物実験モデルの作製
この実施例では、12週齢の雄のラット(品系:Sprague−Dawley(S.D.) Rat;供給源:台湾台北楽斯科生物科技股▲ふん▼有限公司)で糖尿病動物実験モデルを作製した。まず、実験ラットに18時間禁食させてから、ストレプトゾトシン(10mMのクエン酸ナトリウムに溶解した, pH 4.5;Sigma−Aldrich Chemical, St. Louis, MO, U.S.A.)を50mg/kg体重で腹腔内注射(intraperitoneal injection)した。二日間を経て、糖尿病の誘発に成功したかどうかを確認するために、グルコースオキシダーゼ(glucose oxidase)法によって血糖を測定し、血糖濃度が16mmol/L以上のラットを選んだ。この糖尿病になったラットの血糖濃度を毎日監視した。
2. Preparation of an allergen-sensitized animal experimental model In this example, a 12-week-old male rat (product line: Sprague-Dawley (SD)) Rat; source: Taiwan Taipei Music Biotechnology Co., Ltd. Co., Ltd.) created a diabetic animal experiment model. First, experimental rats were refed for 18 hours and then streptozotocin (dissolved in 10 mM sodium citrate, pH 4.5; Sigma-Aldrich Chemical, St. Louis, MO, USA) 50 mg / Intraperitoneal injection with kg body weight. After two days, in order to confirm whether or not the induction of diabetes was successful, blood glucose was measured by the glucose oxidase method, and rats having a blood glucose concentration of 16 mmol / L or more were selected. The blood glucose concentration of this diabetic rat was monitored daily.
健康ラットと糖尿病ラットを5群に分け、それぞれは、健康ラット対照群(6匹;20.60%の脂肪、56.57%の炭水化物及び22.83%のタンパク質を含む標準飼料を給与した。以下、健康対照群と略称する)、糖尿病ラット対照群(6匹;標準飼料を給与し、以下、糖尿病ラットと略称する)、インシュリンを投与した糖尿病ラット対照群(6匹;標準飼料を給与し、インシュリン注射量が4−5U/キログラム/日である。、以下、糖尿病ラット+インシュリンと略称する)、ラクトバチルス・ロイテリGM−263(ADR−1)を経口投与した健康ラット対照群(6匹;標準飼料を給与し、各ラットに対する毎日の飼育菌量が約1×109コロニー形成単位(colony−forming unit;CFU)であった。以下はGM−263と略称する)、ラクトバチルス・ロイテリGM−263(ADR−1)を経口投与した糖尿病ラット(6匹;標準飼料を給与し、各ラットに対する毎日の飼育菌量が約1×109 CFUであった。以下、糖尿病ラット+GM−263と略称する)であった。 Healthy rats and diabetic rats were divided into 5 groups, each fed a healthy rat control group (6 animals; standard diet containing 20.60% fat, 56.57% carbohydrates and 22.83% protein). Hereinafter, abbreviated as healthy control group), diabetic rat control group (6 animals; fed standard diet, hereinafter abbreviated as diabetic rat), diabetic rat control group administered insulin (6 animals; fed standard diet) Insulin injection amount is 4-5 U / kg / day, hereinafter abbreviated as diabetic rat + insulin), Lactobacillus reuteri GM-263 (ADR-1) control group (6 rats) A standard diet was fed, and the daily rearing amount for each rat was about 1 × 10 9 colony-forming units (CFU). Is abbreviated as GM-263), diabetic rats orally administered with Lactobacillus reuteri GM-263 (ADR-1) (6 animals; fed standard diet, and the daily rearing amount of each rat is about 1 × 10 9 CFU, hereinafter abbreviated as diabetic rat + GM-263).
各群のラットは、摂食量には顕著な差異がなく、毎日約25.6±0.77g(p=0.1747)であった。実験材料の全ては0.5mLのリン酸塩緩衝生理食塩水(phosphate−buffered saline;PBS)に調合され、毎日二回投与した。 The rats in each group had no significant difference in food intake and were about 25.6 ± 0.77 g (p = 0.747) daily. All experimental materials were formulated in 0.5 mL phosphate-buffered saline (PBS) and administered twice daily.
ストレプトゾトシンを注射してから28日後、全てのラットを屠殺して、各ラット毎の体重と左腎の重量を記録し、その後、特定タンパク含有量分析を行うために、解剖して腎臓の皮質を取り出した。 Twenty-eight days after streptozotocin injection, all rats were sacrificed and the body weight and left kidney weight of each rat were recorded, followed by dissection and kidney cortex for specific protein content analysis. I took it out.
ラットの飼育温度は25±1℃、相対湿度は65±5%であり、12時間毎の明暗サイクルに維持し、標準実験室レベルの飼料(Labdiet(登録商標) Laboratory Rodent Diet #5001, PMI Nutrition International Inc., U.S.A)で飼育し、飼育期間中に飼料及び水を自由摂取させた。ラットの飼育条件は、全て台湾の衛生研究院より公表されている実験動物管理ガイドにより行った。 Rats were kept at 25 ± 1 ° C. and relative humidity of 65 ± 5%, maintained on a light / dark cycle every 12 hours, and standard laboratory level food (Labdiet® Laboratory Rodent Diet # 5001, PMI Nutrition) International Inc., U.S.A), and freely ingested feed and water during the breeding period. Rats were kept under the laboratory animal management guide published by the Institute of Health in Taiwan.
実 施 例 2:
生理に対するプロバイオティクス菌株GM−263(ADR−1)の影響の評価
1.糖尿病ラットの糖化ヘモグロビンと血糖濃度に対するプロバイオティクス菌株GM−263(ADR−1)の影響の評価
図1Aと図1Bは、本発明の一実施例によるラットの糖化ヘモグロビン(glycated hemoglobin)濃度(図1A)及び血糖濃度(図1B)を示す棒グラフである。図1Aの縦軸は糖化ヘモグロビン濃度(HbA1c)の百分率(%)を示し、図1Bの縦軸は血糖濃度(mg/dl)を示す。図1Aと図1Bの横軸は、それぞれ健康ラット対照群(健康対照群と略称)、ラクトバチルス・ロイテリGM−263(ADR−1)を経口投与した健康ラット対照群(GM−263と略称)、糖尿病ラット対照群(糖尿病ラットと略称)、ラクトバチルス・ロイテリGM−263(ADR−1)を経口投与した糖尿病ラット(糖尿病ラット+GM−263と略称)、インシュリンを投与した糖尿病ラット対照群(糖尿病ラット+インシュリンと略称)を示す。図面記号*は、健康対照群と比べて、p<0.05であることを示し、図面記号#は、糖尿病ラットと比べて、p<0.05であることを示す。
Example 2:
Evaluation of the effect of probiotic strain GM-263 (ADR-1) on physiology 1. Evaluation of the effect of probiotic strain GM-263 (ADR-1) on glycated hemoglobin and blood glucose concentration in diabetic rats FIG. 1A and FIG. 1B FIG. 3 is a bar graph showing the glycated hemoglobin concentration (FIG. 1A) and blood glucose concentration (FIG. 1B) of a rat according to one embodiment of the present invention. The vertical axis in FIG. 1A indicates the percentage (%) of the glycated hemoglobin concentration (HbA1c), and the vertical axis in FIG. 1B indicates the blood glucose concentration (mg / dl). 1A and 1B, the horizontal axis represents a healthy rat control group (abbreviated as a healthy control group) and a healthy rat control group (abbreviated as GM-263) to which Lactobacillus reuteri GM-263 (ADR-1) was orally administered, respectively. Diabetic rat control group (abbreviated as diabetic rat), diabetic rat orally administered Lactobacillus reuteri GM-263 (ADR-1) (abbreviated as diabetic rat + GM-263), diabetic rat control group administered diabetes (diabetic) Rat + abbreviated as insulin). The drawing symbol * indicates p <0.05 compared to the healthy control group, and the drawing symbol # indicates p <0.05 compared to diabetic rats.
図1Aと図1Bの結果から分かるように、ストレプトゾトシンを注射してから28日後、ラクトバチルス・ロイテリGM−263(ADR−1)を経口投与したGM−263群の糖化ヘモグロビン濃度と血糖濃度は、健康対照群に近いが、糖尿病ラットの糖化ヘモグロビン濃度(>7.5%)と血糖濃度(>350mg/dl)は、健康対照群やGM−263群より著しく高い。しかしながら、ラクトバチルス・ロイテリGM−263を経口投与された糖尿病ラット(糖尿病ラット+GM−263)は、その糖化ヘモグロビン濃度と血糖濃度の値が、インシュリンを投与した糖尿病ラット対照群(糖尿病ラット+インシュリン)に近いことから、糖尿病ラットに実施例1のプロバイオティクス菌株GM−263(ADR−1)を経口投与することによって、確かに糖化ヘモグロビンと血糖濃度を低下できることを示した。 As can be seen from the results of FIG. 1A and FIG. 1B, glycated hemoglobin concentration and blood glucose concentration of GM-263 group to which Lactobacillus reuteri GM-263 (ADR-1) was orally administered 28 days after the injection of streptozotocin, Although close to the healthy control group, the glycated hemoglobin concentration (> 7.5%) and blood glucose concentration (> 350 mg / dl) of diabetic rats are significantly higher than the healthy control group and the GM-263 group. However, the diabetic rat (diabetic rat + GM-263) orally administered with Lactobacillus reuteri GM-263 has a diabetic rat control group (diabetic rat + insulin) whose glycated hemoglobin concentration and blood glucose concentration are administered with insulin. Thus, it was shown that glycated hemoglobin and blood glucose concentration can be surely reduced by orally administering the probiotic strain GM-263 (ADR-1) of Example 1 to diabetic rats.
2.糖尿病ラットの体重と腎重に対するプロバイオティクス菌株GM−263(ADR−1)の影響の評価
図2Aと図2Bは、本発明の一実施例によるラットの体重(図2A)と左腎の重量(図2B)を示す棒グラフである。図2Aの縦軸は体重(g)を示し、図2Bの縦軸は左腎の重量(g)を示す。図2Aと図2Bの横軸は、それぞれ健康ラット対照群(健康対照群と略称)、ラクトバチルス・ロイテリGM−263(ADR−1)を経口投与した健康ラット対照群(GM−263と略称)、糖尿病ラット対照群(糖尿病ラットと略称)、ラクトバチルス・ロイテリGM−263(ADR−1)を経口投与した糖尿病ラット(糖尿病ラット+GM−263と略称)、インシュリンを投与した糖尿病ラット対照群(糖尿病ラット+インシュリンと略称)を示す。図面記号*は、健康対照群と比べて、p<0.05であることを示し、図面記号#は、糖尿病ラットと比べて、p<0.05であることを示す。
2. Evaluation of the effect of probiotic strain GM-263 (ADR-1) on body weight and kidney weight of diabetic rats Figures 2A and 2B show the body weight (Figure 2A) and left kidney of a rat according to one embodiment of the present invention. It is a bar graph which shows the weight (FIG. 2B). The vertical axis in FIG. 2A indicates body weight (g), and the vertical axis in FIG. 2B indicates the weight (g) of the left kidney. 2A and 2B, the horizontal axis represents a healthy rat control group (abbreviated as a healthy control group) and a healthy rat control group (abbreviated as GM-263) to which Lactobacillus reuteri GM-263 (ADR-1) was orally administered, respectively. Diabetic rat control group (abbreviated as diabetic rat), diabetic rat orally administered Lactobacillus reuteri GM-263 (ADR-1), diabetic rat control group administered diabetes (abbreviated as GM-263) Rat + abbreviated as insulin). The drawing symbol * indicates p <0.05 compared to the healthy control group, and the drawing symbol # indicates p <0.05 compared to diabetic rats.
図2Aと図2Bの結果から分かるように、ストレプトゾトシンを注射してから28日後、ラクトバチルス・ロイテリGM−263(ADR−1)を経口投与したGM−263群の体重と左腎の重量は、健康対照群に近いが、糖尿病ラットの体重は、健康対照群やGM−263群より著しく低く、糖尿病ラットの左腎の重量は、健康対照群やGM−263群より著しく高かった。上記の内、腎重の変化は腎線維化を評価する指標の一つである。 As can be seen from the results of FIG. 2A and FIG. 2B, 28 days after the injection of streptozotocin, the weight of the GM-263 group to which Lactobacillus reuteri GM-263 (ADR-1) was orally administered and the weight of the left kidney were Although close to the healthy control group, the body weight of the diabetic rats was significantly lower than that of the healthy control group and the GM-263 group, and the weight of the left kidney of the diabetic rats was significantly higher than that of the healthy control group and the GM-263 group. Among the above, changes in renal weight are one of the indices for evaluating renal fibrosis.
しかしながら、ラクトバチルス・ロイテリGM−263(ADR−1)を経口投与された糖尿病ラット(糖尿病ラット+GM−263)は、その体重(図2A)が再び上昇する傾向があり、インシュリンを投与した糖尿病ラット対照群(糖尿病ラット+インシュリン)に近かった。なお、図2Bに示すように、ラクトバチルス・ロイテリGM−263(ADR−1)を経口投与された糖尿病ラット(糖尿病ラット+GM−263)は、その左腎の重量(図2B)が糖尿病ラットより著しく低く、インシュリンを投与した糖尿病ラット対照群(糖尿病ラット+インシュリン)に近いことから、糖尿病ラットに実施例1のプロバイオティクス菌株GM−263(ADR−1)を経口投与することによって、確かに糖尿病による腎線維化の状況を改善できることを示した。 However, diabetic rats (diabetic rats + GM-263) orally administered Lactobacillus reuteri GM-263 (ADR-1) tended to increase their body weight (FIG. 2A) again, and diabetic rats administered insulin. Close to the control group (diabetic rats + insulin). As shown in FIG. 2B, the diabetic rat (diabetic rat + GM-263) orally administered with Lactobacillus reuteri GM-263 (ADR-1) has a weight of the left kidney (FIG. 2B) that is higher than that of the diabetic rat. By being orally administered the probiotic strain GM-263 (ADR-1) of Example 1 to a diabetic rat, it is certainly because it is extremely low and close to the diabetic rat control group (diabetic rat + insulin) administered insulin. It was shown that the condition of renal fibrosis due to diabetes can be improved.
実 施 例 3:
遺伝子制御と細胞内タンパク質の発現に対するプロバイオティクス菌株GM−26
3(ADR−1)の影響の評価
1.腎臓組織の抽出
実施例1で取り出された腎臓皮質を、この組織約50mgに対し、0.1%のドデシル硫酸ナトリウム(sodium dodecyl sulfate;SDS)溶解バッファー(SDS lysis buffer)1mLの割合で加え、氷上に4℃で保持した後、組織ホモジナイザーで砕き、約10分間静置反応させ、腎臓皮質組織を十分に均質化(homogenize)して、腎臓皮質細胞内のタンパク質をさらに溶出した。
Example 3:
Probiotic strain GM-26 for gene regulation and intracellular protein expression
Evaluation of the effect of 3 (ADR-1) 1. Extraction of kidney tissue 0.1% sodium dodecyl sulfate (SDS) was dissolved in about 50 mg of this tissue from the kidney cortex. Add 1 mL of buffer (SDS lysis buffer), hold at 4 ° C. on ice, crush it with a tissue homogenizer, let it stand for about 10 minutes, homogenize the kidney cortex tissue sufficiently, Intracellular proteins were further eluted.
前記SDS溶解バッファーは、50mMの塩化トリスヒドロキシメチルアミノメタン(tris(hydroxymethyl)aminomethane chloride;Tris−Cl, pH 8.0;Sigma−Aldrich Chemical, St. Louis, MO, U.S.A.)溶液、150mMの塩化ナトリウム、0.02%のアジ化ナトリウム(sodium azide;Sigma−Aldrich Chemical, St. Louis, MO, U.S.A.)、1%の界面活性剤NP−40(NONIDET p−40 ethyl phenyl polyethylene glycol;Sigma−Aldrich Chemical, St. Louis, MO, U.S.A.)、0.05%のオルトバナジン酸ナトリウム(sodium orthovanadate ; Na3VO4;Sigma−Aldrich Chemical, St. Louis, MO, U.S.A.)、100μg/mLのフェニルメチルスルホニルフッ化物(phenylmethylsulfonyl fluoride;PMSF;Sigma−Aldrich Chemical, St. Louis, MO, U.S.A.)、1μg/mLのアプロチニン(aprotinin;Sigma−Aldrich Chemical, St. Louis, MO, U.S.A.)、プロテイナーゼ・インヒビター・カクテル(proteinase inhibitor cocktail;Sigma−Aldrich Chemical, St. Louis, MO, U.S.A.)を含んでもよい。 The SDS lysis buffer was 50 mM trishydroxymethylaminomethane chloride (tris (hydroxymethyl) aminomethane chloride; Tris-Cl, pH 8.0; Sigma-Aldrich Chemical, St. Louis, MO, USA) solution. 150 mM sodium chloride, 0.02% sodium azide (Sigma-Aldrich Chemical, St. Louis, MO, USA), 1% surfactant NP-40 (NONIDET p- 40 ethyl phenylene polyglycol; Sigma-Aldrich Chemical, St. Louis, MO, USA, 0.05% sodium orthovanadate (sodiu Na 3 VO 4 ; Sigma-Aldrich Chemical, St. Louis, MO, U.S.A., 100 μg / mL phenylmethylsulfonyl fluoride (PMSFl-Sulfidylfluoride; , MO, U.S.A.), 1 μg / mL aprotinin (Sigma-Aldrich Chemical, St. Louis, MO, U.S.A.), proteinase-inhibitor cocktail cocktail; Aldrich Chemical, St. Louis, MO, U.S.A.).
その後、得られたホモジネート(homogenate)を約12000rpmの回転速度で、4℃において、約20分間遠心した。そして、上澄みを回収して、Bradford法によりタンパク質の濃度を測量した後、−70℃に保存し、その後、評価を行った。 Thereafter, the resulting homogenate was centrifuged at a rotational speed of about 12000 rpm at 4 ° C. for about 20 minutes. Then, the supernatant was collected and the protein concentration was measured by the Bradford method, and then stored at -70 ° C., and then evaluated.
2.ウェスタンブロッティング(Western blotting)
前記のように得られたホモジネートに対して、10%のドデシル硫酸ナトリウム・ポリアクリルアミドゲル電気泳動分析(sodium dodecyl sulfate−polyacrylamide gel electrophoresis;SDS−PAGE;Sigma−Aldrich Chemical, St. Louis, MO, U.S.A.)を行った。100Vの電圧で、タンパク質電気泳動緩衝液(125mM Tris−base; 1.25 M Glycine; 1% SDS)において、電気泳動を約3時間行った。その後、192mMのグリシン(glycine)と20%(v/v)のメタノールを含む25mMのTris−HCl溶液(pH 8.3)により、15分間平衡化した。前記SDS−PAGEの調製と関連設備は、当業者が既に熟知しているので、ここで詳しく述べない。
2. Western blotting
10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis (SDS-PAGE; Sigma-Aldrich Chemical, St. MO, St. MO), homogenate obtained as described above. S.A.) was performed. Electrophoresis was performed for about 3 hours in protein electrophoresis buffer (125 mM Tris-base; 1.25 M Glycine; 1% SDS) at a voltage of 100V. Thereafter, the mixture was equilibrated for 15 minutes with a 25 mM Tris-HCl solution (pH 8.3) containing 192 mM glycine and 20% (v / v) methanol. The preparation of the SDS-PAGE and the related equipment are already familiar to those skilled in the art and will not be described in detail here.
前記SDS−PAGEの電気泳動ゲルは、ウェスタンブロッティング分析(Western blotting assay)を続けて行ってもよい。この実施例において、ウェスタンブロッティングキットによって(例えばBio−Rad Scientific Instruments Transfer Unit)、100Vで、タンパク質電気泳動緩衝液(125mM Tris−base; 1.25 M Glycine; 1% SDS)により、電気泳動を約3時間行い、前記電気泳動ゲルのタンパク質を転写膜に転写した。転写膜としては、例えばProtranTMニトロセルロースメンブレーン(nitrocellulose membrane、孔径0.45μM;Schieicher & Schuell, Kneene, NH, U.S.A.)であってもよい。 The SDS-PAGE electrophoresis gel may be subjected to Western blotting assay. In this example, electrophoresis was approximately performed by Western blotting kit (eg, Bio-Rad Scientific Instruments Transfer Unit) at 100 V with protein electrophoresis buffer (125 mM Tris-base; 1.25 M Glycine; 1% SDS). The electrophoresis gel protein was transferred to a transfer membrane for 3 hours. The transfer membrane may be, for example, Protran ™ nitrocellulose membrane (nitrocellose membrane, 0.45 μM pore size; Schieicher & Schuell, Kneeene, NH, USA).
その後、Blocking溶液(例えば、5%の脱脂粉乳をTBS緩衝液(10mM Tris−Base、100mM NaCl、0.1% Tween−20、pH 7.4)に溶解する)で、室温で約1時間反応後、一次抗体(primary antibody)(抗体結合溶液で希釈し、抗体結合溶液の組成は後述する)を入れ、4℃で一晩反応させた。そして、TBS緩衝液で三回洗浄した。各回の洗浄時間は10分間であった。続いて、TBS緩衝液により4000倍に希釈した二次抗体(secondary antibody)をいれて、37℃で1時間反応させてから、TBS緩衝液で三回洗浄した。各回の洗浄時間は10分間であった。その後、例えば増強化学発光(Enhanced ChemiLuminescence;ECL)反応キット(Amersham Corp., Arlington Heights, IL, U.S.A.)などの冷光呈色剤を転写膜に入れて、冷光法によりLAS−3000のルミノ・イメージアナライザ(例えばFujifilm LAS−3000 chemiluminescence detection system, Tokyo, Japan)に現像させ、結果を分析した。 Thereafter, the reaction is performed in a Blocking solution (for example, 5% nonfat dry milk is dissolved in TBS buffer (10 mM Tris-Base, 100 mM NaCl, 0.1% Tween-20, pH 7.4)) at room temperature for about 1 hour. Thereafter, a primary antibody (primary antibody was diluted with an antibody binding solution and the composition of the antibody binding solution will be described later) was added and allowed to react overnight at 4 ° C. And it wash | cleaned 3 times with TBS buffer. Each washing time was 10 minutes. Subsequently, a secondary antibody diluted with TBS buffer 4000 times was added, reacted at 37 ° C. for 1 hour, and then washed three times with TBS buffer. Each washing time was 10 minutes. Then, for example, a cold photochromic agent such as Enhanced Chemiluminescence (ECL) reaction kit (Amersham Corp., Arlington Heights, IL, USA) is placed in the transfer film, and LAS-3000 is obtained by the cold light method. The results were analyzed using a Lumino Image Analyzer (for example, Fujifilm LAS-3000 chemistry detection system, Tokyo, Japan).
前記一次抗体は、例えば、JAK2、STAT1、PAI−1、P21Waf1/Cip1、α−SMA(上記製品がSanta Cruz Biotechnology, Inc., Santa Cruz, CA, U.S.A.より)、フィブロネクチン(Chemicon, Temecula, CA, U.S.A.)、リン酸化JAK2、リン酸化STAT1(上記製品がUpstate Biotechnology, Inc., Santa Cruz, CA, U.S.A.より)、β−アクチン(β−actin;Sigma−Aldrich Chemical, St. Louis, MO, U.S.A)に対するモノクロナール抗体であってもよい。 Examples of the primary antibody include JAK2, STAT1, PAI-1, P21 Wafl / Cip1 , α-SMA (the above product is from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), fibronectin ( Chemicon, Temecula, CA, U.S.A.), phosphorylated JAK2, phosphorylated STAT1 (the above products are from Upstate Biotechnology, Inc., Santa Cruz, CA, U.S.A.), β-actin (β -Monotin antibodies against -actin; Sigma-Aldrich Chemical, St. Louis, MO, USA).
前記二次抗体は、例えば、ストレプトアビジン−ペルオキシダーゼ(streptavidin−peroxidase)を結合したヤギ抗マウスIgG (goat anti−mouse IgG−HRP)、又はストレプトアビジン−ペルオキシダーゼを結合したヤギ抗ウサギIgG (goat anti−rabbit IgG−HRP) (上記製品がAmersham Corp., Arlington Heights, IL, U.S.A.より)であってもよい。 Examples of the secondary antibody include goat anti-mouse IgG-HRP conjugated with streptavidin-peroxidase, or goat anti-rabbit IgG conjugated with streptavidin-peroxidase (goat anti-IgG). Rabbit IgG-HRP) (The product is from Amersham Corp., Arlington Heights, IL, USA).
前記抗体結合溶液は、例えば、TBS緩衝液(10mM Tris−Base, 100mM NaCl, 0.1% Tween−20, pH 7.4を含む)を含んでもよい。 The antibody binding solution may contain, for example, a TBS buffer (containing 10 mM Tris-Base, 100 mM NaCl, 0.1% Tween-20, pH 7.4).
ウェスタンブロッティングのバンド強度は、デンシトメトリー分析(densitometric analysis)法により定量される。その結果は、目標タンパクとβ−アクチンとのバンド強度の割合で表す。それに、ここでいう毎群のサンプルは、少なくとも三回繰り返したものであり、得られた数値は、「平均値±標準偏差(mean±SD)」で示す。統計分析は、One−way ANOVAにより分析し、Tukey−Kramer型多重比較検定法(Tukey−type multiple comparison test)によってスチューデント化残差(studentized)で各群の差異顕著性を比較した。各群の差異がp<0.05の場合、顕著性差異があると考えられる。 The Western blotting band intensity is quantified by a densitometric analysis method. The result is expressed as a ratio of the band intensity between the target protein and β-actin. In addition, the samples in each group mentioned here were repeated at least three times, and the obtained numerical values are indicated by “mean ± standard deviation (mean ± SD)”. Statistical analysis was performed by One-way ANOVA, and the difference saliency of each group was compared by Studentized residual by the Tukey-Kramer type multiple comparison test (Tukey-type multiple comparison test). If the difference in each group is p <0.05, it is considered that there is a significant difference.
3.糖尿病ラットの腎臓皮質のJAK2とSTAT1のリン酸化に対するプロバイオティクス菌株GM−263(ADR−1)の影響の評価
過去の研究によれば、糖尿病動物の高血糖により腎線維化を誘発した過程において、JAK/STATsシグナル伝導経路(signal transduction pathway)は、重要な遺伝子制御の役割を担っている。そこで、この実施例は、更に糖尿病ラットの腎臓皮質のJAK2とSTAT1のリン酸化に対するプロバイオティクス菌株GM−263(ADR−1)の影響を評価した。
3. Evaluation of the effect of probiotic strain GM-263 (ADR-1) on the phosphorylation of JAK2 and STAT1 in renal cortex of diabetic rats According to past studies, renal fibrosis was induced by hyperglycemia in diabetic animals In the process, the JAK / STATs signal conduction pathway plays an important gene regulatory role. Therefore, this example further evaluated the effect of probiotic strain GM-263 (ADR-1) on the phosphorylation of JAK2 and STAT1 in the renal cortex of diabetic rats.
図3は、本発明の一実施例による糖尿病ラットの腎臓皮質組織を示すウェスタンブロッティング解析図である。その内、レーン1−2は健康対照群を示し、レーン3−4は糖尿病ラット群を示し、レーン5−6はラクトバチルス・ロイテリGM−263(ADR−1)を経口投与した糖尿病ラット群(糖尿病ラット+GM−263)を示し、レーン7−8はインシュリンを投与した糖尿病ラット対照群(糖尿病ラット+インシュリン)を示す。図3は、糖尿病ラットの腎臓皮質組織内のJAK2(約120kDa)、STAT1(約90kDa)、リン酸化JAK2(約120kDa)、STAT1(約90kDa)の発現を分析するものである。また、図5のβ−アクチンは、前記蛋白の発現量を標準化するものである。 FIG. 3 is a western blotting analysis diagram showing kidney cortex tissue of a diabetic rat according to one embodiment of the present invention. Among them, lane 1-2 shows a healthy control group, lane 3-4 shows a diabetic rat group, and lane 5-6 shows a diabetic rat group orally administered with Lactobacillus reuteri GM-263 (ADR-1) ( Diabetic rats + GM-263), and lanes 7-8 show diabetic rat control groups (diabetic rats + insulin) administered insulin. FIG. 3 analyzes the expression of JAK2 (about 120 kDa), STAT1 (about 90 kDa), phosphorylated JAK2 (about 120 kDa), and STAT1 (about 90 kDa) in the renal cortical tissue of diabetic rats. Further, β-actin in FIG. 5 standardizes the expression level of the protein.
図4Aは、β−アクチンの発現量によって標準化された図3のリン酸化JAK2を示す棒グラフであり、縦軸はβ−アクチンの発現量によって標準化されたリン酸化JAK2の相対量(即ち、JAK2リン酸化/β−アクチンの相対発現量、任意単位(arbitary units))を示す。また、健康対照群のJAK2リン酸化/β−アクチンの相対発現量を1.0とする。 4A is a bar graph showing the phosphorylated JAK2 of FIG. 3 normalized by the expression level of β-actin, and the vertical axis indicates the relative amount of phosphorylated JAK2 normalized by the expression level of β-actin (ie, JAK2 phosphorylation). Oxidation / relative expression level of β-actin, arbitrary units). Further, the relative expression level of JAK2 phosphorylation / β-actin in the healthy control group is set to 1.0.
図4Bは、β−アクチンの発現量によって標準化された図3のリン酸化STAT1を示す棒グラフであり、縦軸はβ−アクチンの発現量によって標準化されたリン酸化STAT1の相対量(即ち、リン酸化STAT1/β−アクチンの相対発現量、任意単位)を示す。また、健康対照群のSTAT1リン酸化/β−アクチンの相対発現量を1.0とする。図4Aと図4Bの図面記号*は、健康対照群と比べて、p<0.05であることを示し、図面記号#は、糖尿病ラットと比べて、p<0.05であることを示す。 FIG. 4B is a bar graph showing the phosphorylated STAT1 of FIG. 3 normalized by the expression level of β-actin, and the vertical axis represents the relative amount of phosphorylated STAT1 normalized by the expression level of β-actin (ie, phosphorylation). STAT1 / β-actin relative expression level, arbitrary unit). The relative expression level of STAT1 phosphorylation / β-actin in the healthy control group is 1.0. 4A and 4B, the symbol * indicates that p <0.05 compared to the healthy control group, and the symbol # indicates that p <0.05 compared with the diabetic rat. .
図3、図4A、図4Bの結果からわかるように、ストレプトゾトシンを注射してから28日後、糖尿病ラットのJAK2(図3の上からの二列のレーン3−4)とSTAT1(図3の下からの二列のレーン3−4)のリン酸化の相対量は、いずれも健康対照群(図3のレーン1−2)より著しく高いことから、糖尿病ラットの腎臓皮質のJAK2とSTAT1は高度に活性化されている状態になることが示された。 As can be seen from the results of FIG. 3, FIG. 4A, and FIG. 4B, 28 days after the injection of streptozotocin, JAK2 (lanes 3-4 in the two rows from the top of FIG. 3) and STAT1 (bottom of FIG. 3) Since the relative amount of phosphorylation in lanes 3-4) in two rows from both is significantly higher than in the healthy control group (lane 1-2 in FIG. 3), JAK2 and STAT1 in the renal cortex of diabetic rats are highly It has been shown to be activated.
しかしながら、ラクトバチルス・ロイテリGM−263(ADR−1)を経口投与された糖尿病ラット(糖尿病ラット+GM−263)は、そのJAK2とSTAT1のリン酸化の相対量(図3のレーン5−6)が著しく低下して、インシュリンを投与した糖尿病ラット対照群(糖尿病ラット+インシュリン)(図3のレーン7−8)に近いことから、実施例1のプロバイオティクス菌株GM−263(ADR−1)を経口投与することによって、確かにJAK2とSTAT1のリン酸化を特異的に抑制できることを示した。 However, a diabetic rat (diabetic rat + GM-263) orally administered with Lactobacillus reuteri GM-263 (ADR-1) has a relative amount of phosphorylation of JAK2 and STAT1 (lanes 5-6 in FIG. 3). The probiotic strain GM-263 (ADR-1) of Example 1 was remarkably reduced and was close to the diabetic rat control group (diabetic rat + insulin) (lanes 7-8 in FIG. 3) administered with insulin. It was shown that phosphorylation of JAK2 and STAT1 can be specifically suppressed by oral administration.
4.糖尿病ラットの腎臓皮質の細胞内タンパク質の発現に対するプロバイオティクス菌株GM−263(ADR−1)の影響の評価
図5は、本発明の他の実施例による糖尿病ラットの腎臓皮質組織を示すウェスタンブロッティング解析図であり、レーン1−2は健康対照群を示し、レーン3−4は糖尿病ラット群を示し、レーン5−6はラクトバチルス・ロイテリGM−263(ADR−1)を経口投与した糖尿病ラット群(糖尿病ラット+GM−263)を示し、レーン7−8はインシュリンを投与した糖尿病ラット対照群(糖尿病ラット+インシュリン)を示す。図5は、糖尿病ラットの腎臓皮質組織内のPAI−1(約50kDa)、P21Waf1/Cip1(約20kDa)、α−SMA(約42kDa)、フィブロネクチン(約200kDa)の発現量を分析するものである。また、β−アクチンは、前記タンパクの発現量を標準化するものである。
4. Evaluation of the effect of probiotic strain GM-263 (ADR-1) on the expression of intracellular proteins in the renal cortex of diabetic rats Figure 5 shows the renal cortex tissue of diabetic rats according to another embodiment of the present invention. It is a Western blotting analysis figure, Lane 1-2 shows a healthy control group, Lane 3-4 shows a diabetic rat group, Lane 5-6 orally administered Lactobacillus reuteri GM-263 (ADR-1) A diabetic rat group (diabetic rat + GM-263) is shown, and lanes 7-8 show a diabetic rat control group (diabetic rat + insulin) administered with insulin. FIG. 5 shows the expression levels of PAI-1 (about 50 kDa), P21 Wafl / Cip1 (about 20 kDa), α-SMA (about 42 kDa), and fibronectin (about 200 kDa) in the renal cortical tissue of diabetic rats. is there. In addition, β-actin standardizes the expression level of the protein.
図6Aは、β−アクチンの発現量によって標準化された図5のPAI−1発現量を示す棒グラフであり、縦軸はβ−アクチンの発現量によって標準化されたPAI−1の相対発現量(即ち、PAI−1/β−アクチンの相対発現量、任意単位)を示す。また、健康対照群のPAI−1/β−アクチンの相対発現量を1.0とする。 FIG. 6A is a bar graph showing the expression level of PAI-1 in FIG. 5 normalized by the expression level of β-actin, and the vertical axis shows the relative expression level of PAI-1 normalized by the expression level of β-actin (ie, , Relative expression level of PAI-1 / β-actin, arbitrary unit). The relative expression level of PAI-1 / β-actin in the healthy control group is 1.0.
図6Bは、β−アクチンの発現量によって標準化された図5のP21Waf1/Cip1発現量を示す棒グラフであり、縦軸はβ−アクチンの発現量によって標準化されたP21Waf1/Cip1の相対発現量(即ち、P21Waf1/Cip1/β−アクチンの相対発現量、任意単位)を示す。また、健康対照群のP21Waf1/Cip1/β−アクチンの相対発現量を1.0とする。 6B is a bar graph showing the expression level of P21 Wafl / Cip1 in FIG. 5 normalized by the expression level of β-actin, and the vertical axis shows the relative expression level of P21 Wafl / Cip1 normalized by the expression level of β-actin. (That is, the relative expression level of P21 Waf1 / Cip1 / β-actin, arbitrary unit). The relative expression level of P21 Waf1 / Cip1 / β-actin in the healthy control group is set to 1.0.
図6Cは、β−アクチンの発現量によって標準化された図5のα−SMA発現量を示す棒グラフであり、縦軸はβ−アクチンの発現量によって標準化されたα−SMAの相対発現量(即ちα−SMA/β−アクチンの相対発現量、任意単位)を示す。また、健康対照群のα−SMA/β−アクチンの相対発現量を1.0とする。 6C is a bar graph showing the α-SMA expression level of FIG. 5 normalized by the β-actin expression level, and the vertical axis shows the relative expression level of α-SMA normalized by the β-actin expression level (ie, relative expression level of α-SMA / β-actin, arbitrary unit). Also, the relative expression level of α-SMA / β-actin in the healthy control group is 1.0.
図6Dは、β−アクチンの発現量によって標準化された図5のフィブロネクチン発現量を示す棒グラフであり、縦軸はβ−アクチンの発現量によって標準化されたフィブロネクチンの相対発現量(即ち、フィブロネクチン/β−アクチンの相対発現量、任意単位)を示す。また、健康対照群のフィブロネクチン/β−アクチンの相対発現量を1.0とする。 6D is a bar graph showing the expression level of fibronectin in FIG. 5 normalized by the expression level of β-actin, and the vertical axis shows the relative expression level of fibronectin normalized by the expression level of β-actin (ie, fibronectin / β -Relative expression level of actin, arbitrary unit). The relative expression level of fibronectin / β-actin in the healthy control group is set to 1.0.
図5、図6A乃至図6Dの結果からわかるように、ストレプトゾトシンを注射してから28日後、糖尿病ラットのPAI−1(図5の上から一列目のレーン3−4)、P21Waf1/Cip1(図5の上から二列目のレーン3−4)、α−SMA(図5の上から三列目のレーン3−4)、フィブロネクチン(図5の最後の列のレーン3−4)のリン酸化の相対量は、健康対照群(図5のレーン1−2)より著しく高いことから、糖尿病ラットの腎臓皮質のPAI−1、P21Waf1/Cip1、α−SMA、フィブロネクチンは高度に発現されている状態になり、糖尿病ラットの腎線維化を引き起こしたことを示した。 As can be seen from the results of FIGS. 5 and 6A to 6D, 28 days after the injection of streptozotocin, PAI-1 of the diabetic rat (lane 3-4 in the first row from the top of FIG. 5), P21 Wafl / Cip1 ( Lanes 3-4 in the second row from the top of FIG. 5, α-SMA (lane 3-4 in the third row from the top of FIG. 5), fibronectin (lane 3-4 in the last row of FIG. 5) Since the relative amount of oxidation is significantly higher than that in the healthy control group (lane 1-2 in FIG. 5), PAI-1, P21 Wafl / Cip1 , α-SMA, and fibronectin in the renal cortex of diabetic rats are highly expressed. It was shown that it caused renal fibrosis in diabetic rats.
しかし、ラクトバチルス・ロイテリGM−263(ADR−1)を経口投与された糖尿病ラット(糖尿病ラット+GM−263)は、そのPAI−1、P21Waf1/Cip1、α−SMA、フィブロネクチンの相対量(図5のレーン5−6)が著しく低下し、それにP21Waf1/Cip1とα−SMAの低下程度が、インシュリンを投与した糖尿病ラット対照群(糖尿病ラット+インシュリン)(図5のレーン7−8)よりも低いことから、実施例1のプロバイオティクス菌株GM−263(ADR−1)を経口投与することによって、確かにPAI−1、P21Waf1/Cip1、α−SMA、フィブロネクチンの発現量を特異的に抑制できることを示した。 However, a diabetic rat (diabetic rat + GM-263) orally administered with Lactobacillus reuteri GM-263 (ADR-1) has a relative amount of PAI-1, P21 Wafl / Cip1 , α-SMA and fibronectin (Fig. 5 lanes 5-6) markedly decreased, and P21 Wafl / Cip1 and α-SMA decreased to a diabetic rat control group (diabetic rats + insulin) administered insulin (lanes 7-8 in FIG. 5). Therefore, by orally administering the probiotic strain GM-263 (ADR-1) of Example 1, the expression levels of PAI-1, P21 Wafl / Cip1 , α-SMA, and fibronectin are specific. It was shown that it can be suppressed.
以上をまとめていえば、本発明のプロバイオティクス菌株GM−263(ADR−1)(ラクトバチルスロイテリGM−263(ADR−1))(受託番号はCCTCC M 209263)は、糖尿病による腎線維化の治療に応用できることが証明されており、また、本発明は、乳酸菌の関与可能な制御機構、即ち、JAK2/STAT1のシグナル伝導経路を特異的に抑制し、腎線維化に関するタンパク質(PAI−1、P21Waf1/Cip1、α−SMA、フィブロネクチン)の発現を抑制する制御機構を提示し、それによって、糖尿病による腎線維化を有効に治療でき、それに従い、プロバイオティクス菌株をその他に応用するよう開発する。 In summary, the probiotic strain GM-263 (ADR-1) (Lactobacillus reuteri GM-263 (ADR-1)) of the present invention (accession number is CCTCC M 209263) It has been proved that it can be applied to therapy, and the present invention specifically inhibits a regulatory mechanism in which lactic acid bacteria can participate, that is, a signal conduction pathway of JAK2 / STAT1, and a protein related to renal fibrosis (PAI-1, P21 Waf1 / Cip1 , α-SMA, fibronectin), a regulatory mechanism that suppresses the expression of kidneys, thereby effectively treating renal fibrosis due to diabetes, and developed to apply probiotic strains to others accordingly To do.
ここで補充するのは、本発明は、特定の菌株、特定の分析方式、特定の動物モデル、特定の反応条件、特定の使用方法、特定の材料又は特定の設備等を例として、本発明の、糖尿病による腎線維化の治療におけるプロバイオティクス菌株GM−263(ADR−1)の用途を説明するが、当業者が理解するように、本発明はこれらに限られず、本発明の精神と範囲から逸脱しない限り、本発明のプロバイオティクス菌株GM−263(ADR−1)がその他のプロバイオティクス菌株、その他の分析方式、その他の動物モデル、その他の反応条件、その他の使用方式、その他の同等レベルの材料又はその他の設備等によって行ってもよい。また、本発明のプロバイオティクス菌株GM−263(ADR−1)を、例えば医薬組成物、補助飲食品、食品又はその組成分などの、糖尿病による腎線維化を治療する組成物の製造に用いる場合、プロバイオティクス菌株GM−263(ADR−1)は、生菌又は不活化菌であってもよく、また、凍結乾燥の形であってもよい。なお、本発明のプロバイオティクス菌株GM−263(ADR−1)は、更にその他の成分を含んでもよく、例えばグルコース、マルトデキストリン、乳児用調製粉乳、フラクトオリゴ糖、ステアリン酸マグネシウム、ヨーグルト香料、その他の分離しにくい成分、又は前記の任意の組み合わせを更に含んでもよい。 Here, the present invention supplements the present invention by taking a specific strain, a specific analysis method, a specific animal model, a specific reaction condition, a specific method of use, a specific material or a specific equipment as an example. Although the use of probiotic strain GM-263 (ADR-1) in the treatment of renal fibrosis due to diabetes will be described, as those skilled in the art will appreciate, the present invention is not limited thereto, and the spirit and scope of the present invention Unless deviating from the above, the probiotic strain GM-263 (ADR-1) of the present invention may be used in other probiotic strains, other analytical methods, other animal models, other reaction conditions, other usage methods, You may carry out by the material of an equivalent level, or other facilities. Further, the probiotic strain GM-263 (ADR-1) of the present invention is used for the production of a composition for treating renal fibrosis due to diabetes, such as a pharmaceutical composition, an auxiliary food or drink, a food, or a component thereof. In some cases, the probiotic strain GM-263 (ADR-1) may be live or inactivated, and may be in lyophilized form. The probiotic strain GM-263 (ADR-1) of the present invention may further contain other components, such as glucose, maltodextrin, infant formula, fructooligosaccharide, magnesium stearate, yogurt flavor, and others. These components may be further included, or any combination of the above.
本発明の前記実施例から分かるように、本発明の、糖尿病による腎線維化の治療におけるプロバイオティクス菌株GM−263(ADR−1)の用途は、その利点がプロバイオティクス菌株GM−263(ADR−1)を用いて、糖化ヘモグロビンと血糖濃度を低下させ、体重と腎重を正常な範囲に回復させて、更に、JAK2/STAT1のシグナル伝導経路を特異的に抑制し、腎線維化に関するタンパク質(例えばPAI−1、P21Waf1/Cip1、α−SMA、フィブロネクチン等)の発現を抑制することによって、糖尿病による腎線維化を有効に治療し、それに従い、プロバイオティクス菌株GM−263(ADR−1)をその他に応用するよう開発する。 As can be seen from the above examples of the present invention, the use of probiotic strain GM-263 (ADR-1) in the treatment of renal fibrosis due to diabetes according to the present invention has the advantage of probiotic strain GM-263 ( ADR-1) is used to reduce glycated hemoglobin and blood glucose levels, restore body weight and kidney weight to normal ranges, and specifically inhibit the signal conduction pathway of JAK2 / STAT1, and By inhibiting the expression of proteins (eg PAI-1, P21 Waf1 / Cip1 , α-SMA, fibronectin, etc.), renal fibrosis due to diabetes is effectively treated and probiotic strain GM-263 (ADR) -1) will be developed for other applications.
本発明を複数の実施例によって以上のように開示したが、これは本発明を限定するものではなく、当業者であれば、本発明の精神及び範囲から逸脱しない限り、各種の変更及び修飾することができる。本発明は、特許請求の範囲の記載によって限定される。 Although the present invention has been disclosed in the foregoing by a plurality of embodiments, this is not intended to limit the present invention, and various changes and modifications can be made by those skilled in the art without departing from the spirit and scope of the present invention. be able to. The present invention is limited by the description of the scope of claims.
Claims (4)
In addition, at least one other strain is included, and the other strains are Lactobacillus acidophilus, Lactobacillus plantarum, Bifidobacterium longum, Lactobacillus lumfum. , Lactobacillus bulgaricus, Streptococcus thermophilus, Lactobacillus cremoris, Lactobacillus cremoris Lacaris para ps casei subsp. paracasei), Lactobacillus rhamnosus · GG (Lactobacillus rhamnosus GG) and prophylactic pharmaceutical compositions of renal fibrosis due to diabetes according to claim 1 selected from the group consisting of any combination of the.
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