WO2016080449A1 - Endoplasmic reticulum stress inhibitor - Google Patents

Endoplasmic reticulum stress inhibitor Download PDF

Info

Publication number
WO2016080449A1
WO2016080449A1 PCT/JP2015/082424 JP2015082424W WO2016080449A1 WO 2016080449 A1 WO2016080449 A1 WO 2016080449A1 JP 2015082424 W JP2015082424 W JP 2015082424W WO 2016080449 A1 WO2016080449 A1 WO 2016080449A1
Authority
WO
WIPO (PCT)
Prior art keywords
bifidobacterium
endoplasmic reticulum
ferm
reticulum stress
strain
Prior art date
Application number
PCT/JP2015/082424
Other languages
French (fr)
Japanese (ja)
Inventor
拓哉 秋山
憲司 大石
アンディ ウラールト
Original Assignee
株式会社ヤクルト本社
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 株式会社ヤクルト本社 filed Critical 株式会社ヤクルト本社
Priority to JP2016560271A priority Critical patent/JP6670251B2/en
Publication of WO2016080449A1 publication Critical patent/WO2016080449A1/en

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A61K35/741—Probiotics
    • A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
    • A61K35/745—Bifidobacteria

Definitions

  • the present invention relates to an inhibitor of endoplasmic reticulum stress involved in cardiomyopathy and the like.
  • Patent Document 1 As preventive and therapeutic agents for diseases caused by endoplasmic reticulum stress, ASK1 inhibitors (Patent Document 1), fat-soluble extract components derived from mulberry yellow (Patent Document 2), Yokukansan (Patent Document 3) and the like have been reported. Yes.
  • the subject of this invention is providing the endoplasmic reticulum stress inhibitor which has the safe and outstanding effect.
  • the present inventors searched for endoplasmic reticulum stress inhibitors by paying attention to probiotics, and found that Bifidobacterium bacteria have a strong endoplasmic reticulum stress inhibitory action, thereby completing the present invention.
  • the present invention provides the following [1] to [12].
  • An endoplasmic reticulum stress inhibitor containing a Bifidobacterium genus and / or a treated product thereof as an active ingredient.
  • the genus Bifidobacterium is at least one selected from Bifidobacterium breve, Bifidobacterium adrecentis, Bifidobacterium bifidum and Bifidobacterium longum
  • the endoplasmic reticulum stress inhibitor as described.
  • Bifidobacterium bacteria are Bifidobacterium breve YIT12272 strain (FERM BP-11320), Bifidobacterium breve YIT10001 strain (FERM BP-8205), Bifidobacterium adrecentis YIT4011 strain Endoplasmic reticulum stress according to [1] or [2], which is at least one selected from (ATCC 15703), Bifidobacterium bifidum YIT10347 strain (FERM BP-10613) and Bifidobacterium longum YIT4021 strain (ATCC15707) Inhibitor.
  • the genus Bifidobacterium is one or more selected from Bifidobacterium breve, Bifidobacterium adrecentis, Bifidobacterium bifidum and Bifidobacterium longum [4] Use of description.
  • Bifidobacterium genus bacteria are Bifidobacterium breve YIT12272 strain (FERM BP-11320), Bifidobacterium breve YIT10001 strain (FERM BP-8205), Bifidobacterium adrecentis YIT4011 strain The use according to [4] or [5], which is one or more selected from (ATCC 15703), Bifidobacterium bifidum YIT10347 strain (FERM BP-10613) and Bifidobacterium longum YIT4021 strain (ATCC15707).
  • the genus Bifidobacterium is one or more selected from Bifidobacterium breve, Bifidobacterium adrecentis, Bifidobacterium bifidum and Bifidobacterium longum [7] The bacterium described above and / or a treated product thereof.
  • Bifidobacterium genus bacteria include Bifidobacterium breve YIT12272 strain (FERM BP-11320), Bifidobacterium breve YIT10001 strain (FERM BP-8205), Bifidobacterium adrecentis YIT4011 strain (ATCC15703), Bifidobacterium bifidum YIT10347 strain (FERM BP-10613) and Bifidobacterium longum YIT4021 strain (ATCC15707), which is one or more selected from [7] or [8] Or a treated product thereof.
  • a method for suppressing endoplasmic reticulum stress comprising administering an effective amount of a Bifidobacterium bacterium and / or a treated product thereof.
  • the genus Bifidobacterium is one or more selected from Bifidobacterium breve, Bifidobacterium adrecentis, Bifidobacterium bifidum and Bifidobacterium longum [10] The method described.
  • Bifidobacterium bacteria are Bifidobacterium breve YIT12272 strain (FERM BP-11320), Bifidobacterium breve YIT10001 strain (FERM BP-8205), Bifidobacterium adrecentis YIT4011 strain
  • the endoplasmic reticulum stress inhibitor of the present invention is useful as a preventive and therapeutic agent for cardiomyopathy and the like because of its high safety and strong endoplasmic reticulum stress inhibitory action.
  • FIG. 6 shows Western blotting of Grp78 expression in Caco-2 monolayers after tunicamycin stimulation.
  • the lower row shows Western blotting of ⁇ actin (loading control) expression.
  • 2 shows the effect of Bifidobacterium (YIT10001 strain) on the enhancement of membrane permeability of Caco-2 monolayers caused by endoplasmic reticulum stress (stunicamycin stimulation from the apical side).
  • 2 shows the effect of Bifidobacterium (YIT10001 strain) on the enhancement of membrane permeability of Caco-2 monolayers caused by endoplasmic reticulum stress (stimulated tunicamycin from the basal side).
  • 2 shows the effect of various Bifidobacteria on enhancement of membrane permeability of Caco-2 monolayers caused by endoplasmic reticulum stress (stunicamycin stimulation from the apical side).
  • the effect of Bifidobacterium (YIT4001 strain, YIT10347 strain) on the expression of endoplasmic reticulum stress marker (CHOP) is shown.
  • the lower row shows Western blotting of ⁇ actin (loading control) expression.
  • the effect on the expression of UGGT mRNA in Caco-2 monolayer of various Bifidobacterium is shown.
  • the active ingredient of the endoplasmic reticulum stress inhibitor of the present invention is a bacterium belonging to the genus Bifidobacterium and / or a treated product thereof.
  • the bacteria belonging to the genus Bifidobacterium at least one selected from Bifidobacterium breve, Bifidobacterium adrecentis, Bifidobacterium bifidum and Bifidobacterium longum is preferable.
  • Bifidobacterium breve YIT12272 (FERM BP-11320), Bifidobacterium breve YIT10001 (FERM BP-8205), Bifidobacterium adrecentis YIT4011
  • the method for preparing the genus Bifidobacterium used in the present invention is not particularly limited, and may be performed according to a conventional method.
  • the Bifidobacterium genus bacterium used in the present invention is cultured by inoculating the inoculum of the bacterium into a proliferable medium, and using a means for isolating and purifying the microbial cells such as centrifugation and filtration after the culture.
  • the bacterium can be used as it is, the lyophilized microbial cell, a dead cell obtained by subjecting the microbial cell to heat treatment or alcohol treatment, and a culture containing the microbial cell.
  • the culture containing the bacteria is subjected to a treatment such as heat treatment or the like. May be used as an active ingredient of the endoplasmic reticulum stress inhibitor of the present invention.
  • the medium capable of growing Bifidobacterium is not particularly limited, and is a nutrient medium composed of various organic and inorganic nutrient sources, such as GAM medium, MRS medium, BL medium, and the like. Can be mentioned.
  • animal milk such as cow's milk, goat milk, skim milk, milk powder, skim milk powder, milk products such as fresh cream, soy milk, processed soybean products such as soy flour, etc. can be used as a suitable medium. These may be used as they are or after diluting or concentrating to an appropriate concentration as necessary.
  • the pH of the medium is not particularly limited.
  • Bifidobacterium genus bacteria may not always have good growth depending on the type of medium. Therefore, a known bifido that can be used as a yeast extract, soybean peptide, or other fermentation aid in the medium as necessary. It is preferable to add a growth promoting substance for bacteria belonging to the genus Bacteria and a reducing agent such as vitamin C.
  • the culture of Bifidobacterium using the above-mentioned medium is not particularly limited as long as normal culture conditions are applied as they are. That is, various conditions such as temperature, time, and culture atmosphere suitable for the Bifidobacterium inoculated into the medium may be appropriately set.
  • the culture temperature may be 25 to 46 ° C., preferably 35 to 42 ° C.
  • the culture time may be 6 to 120 hours, preferably 24 to 72 hours.
  • the culture atmosphere is preferably carried out under anaerobic conditions, and the culture method is not particularly limited, such as standing, stirring, shaking, etc., and any of them may be selected.
  • Bifidobacterium genus bacteria used as an active ingredient of the endoplasmic reticulum stress inhibitor of the present invention many of these microorganisms have been conventionally used for the production of various fermented foods such as yogurt, so they are always taken orally. However, it is safe and has an excellent endoplasmic reticulum stress-suppressing action as shown in Examples below, and therefore can be used as a prophylactic and therapeutic drug for diseases caused by endoplasmic reticulum stress, such as cardiomyopathy.
  • the Bifidobacterium bacterium used as an active ingredient of the endoplasmic reticulum stress inhibitor of the present invention has an action of suppressing the expression of various endoplasmic reticulum stress response factors (CHOP, GRP78, ERdj4, XBP1s, etc.), It can be used as an endoplasmic reticulum stress response factor expression inhibitor, or an expression inhibitor of one or more proteins selected from CHOP, GRP78, ERdj4 and XBP1s.
  • Bifidobacterium bacteria also enhance the mRNA expression of UDP-glucose: glycoprotein glucosyltransferase (UGGT), an enzyme that reduces the unfolded glycoprotein in the endoplasmic reticulum back into the folding cycle. Therefore, the mechanism of the endoplasmic reticulum oxidative stress suppression action by Bifidobacterium is considered to be due to the UGGT expression promoting action.
  • Bifidobacterium bacteria are useful as UGGT expression promote
  • the desired effect can be obtained by ingesting the Bifidobacterium genus bacterium used in the present invention orally regardless of the state of live cells and / or dead cells. Therefore, the Bifidobacterium genus bacteria used in the present invention do not necessarily need to be used as viable cells, and the desired cells described above can be used even if cells that have been completely or partially killed due to internal or external factors due to storage or the like are used. Since an effect can be acquired, it can be set as the form for medicine etc. which gave various processing (processing). Moreover, Bifidobacterium genus bacteria can be used also as food-drinks for endoplasmic reticulum stress suppression.
  • the endoplasmic reticulum stress-suppressing agent of the present invention can be made into pharmaceutical compositions of various dosage forms together with a pharmaceutically acceptable carrier according to a conventional method. Moreover, as a form of food / beverage products, functional food, health food, food for specified health, etc. are mentioned.
  • lactose sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid and other excipients, water, ethanol, propanol, Glucose solution, starch solution, gelatin solution, binders such as carboxymethylcellulose, methylcellulose, potassium phosphate, polyvinylpyrrolidone, sodium alginate, catechin powder, sodium bicarbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, etc.
  • binders such as carboxymethylcellulose, methylcellulose, potassium phosphate, polyvinylpyrrolidone, sodium alginate, catechin powder, sodium bicarbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, etc.
  • Granules, tablets, capsules and the like can be produced by conventional methods by adding a disintegrant, a humectant such as glycerin and starch, a lubricant such as purified talc, stearate and polyethylene glycol.
  • the tablets can be made into tablets with ordinary coatings as necessary, for example, sugar-coated tablets, gelatin-encapsulated tablets, enteric-coated tablets, film-coated tablets, double tablets, or multiple tablets.
  • Specific examples of the food and drink include yogurt and beverages.
  • the endoplasmic reticulum stress inhibitor of the present invention may contain lactic acid bacteria that can be taken orally and give a beneficial function to the living body.
  • the amount of Bifidobacterium genus used per day in the endoplasmic reticulum stress-suppressing agent of the present invention varies depending on the subject's symptoms, age, weight, etc., and thus cannot be generally determined.
  • the number of dead bacteria may be about 10 3 to 10 13 or the amount obtained by treating the number of cells as a treated cell.
  • Example 1 A Materials and Methods (1) Preparation of heat-killed cells Bifidobacterium breve YIT10001 strain, Bifidobacterium breve YIT12272, Bifidobacterium adrecentis YIT4011 T , Bifidobacterium bifidum YIT10347 and Bifidobacterium Each strain of Um longum YIT4021 T was cultured overnight, and the cells were collected by centrifugation and then washed twice with sterile water. The washed cells were suspended in sterilized water and heated in a boiling water bath for 30 minutes to prepare heated dead cells.
  • a part of the suspension containing the dead cells was stained with DAPI (4 ′, 6-diamidino-2-phenylindole), and the number of bacteria was measured using a fluorescence microscope.
  • the heated dead cells were lyophilized, suspended in PBS to a concentration of 5 10 cells / mL, and aliquoted and stored at ⁇ 80 ° C.
  • TEER Cell culture and transepithelial electrical resistance test Human colon cancer-derived epithelial cell line (Caco-2) was cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% (v / v) fetal bovine serum ( FBS), 100 U / mL penicillin, 100 ⁇ g / mL streptomycin, 1% non-essential amino acid, 25 mM Hepes, 1 mM sodium pyruvate, 2 mM L-glutamine at 37 ° C., 5% (v / v) CO 2 Incubated under high humidity.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • the transmembrane electrical resistance (TEER) test was performed by partially modifying the protocol of the Biocoat HTS Caco-2 assay system (Beckton Dickinson).
  • Caco-2 cells were seeded on a fibrous collagen membrane-treated culture insert (pore 1 ⁇ m, surface area 0.3 cm 2 ) at a cell density of 10 5 or 2 ⁇ 10 5 / insert and suspended in the above medium. did.
  • the apical (corresponding to the luminal side of intestinal epithelial cells) and basal (corresponding to the lamina intestinal epithelial cells) medium was replaced with Entero-Stim differentiation medium supplemented with Mito + Serum Extender.
  • Anti-CHOP antibody (1: 1000, Cell Signaling
  • anti-GRP78 antibody (1: 1000, Cell Signaling
  • anti-XBP1s antibody (1: 1000, BioLegend)
  • Anti- ⁇ -actin antibody (1: 1000, Santa Cruz).
  • Blocked PVDF was incubated with each primary antibody at 4 ° C. overnight, washed, and then incubated with an HRP-conjugated secondary antibody (Abcam) derived from mouse, rabbit or goat. Protein bands were detected with ECL (Enhanced chemiluminescent: Pierce) and ChemiDoc MP system (Biorad).
  • the obtained cDNA was prepared to 5 ng / ⁇ L using nuclease free H 2 O, and 2 ⁇ L (10 ng) was subjected to TaqMan gene expression assay (Applied Biosystems) using a 384-well plate to perform RT-qPCR.
  • the expression level of each mRNA was compared by calculating the relative expression level with respect to Tbp, which is a reference gene, by the ⁇ Ct method.
  • the probes used for the analysis were as follows: Tbp; Hs00427620_mL, GRP78; Hs00607129_gH, CHOP; Hs00358796_gL, ERdj4; Hs01052402_mL.
  • Bifidobacterium genus bacteria other than Bifidobacterium breve YIT10001 Effect of Bifidobacterium genus bacteria other than Bifidobacterium breve YIT10001 on endoplasmic reticulum stress
  • Bifidobacterium breve YIT12272, Bifidobacterium adrecentis YIT4011, Bifidobacterium bifidum YIT10347, and Bifidobacterium longum YIT4021 were examined for their inhibitory effects on the enhancement of membrane permeability caused by endoplasmic reticulum stress.
  • FIG. 5 it was found that the Bifidobacterium genus shows an excellent endoplasmic reticulum stress inhibitory action.
  • CHOP protein a kind of endoplasmic reticulum stress marker
  • Example 2 (UGGT expression promoting action) (1) The Caco-2 monolayer was dissolved in TRIzol (Life technologies) and then subjected to RNeasy kit (Qiagen) to extract RNA. The concentration of the obtained RNA was measured with Nanodrop (Thermo Scientific), and it was confirmed by performing PCR on ⁇ -actin that there was no contamination of gDNA (Genomic DNA). Using 1 ⁇ g of RNA as a template, cDNA was synthesized using iScript cDNA synthesis kit (BioRad).
  • the obtained cDNA was prepared to 5 ng / ⁇ l using nuclease free H 2 O, and 2 ⁇ l (10 ng) was subjected to TaqMan gene expression assay (Applied Biosystems) using a 384-well plate, and RT-qPCR was performed. The expression level of each mRNA was compared by calculating the relative expression level with respect to the reference gene Tbp by the ⁇ Ct method.
  • the probes used for the analysis were as follows: Tbp; Hs00427620_m1, UGGT; Hs00917255_m1.
  • glucose at the oligosaccharide end is removed by an enzyme called Glucosidase II, and the protein dissociates from the molecular chaperone.
  • Glucosidase II an enzyme that dissociates from the molecular chaperone.
  • the protein is excreted from the endoplasmic reticulum, and in some cases after further modification in the Golgi apparatus, it is then extracellularly or onto the cell membrane. And will be transported.
  • the glycoprotein is transported to the degradation mechanism with the help of a defective proteolytic factor (ii) or (iii) by an enzyme called UGGT (UDP-glucose: glycoprotein glucosyltransferase)
  • UGGT UDP-glucose: glycoprotein glucosyltransferase

Abstract

Provided is an endoplasmic reticulum stress inhibitor that is stable and has an excellent effect. An endoplasmic reticulum stress inhibitor, wherein bacteria belonging to the Bifidobacterium genus, and/or treated products of said bacteria, are an active ingredient.

Description

小胞体ストレス抑制剤ER stress inhibitor
　本発明は、心筋症等に関与している小胞体ストレスの抑制剤に関する。 The present invention relates to an inhibitor of endoplasmic reticulum stress involved in cardiomyopathy and the like.
　種々の原因により高次構造に折り畳まれなかった異常蛋白(unfolded protein)が小胞体に集積すると小胞体ストレス反応が起こり、分子シャペロンなどの産生増加、一般の蛋白の翻訳抑制、ユビキチン－プロテアソーム系による異常蛋白の分解が亢進する。通常は、こうして小胞体への異常蛋白の集積が軽減されるが、この蛋白質処理管理機構の処理能力以上に異常蛋白が小胞体に集積すると、細胞内外で異常蛋白の凝集、集積が起こり、細胞機能不全、細胞死が起こると考えられている。このような小胞体ストレス反応は、心筋症等に深く関与していることが知られている。 When an unfolded protein that is not folded into a higher-order structure due to various causes accumulates in the endoplasmic reticulum, an endoplasmic reticulum stress reaction occurs, resulting in increased production of molecular chaperones, suppression of general protein translation, and ubiquitin-proteasome Abnormal protein degradation increases. Normally, abnormal protein accumulation in the endoplasmic reticulum is reduced in this way, but if abnormal protein accumulates in the endoplasmic reticulum beyond the processing capacity of this protein processing control mechanism, abnormal protein aggregation and accumulation occurs inside and outside the cell, and the cell It is thought that dysfunction and cell death occur. It is known that such an endoplasmic reticulum stress reaction is deeply involved in cardiomyopathy and the like.
　小胞体ストレスに起因する疾患の予防治療薬としては、ＡＳＫ１阻害剤（特許文献１）、桑黄由来の脂溶性抽出成分（特許文献２）、抑肝散（特許文献３）等が報告されている。 As preventive and therapeutic agents for diseases caused by endoplasmic reticulum stress, ASK1 inhibitors (Patent Document 1), fat-soluble extract components derived from mulberry yellow (Patent Document 2), Yokukansan (Patent Document 3) and the like have been reported. Yes.
国際公開第２００２／３８１７９号International Publication No. 2002/38179 特開２００６－３４２０７７号公報JP 2006-342077 A 特開２０１１－０３７７２２号公報JP 2011-037722 A
　しかしながら、前記の小胞体ストレス抑制剤は、その効果が明らかでなかったり、安全性の面で不明なものが多かった。また、プロバイオティクスが小胞体ストレス抑制作用を有することは全く報告されていなかった。
　従って、本発明の課題は、安全でかつ優れた効果を有する小胞体ストレス抑制剤を提供することにある。 However, many of the aforementioned endoplasmic reticulum stress inhibitors are not clear in their effects or are unknown in terms of safety. In addition, it has never been reported that probiotics have an endoplasmic reticulum stress inhibitory action.
Therefore, the subject of this invention is providing the endoplasmic reticulum stress inhibitor which has the safe and outstanding effect.
　そこで本発明者は、プロバイオティクスに着目して小胞体ストレス抑制剤を探索したところ、ビフィドバクテリウム属細菌が強い小胞体ストレス抑制作用を有することを見出し、本発明を完成した。 Therefore, the present inventors searched for endoplasmic reticulum stress inhibitors by paying attention to probiotics, and found that Bifidobacterium bacteria have a strong endoplasmic reticulum stress inhibitory action, thereby completing the present invention.
　すなわち、本発明は、次の〔１〕～〔１２〕を提供するものである。
〔１〕ビフィドバクテリウム属細菌及び／又はその菌体処理物を有効成分とする小胞体ストレス抑制剤。
〔２〕ビフィドバクテリウム属細菌が、ビフィドバクテリウム・ブレーベ、ビフィドバクテリウム・アドレセンティス、ビフィドバクテリウム・ビフィダム及びビフィドバクテリウム・ロンガムから選ばれる１種以上である〔１〕記載の小胞体ストレス抑制剤。
〔３〕ビフィドバクテリウム属細菌が、ビフィドバクテリウム・ブレーベＹＩＴ１２２７２株（ＦＥＲＭ　ＢＰ－１１３２０）、ビフィドバクテリウム・ブレーベＹＩＴ１０００１株（ＦＥＲＭ　ＢＰ－８２０５）、ビフィドバクテリウム・アドレセンティスＹＩＴ４０１１株（ＡＴＣＣ１５７０３）、ビフィドバクテリウム・ビフィダムＹＩＴ１０３４７株（ＦＥＲＭ　ＢＰ－１０６１３）及びビフィドバクテリウム・ロンガムＹＩＴ４０２１株（ＡＴＣＣ１５７０７）から選ばれる１種以上である〔１〕又は〔２〕記載の小胞体ストレス抑制剤。
〔４〕小胞体ストレス抑制剤製造のための、ビフィドバクテリウム属細菌及び／又はその菌体処理物の使用。
〔５〕ビフィドバクテリウム属細菌が、ビフィドバクテリウム・ブレーベ、ビフィドバクテリウム・アドレセンティス、ビフィドバクテリウム・ビフィダム及びビフィドバクテリウム・ロンガムから選ばれる１種以上である〔４〕記載の使用。
〔６〕ビフィドバクテリウム属細菌が、ビフィドバクテリウム・ブレーベＹＩＴ１２２７２株（ＦＥＲＭ　ＢＰ－１１３２０）、ビフィドバクテリウム・ブレーベＹＩＴ１０００１株（ＦＥＲＭ　ＢＰ－８２０５）、ビフィドバクテリウム・アドレセンティスＹＩＴ４０１１株（ＡＴＣＣ１５７０３）、ビフィドバクテリウム・ビフィダムＹＩＴ１０３４７株（ＦＥＲＭ　ＢＰ－１０６１３）及びビフィドバクテリウム・ロンガムＹＩＴ４０２１株（ＡＴＣＣ１５７０７）から選ばれる１種以上である〔４〕又は〔５〕記載の使用。
〔７〕小胞体ストレスを抑制するための、ビフィドバクテリウム属細菌及び／又はその菌体処理物。
〔８〕ビフィドバクテリウム属細菌が、ビフィドバクテリウム・ブレーベ、ビフィドバクテリウム・アドレセンティス、ビフィドバクテリウム・ビフィダム及びビフィドバクテリウム・ロンガムから選ばれる１種以上である〔７〕記載の細菌及び／又はその菌体処理物。
〔９〕ビフィドバクテリウム属細菌が、ビフィドバクテリウム・ブレーベＹＩＴ１２２７２株（ＦＥＲＭ　ＢＰ－１１３２０）、ビフィドバクテリウム・ブレーベＹＩＴ１０００１株（ＦＥＲＭ　ＢＰ－８２０５）、ビフィドバクテリウム・アドレセンティスＹＩＴ４０１１株（ＡＴＣＣ１５７０３）、ビフィドバクテリウム・ビフィダムＹＩＴ１０３４７株（ＦＥＲＭ　ＢＰ－１０６１３）及びビフィドバクテリウム・ロンガムＹＩＴ４０２１株（ＡＴＣＣ１５７０７）から選ばれる１種以上である〔７〕又は〔８〕記載の細菌及び／又はその菌体処理物。
〔10〕ビフィドバクテリウム属細菌及び／又はその菌体処理物の有効量を投与することを特徴とする、小胞体ストレスの抑制方法。
〔11〕ビフィドバクテリウム属細菌が、ビフィドバクテリウム・ブレーベ、ビフィドバクテリウム・アドレセンティス、ビフィドバクテリウム・ビフィダム及びビフィドバクテリウム・ロンガムから選ばれる１種以上である〔１０〕記載の方法。
〔12〕ビフィドバクテリウム属細菌が、ビフィドバクテリウム・ブレーベＹＩＴ１２２７２株（ＦＥＲＭ　ＢＰ－１１３２０）、ビフィドバクテリウム・ブレーベＹＩＴ１０００１株（ＦＥＲＭ　ＢＰ－８２０５）、ビフィドバクテリウム・アドレセンティスＹＩＴ４０１１株（ＡＴＣＣ１５７０３）、ビフィドバクテリウム・ビフィダムＹＩＴ１０３４７株（ＦＥＲＭ　ＢＰ－１０６１３）及びビフィドバクテリウム・ロンガムＹＩＴ４０２１株（ＡＴＣＣ１５７０７）から選ばれる１種以上である〔１０〕又は〔１１〕記載の方法。 That is, the present invention provides the following [1] to [12].
[1] An endoplasmic reticulum stress inhibitor containing a Bifidobacterium genus and / or a treated product thereof as an active ingredient.
[2] The genus Bifidobacterium is at least one selected from Bifidobacterium breve, Bifidobacterium adrecentis, Bifidobacterium bifidum and Bifidobacterium longum [1] The endoplasmic reticulum stress inhibitor as described.
[3] Bifidobacterium bacteria are Bifidobacterium breve YIT12272 strain (FERM BP-11320), Bifidobacterium breve YIT10001 strain (FERM BP-8205), Bifidobacterium adrecentis YIT4011 strain Endoplasmic reticulum stress according to [1] or [2], which is at least one selected from (ATCC 15703), Bifidobacterium bifidum YIT10347 strain (FERM BP-10613) and Bifidobacterium longum YIT4021 strain (ATCC15707) Inhibitor.
[4] Use of a bacterium belonging to the genus Bifidobacterium and / or a treated product thereof for producing an endoplasmic reticulum stress inhibitor.
[5] The genus Bifidobacterium is one or more selected from Bifidobacterium breve, Bifidobacterium adrecentis, Bifidobacterium bifidum and Bifidobacterium longum [4] Use of description.
[6] Bifidobacterium genus bacteria are Bifidobacterium breve YIT12272 strain (FERM BP-11320), Bifidobacterium breve YIT10001 strain (FERM BP-8205), Bifidobacterium adrecentis YIT4011 strain The use according to [4] or [5], which is one or more selected from (ATCC 15703), Bifidobacterium bifidum YIT10347 strain (FERM BP-10613) and Bifidobacterium longum YIT4021 strain (ATCC15707).
[7] A Bifidobacterium bacterium and / or a treated product thereof for suppressing endoplasmic reticulum stress.
[8] The genus Bifidobacterium is one or more selected from Bifidobacterium breve, Bifidobacterium adrecentis, Bifidobacterium bifidum and Bifidobacterium longum [7] The bacterium described above and / or a treated product thereof.
[9] Bifidobacterium genus bacteria include Bifidobacterium breve YIT12272 strain (FERM BP-11320), Bifidobacterium breve YIT10001 strain (FERM BP-8205), Bifidobacterium adrecentis YIT4011 strain (ATCC15703), Bifidobacterium bifidum YIT10347 strain (FERM BP-10613) and Bifidobacterium longum YIT4021 strain (ATCC15707), which is one or more selected from [7] or [8] Or a treated product thereof.
[10] A method for suppressing endoplasmic reticulum stress, comprising administering an effective amount of a Bifidobacterium bacterium and / or a treated product thereof.
[11] The genus Bifidobacterium is one or more selected from Bifidobacterium breve, Bifidobacterium adrecentis, Bifidobacterium bifidum and Bifidobacterium longum [10] The method described.
[12] Bifidobacterium bacteria are Bifidobacterium breve YIT12272 strain (FERM BP-11320), Bifidobacterium breve YIT10001 strain (FERM BP-8205), Bifidobacterium adrecentis YIT4011 strain The method according to [10] or [11], wherein the method is one or more selected from (ATCC 15703), Bifidobacterium bifidum YIT10347 strain (FERM BP-10613) and Bifidobacterium longum YIT4021 strain (ATCC15707).
　本発明の小胞体ストレス抑制剤は、安全性が高く、かつ強い小胞体ストレス抑制作用を有していることから、心筋症等の予防治療剤として有用である。 The endoplasmic reticulum stress inhibitor of the present invention is useful as a preventive and therapeutic agent for cardiomyopathy and the like because of its high safety and strong endoplasmic reticulum stress inhibitory action.
ツニカマイシン１０μｇ／ｍＬ添加によるＣａｃｏ－２単層のＴＥＥＲ値の変化を示す。The change of the TEER value of the Caco-2 monolayer by the addition of 10 μg / mL of tunicamycin is shown. ツニカマイシン刺激後のＣａｃｏ－２単層におけるＧｒｐ７８の発現のウエスタンブロッティングを示す。下段はβＡｃｔｉｎ（ローディング・コントロール）の発現のウエスタンブロッティング。FIG. 6 shows Western blotting of Grp78 expression in Caco-2 monolayers after tunicamycin stimulation. The lower row shows Western blotting of βactin (loading control) expression. 小胞体ストレスによって引き起こされるＣａｃｏ－２単層の膜透過性の亢進に対するビフィドバクテリウム（ＹＩＴ１０００１株）の効果を示す（頂端側からツニカマイシン刺激）。2 shows the effect of Bifidobacterium (YIT10001 strain) on the enhancement of membrane permeability of Caco-2 monolayers caused by endoplasmic reticulum stress (stunicamycin stimulation from the apical side). 小胞体ストレスによって引き起こされるＣａｃｏ－２単層の膜透過性の亢進に対するビフィドバクテリウム（ＹＩＴ１０００１株）の効果を示す（基底側からツニカマイシン刺激）。2 shows the effect of Bifidobacterium (YIT10001 strain) on the enhancement of membrane permeability of Caco-2 monolayers caused by endoplasmic reticulum stress (stimulated tunicamycin from the basal side). 小胞体ストレスによって引き起こされるＣａｃｏ－２単層の膜透過性の亢進に対する各種ビフィドバクテリウムの効果を示す（頂端側からツニカマイシン刺激）。2 shows the effect of various Bifidobacteria on enhancement of membrane permeability of Caco-2 monolayers caused by endoplasmic reticulum stress (stunicamycin stimulation from the apical side). 小胞体ストレスマーカー（ＣＨＯＰ）の発現に対するビフィドバクテリウム（ＹＩＴ４００１株、ＹＩＴ１０３４７株）の効果を示す。下段はβＡｃｔｉｎ（ローディング・コントロール）の発現のウエスタンブロッティング。The effect of Bifidobacterium (YIT4001 strain, YIT10347 strain) on the expression of endoplasmic reticulum stress marker (CHOP) is shown. The lower row shows Western blotting of βactin (loading control) expression. 各種ビフィドバクテリウムのＣａｃｏ－２単層内のＵＧＧＴ　ｍＲＮＡの発現に対する効果を示す。The effect on the expression of UGGT mRNA in Caco-2 monolayer of various Bifidobacterium is shown.
　本発明の小胞体ストレス抑制剤の有効成分は、ビフィドバクテリウム属細菌及び／又はその菌体処理物である。 The active ingredient of the endoplasmic reticulum stress inhibitor of the present invention is a bacterium belonging to the genus Bifidobacterium and / or a treated product thereof.
　ビフィドバクテリウム属細菌としては、ビフィドバクテリウム・ブレーベ、ビフィドバクテリウム・アドレセンティス、ビフィドバクテリウム・ビフィダム及びビフィドバクテリウム・ロンガムから選ばれる１種以上が好ましい。これらのビフィドバクテリウム属細菌のうち、さらにビフィドバクテリウム・ブレーベＹＩＴ１２２７２株（ＦＥＲＭ　ＢＰ－１１３２０）、ビフィドバクテリウム・ブレーベＹＩＴ１０００１株（ＦＥＲＭ　ＢＰ－８２０５）、ビフィドバクテリウム・アドレセンティスＹＩＴ４０１１株（ＡＴＣＣ１５７０３）、ビフィドバクテリウム・ビフィダムＹＩＴ１０３４７株（ＦＥＲＭ　ＢＰ－１０６１３）及びビフィドバクテリウム・ロンガムＹＩＴ４０２１株（ＡＴＣＣ１５７０７）から選ばれる１種以上が好ましい。 As the bacteria belonging to the genus Bifidobacterium, at least one selected from Bifidobacterium breve, Bifidobacterium adrecentis, Bifidobacterium bifidum and Bifidobacterium longum is preferable. Among these bacteria belonging to the genus Bifidobacterium, Bifidobacterium breve YIT12272 (FERM BP-11320), Bifidobacterium breve YIT10001 (FERM BP-8205), Bifidobacterium adrecentis YIT4011 One or more selected from the strain (ATCC15703), Bifidobacterium bifidum YIT10347 strain (FERM BP-10613) and Bifidobacterium longum YIT4021 strain (ATCC15707) are preferred.
　本発明で用いるビフィドバクテリウム属細菌の調製方法は、特に限定されるものではなく、常法に従って行えばよい。
　例えば、本発明に用いるビフィドバクテリウム属細菌は、当該細菌の種菌を増殖可能な培地に接種して培養し、培養後遠心分離やろ過等の菌体を単離・精製する手段を用いて調製することができる。また、当該細菌は、得られる生菌体をそのままで使用できる他、菌体を凍結乾燥した物、生菌体に加熱処理やアルコール処理等を施した死菌体、当該菌体を含む培養物、菌体からの抽出物やその分画物或いは更にそれらを粉砕等により加工した物ならびにこれらの混合物等に調製し、使用できる。
　また、ビフィドバクテリウム属細菌の調製の際に増殖可能な培地として経口的に摂取可能な培地を用いる場合には、当該細菌を含む培養物をそのまま或いは加熱処理などの加工処理を施したものを本発明の小胞体ストレス抑制剤の有効成分として使用してもよい。 The method for preparing the genus Bifidobacterium used in the present invention is not particularly limited, and may be performed according to a conventional method.
For example, the Bifidobacterium genus bacterium used in the present invention is cultured by inoculating the inoculum of the bacterium into a proliferable medium, and using a means for isolating and purifying the microbial cells such as centrifugation and filtration after the culture. Can be prepared. In addition, the bacterium can be used as it is, the lyophilized microbial cell, a dead cell obtained by subjecting the microbial cell to heat treatment or alcohol treatment, and a culture containing the microbial cell. In addition, it can be prepared and used as an extract from bacterial cells, a fraction thereof, a product obtained by further processing them by pulverization, or a mixture thereof.
In addition, when a medium that can be taken orally is used as a medium that can be grown during the preparation of Bifidobacterium, the culture containing the bacteria is subjected to a treatment such as heat treatment or the like. May be used as an active ingredient of the endoplasmic reticulum stress inhibitor of the present invention.
　ここで、ビフィドバクテリウム属細菌の増殖可能な培地としては、特に制限されるものではなく、有機・無機の各種栄養源から構成される栄養培地、例えば、ＧＡＭ培地、ＭＲＳ培地、ＢＬ培地等を挙げることができる。また、これら以外にも、牛乳、山羊乳等の獣乳や脱脂乳、粉乳、脱脂粉乳、生クリーム等の乳製品、豆乳、大豆粉等の大豆加工品を好適な培地として用いることができ、これらはそのまま或いは必要に応じて適当な濃度に希釈または濃縮等して使用すればよい。なお、培地のｐＨは、特に制限されない。 Here, the medium capable of growing Bifidobacterium is not particularly limited, and is a nutrient medium composed of various organic and inorganic nutrient sources, such as GAM medium, MRS medium, BL medium, and the like. Can be mentioned. In addition to these, animal milk such as cow's milk, goat milk, skim milk, milk powder, skim milk powder, milk products such as fresh cream, soy milk, processed soybean products such as soy flour, etc. can be used as a suitable medium. These may be used as they are or after diluting or concentrating to an appropriate concentration as necessary. The pH of the medium is not particularly limited.
　一般に、ビフィドバクテリウム属細菌は、培地の種類によって、増殖性が必ずしも良好でない場合があるため、必要に応じて前記培地に、酵母エキス、大豆ペプチド、その他発酵助剤となり得る公知のビフィドバクテリウム属細菌の生育促進物質やビタミンＣ等の還元剤を添加することが好ましい。 In general, Bifidobacterium genus bacteria may not always have good growth depending on the type of medium. Therefore, a known bifido that can be used as a yeast extract, soybean peptide, or other fermentation aid in the medium as necessary. It is preferable to add a growth promoting substance for bacteria belonging to the genus Bacteria and a reducing agent such as vitamin C.
　また、前記培地を用いたビフィドバクテリウム属細菌の培養は、通常の培養条件をそのまま適用すればよく、特に限定されるものではない。すなわち、培地に接種するビフィドバクテリウム属細菌に適した温度、時間、培養雰囲気等の各種条件を適宜設定して行えばよい。例えば、培養温度は、２５～４６℃、好ましくは３５～４２℃、培養時間は６～１２０時間、好ましくは２４～７２時間とすればよい。また、培養雰囲気は、嫌気的な条件で行うことが好ましく、培養方法については静置、攪拌、振盪等、特に制限されることはなく、いずれを選択してもよい。 Further, the culture of Bifidobacterium using the above-mentioned medium is not particularly limited as long as normal culture conditions are applied as they are. That is, various conditions such as temperature, time, and culture atmosphere suitable for the Bifidobacterium inoculated into the medium may be appropriately set. For example, the culture temperature may be 25 to 46 ° C., preferably 35 to 42 ° C., and the culture time may be 6 to 120 hours, preferably 24 to 72 hours. The culture atmosphere is preferably carried out under anaerobic conditions, and the culture method is not particularly limited, such as standing, stirring, shaking, etc., and any of them may be selected.
　本発明の小胞体ストレス抑制剤の有効成分として用いるビフィドバクテリウム属細菌について、これらの微生物の多くは、従来、ヨーグルト等の各種発酵食品の製造に用いられているので、常時経口的に摂取しても安全なものであり、後記実施例に示すように、優れた小胞体ストレス抑制作用を有することから、小胞体ストレスに起因する疾患、例えば心筋症等の予防治療薬として利用できる。 About the Bifidobacterium genus bacteria used as an active ingredient of the endoplasmic reticulum stress inhibitor of the present invention, many of these microorganisms have been conventionally used for the production of various fermented foods such as yogurt, so they are always taken orally. However, it is safe and has an excellent endoplasmic reticulum stress-suppressing action as shown in Examples below, and therefore can be used as a prophylactic and therapeutic drug for diseases caused by endoplasmic reticulum stress, such as cardiomyopathy.
　本発明の小胞体ストレス抑制剤の有効成分として用いるビフィドバクテリウム属細菌は、種々の小胞体ストレス応答因子（ＣＨＯＰ、ＧＲＰ７８、ＥＲｄｊ４、ＸＢＰ１ｓ等）の発現を抑制する作用を有することから、小胞体ストレス応答因子発現抑制剤、またはＣＨＯＰ、ＧＲＰ７８、ＥＲｄｊ４およびＸＢＰ１ｓから選ばれる１種以上のタンパクの発現抑制剤等として使用することができる。
　また、ビフィドバクテリウム属細菌は、小胞体内の折り畳み不全の糖タンパクを再び折り畳みサイクルへと還元させる酵素であるＵＤＰ－ｇｌｕｃｏｓｅ：ｇｌｙｃｏｐｒｏｔｅｉｎ　ｇｌｕｃｏｓｙｌｔｒａｎｓｆｅｒａｓｅ（ＵＧＧＴ）のｍＲＮＡ発現を亢進する。従って、ビフィドバクテリウム属細菌による小胞体酸化ストレス抑制作用の機序は、ＵＧＧＴ発現促進作用によるものと考えられる。また、ビフィドバクテリウム属細菌は、ＵＧＧＴ発現促進剤として有用である。 Since the Bifidobacterium bacterium used as an active ingredient of the endoplasmic reticulum stress inhibitor of the present invention has an action of suppressing the expression of various endoplasmic reticulum stress response factors (CHOP, GRP78, ERdj4, XBP1s, etc.), It can be used as an endoplasmic reticulum stress response factor expression inhibitor, or an expression inhibitor of one or more proteins selected from CHOP, GRP78, ERdj4 and XBP1s.
Bifidobacterium bacteria also enhance the mRNA expression of UDP-glucose: glycoprotein glucosyltransferase (UGGT), an enzyme that reduces the unfolded glycoprotein in the endoplasmic reticulum back into the folding cycle. Therefore, the mechanism of the endoplasmic reticulum oxidative stress suppression action by Bifidobacterium is considered to be due to the UGGT expression promoting action. Bifidobacterium bacteria are useful as UGGT expression promoters.
　また、本発明に用いるビフィドバクテリウム属細菌は、生菌体及び／又は死菌体の状態に関係なく、経口的に摂取することにより、上記所望の効果を得ることができる。そのため、本発明で用いるビフィドバクテリウム属細菌は、必ずしも全て生菌体として用いる必要はなく、保存等による内外的な要因により、全部または一部が死滅した菌体を用いても上記所望の効果を得ることができるので、種々の加工（処理）を施した医薬用等の形態とすることができる。また、ビフィドバクテリウム属細菌は、小胞体ストレス抑制用の飲食品としても使用することができる。 In addition, the desired effect can be obtained by ingesting the Bifidobacterium genus bacterium used in the present invention orally regardless of the state of live cells and / or dead cells. Therefore, the Bifidobacterium genus bacteria used in the present invention do not necessarily need to be used as viable cells, and the desired cells described above can be used even if cells that have been completely or partially killed due to internal or external factors due to storage or the like are used. Since an effect can be acquired, it can be set as the form for medicine etc. which gave various processing (processing). Moreover, Bifidobacterium genus bacteria can be used also as food-drinks for endoplasmic reticulum stress suppression.
　本発明の小胞体ストレス抑制剤は、常法に従って、薬学的に許容される担体とともに種々の剤型の医薬組成物とすることができる。また、飲食品の形態としては、機能性食品、健康食品、特定保健用食品等が挙げられる。
　例えば、上記菌株またはその加工物（処理物）等に、乳糖、白糖、塩化ナトリウム、ブドウ糖、尿素、デンプン、炭酸カルシウム、カオリン、結晶セルロース、ケイ酸等の賦形剤、水、エタノール、プロパノール、ブドウ糖液、デンプン液、ゼラチン溶液、カルボキシメチルセルロース、メチルセルロース、リン酸カリウム、ポリビニルピロリドン等の結合剤、アルギン酸ナトリウム、カテキン末、炭酸水素ナトリウム、炭酸カルシウム、ポリオキシエチレンソルビタン脂肪酸エステル類、ラウリル硫酸ナトリウム等の崩壊剤、グリセリン、デンプン等の保湿剤、精製タルク、ステアリン酸塩、ポリエチレングリコール等の滑沢剤等を加え、常法により顆粒剤、錠剤、カプセル剤等を製造することができる。更に、錠剤については、必要に応じ通常の剤皮を施した錠剤、例えば、糖衣錠、ゼラチン被包錠、腸溶被錠、フィルムコーティング錠或いは二重錠、多重錠とすることもできる。飲食品の具体的な形態としては、ヨーグルト、飲料等が挙げられる。 The endoplasmic reticulum stress-suppressing agent of the present invention can be made into pharmaceutical compositions of various dosage forms together with a pharmaceutically acceptable carrier according to a conventional method. Moreover, as a form of food / beverage products, functional food, health food, food for specified health, etc. are mentioned.
For example, in the above strain or processed product (processed product) thereof, lactose, sucrose, sodium chloride, glucose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid and other excipients, water, ethanol, propanol, Glucose solution, starch solution, gelatin solution, binders such as carboxymethylcellulose, methylcellulose, potassium phosphate, polyvinylpyrrolidone, sodium alginate, catechin powder, sodium bicarbonate, calcium carbonate, polyoxyethylene sorbitan fatty acid esters, sodium lauryl sulfate, etc. Granules, tablets, capsules and the like can be produced by conventional methods by adding a disintegrant, a humectant such as glycerin and starch, a lubricant such as purified talc, stearate and polyethylene glycol. Furthermore, the tablets can be made into tablets with ordinary coatings as necessary, for example, sugar-coated tablets, gelatin-encapsulated tablets, enteric-coated tablets, film-coated tablets, double tablets, or multiple tablets. Specific examples of the food and drink include yogurt and beverages.
　更に、本発明の小胞体ストレス抑制剤は、経口摂取が可能であって生体に有益な機能を与える乳酸菌を含むものであってもよい。 Furthermore, the endoplasmic reticulum stress inhibitor of the present invention may contain lactic acid bacteria that can be taken orally and give a beneficial function to the living body.
　本発明の小胞体ストレス抑制剤におけるビフィドバクテリウム属細菌の１日当たりの使用量は、対象者の症状、年齢、体重等によっても異なるため、一概に決定することは出来ないが、生菌および／または死菌の菌数としておよそ１０3～１０13個程度或いは菌体処理物として前記菌数の菌体を処理して得られる量とすればよい。 The amount of Bifidobacterium genus used per day in the endoplasmic reticulum stress-suppressing agent of the present invention varies depending on the subject's symptoms, age, weight, etc., and thus cannot be generally determined. The number of dead bacteria may be about 10 3 to 10 13 or the amount obtained by treating the number of cells as a treated cell.
　以下に、実施例を挙げて本発明を更に詳細に説明するが、本発明はこれらの実施例になんら制約されるものではない。 Hereinafter, the present invention will be described in more detail with reference to examples, but the present invention is not limited to these examples.
実施例１
Ａ．材料と方法
（１）加熱死菌体の調製
　ビフィドバクテリウム・ブレーベＹＩＴ１０００１株、ビフィドバクテリウム・ブレーベＹＩＴ１２２７２、ビフィドバクテリウム・アドレセンティスＹＩＴ４０１１T、ビフィドバクテリウム・ビフィダムＹＩＴ１０３４７およびビフィドバクテリウム・ロンガムＹＩＴ４０２１Tの各菌株を一晩培養し、遠心分離で菌体を回収後、滅菌水を用いて２回洗浄した。洗浄後の菌体を滅菌水中に懸濁し、沸騰水浴中で３０分間加熱することにより加熱死菌体を調製した。加熱死菌体を含む懸濁液の一部をＤＡＰＩ（４’，６－diamidino－２－phenylindole）で染色し、蛍光顕微鏡を用いて細菌数を測定した。加熱死菌体は凍結乾燥し、５10ｃｅｌｌｓ／ｍＬの濃度となるようにＰＢＳに懸濁後、小分けにして－８０℃で保管した。 Example 1
A. Materials and Methods (1) Preparation of heat-killed cells Bifidobacterium breve YIT10001 strain, Bifidobacterium breve YIT12272, Bifidobacterium adrecentis YIT4011 T , Bifidobacterium bifidum YIT10347 and Bifidobacterium Each strain of Um longum YIT4021 T was cultured overnight, and the cells were collected by centrifugation and then washed twice with sterile water. The washed cells were suspended in sterilized water and heated in a boiling water bath for 30 minutes to prepare heated dead cells. A part of the suspension containing the dead cells was stained with DAPI (4 ′, 6-diamidino-2-phenylindole), and the number of bacteria was measured using a fluorescence microscope. The heated dead cells were lyophilized, suspended in PBS to a concentration of 5 10 cells / mL, and aliquoted and stored at −80 ° C.
（２）細胞培養および経上皮膜間電気抵抗（ＴＥＥＲ）試験
　ヒト結腸癌由来上皮細胞株（Ｃａｃｏ－２）は、ダルベッコ変法イーグル培地（ＤＭＥＭ）に１０％（ｖ／ｖ）ウシ胎児血清（ＦＢＳ）、１００Ｕ／ｍＬペニシリン、１００μｇ／ｍＬストレプトマイシン、１％非必須アミノ酸、２５ｍＭ　Ｈｅｐｅｓ、１ｍＭ　ピルビン酸ナトリウム、２ｍＭ　Ｌ－グルタミンを添加した培地にて、３７℃、５％（ｖ／ｖ）ＣＯ2、多湿下で培養した。経上皮膜間電気抵抗（ＴＥＥＲ）試験は、Ｂｉｏｃｏａｔ　ＨＴＳ　Ｃａｃｏ－２アッセイシステム（Beckton Dickinson社）のプロトコルを一部改変して行った。初日にＣａｃｏ－２細胞を線維性コラーゲン皮膜処置済みカルチャーインサート（孔１μｍ、表面積０．３ｃｍ2）に１０5または２×１０5／インサートの細胞密度で、上述の培地に懸濁した細胞を播種した。３日目に頂端側（腸上皮細胞の管腔側に相当）および基底側（腸上皮細胞の粘膜固有層側に相当）の培地を、Ｍｉｔｏ＋Ｓｅｒｕｍ　Ｅｘｔｅｎｄｅｒを添加したＥｎｔｅｒｏ－Ｓｔｉｍ分化培地に交換した。４日目に同培地を新鮮な培地に交換し、各種ビフィズス菌の加熱死菌体と共に前培養を開始した。前培養１２時間後（５日目）にＴＥＥＲ値を測定することで分化が正常に完了したことを確認し、実験を開始した。 (2) Cell culture and transepithelial electrical resistance (TEER) test Human colon cancer-derived epithelial cell line (Caco-2) was cultured in Dulbecco's modified Eagle's medium (DMEM) with 10% (v / v) fetal bovine serum ( FBS), 100 U / mL penicillin, 100 μg / mL streptomycin, 1% non-essential amino acid, 25 mM Hepes, 1 mM sodium pyruvate, 2 mM L-glutamine at 37 ° C., 5% (v / v) CO 2 Incubated under high humidity. The transmembrane electrical resistance (TEER) test was performed by partially modifying the protocol of the Biocoat HTS Caco-2 assay system (Beckton Dickinson). On the first day, Caco-2 cells were seeded on a fibrous collagen membrane-treated culture insert (pore 1 μm, surface area 0.3 cm 2 ) at a cell density of 10 5 or 2 × 10 5 / insert and suspended in the above medium. did. On day 3, the apical (corresponding to the luminal side of intestinal epithelial cells) and basal (corresponding to the lamina propria side of intestinal epithelial cells) medium was replaced with Entero-Stim differentiation medium supplemented with Mito + Serum Extender. On the fourth day, the same medium was replaced with a fresh medium, and pre-culture was started together with heat-killed cells of various bifidobacteria. The experiment was started after confirming that the differentiation was completed normally by measuring the TEER value 12 hours after preculture (day 5).
（３）小胞体ストレスの誘導
　１ｍｇ／ｍＬツニカマイシン（Sigma社）ストック溶液は２５ｍＭ　水酸化ナトリウムを用いて調製した。ツニカマイシンを１０μｇ／ｍＬの濃度でＣａｃｏ－２単層に添加することにより小胞体ストレスを誘導し、Ｃａｃｏ－２単層のＴＥＥＲ値を１２時間毎に４８時間測定した。なお、対照ウェルには溶媒を等量添加した。 (3) Induction of Endoplasmic Reticulum Stress A 1 mg / mL tunicamycin (Sigma) stock solution was prepared using 25 mM sodium hydroxide. Endoplasmic reticulum stress was induced by adding tunicamycin to Caco-2 monolayer at a concentration of 10 μg / mL, and the TEER value of Caco-2 monolayer was measured every 12 hours for 48 hours. An equal amount of solvent was added to the control well.
（４）イムノブロッティング
　Ｃａｃｏ－２単層をLaemmli bufferで溶解し、１５分間煮沸した。ＳＤＳポリアクリルアミドゲル（Biorad社）にサンプルを等量ずつ供し、変性電気泳動法によりタンパクを分離した。分離されたタンパクをウェット式電気泳動法によりＰＶＤＦ（Millipore社）に転写した後、０．１％（ｖ／ｖ）Ｔｗｅｅｎ－２０入りＴＢＳ溶液に５％（ｗ／ｖ）ウシ血清アルブミンを添加した溶液中でブロッキング処理を行った。試験に用いた一次抗体と濃度は以下の通り：抗ＣＨＯＰ抗体（１：１０００、Cell Signaling社）、抗ＧＲＰ７８抗体（１：１０００、Cell Signaling社）、抗ＸＢＰ１ｓ抗体（１：１０００、BioLegend社）、抗β－ａｃｔｉｎ抗体（１：１０００、Santa Cruz社）。ブロッキング処理したＰＶＤＦを各一次抗体と４℃で一晩インキュベートし、洗浄した後、マウス、ウサギまたはヤギ由来のＨＲＰ結合二次抗体（Abcam社）とインキュベートした。タンパクのバンドはＥＣＬ（Enhanced chemiluminescent:Pierce社）およびＣｈｅｍｉＤｏｃ　ＭＰシステム（Biorad社）で検出した。 (4) Immunoblotting A Caco-2 monolayer was dissolved in Laemmli buffer and boiled for 15 minutes. An equal amount of sample was applied to SDS polyacrylamide gel (Biorad), and proteins were separated by denaturing electrophoresis. After the separated protein was transferred to PVDF (Millipore) by wet electrophoresis, 5% (w / v) bovine serum albumin was added to a TBS solution containing 0.1% (v / v) Tween-20. Blocking treatment was performed in the solution. Primary antibodies and concentrations used in the test are as follows: anti-CHOP antibody (1: 1000, Cell Signaling), anti-GRP78 antibody (1: 1000, Cell Signaling), anti-XBP1s antibody (1: 1000, BioLegend) Anti-β-actin antibody (1: 1000, Santa Cruz). Blocked PVDF was incubated with each primary antibody at 4 ° C. overnight, washed, and then incubated with an HRP-conjugated secondary antibody (Abcam) derived from mouse, rabbit or goat. Protein bands were detected with ECL (Enhanced chemiluminescent: Pierce) and ChemiDoc MP system (Biorad).
（５）各mRNAを標的とした定量的PCR
　Caco-2単層をTRIzol（Life technologies社）で溶解後、RNeasy kit（Qiagen社）に供することでRNAを抽出した。得られたRNAの濃度をNanodrop（Thermo Scientific社）で測定し、gDNA（Genomic DNA）のコンタミネーションが無いことをβ-actinに対するPCRを実施することで確認した。１μｇのRNAを鋳型とし、iScript cDNA synthesis kit（BioRad社）を用いてcDNAを合成した。得られたcDNAをnuclease free H2Oを用いて５ｎｇ/μＬに調製し、２μＬ（１０ｎｇ）を３８４穴プレートを用いたTaqMan gene expression assay（Applied Biosystems 社）に供してRT-qPCRを実施した。各mRNAの発現レベルは、ΔΔCt 法により、レファレンス遺伝子であるTbpに対する相対的な発現量を求めることで比較した。解析に用いたプローブは以下の通り：Tbp; Hs00427620_mL、GRP78; Hs00607129_gH、CHOP; Hs00358796_gL、ERdj4; Hs01052402_mL。 (5) Quantitative PCR targeting each mRNA
After dissolving the Caco-2 monolayer with TRIzol (Life technologies), RNA was extracted by using an RNeasy kit (Qiagen). The concentration of the obtained RNA was measured with Nanodrop (Thermo Scientific), and it was confirmed by performing PCR on β-actin that there was no gDNA (Genomic DNA) contamination. Using 1 μg of RNA as a template, cDNA was synthesized using iScript cDNA synthesis kit (BioRad). The obtained cDNA was prepared to 5 ng / μL using nuclease free H 2 O, and 2 μL (10 ng) was subjected to TaqMan gene expression assay (Applied Biosystems) using a 384-well plate to perform RT-qPCR. The expression level of each mRNA was compared by calculating the relative expression level with respect to Tbp, which is a reference gene, by the ΔΔCt method. The probes used for the analysis were as follows: Tbp; Hs00427620_mL, GRP78; Hs00607129_gH, CHOP; Hs00358796_gL, ERdj4; Hs01052402_mL.
Ｂ．結果
（１）ビフィドバクテリウム属細菌の小胞体ストレスに対する効果
　ｉ）小胞体ストレス誘導がＣａｃｏ－２単層の膜透過性に及ぼす影響についての検証
　Ｃａｃｏ－２単層の膜透過性が小胞体ストレスにより亢進するか否かを検証した。小胞体ストレス誘導物質であるツニカマイシンを１０μｇ／ｍＬの濃度でＣａｃｏ－２単層の頂端側に添加した結果、ＴＥＥＲ値が漸次低下し、４８時間インキュベート後には初期値の３２％に至った（図１）。本結果は、頂端側への１０μｇ／ｍＬのツニカマイシン添加により、Ｃａｃｏ－２単層の膜透過性が亢進したことを示している。小胞体ストレスの誘導が、実際の腸管上皮の透過性に影響を及ぼす可能性が示唆された。 B. Results (1) Effect of Bifidobacterium on endoplasmic reticulum stress i) Verification of the effect of endoplasmic reticulum stress induction on the membrane permeability of Caco-2 monolayer The membrane permeability of Caco-2 monolayer is the endoplasmic reticulum It was verified whether it was enhanced by stress. As a result of adding tunicamycin, an endoplasmic reticulum stress inducer, to the apical side of the Caco-2 monolayer at a concentration of 10 μg / mL, the TEER value gradually decreased and reached 32% of the initial value after 48 hours of incubation (FIG. 1). This result shows that the membrane permeability of the Caco-2 monolayer was enhanced by the addition of 10 μg / mL tunicamycin to the apical side. It was suggested that induction of endoplasmic reticulum stress may affect the permeability of actual intestinal epithelium.
　ツニカマイシンの刺激によるＣａｃｏ－２単層の膜透過性の亢進が、小胞体ストレスマーカーの変動と伴っているかを検証するため、Ｇｒｐ７８（小胞体ストレスマーカーの一種）の発現をウェスタンブロッティングで評価した。Ｃａｃｏ－２単層を１０μｇ／ｍＬのツニカマイシンで頂端側から種々の時間刺激した後、総細胞の溶解液を調製した。この溶解液を、βＡｃｔｉnの発現をローディング・コントロール（ウェスタンブロッティングにおける陽性対照）としてウェスタンブロッティングで分析した結果、ツニカマイシン刺激の開始から２４時間後および４８時間後にＧｒｐ７８タンパクの発現量は顕著に増大した（図２）。なお、図１に示した通り、これらの刺激時間では、Ｃａｃｏ－２単層の透過性の亢進が認められている。 In order to verify whether the increase in membrane permeability of Caco-2 monolayers by stimulation with tunicamycin was accompanied by changes in the endoplasmic reticulum stress marker, the expression of Grp78 (a type of endoplasmic reticulum stress marker) was evaluated by Western blotting. A Caco-2 monolayer was stimulated with 10 μg / mL tunicamycin from the apical side for various times, and a total cell lysate was prepared. This lysate was analyzed by Western blotting for βActin expression as a loading control (positive control in Western blotting). As a result, the expression level of Grp78 protein was remarkably increased 24 hours and 48 hours after the start of tunicamycin stimulation ( Figure 2). As shown in FIG. 1, the Caco-2 monolayer increased in permeability during these stimulation times.
　以上の結果は、Ｃａｃｏ－２単層を１０μｇ／ｍＬツニカマイシンで刺激したときの膜透過性の亢進が小胞体ストレスの誘導と関連していることを示している。 These results indicate that the increase in membrane permeability when Caco-2 monolayer was stimulated with 10 μg / mL tunicamycin is associated with the induction of endoplasmic reticulum stress.
　ii）小胞体ストレス誘導によって引き起こされるＣａｃｏ－２単層の膜透過性の亢進にビフィドバクテリウム属細菌が与える効果の検証
　ｉ）では、小胞体ストレスを誘導するとＣａｃｏ－２単層の膜透過性が亢進することを明らかにした。続いて、Ｃａｃｏ－２単層をビフィドバクテリウム・ブレーベＹＩＴ１０００１株と前培養することにより、この過程がどのような影響を受けるか検証した。加熱死菌体の添加量を種々の濃度で検討し、１０9／ｍＬを至適試験菌数と決定した。Ｃａｃｏ－２単層に同濃度（１０9／ｍＬ）のビフィドバクテリウム・ブレーベＹＩＴ１０００１株の加熱死菌体を添加し１２時間前培養した後、ツニカマイシンを１０μｇ／ｍＬの濃度でＣａｃｏ－２単層の頂端側に添加し、小胞体ストレスの誘導を開始した。菌体と前培養していない対照のＣａｃｏ－２単層では、ツニカマイシン添加４８時間後にＴＥＥＲ値が初期値の２７％にまで低下した。一方、ビフィドバクテリウム・ブレーベＹＩＴ１０００１株と前培養したＣａｃｏ－２単層ではツニカマイシン添加４８時間後においてもＴＥＥＲ値が初期値の４６％に留まった（図３）。以上より、ツニカマイシンを４８時間作用させて誘導した小胞体ストレスによるＣａｃｏ－２単層の膜透過性の亢進が、ビフィドバクテリウム・ブレーベＹＩＴ１０００１株加熱死菌体との１２時間の前培養により有意に抑制されることが明らかとなった。データは示さないが、Ｃａｃｏ－２単層をビフィドバクテリウム・ブレーベＹＩＴ１０００１株と前培養後にツニカマイシンの溶解に用いた溶媒のみを添加してもＴＥＥＲ値に変化は認められなかった。本結果は、ビフィドバクテリウム・ブレーベＹＩＴ１０００１株が平常時のＣａｃｏ－２単層の膜透過性に影響を与えないことを示している。 ii) Examination of the effect of Bifidobacterium on the enhancement of membrane permeability of Caco-2 monolayer caused by endoplasmic reticulum stress induction In i), when endoplasmic reticulum stress is induced, the membrane permeability of Caco-2 monolayer It was revealed that sex was enhanced. Subsequently, the influence of this process was verified by pre-culturing the Caco-2 monolayer with the Bifidobacterium breve YIT10001 strain. The addition amount of the heated dead cells was examined at various concentrations, and 10 9 / mL was determined as the optimum number of test bacteria. Heat-dead cells of Bifidobacterium breve YIT10001 strain at the same concentration (10 9 / mL) were added to the Caco-2 monolayer and pre-cultured for 12 hours, and then tunicamycin was added at a concentration of 10 μg / mL. Added to the apical side of the layer to initiate the induction of endoplasmic reticulum stress. In the control Caco-2 monolayer that was not precultured with the cells, the TEER value decreased to 27% of the initial value 48 hours after the addition of tunicamycin. On the other hand, in the Caco-2 monolayer pre-cultured with Bifidobacterium breve YIT10001, the TEER value remained at 46% of the initial value even 48 hours after the addition of tunicamycin (FIG. 3). From the above, enhancement of membrane permeability of Caco-2 monolayer by endoplasmic reticulum stress induced by tunicamycin acting for 48 hours was significant by pre-culture for 12 hours with Bifidobacterium breve YIT10001 heated dead cells. It became clear that it was suppressed. Although no data is shown, no change was observed in the TEER value even when the Caco-2 monolayer was added to the Bifidobacterium breve YIT10001 strain alone and the solvent used to dissolve tunicamycin was added after pre-culture. This result shows that the Bifidobacterium breve YIT10001 strain does not affect the membrane permeability of the normal Caco-2 monolayer.
　iii)ツニカマイシンで誘導したＣａｃｏ－２単層の膜透過性の亢進に対するビフィドバクテリウム・ブレーベＹＩＴ１０００１株の抑制効果メカニズムの検証
　ツニカマイシン刺激によるＣａｃｏ－２単層の膜透過性の亢進に対するビフィドバクテリウム・ブレーベＹＩＴ１０００１株の抑制効果のメカニズムを評価するため、本菌が単層頂端側においてツニカマイシンの取り込みを阻害している可能性を検討した。ツニカマイシンをＣａｃｏ－２単層の基底側に添加し、頂端側の菌体がツニカマイシンの取り込みに与え得る影響を排除してＴＥＥＲ試験を行った。１０9／ｍＬのビフィドバクテリウム・ブレーベＹＩＴ１０００１株とＣａｃｏ－２単層を前培養した場合には、基底側からのツニカマイシンの刺激により誘導された膜透過性の亢進を抑制する傾向（ビフィドバクテリウム・ブレーベＹＩＴ１０００１株処置および対照でのツニカマイシン刺激４８時間後の各ＴＥＥＲ値は初期値の６９％および５５％）が認められた（図４）。ビフィドバクテリウム・ブレーベＹＩＴ１０００１株が空間的に小胞体ストレス誘導物質から離れていてもツニカマイシン刺激による膜透過性の亢進を抑制したことから、本菌の効果が菌体による化学物質の取り込みの阻害によるものではないことが示唆された。 iii) Verification of the inhibitory effect mechanism of Bifidobacterium breve YIT10001 on enhancement of membrane permeability of Caco-2 monolayer induced by tunicamycin Bifidobacteria on enhancement of membrane permeability of Caco-2 monolayer by tunicamycin stimulation In order to evaluate the mechanism of the inhibitory effect of the Umbreve YIT10001 strain, the possibility that this bacterium inhibits the uptake of tunicamycin on the monolayer apex side was examined. Tunermycin was added to the basal side of the Caco-2 monolayer, and the TEER test was performed excluding the effect that apical cells could have on tunicamycin uptake. In the case where 10 9 / mL Bifidobacterium breve YIT10001 strain and Caco-2 monolayer were precultured, the tendency to suppress the increase in membrane permeability induced by tunicamycin stimulation from the basal side (Bifido Bacterium breve YIT10001 treatment and the control were found to have a TEER value of 69% and 55% of the initial value 48 hours after tunicamycin stimulation (FIG. 4). Since Bifidobacterium breve YIT10001 strain spatially separated from the endoplasmic reticulum stress inducer, the enhancement of membrane permeability induced by tunicamycin was suppressed, so the effect of this bacterium was the inhibition of chemical uptake by the microbial cell It was suggested that it was not due to.
（２）ビフィドバクテリウム・ブレーベＹＩＴ１０００１株以外のビフィドバクテリウム属細菌の小胞体ストレスに対する作用
　前記のii）と同様にして、ビフィドバクテリウム・ブレーベＹＩＴ１２２７２株、ビフィドバクテリウム・アドレセンティスＹＩＴ４０１１株、ビフィドバクテリウム・ビフィダムＹＩＴ１０３４７株及びビフィドバクテリウム・ロンガムＹＩＴ４０２１株の小胞体ストレスによる膜透過性の亢進に対する抑制作用を検討した。
　その結果、図５に示すように、ビフィドバクテリウム属細菌が優れた小胞体ストレス抑制作用を示すことが判明した。 (2) Effect of Bifidobacterium genus bacteria other than Bifidobacterium breve YIT10001 on endoplasmic reticulum stress In the same manner as in ii) above, Bifidobacterium breve YIT12272, Bifidobacterium adrecentis YIT4011, Bifidobacterium bifidum YIT10347, and Bifidobacterium longum YIT4021 were examined for their inhibitory effects on the enhancement of membrane permeability caused by endoplasmic reticulum stress.
As a result, as shown in FIG. 5, it was found that the Bifidobacterium genus shows an excellent endoplasmic reticulum stress inhibitory action.
　また、ビフィドバクテリウム属細菌が小胞体ストレスマーカーの発現を抑制するか否かを検証するため、ＣＨＯＰタンパク（小胞体ストレスマーカーの一種）の発現をウェスタンブロットで解析した。その結果、１０9／ｍＬのビフィドバクテリウム・アドレセンティスＹＩＴ４０１１株及びビフィドバクテリウム・ビフィダムＹＩＴ１０３４７株と１２時間前培養したＣａｃｏ－２単層内では、ツニカマイシン刺激開始から６時間後および１２時間後のＣＨＯＰタンパクの発現が抑制されることが明らかとなった（図６）。
　さらに、データは示さなかったが、本発明の菌株が、他の小胞体ストレスマーカー（ＧＲＰ７８、ＸＢＰ１ｓ）の発現を抑制することを確認した。 In addition, in order to verify whether Bifidobacterium bacteria suppress the expression of the endoplasmic reticulum stress marker, the expression of CHOP protein (a kind of endoplasmic reticulum stress marker) was analyzed by Western blot. As a result, in a Caco-2 monolayer pre-cultured with 10 9 / mL of Bifidobacterium adrecentis YIT4011 and Bifidobacterium bifidum YIT10347, 6 hours and 12 hours after the start of tunicamycin stimulation It was revealed that the later expression of CHOP protein was suppressed (FIG. 6).
Furthermore, although data were not shown, it confirmed that the strain of this invention suppressed the expression of another endoplasmic reticulum stress marker (GRP78, XBP1s).
（３）種々の小胞体ストレスマーカーの発現に対する作用
　データは示さなかったが、各mRNAを標的とした定量的PCRの結果においても、本発明の菌株が、種々の小胞体ストレスマーカー（ＣＨＯＰ、ＧＲＰ７８、ＥＲｄｊ４）の発現を抑制することを確認した。 (3) Action on the expression of various endoplasmic reticulum stress markers Although data was not shown, the strain of the present invention also showed various endoplasmic reticulum stress markers (CHOP, GRP78) in the results of quantitative PCR targeting each mRNA. , It was confirmed to suppress the expression of ERdj4).
実施例２（ＵＧＧＴ発現促進作用）
（１）Ｃａｃｏ－２単層をＴＲＩｚｏｌ（Ｌｉｆｅ　ｔｅｃｈｎｏｌｏｇｉｅｓ社）で溶解後、ＲＮｅａｓｙ　ｋｉｔ（Ｑｉａｇｅｎ社）に供することでＲＮＡを抽出した。得られたＲＮＡの濃度をＮａｎｏｄｒｏｐ（Ｔｈｅｒｍｏ　Ｓｃｉｅｎｔｉｆｉｃ社）で測定し、ｇＤＮＡ（Genomic DNA）のコンタミネーションが無いことをβ－ａｃｔｉｎに対するＰＣＲを実施することで確認した。１μｇのＲＮＡを鋳型とし、ｉＳｃｒｉｐｔ　ｃＤＮＡ　ｓｙｎｔｈｅｓｉｓ　ｋｉｔ（ＢｉｏＲａｄ社）を用いてｃＤＮＡを合成した。得られたｃＤＮＡをｎｕｃｌｅａｓｅ　ｆｒｅｅ　Ｈ2Ｏを用いて５ｎｇ／μｌに調製し、２μｌ（１０ｎｇ）を３８４穴プレートを用いたＴａｑＭａｎ　ｇｅｎｅ　ｅｘｐｒｅｓｓｉｏｎ　ａｓｓａｙ（Ａｐｐｌｉｅｄ　Ｂｉｏｓｙｓｔｅｍｓ社）に供してＲＴ－ｑＰＣＲを実施した。各ｍＲＮＡの発現レベルは、ΔΔＣｔ法により、レファレンス遺伝子であるＴｂｐに対する相対的な発現量を求めることで比較した。解析に用いたプローブは以下の通り：Tbp; Hs00427620_m1、UGGT; Hs00917255_m1。 Example 2 (UGGT expression promoting action)
(1) The Caco-2 monolayer was dissolved in TRIzol (Life technologies) and then subjected to RNeasy kit (Qiagen) to extract RNA. The concentration of the obtained RNA was measured with Nanodrop (Thermo Scientific), and it was confirmed by performing PCR on β-actin that there was no contamination of gDNA (Genomic DNA). Using 1 μg of RNA as a template, cDNA was synthesized using iScript cDNA synthesis kit (BioRad). The obtained cDNA was prepared to 5 ng / μl using nuclease free H 2 O, and 2 μl (10 ng) was subjected to TaqMan gene expression assay (Applied Biosystems) using a 384-well plate, and RT-qPCR was performed. The expression level of each mRNA was compared by calculating the relative expression level with respect to the reference gene Tbp by the ΔΔCt method. The probes used for the analysis were as follows: Tbp; Hs00427620_m1, UGGT; Hs00917255_m1.
（２）糖タンパク質の折り畳みは、小胞体内の「Ｃａｌｎｅｘｉｎ／Ｃａｌｒｅｔｉｃｕｌｉｎ（ＣＮＸ／ＣＲＴ）サイクル」と呼ばれる経路上で行われることが知られている。小胞体内に糖タンパク質の基となるペプチド鎖が合成されると、第一に、Ｎ－ｘ－Ｓ／Ｔというアミノ酸配列のアスパラギン（Ｎ）に、グルコース残基を末端に持つマンノース糖鎖（Ｇｌｃ１Ｍａｎ５－９ＧｌｃＮＡｃ２）が付加される。続いて、本オリゴ糖が付加されたペプチド鎖は、ＣａｌｎｅｘｉｎやＣａｌｒｅｔｉｃｕｌｉｎをはじめとする分子シャペロンによる認識を受け、折り畳みが開始される。折り畳みがある程度完了すると、オリゴ糖末端のグルコースはＧｌｕｃｏｓｉｄａｓｅ　ＩＩと呼ばれる酵素により除去され、タンパク質は分子シャペロンから解離する。この段階で（ｉ）糖タンパクの折り畳みが正常に完了していた場合、タンパク質は小胞体外へと排出され、場合によってはゴルジ体にて更なる修飾を受けた後、細胞外や細胞膜上へと輸送される。一方、折り畳みが不完全だった場合、糖タンパク質は(ii）不良タンパク分解因子の補助を受けながら分解機構へと輸送されるか、（iii）ＵＧＧＴ（ＵＤＰ－ｇｌｕｃｏｓｅ：ｇｌｙｃｏｐｒｏｔｅｉｎ　ｇｌｕｃｏｓｙｌｔｒａｎｓｆｅｒａｓｅ）と呼ばれる酵素によりオリゴ糖鎖の末端に再びグルコース残基が付加されることで、再度タンパク質の折り畳みサイクルへと還元される。このように、Ｇｌｕｃｏｓｉｄａｓｅ　ＩＩおよびＵＧＧＴにより駆動されるサイクルが、ＣＮＸ／ＣＲＴサイクルである。 (2) It is known that glycoprotein folding is performed on a pathway called “Calnexin / Caleticulin (CNX / CRT) cycle” in the endoplasmic reticulum. When a peptide chain that is the basis of a glycoprotein is synthesized in the endoplasmic reticulum, first, asparagine (N) having an amino acid sequence of NxS / T is bound to a mannose sugar chain having a glucose residue at its end ( Glc1Man5-9GlcNAc2) is added. Subsequently, the peptide chain to which the oligosaccharide has been added is recognized by molecular chaperones such as Calnexin and Calreticulin, and starts to fold. When folding is completed to some extent, glucose at the oligosaccharide end is removed by an enzyme called Glucosidase II, and the protein dissociates from the molecular chaperone. At this stage (i) if glycoprotein folding has been completed normally, the protein is excreted from the endoplasmic reticulum, and in some cases after further modification in the Golgi apparatus, it is then extracellularly or onto the cell membrane. And will be transported. On the other hand, when the folding is incomplete, the glycoprotein is transported to the degradation mechanism with the help of a defective proteolytic factor (ii) or (iii) by an enzyme called UGGT (UDP-glucose: glycoprotein glucosyltransferase) By re-adding a glucose residue to the end of the oligosaccharide chain, it is reduced again to the protein folding cycle. Thus, the cycle driven by Glucosidase II and UGGT is the CNX / CRT cycle.
　実施例１と同条件でビフィズス菌の加熱死菌体と１２時間前培養したＣａｃｏ－２単層内におけるＵＧＧＴのｍＲＮＡ発現を解析した結果、Ｂ．ｂｒｅｖｅ　ＹＩＴ　１２２７２、Ｂ．ａｄｏｌｅｓｃｅｎｔｉｓ　ＹＩＴ　４０１１、Ｂ．ｂｉｆｉｄｕｍ　ＹＩＴ　１０３４７がＵＧＧＴのｍＲＮＡ発現を亢進させることが明らかとなった（図７）。本結果は、ビフィズス菌の加熱死菌体と共培養したＣａｃｏ－２単層内では、ＵＧＧＴの発現が誘導されることで、折り畳み不全のタンパクを折り畳み機構へと還元させる経路が増強されていることを示唆している。ビフィズス菌は、本効果を一因としてＣａｃｏ－２単層の小胞体ストレスに対する耐性を向上させ、単層内での同ストレスを抑制したことが推察された。 As a result of analysis of mRNA expression of UGGT in a Caco-2 monolayer pre-cultured for 12 hours with a heat-killed Bifidobacterium cell under the same conditions as in Example 1. breve YIT 12272, B.E. adolescentis YIT 4011, B.E. It was revealed that bifidum YIT 10347 enhances UGGT mRNA expression (FIG. 7). This result shows that, in Caco-2 monolayers co-cultured with heat-killed cells of bifidobacteria, the expression of UGGT is induced, thereby enhancing the pathway for reducing a protein with a folding failure to a folding mechanism. Suggests that. It was inferred that Bifidobacteria improved the resistance of Caco-2 monolayer to endoplasmic reticulum stress and suppressed the stress in the monolayer due to this effect.

Claims (12)

  1. 　ビフィドバクテリウム属細菌及び／又はその菌体処理物を有効成分とする小胞体ストレス抑制剤。 An endoplasmic reticulum stress inhibitor comprising a Bifidobacterium genus and / or a treated product thereof as an active ingredient.
  2. 　ビフィドバクテリウム属細菌が、ビフィドバクテリウム・ブレーベ、ビフィドバクテリウム・アドレセンティス、ビフィドバクテリウム・ビフィダム及びビフィドバクテリウム・ロンガムから選ばれる１種以上である請求項１記載の小胞体ストレス抑制剤。 2. The small Bifidobacterium bacterium is one or more selected from Bifidobacterium breve, Bifidobacterium adrecentis, Bifidobacterium bifidum and Bifidobacterium longum. ER stress inhibitor.
  3. 　ビフィドバクテリウム属細菌が、ビフィドバクテリウム・ブレーベＹＩＴ１２２７２株（ＦＥＲＭ　ＢＰ－１１３２０）、ビフィドバクテリウム・ブレーベＹＩＴ１０００１株（ＦＥＲＭ　ＢＰ－８２０５）、ビフィドバクテリウム・アドレセンティスＹＩＴ４０１１株（ＡＴＣＣ１５７０３）、ビフィドバクテリウム・ビフィダムＹＩＴ１０３４７株（ＦＥＲＭ　ＢＰ－１０６１３）及びビフィドバクテリウム・ロンガムＹＩＴ４０２１株（ＡＴＣＣ１５７０７）から選ばれる１種以上である請求項１又は２記載の小胞体ストレス抑制剤。 Bifidobacterium genus bacteria include Bifidobacterium breve YIT12272 (FERM BP-11320), Bifidobacterium breve YIT10001 (FERM BP-8205), Bifidobacterium adrecentis YIT4011 (ATCC15703) The endoplasmic reticulum stress inhibitor according to claim 1 or 2, which is one or more selected from Bifidobacterium bifidum strain YIT10347 (FERM BP-10613) and Bifidobacterium longum YIT4021 (ATCC15707).
  4. 　小胞体ストレス抑制剤製造のための、ビフィドバクテリウム属細菌及び／又はその菌体処理物の使用。 Use of Bifidobacterium bacteria and / or treated cells thereof for the production of an endoplasmic reticulum stress inhibitor.
  5. 　ビフィドバクテリウム属細菌が、ビフィドバクテリウム・ブレーベ、ビフィドバクテリウム・アドレセンティス、ビフィドバクテリウム・ビフィダム及びビフィドバクテリウム・ロンガムから選ばれる１種以上である請求項４記載の使用。 The use according to claim 4, wherein the Bifidobacterium is one or more selected from Bifidobacterium breve, Bifidobacterium adrecentis, Bifidobacterium bifidum and Bifidobacterium longum. .
  6. 　ビフィドバクテリウム属細菌が、ビフィドバクテリウム・ブレーベＹＩＴ１２２７２株（ＦＥＲＭ　ＢＰ－１１３２０）、ビフィドバクテリウム・ブレーベＹＩＴ１０００１株（ＦＥＲＭ　ＢＰ－８２０５）、ビフィドバクテリウム・アドレセンティスＹＩＴ４０１１株（ＡＴＣＣ１５７０３）、ビフィドバクテリウム・ビフィダムＹＩＴ１０３４７株（ＦＥＲＭ　ＢＰ－１０６１３）及びビフィドバクテリウム・ロンガムＹＩＴ４０２１株（ＡＴＣＣ１５７０７）から選ばれる１種以上である請求項４又は５記載の使用。 Bifidobacterium genus bacteria include Bifidobacterium breve YIT12272 (FERM BP-11320), Bifidobacterium breve YIT10001 (FERM BP-8205), Bifidobacterium adrecentis YIT4011 (ATCC15703) The use according to claim 4 or 5, which is one or more selected from Bifidobacterium bifidum YIT10347 strain (FERM BP-10613) and Bifidobacterium longum YIT4021 strain (ATCC15707).
  7. 　小胞体ストレスを抑制するための、ビフィドバクテリウム属細菌及び／又はその菌体処理物。 Bifidobacterium genus and / or a treated product thereof for suppressing endoplasmic reticulum stress.
  8. 　ビフィドバクテリウム属細菌が、ビフィドバクテリウム・ブレーベ、ビフィドバクテリウム・アドレセンティス、ビフィドバクテリウム・ビフィダム及びビフィドバクテリウム・ロンガムから選ばれる１種以上である請求項７記載の細菌及び／又はその菌体処理物。 8. The bacterium according to claim 7, wherein the bacterium belonging to the genus Bifidobacterium is at least one selected from Bifidobacterium breve, Bifidobacterium adrecentis, Bifidobacterium bifidum and Bifidobacterium longum. And / or a treated product thereof.
  9. 　ビフィドバクテリウム属細菌が、ビフィドバクテリウム・ブレーベＹＩＴ１２２７２株（ＦＥＲＭ　ＢＰ－１１３２０）、ビフィドバクテリウム・ブレーベＹＩＴ１０００１株（ＦＥＲＭ　ＢＰ－８２０５）、ビフィドバクテリウム・アドレセンティスＹＩＴ４０１１株（ＡＴＣＣ１５７０３）、ビフィドバクテリウム・ビフィダムＹＩＴ１０３４７株（ＦＥＲＭ　ＢＰ－１０６１３）及びビフィドバクテリウム・ロンガムＹＩＴ４０２１株（ＡＴＣＣ１５７０７）から選ばれる１種以上である請求項７又は８記載の細菌及び／又はその菌体処理物。 Bifidobacterium genus bacteria include Bifidobacterium breve YIT12272 (FERM BP-11320), Bifidobacterium breve YIT10001 (FERM BP-8205), Bifidobacterium adrecentis YIT4011 (ATCC15703) The bacterium according to claim 7 or 8, and / or treatment of the microbial cell thereof, wherein the bacterium is one or more selected from Bifidobacterium bifidum YIT10347 (FERM BP-10613) and Bifidobacterium longum YIT4021 (ATCC15707) object.
  10. 　ビフィドバクテリウム属細菌及び／又はその菌体処理物の有効量を投与することを特徴とする、小胞体ストレスの抑制方法。 A method for suppressing endoplasmic reticulum stress, comprising administering an effective amount of a Bifidobacterium bacterium and / or a treated product thereof.
  11. 　ビフィドバクテリウム属細菌が、ビフィドバクテリウム・ブレーベ、ビフィドバクテリウム・アドレセンティス、ビフィドバクテリウム・ビフィダム及びビフィドバクテリウム・ロンガムから選ばれる１種以上である請求項１０記載の方法。 The method according to claim 10, wherein the Bifidobacterium genus bacterium is one or more selected from Bifidobacterium breve, Bifidobacterium adrecentis, Bifidobacterium bifidum and Bifidobacterium longum. .
  12. 　ビフィドバクテリウム属細菌が、ビフィドバクテリウム・ブレーベＹＩＴ１２２７２株（ＦＥＲＭ　ＢＰ－１１３２０）、ビフィドバクテリウム・ブレーベＹＩＴ１０００１株（ＦＥＲＭ　ＢＰ－８２０５）、ビフィドバクテリウム・アドレセンティスＹＩＴ４０１１株（ＡＴＣＣ１５７０３）、ビフィドバクテリウム・ビフィダムＹＩＴ１０３４７株（ＦＥＲＭ　ＢＰ－１０６１３）及びビフィドバクテリウム・ロンガムＹＩＴ４０２１株（ＡＴＣＣ１５７０７）から選ばれる１種以上である請求項１０又は１１記載の方法。 Bifidobacterium genus bacteria include Bifidobacterium breve YIT12272 (FERM BP-11320), Bifidobacterium breve YIT10001 (FERM BP-8205), Bifidobacterium adrecentis YIT4011 (ATCC15703) The method according to claim 10 or 11, which is one or more selected from Bifidobacterium bifidum YIT10347 strain (FERM BP-10613) and Bifidobacterium longum YIT4021 strain (ATCC15707).
PCT/JP2015/082424 2014-11-19 2015-11-18 Endoplasmic reticulum stress inhibitor WO2016080449A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2016560271A JP6670251B2 (en) 2014-11-19 2015-11-18 ER stress inhibitor

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2014-234388 2014-11-19
JP2014234388 2014-11-19

Publications (1)

Publication Number Publication Date
WO2016080449A1 true WO2016080449A1 (en) 2016-05-26

Family

ID=56013983

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2015/082424 WO2016080449A1 (en) 2014-11-19 2015-11-18 Endoplasmic reticulum stress inhibitor

Country Status (2)

Country Link
JP (1) JP6670251B2 (en)
WO (1) WO2016080449A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019087280A1 (en) * 2017-10-31 2019-05-09 森永乳業株式会社 Muscle-building composition

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0859492A (en) * 1994-08-26 1996-03-05 Yakult Honsha Co Ltd Antidiabetic drug
JPH1132763A (en) * 1997-07-14 1999-02-09 Yakult Honsha Co Ltd Monoclonal antibody for bifidobacterium bifidum
JP2009242430A (en) * 2009-07-17 2009-10-22 Snow Brand Milk Prod Co Ltd Prevention, improvement and therapeutic agent of metabolic disorder disease with age
WO2012043532A1 (en) * 2010-10-01 2012-04-05 株式会社ヤクルト本社 Fermented food and method for producing same
WO2013144080A1 (en) * 2012-03-30 2013-10-03 Nestec S.A. 4-oxo-2-pentenoic acid and cardiovascular health

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0859492A (en) * 1994-08-26 1996-03-05 Yakult Honsha Co Ltd Antidiabetic drug
JPH1132763A (en) * 1997-07-14 1999-02-09 Yakult Honsha Co Ltd Monoclonal antibody for bifidobacterium bifidum
JP2009242430A (en) * 2009-07-17 2009-10-22 Snow Brand Milk Prod Co Ltd Prevention, improvement and therapeutic agent of metabolic disorder disease with age
WO2012043532A1 (en) * 2010-10-01 2012-04-05 株式会社ヤクルト本社 Fermented food and method for producing same
WO2013144080A1 (en) * 2012-03-30 2013-10-03 Nestec S.A. 4-oxo-2-pentenoic acid and cardiovascular health

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
HORMANNSPERGER G. ET AL.: "Molecular crosstalk of probiotic bacteria with the intestinal immune system: clinical relevance in the context of inflammatory bowel disease", INTERNATIONAL JOURNAL OF MEDICAL MICROBIOLOGY, vol. 300, no. 1, 2010, pages 63 - 73, XP026807104 *
LE, THI KIM CHUNG ET AL.: "Oral administration of Bifidobacterium spp. improves insulin resistance, induces adiponectin, and prevents inflammatory adipokine expressions", BIOMEDICAL RESEARCH, vol. 35, no. 5, October 2014 (2014-10-01), pages 303 - 310 *
NAOHIRO EGAWA ET AL.: "Shohotai Stress to Shikkan Shohotai Stress to Shinkei Shikkan", GEKKAN MEDICAL SCIENCE DIGEST, vol. 33, no. 13, 2007, pages 1217 - 1220 *
NOZOMI KONO ET AL.: "Saturated fatty acid and unfolded protein response", JOURNAL OF CLINICAL AND EXPERIMENTAL MEDICINE, vol. 248, no. 13, March 2014 (2014-03-01), pages 1178 - 1183 *
TAKANORI KUMAGAI ET AL.: "ER stress and kidney diseases", THE CELL, vol. 45, no. 4, 2013, pages 184 - 188 *
TETSUO MINAMINO ET AL.: "ER stress in cardiovascular diseases", THE CELL, vol. 45, no. 4, 2013, pages 180 - 183 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019087280A1 (en) * 2017-10-31 2019-05-09 森永乳業株式会社 Muscle-building composition
CN111212575A (en) * 2017-10-31 2020-05-29 森永乳业株式会社 Composition for muscle building
JPWO2019087280A1 (en) * 2017-10-31 2020-10-22 森永乳業株式会社 Composition for muscle gain
JP7193469B2 (en) 2017-10-31 2022-12-20 森永乳業株式会社 Muscle building composition

Also Published As

Publication number Publication date
JPWO2016080449A1 (en) 2017-08-24
JP6670251B2 (en) 2020-03-18

Similar Documents

Publication Publication Date Title
EP2615163B1 (en) Composition comprising probiotics having aryl hydrocarbon receptor activating potency for use as anti-inflammatory agents
JP2019500004A (en) Use of pasteurized Ackermancia to treat metabolic disorders
CN106103696B (en) Novel lactobacillus paracasei strain
JP5066091B2 (en) Bacterial strain having antiallergic activity and beverage, food and antiallergic agent containing the bacterial body
US10463704B2 (en) Composition comprising Lactobacillus plantarum
JP5688376B2 (en) Oral DNA damage repair promoter and elastase activity inhibitor
MX2012009799A (en) Method for constructing novel bacterium belonging to the genus bifidobacterium.
WO2022068545A1 (en) Bifidobacterium longum for preventing and/or treating idiopathic hypertension
Motey et al. Probiotic potential of Saccharomyces cerevisiae and Kluyveromyces marxianus isolated from West African spontaneously fermented cereal and milk products
JP6679202B2 (en) Type IV allergy composition
TW201540307A (en) Anti-glycosylation agent, life-extending agent and anti-aging agent
US11752178B2 (en) Methods for the isolation of microbes with enhanced persistance and compositions with such microbes
JP6670251B2 (en) ER stress inhibitor
US20230330165A1 (en) Muscle atrophy prevention agent
WO2006077954A1 (en) Remedy for neurological disease
JPH0930981A (en) Immunopotentiative composition
WO2016080448A1 (en) Preventive and therapeutic agent for celiac disease
US8871195B2 (en) Bacteria thyA(−) mutants with increased vitamin K
TW201945016A (en) Composition for type I allergy
JP6381869B2 (en) Lactic acid bacteria derived from Tsuda turnip which have liver triglyceride reducing effect
US11207359B2 (en) Cholesterol reducing probiotic strain and use thereof
RU2797466C2 (en) Faecalibacterium prausnitzii and desulfovibrio piger for use in the treatment or prevention of diabetes and intestinal diseases
TW202340451A (en) Probiotic compositions for treatment of hair loss
KR20230121587A (en) Composition for preventing or treating fatty liver containing enterococcus faecalis, its culture broth or heat killed enterococcus faecalis
CN117618473A (en) Application of PB (bacterial strain) in preventing, improving and treating metabolic diseases

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15862043

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2016560271

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15862043

Country of ref document: EP

Kind code of ref document: A1