PART Ⅰ: Kev tswj hwm ntawm Synbiotic Lactobacillus Bulgaricus 6c3 Strain, Inulin Thiab Fructooligosaccharide txo qis cov concentration ntawm Indoxyl Sulfate thiab raum puas
Mar 23, 2022
ali.ma@wecistanche.com
AlonsoJerez-Morales, Jose S.Merino, Sindy T.Diaz-Castillo & et al.
1. Taw qhia
Mob raum mob(CKD) yog suav tias yog kev hloov pauv mus tas liraumqauv, nws txoj haujlwm, lossis, nyob rau hauv qhov hnyav tshaj plaws, ob qho tib si. CKD cuam tshuam ncaj qha rau kev noj qab haus huv thiab lub neej zoo ntawm tus neeg [1,2]. Hauv qib physiological, CKD ua rau muaj kev hloov pauv nquag pom yog nce qib ntshav ntawm creatinine, cystatin Cor ureic nitrogen [2]. Kev soj ntsuam nruj thiab kev paub txog cov pej xeem tau nce CKD kev kuaj mob mus txog, piv txwv li, kom txiav txim siab tias 15 feem pua ntawm cov neeg Asmeskas tau cuam tshuam los ntawm cov kab mob no [3]. Kev loj hlob ntawm tus kab mob, nrog rau cov teebmeem ntawm cov metabolites uas tau hais los saum toj no, muaj kev cuam tshuam loj heev hauv ntau lub cev ntawm lub cev, nrog rau cov hlab plawv thiab lub plab zom mov, ua rau muaj cov metabolites cuam tshuam rau kev tuag ntau dua qub. thiab cuam tshuam assimilation, motility, thiab permeability tom kawg [4,5]. Kev hloov hauv plab hnyuv provoke ib qho kev nqus ntawm cov metabolites hauv cov hnyuv uas ua rau muaj kev cuam tshuam ntawm cov kab mob hauv lub cev, uas ua rau muaj feem cuam tshuam rau cov kab mob microbiota ib txwm thiab nws cov haujlwm metabolic thiab tuaj yeem ua rau plab hnyuv dysbiosis [6].
Nyem rau cistanche phiv thiab Cistanche khoom rau lub raum
Kev koom tes ntawm CKD thiab plab hnyuv dysbiosis tej zaum yuav tsis tau piav qhia tsuas yog los ntawm kev hloov pauv hauv kev noj zaub mov, tab sis kuj, ntxiv rau, los ntawm kev nce ntawm luminal pH, theem nrab mus rau kev nce siab ntawm ammonium [7]. Cov neeg mob CKD kawg tau cim qhov sib txawv ntawm ntau cov kab mob hauv lawv cov quav thaum piv rau cov neeg noj qab haus huv [8]. Tshwj xeeb, ntawm cov kab mob genus, CKD cov neeg mob pom qhov nce ntawm Enterorhabdus, Blautia, Ruminococcus, Parasutterella, Bacteroides, Clostridium (sensu stricto), Escherichia, Shigella, thiab Allobaculum thiab txo qis ntawm Akkermansia, Bifidobacterium, ClostridiumLactocus. , Leuconostoc, Alkaliphilus, thiab Pseudomonas [9-11]. Los ntawm lub raum tsis zoo los yog los ntawm txoj hnyuv dysbiosis, muaj qhov txo qis ntawm cov kab mob uas muaj peev xwm ua kom metabolize thiab inhibit cov kab mob uas tsim cov tshuaj lom metabolites, uas ua rau lawv nce ntxiv [12]. Wong et al. [8] pom tau hais tias cov neeg mob raug kev txom nyem siab heev CKD tau pom tias muaj cov kab mob ntau ntxiv uas muaj cov enzymes xws li urease, uricase, thiab cov tsim p-cresol thiab indole.
Kev sib sau ntawm uremic retention metabolites (URMs) hauv cov ntshav, suav nrogindoxyl sulfate(IS), p-cresyl sulfate (pCS), triethylamine, thiab trimethylamine N-oxide (TMAO) [5] yog postulated li accelerators ntawm CKD [13]. YOG(Indoxyl sulfate), qhov zoo tshaj plaws-paub uremic indole, yog secreted los ntawm proximal convoluted tubules ntawm lubraumThiab nws pib los ntawm cov metabolism hauv tryptophan los ntawm cov kab mob tryptophanase. Cov pov thawj los ntawm cov qauv tsiaj qhia tau hais tias IS(Indoxyl sulfate)tuaj yeem cuam tshuam rau lub raum kev ua haujlwm thiab nws tuaj yeem ua rau cov hlwb ntawm cov tubules tsim oxidative kev nyuaj siab hauv endothelial hlwb, inhibiting kho mechanisms, thiab nyiam interstitial tubular fibrosis vim nws ua rau cov genic qhia ntawm TGF -1, ntawm cov ntaub so ntswg inhibitor ntawm metalloproteinases (TIMP. -1) thiab collagen [14]. Txawm li cas los xij, qhov tsis txaus ntawm cov hnyuv microbiota, thiab kev tsim cov tshuaj lom metabolites tuaj yeem hloov kho. Nws tau raug piav qhia tias kev ntxiv nrog qee yam probiotics thiab prebiotics tuaj yeem txo qis kev tsim tawm ntawm qee yam uremic co toxins thiab kho cov microbiota li qub [5]. Piv txwv li, cov kev tshawb fawb meta-analysis tau xaus lus tias cov neeg mob CKD tau txais cov tshuaj ntxiv nrog cov kab mob probiotic nce lawv cov tshuaj tiv thaiv cy-tokines thiab txo lawv cov qib ntawm uremic toxins [15,16]. Qee cov kev tshawb fawb tau tshaj tawm tias kev tswj hwm ntawm synbiotics, kev sib xyaw ua ke ntawm probiotics thiab prebiotics, muaj peev xwm txo qis kev loj hlob ntawm CKD nrog cov txiaj ntsig zoo [12].Hauv cov ntsiab lus ntawm kev ua haujlwm tam sim no, inulin-hom fructans yog cov tshuaj prebiotics vim lawv tau pom. lawv lub peev xwm los txo qhov kev loj hlob ntawm CKD [17] thiab txo URMs, xws li pCS thiab IS(Indoxyl sulfate)[18]. Panza et al. (2017) tau muab cov tshuaj probiotics L.acidophlilus thiab Bifidobacterium longum ntxiv rau fructooligosaccharides (FOS) hauv cov neeg mob uas muaj theem 2-5 CKD thiab tshaj tawm tias cov neeg mob tsis kho tau muaj pCS ntau dua thiab yog(Indoxyl sulfate)hauv cov ntshav tshaj cov neeg mob uas tau txais kev kho mob synbiotic [19]. Hauv kev ua haujlwm no, peb thawj zaug ntsuas lub peev xwm ntawm cov kab mob probiotic kom txo tau IS(Indoxyl sulfate)nyob rau hauv vitro los xaiv qhov pheej hmoo tshaj plaws. Tom qab ntawd, cov kab mob no tau ua ke nrog cov prebiotics inulin thiab fructooligosaccharides (FOS) kom tau txais cov tshuaj synbiotic. Cov prebiotics no tau raug xaiv vim lawv tau pom tias lawv tuaj yeem txo pCS thiab IS(Indoxyl sulfate)concentration hauv ntshav [17,18]. Thaum kawg, qhov ua tau zoo ntawm putative synbiotic raug kuaj hauv vivo, ntsuas ntshav IS(Indoxyl sulfate)thiab qib creatinine thiabraumthiab lub plawv puas siv tus qauv nas (Sprague-Dawley nas) suav nrograumdeficient (nephrectomized) thiab cov tsiaj tsis kho.
2. Cov txiaj ntsig
2.1.Isolation ntawm Strains muaj peev xwm txo tau IS(Indoxyl sulfate)Hauv Vitro
Los ntawm 36 cov qauv ntawm cov khoom siv mis nyuj, 84 hom kab mob raug cais tawm hauv MRS agar ntxiv nrog bromophenol xiav (MRS-BFB), siv los ua qhov sib txawv nruab nrab. Qhov nruab nrab no tso cai rau kev sib txawv ntawm cov kab mob lactic acid. Ntawm pH 3 pawg yuav xim daj thiab ntawm pH ze rau 5 lawv yuav xim xiav [19]. Tag nrho 84 hom kab mob cais tau raug sim hauv vitro los txiav txim siab lawv lub peev xwm los kho qhov txo qis ntawm IS(Indoxyl sulfate). Rau qhov no, hom kab mob tau coj mus rau hauv MRS broth ntxiv nrog 2.8 mM IS(Indoxyl sulfate)rau 48h, thiab tom qab ntawd IS(Indoxyl sulfate)tau ntsuas los ntawm kev ua tau zoo ntawm cov kua chromatography (HPLC) Cov kab mob lc2, 6c3, thiab VIc2 tuaj yeem txo qis (p<0.05) the="" concentration="" of="" the="" compound="" (table="" 1).="" furthermore,="" the="" three="" strains="" were="" cultured="" together="" for="" 48="" h="" in="" the="" presence="" of="">(Indoxyl sulfate)los soj ntsuam seb lawv puas tau sib koom ua ke kom txo tau IS(Indoxyl sulfate). Thaum tag nrho peb hom kab lis kev cai raug coj los ua ke nyob rau hauv lub xub ntiag ntawm IS(Indoxyl sulfate), qhov concentration ntawm IS(Indoxyl sulfate)kuj tseem txo qis tab sis tsawg dua qhov ua tiav los ntawm cov kab mob ib leeg, txiav txim siab qhov muaj peev xwm ntawm kev sib koom ua ke (Table1).
Table 1.Hauv vitro txo, yog tiasindoxyl sulfate(IS), raug soj ntsuam los ntawm high-performance kua chromatography (HPLC), kho los ntawm kab mob.
2.2. Kev txheeb xyuas cov kab mob uas tau xaiv
Peb hom kev xaiv vim lawv muaj peev xwm txo tau IS(Indoxyl sulfate)tau txheeb xyuas los ntawm 16S rRNA sequencing tom qab PCR amplification ntawm DNA.Strain 1c2 yog ib tug Gram-positive saw coccus uas, nyob rau hauv lub molecular tsom xam, amplified nrog lub LacZ primers rau Streptococcus thermophilus. Hom 6c3 thiab VIc2 yog ob qho tib si Gram-positive bacilli thiab nthuav dav nrog LbG primers los txheeb xyuas cov genus Lactobacillus. Kev tsom xam ntawm 16SrRNA sequencing tso cai rau txheeb xyuas cov kab mob lc2 li Streptococcus thermophilus (97.3 feem pua zoo; Nkag mus rau: AY188354.1), strain 6c3 li Lactobacillus bulgaricus (99.7 feem pua zoo sib xws; Access Number: CR954254.1) 99.5 feem pua zoo ib yam; Tus Naj Npawb: AY773947.1).Tsuas yog ib qho ntawm peb hom raug xaiv los npaj cov tshuaj synbiotic thiab ua raws li cov kev ntsuam xyuas hauv qab no. Streptococcus thermophilus 1c2 strain raug muab pov tseg vim nws pom tias muaj kev loj hlob tsawg. Los ntawm ob hom kab mob ntxiv, Lactobacillus bul-garicus 6c3 strain raug xaiv vim tias nws pom zoo probiotic functionality thiab kev nyab xeeb zoo dua li Lactobacillus acidophilus VIIc2 hom.
2.3. Cov nyhuv ntawm Synbiotic hauv ntshav Creatinine thiab IS(Indoxyl sulfate)Qib
Lub probiotic L. bulgaricus 6c3 tau lyophilized nyob rau hauv koom nrog cov prebiotics Inuline thiab FOS, yog qhov concentration ntawm probiotic 10 degree CFU / g ntawm lyophilized khoom. Kaum grams ntawm synbiotic tau muab tshuaj txhua hnub, hauv cov dej haus, rau cov pab pawg ntawm nephrectomized synbiotic kho Sprague-Dawley nas (Lac). Cov pab pawg nephrectomized untreated (Nef) thiab pawg tswj tsis-nephrectomized tsis tau txais cov syn-biotic. Serum creatinine thiab IS(Indoxyl sulfate)theem ntawm cov nas raug ntsuas los ntawm HPLC thaum pib (ib lub lis piam tom qab nephrectomy) thiab qhov kawg ntawm qhov kev xeem. Ntxiv mus, cov hematocrits ntawm cov tsiaj ntawm tag nrho cov pab pawg neeg raug kaw. Qhov sib txawv ntawm cov ntshav creatinine qib tom kawg ntawm qhov kev ntsuam xyuas tau txawv heev (p<0.05) in="" the="" nef="" and="" lac="" groups,increasing="" 127%and="" 121%,="" respectively,="" when="" compared="" to="" their="" initial="" values(figure="" 1).="" the="" control="" group="" showed="" a="" non-significant="" creatinine="" increase="" of="" only="" 2%.="" the="" increase="" of="" creatinine="" serum="" levels="" in="" the="" nef="" and="" lac="" groups="" was="" significantly="" higher="" than="" that="" of="" the="" control="" group="" but="" not="" significantly="" different="" between="" the="" nef="" and="" lac="" groups.="" nevertheless,="" all="" three="" groups="" maintained="" their="" creatinine="" levels="" within="" de="" normal="" limits="" for="" these="" rats.="" on="" the="" other="" hand,="" serum="">(Indoxyl sulfate)Cov qib nce siab tsuas yog hauv pawg Nef sib raug rau nephrectomized (lub raum tsis zoo) cov tsiaj tsis tau tswj hwm cov synbiotic (38.8 feem pua) (p<0.05).>(Indoxyl sulfate)qib tau pom qhov tsis tseem ceeb 0.5 feem pua nce hauv pawg tswj hwm thiab qhov tsis tseem ceeb 0.8 feem pua ntawm cov pab pawg Lac (Daim duab 2). Thaum cov pab pawg ntawm nephrectomized tsiaj tau muab piv, cov pab pawg tsis tau noj cov synbiotic (Nef) tau pom tias muaj qib siab ntawm IS.(Indoxyl sulfate)nyob rau hauv kev sib piv nrog cov pab pawg neeg uas noj cov synbiotic (Lac). Tsis muaj qhov sib txawv tseem ceeb ntawm pawg Lac thiab pawg tswj hwm. Kev tsom xam ntawm cov ntaub ntawv tshawb fawb txog kev sib raug zoo ntawm creatinine thiab IS(Indoxyl sulfate)qib tau pom tias qhov kev sib raug zoo no tsis muaj (Cov Khoom Siv Ntxiv: Figures S1-S3). Thaum kawg, cov txiaj ntsig ntawm hematocrits tsis pom qhov sib txawv ntawm peb pawg (Daim duab 3).
Daim duab 1. (a) Serum creatinine qib ntawm Sprague-Dawley nas thaum pib thiab qhov kawg ntawm 16 lub lis piam kev tswj hwm ntawm synbiotic. (b) Qhov sib txawv ntawm txoj kev rau qhov kawg ntawm creatinine concentrations nrog 95 feem pua Tukey ib txhij kev ntseeg siab ib ntus (Yog tias ib ntus tsis muaj xoom, qhov sib txuas txhais tau txawv heev). Nef: nephrectomized nas tsis tau txais cov synbiotic, Lac: nephrectomized nas tau txais cov synbiotic, Tswj: sham-nephrectomized nas.
Daim duab 2.(a) Serumindoxyl sulfate(IS) theem ntawm Sprague-Dawley nas thaum pib thiab thaum kawg ntawm 16 lub lis piam kev tswj hwm ntawm cov synbiotic.(b) Qhov txawv ntawm qhov txhais tau tias yog qhov kawg(Indoxyl sulfate)concentrations nrog 95 feem pua Tukey ib txhij kev ntseeg siab ib ntus (Yog hais tias ib ntus tsis muaj xoom, qhov sib txuas txhais tau txawv heev). Nef: nephrectomized nas tsis tau txais cov synbiotic, Lac: nephrectomized nas tau txais cov synbiotic, Tswj: sham-nephrectomized nas.
2.4.Qhov Kev Tshawb Fawb Txog Kev Tshawb Fawb Qhia pom tias Kev Tswj Xyuas Sybiotic txo qis kev nce qib ntawm CKD
Thaum kawg ntawm qhov kev sim, nas tau euthanized kom tau txais cov qauv ntawm sab lauglub raumthiab lub siab yuav tsum tau ua rau kev soj ntsuam histological. Qhov chaw fibrotic tau txiav txim siab los ntawm Masson's trichrome (MT) stain siv cov cuab yeej informatic Image-Pro Plus 3. (Table 2) thiab los ntawm ob-photon excitation microscopy, ib txoj kev uas kuaj pom collagen los ntawm emission ntawm ob-photons, ua rau cov collagen tsim ib tug thib ob harmonic, uas yuav pom tau los ntawm confocal microscopy tau txais ib daim duab tshwj xeeb ze rau lub koom haum thiab kev faib tawm ntawm collagen nyob rau hauv cov ntaub so ntswg. Thaum muab piv rau thaj chaw fibrotic ntawm ob lub raum, soj ntsuam los ntawm MT stain, nws tuaj yeem pom tias thaj chaw fibrotic, yog 25 feem pua loj dua hauv pawg Nef tab sis tsuas yog 12 feem pua loj dua hauv pawg Lac, tswj cov snbiotic thaum piv rau sham- nephrectomized tswj tsiaj. Cov kab mob tau pom nrog cov txheej txheem staining no tau nyob ntawm cov txheej txheem parietal ntawm Bowman's capsule thiab hauv qab daus daim nyias nyias ntawm cov proximal thiab distal convoluted tubules ntawm cov tsiaj ntawm pawg Nef thiab Lac. Nyob rau hauv cov ntaub ntawv ntawm pawg Nef, suav nrog cov tsiaj tsis tau tswj cov synbiotic, qhov poob ntawm cytoplasm ntawm cov hlwb ntawm cov tubules convoluted tau pom nrog kev loj hlob ntawm cov ntaub so ntswg fibrotic. Thaum cov ntaub so ntswg ntawm lub raum tau soj ntsuam los ntawm ob-photon excitation microscopy, cov collagen tau pom nyob rau hauv tag nrho cov qauv uas muaj ib tug mesh-hom conformation. Cov pab pawg Nef thiab Lac tau pom cov kab mob ntau dua li pawg tswj hwm, uas tsuas pom ib qho chaw fibrotic (Daim duab 4). Tsis muaj qhov txhab tau pom nyob rau hauv lub raum corpuscles hauv ib qho qauv thaum pom nyob rau hauv ob-photon excitation microscopy.
Rooj 2.Feem pua ntawm thaj chaw fibrotic hauv Masson's trichrome stainedraumntu histological. Cov txiaj ntsig tau nthuav tawm raws li qhov nruab nrab ntxiv qhov yuam kev.
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| Daim duab 3.Hematocrit qhov tseem ceeb ntawm Sprague-Dawley nas thaum kawg ntawm 16 lub lis piam kev tswj hwm ntawm synbiotic. Nef: nephrectomized nas tsis tau txais cov synbiotic, Lac: nephrectomized nas tau txais cov synbiotic, Tswj: sham-nephrectomized nas. | Daim duab 4.Histological seem ntawm ob lub raum ntawm Sprague-Dawley nas.(AC): Masson's trichrome stain ntawm nephrons. Fibrotic thaj chaw raug pom nyob rau hauv xiav ( xub xub).(DF): tib cov ntaub so ntswg pom los ntawm ob-photon excitation microscopy. Fibrotic ntaub so ntswg yog pom nyob rau hauv ntsuab. Nef: nephrectomized nas tsis tau txais cov synbiotic, Lac: nephrectomized nas tau txais cov synbiotic, Tswj: sham-nephrectomized nas. Scale bar∶ 100 μum rau (AC) micrographs thiab 50 μm rau (DF) micrographs. |
2.5.Kev tswj hwm ntawm Synbiotic Tsis Ua Rau Cardic Fibrosis
Nyob rau hauv lub plawv, MT stain qhia pom muaj cov chaw fibrotic nyob rau hauv cov ntaub so ntswg ntawm nephrectomized nas, thaum cov cheeb tsam no tsis nyob rau hauv cov tsiaj tswj. Qhov induction ntawm CKD nyob rau hauv nas generates cardiac fibrosis uas tsis tau tiv thaiv los ntawm kev tswj ntawm synbiotic kho nyob rau hauv nephrectomized nas (Daim duab 5).
Daim duab 5.Masson's trichrome(MT) staining ntawm lub plawv ntawm Sprague-Dawley nas.Fibrotic chaw yog stained nyob rau hauv xiav ( xub). Nef: nephrectomized nas tsis tau txais cov synbiotic, Lac: nephrectomized nas tau txais cov synbiotic. Tswj: sham-nephrectomized nas. Scale bar 100 um.
