Probiotics Prevent Dysbiosis and the Rise in Blood Pressure in Genetic Hypertension: Role of Short-Chain Fatty Acids

RESEARCH ARTICLE www.mn -jou nal.com P obio ics P e en Dysbiosis and he Rise in Blood P essu e in Gene ic Hype ension: Role o Sho -Chain Fa y Acids Iñaki Robles-Ve a, Ma a To al, Nés o de la Visi ación, Manuel Sánchez, Manuel Gómez-Guzmán, Miguel Rome o, Tao Yang, José L. Izquie do-Ga cia, Rosa io Jiménez, Jesús Ruiz-Cabello, Edua do Gue a-He nández, Mohan K. Raizada, F ancisco Pé ez-Vizcaíno, and Juan Dua e* Scope: The objec i e o his s udy is o de e mine he ca dio ascula effec s o he p obio ics Bi idobac e ium b e e CECT7263 (BFM) and Lac obacillus e men um CECT5716 (LC40), and he sho chain a y acids bu y a e, and ace a e in spon aneously hype ensi e a s (SHR). Me hods and esul s: Ten fi e-week old Wis a Kyo o a s (WKY) and fi y aged-ma ched SHR a e andomly dis ibu ed in o six g oups: con ol WKY, con ol SHR, ea ed SHR-LC40, ea ed SHR-BMF, ea ed SHR-bu y a e, and ea ed SHR- ace a e. Ch onic ea men s wi h LC40 o BFM inc ease bu y a e-p oducing bac e ia and p e en he blood p essu e inc ease in SHR. O al ea men wi h bu y a e o ace a e also p e en s he inc ease in bo h blood p essu e and Fi mi- cu es/Bac e oide es (F/B) a io. All ea men s es o e he Th17/T eg balance in mesen e ic lymph nodes, no malized endo oxemia, and p e en he impai men o endo helium-dependen elaxa ion o ace ylcholine, as a esul o educed NADPH oxidase-d i en eac i e oxygen species p oduc ion. These p o ec i e effec s migh be media ed by bo h he e- duc ion in ascula lipopolysaccha ide (LPS)/ oll-like ecep o 4 (TLR4) pa hway and he inc ease in T eg infil a ion in he ascula u e. Conclusion: The p obio ics LC40 and BFM p e en dysbiosis and he de elopmen o endo helial dys unc ion and high blood p essu e in gene ic hype ension. These effec s seem o be ela ed o endo oxemia educ ion and o inc ease T eg accumula ion in he ascula u e. I. Robles-Ve a, N. de la Visi ación, D . M. Sánchez, D . M. Gómez-Guzmán, D . M. Rome o, D . R. Jiménez, P o . J. Dua e Depa men o Pha macology School o Pha macy and Cen e o Biomedical Resea ch (CIBM) Uni e si y o G anada 18071 G anada, Spain E-mail: jmdua e@ug .es D . M. To al Gene Regula ion in Ca dio ascula Remodeling and Inflamma ion G oup Cen o Nacional de In es igaciones Ca dio ascula es (CNIC) 28029 Mad id, Spain D . M. To al, D . R. Jiménez, P o . J. Dua e CIBERCV Spain D . M. Sánchez, D . M. Gómez-Guzmán, D . M. Rome o, D . R. Jiménez, P o . J. Dua e Ins i u o de In es igación Biosani a ia de G anada 18016 G anada, Spain The ORCID iden ifica ion numbe (s) o he au ho (s) o his a icle can be ound unde h ps://doi.o g/10.1002/mn .201900616 DOI: 10.1002/mn .201900616 D .T.Yang,P o .M.K.Raizada Depa men o Physiology and Func ional Genomics Uni e si y o Flo ida Gaines ille 32610, FL, USA D .T.Yang Mic obiome Conso ium and Cen e o Hype ension and P ecision Medicine, Depa men o Physiology and Pha macology Uni e si y o Toledo College o Medicine and Li e Sciences Toledo, Ohio 43606 D . J. L. Izquie do-Ga cia, P o . J. Ruiz-Cabello CIC biomaGUNE Donos ia-San Sebas ián 20014, Spain P o . E. Gue a-He nández Depa men o Nu i ion and B oma ology Uni e si y o G anada 18071 G anada Spain P o . F. Pé ez-Vizcaíno Depa amen o de Fa macología y Toxicología Facul ad de Medicina Uni e sidad Complu ense de Mad id Cibe En e medades Respi a o ias (Cibe es) Ins i u o de In es igación Sani a ia G ego io Ma añón (IISGM) 28040 Mad id, Spain Mol. Nu . Food Res. 2020,64, 1900616 © 2020 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim 1900616 (1 o 13) www.ad ancedsciencenews.com www.mn -jou nal.com 1. In oduc ion Hype ension is among he mos p e alen isk ac o s o ca dio- ascula e en s such as s oke and myoca dial in a c ion. Up o p esen da e, an e e -inc easing numbe o s udies ha e shown a link be ween gu mic obial signa u es and hype ension in bo h animal models and human pa ien s.[1–6] In gene al, bo h mani es dysbiosis as a esul o dec eases in e enness, mic obial di e si y, ichness, and an inc eased Fi micu es/Bac e oide es (F/B) a io in he enin-dependen o m o hype ension in bo h essen ial hype ensi e pa ien s and animals.[1,3,4] On he o he hand, no significan changes in F/B we e p esen in enin-independen hype ensi e mice.[5] Angio ensin II-in used ge m- ee mice showed ha changes in gu mic obio a a e in ol ed in an- gio ensin II-induced ascula dys unc ion and hype ension.[7] In addi ion, ecal ansplan a ion om spon aneously hype ensi e- s oke p one a s[8] o SHR[9,10] o Wis a Kyo o a s (WKY) o om hype ensi e subjec s[4] o ge m- ee mice, espec i ely, in- duces an inc ease in blood p essu e. These obse a ions sugges ha gu mic obio a egula es blood p essu e. Howe e , he exac oles o bac e ia and gu heal h in hype ension ha e no been elucida ed. Recen ly, we demons a ed he key ole o T-cell ac i- a ion in he gu immune sys em and ascula T-cells accumu- la ion in he hype ensi e esponse igge ed by ecal mic obio a ansplan a ion om SHR o WKY a s.[10] The gu mic obio a communica es wi h dis al o gans h ough he p oduc ion o a high numbe o me aboli es ha can be abso bed in o he sys emic ci cula ion and exe biological effec s.[11] The signaling molecules a e bac e ial me abolic p od- uc s, including sho chain a y acids (SCFAs),[12] and bac e ial wall componen s such as lipopolysaccha ide (LPS).[13] In ac , gu dysbiosis in hype ension has been cha ac e ized by an inc ease in lac a e-p oducing bac e ia, and a dec ease in ace a e- and bu y a e-p oducing bac e ial popula ions.[1,10] SCFAs can impac enin sec e ion and blood p essu e egula ion s imula ing hos G-p o ein–coupled ecep o pa hways.[12] In ac , bu y a e has been shown o a enua e angio ensin II-induced hype ension in mice,[14,15] and bo h, ace a e supplemen a ion o a die ich in fibe , which subs an ially inc eases he p oduc ion o SCFAs such as ace a e, p e en ed he de elopmen o hype ension in deoxy- co icos e one ace a e (DOCA)-sal .[5] Inc eased bac e ial p oduc- ion o SCFAs is associa ed wi h educed ci cula ing CD4+im- mune cells.[15,16] Bac e ial LPS, h ough oll-like ecep o (TLR)4 ac i a ion, con ibu es o he low-g ade ascula inflamma ion and, ul ima ely, he inc eased blood p essu e p esen in SHR.[17] Thus, i is highly p obable ha gu mic obio a is unc- ionally in ol ed in blood p essu e con ol. In ac , a me a- analysis demons a ed a significan educ ion in blood p essu e in p obio ic- ea ed pa ien s.[18] Fu he mo e, a beneficial ole o Lac obacillus p obio ics and kefi in blood p essu e egula ion and ascula p o ec ion ha e been desc ibed in SHR wi h s abi- lized hype ension,[19–21] as a esul o es o ing he imbalance in eac i e oxygen species (ROS)/ni ic oxide (NO) in he a e- ial wall. Howe e , i p obio ics consump ion could p e en gu dysbiosis, educing gu immune sys em T-cell ac i a ion and as- cula T-cells infil a ion in SHR, and i SCFAs play any ole in his effec is unknown. We hypo hesized ha o al supplemen a- ion wi h p obio ics would p e en he aise in blood p essu e in SHR by changing SCFAs-p oducing bac e ia popula ions, hus al e ing he gu communica ion wi h local seconda y lymph o - gans and dis al o gans. Thus, he objec i e o his s udy was o e alua e he ca dio ascula effec s o p obio ics Bifidobac e ium b e e CECT7263 (BFM), and Lac obacillus e men um CECT5716 (LC40), and bu y a e and ace a e in gene ic hype ension. 2. Resul s 2.1. P obio ics and SCFA P e en ed he Raise on Blood P essu e, and Ca diac Hype ophy in SHR As expec ed, a significan ime-dependen inc ease in SBP (≈62 mm Hg) was obse ed in SHR om 5 o 18 weeks old (Figu e 1A,B). Long- e m adminis a ion o bo h p obio ics p e- en ed he aise in SBP (28.4 ±7.8%, and 23.6 ±7.0% by LC40 and BFM, p<0.01 and p<0.5 e sus un ea ed SHR, espec- i ely) (Figu e 1A). Simila ly, bo h ace a e and bu y a e consump- ion also inhibi ed he de elopmen o high BP (17.2 ±5.4%, and 21.4 ±7.0%, espec i ely, p<0.05 e sus un ea ed SHR) (Figu e 1B). The an ihype ensi e effec o hese ea men s was confi med a he end o he expe imen by a di ec p essu e eco ding in he ca o id (Figu e 1C). Howe e , no significan changes in hea a e we e ound among all expe imen al g oups (Figu e 1D). In e es ingly, none o he ea men s was able o change SBP in no mo ensi e WKY a s (Figu e S1, Suppo ing In o ma ion). Bo h WKY and SHR con ol g oups expe ienced an inc ease in body weigh be ween 5–18 weeks old (311.7 ±11.6% and 283.8 ±12.0%, espec i ely). The ea men s did no change weigh gain and we we e no able o find a s a is ical diffe ence among all expe imen al g oups in final body weigh (Table S2, Suppo ing In o ma ion). Absolu e hea weigh (HW) and le en icle weigh (LVW) and hei ela i e alues exp essed as a a io o ibia leng h (TL) we e highe (13%, 24%, 18%, and 26%, espec i ely) in SHR con ol g oup as compa ed wi h WKY con- ol g oup. The p obio ics LC40 and BFM and bu y a e and ac- e a e significan ly educed LVW/TL index, by 7%, 8%, 7%, and 8% espec i ely (Table S2, Suppo ing In o ma ion). 2.2. P obio ics and SCFA Reduced Gu Dysbiosis in SHR The bac e ial communi ies composi ion was e alua ed calcula - ing majo ecological pa ame e s, including Chao ichness, Pielou e enness, and he numbe o obse ed species. Significan diffe - ences among expe imen al g oups we e no ound (Figu e 2A). The analysis o he phyla composi ion (Figu e 2B;Table S3, Sup- po ing In o ma ion) showed ha Fi micu es and Bac e oide es we e he mos abundan phylum in a eces. The p opo ion o bac e ia om he Fi micu es phylum was significan ly highe (≈18%, p<0.05) in SHR han in WKY and bo h bu y a e and ace a e consump ion no malized he p opo ion o bac e ia be- longing o his phylum. Mo eo e , bac e ia om Bac e oide es phylum we e dec eased (≈−37%, p<0.01) in SHR and BFM, bu y a e and ace a e significan ly inc eased his p opo ion simi- la o WKY a s. LC40 ended o educe Fi micu es and inc ease Bac e oide es bu hese changes we e no s a is ically significan . The F/B a io, a signa u e o gu dysbiosis in hype ension,[1] was ≈ wo old highe in SHR han in WKY, and his a io Mol. Nu . Food Res. 2020,64, 1900616 © 2020 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim 1900616 (2 o 13) 16134133, 2020, 6, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/mn .201900616 by Uni e sidad De G anada, Wiley Online Lib a y on [08/01/2024]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License www.ad ancedsciencenews.com www.mn -jou nal.com Figu e 1. P obio ic ea men s p e en ele a ed blood p essu e in spon aneously hype ensi e a s (SHR). A, B) Time cou se o sys olic blood p essu e (SBP), measu ed by ail-cuff ple hysmog aphy in all expe imen al g oups. Mean a e ial blood p essu e (MABP), measu ed by in a-a e ial eco ding in o le ca o id a e y, a he end o he C) expe imen al pe iod and D) hea a e (HR). Resul s a e shown as mean ±SEM (n=7–10). **p<0.01 compa ed wi h Wis a Kyo o (WKY) g oup. #p<0.05 and ##p<0.01 compa ed wi h he non- ea ed SHR g oup. LC40, Lac obacillus e men um CECT5716; BFM, Bifidobac e ium b e e CECT7263. e u ned o no mal alues by he ac ion o BFM, bu y a e and ac- e a e, whe eas LC40 ended o educe i bu no significan ly (p= 0.352) ((Figu e 2C). In addi ion, significan lowe pe cen ages o ace a e- and bu y a e-p oducing bac e ia (≈−50% and −42%, e- spec i ely, p<0.05), and a ≈fi e old, p<0.05, highe pe cen - age o lac a e-p oducing bac e ia we e ound in SHR compa ed o WKY (Figu e 2D). Bo h LC40 and BFM inc eased ≈ wo-, and wo old, espec i ely, p<0.05, bu y a e-p oducing bac e ia in SHR bu had no effec on ace a e- and lac a e-p oducing bac e ia. Ace a e inc eased ≈ wo old, p<0.05 ace a e-p oducing bac e ia while bu y a e hal ed lac a e-p oducing bac e ia (Figu e 2D). Ac- e a e con en in eces was educed 2.7 old in SHR as compa ed o WKY, which was unchanged by BFM, LC40, and bu y a e ea - men . Howe e , ace a e ea men inc eased 6.3- old he ace a e concen a ion in eces. Bu y a e con en in eces was simila be- ween WKY and SHR, bu bo h BFM and bu y a e ea men in SHR inc eased by 3.6- and 2.4- old, espec i ely, bu y a e concen- a ion (Figu e S2, Suppo ing In o ma ion). A he amily le el, a significan inc ease o ≈ h ee old in Lac- obacillaceae, ≈ wo old Tu icibac e iaceae,>100- old Pep os ep o- coccaceae,and≈ ou old Anae oplasma aceae,anda≈40% educ- ion o S24-7 we e ound in SHR eces as compa ed o WKY (Figu e 3A). Bo h p obio ics and ace a e ea men s p e en ed (p<0.05) he inc ease in Pep os ep ococcaceae in SHR. A he genus le el, Lac obacillus and Tu icibac e we e inc eased (≈ h ee, and wo old, espec i ely, p<0.05) and S24-7 g we e educed ≈ 40%, p<0.05 in SHR as compa ed o WKY (Figu e 2B) as p e i- ously desc ibed.[10] Bo h p obio ics and SCFAs ea men s ended o educe Lac obacillus and inc ease S24-7 g, bu only ace a e e- duced significan ly (p<0.05) by ≈2.5- old he abundance o Lac- obacillus (Figu e 3C). The ea men s did no al e he Tu icibac- e p opo ion in SHR (Figu e 3C). The a io Lac obacillus/ S24-7 g posi i ely co ela ed wi h SBP ( 2=0.1375, p<0.05), and was no - malized by all ea men s (Figu e 3D). 2.3. P obio ics Reduced Endo oxemia, Inc eased Bu y a e bu did no Change Ci cula ing Ace a e and Lac a e Le els in SHR An app oxima ely wo- hi d educ ion in mRNA le els o ba ie - o ming junc ion p o eins (zonula occludens-1 (ZO-1) and oc- cludin) in he colon o SHR compa ed o WKY we e ound (Figu e 4A). Bo h p obio ic ea men s es o ed ZO-1 and oc- cludin mRNA le els, sugges ing a possible p ese ed ba ie unc ion. As expec ed, bo h ace a e and bu y a e ea men s a - ec ed mRNA le els encoding igh junc ion p o eins in SHR, no malizing ZO-1, and inc easing wo- and fi e old, espec i ely, occludin. We ha e also ound down egula ion o mucin (MUC)-2 and MUC-3 ansc ip s by ≈90%, and 75%, espec i ely, in SHR, which we e significan ly inc eased by bo h SCFAs (≈10- and 30- old MUC-2, and ≈15- and 12- old MUC-3, bu y a e and ace a e, espec i ely) bu unaffec ed by LC40 and BFM (Figu e 4B). We measu ed endo oxin le els in plasma, and ound hem o be ≈ 70% significan ly (p<0.01) highe in SHR compa ed wi h he WKY g oup (Figu e 4C). In e es ingly, he long- e m ea men wi h bo h p obio ics and SCFAs significan ly p e en ed endo ox- emia in SHR. These esul s sugges ha in es inal pe meabili y is inc eased in SHR and allow bac e ial componen s (e.g., LPS) o en e he blood s eam. We also ound ha colonic exp es- sion o IL-18 (Figu e 4D) was ≈80% lowe (p<0.05) in SHR as Mol. Nu . Food Res. 2020,64, 1900616 © 2020 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim 1900616 (3 o 13) 16134133, 2020, 6, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/mn .201900616 by Uni e sidad De G anada, Wiley Online Lib a y on [08/01/2024]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License www.ad ancedsciencenews.com www.mn -jou nal.com Figu e 2. P obio ic ea men s p e en gu dysbiosis in spon aneously hype ensi e a s (SHR). A) Bac e ial 16S ibosomal DNA we e amplified and sequenced o e alua e h ee majo ecological pa ame e s, Chao ichness, Pielou e enness, and he numbe o obse ed species. B) Phylum b eakdown o he se en mos abundan bac e ial communi ies. C) The Fi micu es/Bac e oide es a io (F/B a io) was calcula ed as a bioma ke o gu dysbiosis. D) Rela i e p opo ions o ace a e-, bu y a e-, and lac a e-p oducing bac e ia in he gu mic obio a om Wis a Kyo o (WKY), and SHR g oups. Resul s a e shown as mean ±SEM (n=5–6). *p<0.05 and **p<0.01 compa ed wi h WKY g oup. #p<0.05 compa ed wi h he non- ea ed SHR g oup. LC40, Lac obacillus e men um CECT5716; BFM, Bifidobac e ium b e e CECT7263. compa ed o WKY. All ea men s inc eased mRNA le els o IL18 in colon (Figu e 4D). As we ound significan changes in SCFAs-p oducing bac- e ia in eces, we nex analyzed he colonic exp ession o he anspo e s o SCFAs, monoca boxyla e anspo e s (MCT)1 and MCT4 (Figu e 4E), and he plasma le els o SCFA om he NMR spec a (Figu e 4F). Bo h MCT1 and MCT4 ansc ip s le - els we e educed in SHR by ≈60%, p<0.05 and 65%, p< 0.01, espec i ely, as compa ed o WKY and we e unchanged by all ea men s, excep o BFM ha inc eased ≈ ou old MCT- 1 and bu y a e ha inc eased ≈se en old MCT-4. Lac a e was significan ly dec eased in plasma (≈−22%, p<0.05), wi hou changes in ace a e le els in plasma om SHR as compa ed o WKY. P obio ic ea men s did no significan ly change he plasma le els o hese SCFAs. In addi ion, nei he ace a e no bu y a e ch onic consump ion al e ed he le els o ace a e and lac a e in SHR. We we e no able o de ec bu y a e le els in he NMR spec a. Howe e , plasma bu y a e concen a ions, mea- su ed by gas ch oma og aphy, we e educed in SHR as com- pa ed WKY, and inc eased by LC40, BFM, and bu y a e ea men (Figu e 4G). Unsupe ised classifica ion s udies wi h PCA we e ca ied ou o analyze he diffe ences be ween spec a om SHR and WKY a s. The plasma spec a p o ided nea ly pe ec disc imina ion be ween he wo g oups (Figu e S3A, Suppo ing In o ma ion). Me abolic diffe ences be ween WKY and SHR a e highligh ed in ep esen a i e plasma spec a (Figu e S3B, Suppo ing In o - ma ion). The chemical shi s o he iden ified me aboli es a e lis ed in Table S4, Suppo ing In o ma ion. SHR showed highe concen a ion o leucine, phenylalanine, c ea inine, and glucose (≈23%, 64%, 23%, and 43%, espec i ely), and ≈20% lowe con- cen a ion o alipha ic chains, as compa ed o WKY. Bo h p obi- o ic LC40 and BFM no malized plasma phenylalanine, wi hou affec ing o he me aboli es. Plasma c ea inine concen a ion was inc eased ≈25% by bu y a e ea men . 2.4. P obio ics Res o ed T Cell Popula ions Changes in Lymphoid O gans in SHR We ound ha he numbe o o al T cells in MLNs was simila in SHR compa ed o WKY (Figu e S4A, Suppo ing In o ma ion). Mol. Nu . Food Res. 2020,64, 1900616 © 2020 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim 1900616 (4 o 13) 16134133, 2020, 6, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/mn .201900616 by Uni e sidad De G anada, Wiley Online Lib a y on [08/01/2024]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License www.ad ancedsciencenews.com www.mn -jou nal.com Figu e 3. P obio ics ea men s con ibu e gu mic obio a homeos asis in spon aneously hype ensi e a s (SHR). Hea map showing he bac e ial amilies and gene a mos diffe ing in abundance be ween he expe imen al g oups. Samples clus e ed by ea men g oup showed ha he ea men s esul ed in dis inc popula ions o bac e ia a he amily and genus le el. A, B) The hea map colo s ep esen he ela i e pe cen age o mic obial amily assigned wi hin each sample as compa ed o SHR. C) Main significan ly modified bac e ial gene a in he gu mic obio a in Wis a Kyo o (WKY) and SHR g oups. D) Ra io Lac obacillus/ S24-7 g and co ela ed wi h sys olic blood p essu e (SBP). Resul s a e shown as mean ±SEM (n=5–6). *p<0.05 and **p<0.01 compa ed wi h WKY g oup. #p<0.05 and ##p<0.01 compa ed wi h he non- ea ed SHR g oup. LC40, Lac obacillus e men um CECT5716; BFM, Bifidobac e ium b e e CECT7263. Howe e , an ≈30% lowe T cell pe cen age was de ec ed in spleen om SHR as compa ed o WKY (Figu e S4B, Suppo ing In- o ma ion). No change was obse ed by ea men s in o al T cells om bo h seconda y lymph o gans. The pe cen age o T eg (CD4+/FoxP3+) was educed ≈65%, whe eas Th17 (CD4+/IL- 17+) lymphocy es we e ≈ wo old significan ly inc eased in bo h MLNs (Figu e 5A) and spleen (Figu e 5B) in SHR compa ed o WKY. All ea men s inc eased T eg and educed Th17 o le els simila o ha ound in WKY in bo h seconda y lymph o gans, wi h he excep ion o ch onic ace a e in spleen. 2.5. P obio ics T ea men Imp o es Endo helial Func ion, Oxida i e S ess, and T Cells Infil a ion in SHR A educed endo helium-dependen asodila o esponse o ace ylcholine when s imula ing wi h phenyleph ine in ao ae om con ol SHR was shown as compa ed wi h ao ae om con ol WKY (Emax =59 ±4% e sus 84 ±3%, espec i ely). Bo h LC40 and BFM ea men s inc eased he elaxa ion in- duced by ace ylcholine in SHR a s (Emax =70 ±5%, and 69 ±2%, espec i ely, p<0.05 e sus SHR con ol) (Figu e 6A). Simila ly, ch onic bu y a e and ace a e also imp o ed he e- laxa ion o ace ylcholine (Emax =74 ±5%, and 63 ±3%, e- spec i ely, p<0.05 e sus SHR con ol) (Figu e 6A). This elaxa ion was unal e ed by ch onic in e en ions wi h bo h p o- bio ics and SCFAs in WKY a s (Figu e S5, Suppo ing In o ma- ion). In all expe imen al g oups, he ace ylcholine-induced e- laxa ion was ully inhibi ed by L-NAME (da a no shown), which shows ha in his essel elaxa ion induced by ace ylcholine in bo h WKY and SHR was comple ely dependen on NO de i ed om endo helium. The endo helium-independen asodila o e- sponses o ni op usside, which di ec ly ac i a es soluble guany- lyl cyclase in ascula smoo h muscle, we e no diffe en among g oups (da a no shown), showing no change in he signaling o NO in ascula smoo h muscle. No significan changes in NOS ac i i y and a ginase ac i i y in ao a om all expe imen- al g oups we e obse ed (Figu e S6, Suppo ing In o ma ion). Mol. Nu . Food Res. 2020,64, 1900616 © 2020 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim 1900616 (5 o 13) 16134133, 2020, 6, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/mn .201900616 by Uni e sidad De G anada, Wiley Online Lib a y on [08/01/2024]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License www.ad ancedsciencenews.com www.mn -jou nal.com Figu e 4. P obio ic ea men s imp o e al e ed gu in eg i y in spon aneously hype ensi e a s (SHR). A) Colonic mRNA le els o occludin, and zonula occludens-1 (ZO-1). B) Mucin (MUC)-2, and MUC-3. C) Plasma endo oxin concen a ions (LPS, EU mL−1, endo oxin uni s mL−1. D) Tissue epai cy okine IL-18. Exp ession o he E) sho -chain a y acids (SCFAs) monoca boxyla e anspo e s, MCT-1 and MCT-4, F) he plasma le els o SCFAs om he nuclea magne ic esonance (NMR) spec a, and G) plasma bu y a e le els by GC in Wis a Kyo o (WKY) and SHR g oups. Resul s a e shown as mean ±SEM (n=7–10). *p<0.05 and **p<0.01 compa ed wi h WKY g oup. #p<0.05 and ##p<0.01 compa ed wi h he non- ea ed SHR g oup. LC40, Lac obacillus e men um CECT5716; BFM, Bifidobac e ium b e e CECT7263. ROS p oduc ion om he NADPH oxidase is a c ucial ele- men in endo helial dys unc ion in SHR. In ac , we ound ha he p esence o he selec i e NADPH oxidase inhibi o VAS2870 in he o gan chambe inc eased he elaxan esponse o ace yl- choline in un ea ed SHR (Emax =80 ±4%), eaching simila e- laxa ion pe cen ages o hose ound in WKY (Emax =85 ±3%). In he p esence o his agen we we e no able o find any diffe - ences be ween g oups, as compa ed o SHR g oup (Figu e 6B). In ag eemen wi h his, NADPH oxidase ROS p oduc ion was in- c eased ≈60%, p<0.01, in ao ic ings om SHR as compa ed wi h WKY a s (Figu e 6C). Bo h p obio ics p e en ed his in- c ease in NADPH oxidase ac i i y in SHR. In ao ic issue om SHR, a significan inc ease in mRNA le els o NADPH oxidase subuni s, NOX-1, NOX-4, p47phox, and p22phox (≈2-, 8.5-, 8,8-, and 3.5- old, espec i ely) was obse ed as compa ed wi h WKY a s (Figu e 6D). Again, bo h p obio ics no malized he gene exp es- sion o NADPH oxidase subuni s in SHR. TLR4 mRNA le els in ao ic homogena es we e ≈eigh old highe in SHR as compa ed wi h WKY (Figu e 6E). In SHR, p obio ics and SCFAs es o ed he TLR4 mRNA le els o simila alues o hose o WKY. The infil a ion o o al T cells was simila in ao a om SHR han om hei no mo ensi e coun e pa s, and was unchanged by all ea men s (Figu e 7A). Howe e , we ound educed by ≈75% he T eg (FoxP3+/CD4 +) popula ions wi hou significan change in he Th17 (IL-17+/CD4 +) popula ions in ao as om he SHR g oup as compa ed o WKY a s (Figu e 7B). LC40, BFM, and bu y a e, bu no ace a e, ea men s es o ed he ao ic accumula ion o T eg wi h no significan changes in Th17 infil- a ion (Figu e 7B). 3. Discussion The main new findings o his s udy a e he ollowing: 1) ch onic ea men s wi h he p obio ics LC40 o BFM p e en ed bo h gu dysbiosis ( educed F/B a io, inc eased bu y a e-p oducing bac- e ia) and he blood p essu e inc ease in SHR; 2) O al ea men wi h he SCFAs, bu y a e o ace a e, also p e en ed he aise in bo h blood p essu e and F/B a io; 3) SBP is di ec ly co ela ed o he Lac obacillus/S24-7 g a io, and he SBP educ ion induced Mol. Nu . Food Res. 2020,64, 1900616 © 2020 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim 1900616 (6 o 13) 16134133, 2020, 6, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/mn .201900616 by Uni e sidad De G anada, Wiley Online Lib a y on [08/01/2024]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License www.ad ancedsciencenews.com www.mn -jou nal.com Figu e 5. P obio ic ea men s es o e T cell imbalance in spon aneously hype ensi e a s (SHR). Regula o y T (T eg) cells, and T-helpe (Th) 17 cells measu ed in A) mesen e ic lymphoid nodes and B) spleen in Wis a Kyo o (WKY) and SHR g oups. Resul s a e shown as mean ±SEM (n=7–10). *p<0.05 and **p<0.01 compa ed wi h WKY g oup. #p<0.05 and ##p<0.01 compa ed wi h he non- ea ed SHR g oup. LC40, Lac obacillus e men um CECT5716; BFM, Bifidobac e ium b e e CECT7263. by all ea men s was linked o a dec ease in his a io; 4) All ea men s es o ed he Th17/T eg balance in MLNs, and no - malized endo oxemia; 5) All ea men s p e en ed he impai - men o endo helium-dependen elaxa ion o ace ylcholine, as a esul o educed NADPH oxidase-d i en ROS p oduc ion; 6) All ea men s we e unable o change SBP and endo helial unc ion in no mo ensi e WKY a s; 7) The p o ec i e effec s induced by ea men s in ascula oxida i e s ess and endo helial unc ion seem o be independen o ace a e and lac a e plasma le els and migh be media ed by he educ ion in ascula LPS/TLR4 pa h- way, he inc ease in T eg infil a ion in he ascula u e in a s wi h gene ic hype ension, and o LC40, BFM, and bu y a e by he inc ease in plasma bu y a e concen a ion. Mul iple s udies ha e demons a ed he associa ion be ween gu dysbiosis and hype ension.[1,2,4,6,10] Ou esul s a e consis- en wi h he main ea u es o dysbio ic mic obio a desc ibed in SHR:[1,10,25] a) an inc eased F/B a io, and b) a educ ion in ace a e- and bu y a e-p oducing bac e ia, wi h highe p opo ion o lac a e-p oducing bac e ia. We ha e p e iously demons a ed ha when SHR wi h es ablished hype ension we e ea ed o 5 weeks wi h he p obio ics, LC40 o Lac obacillus co yni o mis CECT5711 plus Lac obacillus gasse i CECT5714 (1:1) an imp o e- men o endo helial dys unc ion and a dec ease in blood p es- su e we e ound.[19] These ca dio ascula p o ec i e effec s in- duced by Lac obacillus s ains we e associa ed wi h changes in some bac e ial gene a (inc eased Lac obacillus spp and educed Bac e oides and Clos idium ssp) measu ed by qRT-PCR. Da a o he mic obio a composi ion om he p esen s udy is difficul o compa e wi h his p e ious wo k because we now pe o med 16s ibosomal DNA sequencing. Recen ly, we ound a posi i e and nega i e co ela ion o SBP wi h Tu icibac e and S24-7 g bac e- ial abundance, espec i ely.[10] In ag eemen wi h his s udy we showed ha Tu icibac e was inc eased and S24-7 g was educed in SHR as compa ed o WKY. Howe e , he pa hological alue o he lac a e-p oducing genus Tu icibac e o induce a hype en- si e pheno ype has no been es ablished. In he p esen s udy, we ound a significan di ec co ela ion be ween Lac obacillus/S24- 7g a io and SBP. The unclassified genus belonging o he S24-7 Mol. Nu . Food Res. 2020,64, 1900616 © 2020 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim 1900616 (7 o 13) 16134133, 2020, 6, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/mn .201900616 by Uni e sidad De G anada, Wiley Online Lib a y on [08/01/2024]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License www.ad ancedsciencenews.com www.mn -jou nal.com Figu e 6. P o ec i e effec s o p obio ic ea men s in endo helial unc ion and oxida i e s ess in spon aneously hype ensi e a s (SHR). Vascula e- laxan esponses induced by ace ylcholine (Ach), in endo helium-in ac ao ae p e-con ac ed by phenyleph ine (Phe) in he A) absence and in he B) p esence o he NADPH oxidase inhibi o VAS2870 (5 𝜇m) in Wis a Kyo o (WKY) and SHR g oups. NADPH oxidase ac i i y measu ed by C) lucigeninen- hanced chemiluminescence, and ao ic mRNA le els exp ession o NADPH oxidase subuni s D) NOX-4, p47phox, NOX-1, and p22phox, and E) oll-like ecep o (TLR)-4 in Wis a Kyo o (WKY) and SHR g oups. Resul s a e shown as mean ±SEM (n=7–10). *p<0.05 and **p<0.01 compa ed wi h WKY g oup. #p<0.05 and ##p<0.01 compa ed wi h he non- ea ed SHR g oup. LC40, Lac obacillus e men um CECT5716; BFM, Bifidobac e ium b e e CECT7263. amily, he uniden ified axon om o de Bac e oidales,was ound in highe numbe s in non-diabe ic mice also co ela ed posi i ely wi h splenic FoxP3+CD4+T eg cells and he delayed diabe es on- se age.[26] The p obio ics LC40, a s ain o Lac obacillus e men- um, and BFM, a s ain o Bifidobac e ium b e e, did no change he ela i e abundance o i s own gene a in he gu , Lac obacillus o Bifidobac e ium, espec i ely, which indica es ha hei effec is no jus due o a eplacemen o o he bac e ia bu a he o a posi- i e effec on he whole bac e ial communi y. The main change a gene a le el induced by ch onic consump ion o LC40 and BFM, o SCFAs was a educed Lac obacillus/S24-7 g a io. Howe e , we did no pe o m any expe imen add essed o cla i y i his educ- ion is mechanis ically in ol ed on he an ihype ensi e effec s o hese ea men s. Dysbio ic mic obio a om SHR inc eased blood p essu e, a leas in pa , as a consequence o i s effec s on T-cell ac i a ion in he gu immune sys em and in ascula T-cells accumula ion.[9] In e es ingly, bo h LC40 and BFM es o ed he p opo ion o bu y a e-p oducing bac e ia, which we e ound educed in SHR. SCFAs, such as bu y a e, ace a e, and p opiona e, can influence immune unc ion in his pa o he in es ine. In pa icula , he numbe and unc ion o pe iphe al T eg cells in he colon a e enhanced by SCFAs.[27–29] In ac , we ound inc eased T eg cells popula ions in MLNs om SHR ea ed wi h p obio ics ha in- c eased bu y a e-p oducing bac e ia and bu y a e con en in e- ces o wi h o al ace a e o bu y a e. Mo eo e , significan changes in he p opo ion o T eg cells we e ound in spleen om SHR- ea ed g oups as compa ed o SHR, wi h he excep ion o ace a e- ea ed SHR. This lack o effec s could be ela ed o he su p is- ing absence o significan change in he ace a e plasma le els, despi e o al consump ion o ace a e. This could be pa ially ex- plained by: a) he low plasma hal -li e o ace a e;[30] b) educed ex- p ession o MCT1 o ac i e anspo o SCFAs; and c) educed passi e diffusion o ace a e because he exp ession o genes Mol. Nu . Food Res. 2020,64, 1900616 © 2020 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim 1900616 (8 o 13) 16134133, 2020, 6, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/mn .201900616 by Uni e sidad De G anada, Wiley Online Lib a y on [08/01/2024]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License www.ad ancedsciencenews.com www.mn -jou nal.com Figu e 7. P obio ic ea men s imp o e T cell infil a ion in spon aneously hype ensi e a s (SHR). A) To al T cells, egula o y T (T eg) cells, and B) T-helpe (Th) 17 cells measu ed in ao ae o Wis a Kyo o (WKY) and SHR g oups. Resul s a e shown as mean ±SEM (n=7–10). *p<0.05 compa ed wi h WKY g oup. #p<0.05 compa ed wi h he non- ea ed SHR g oup. LC40, Lac obacillus e men um CECT5716; BFM, Bifidobac e ium b e e CECT7263. associa ed wi h igh junc ion such as occluding and ZO-1 we e up egula ed by ea men wi h ace a e. The mos impo an dis- c epancy be ween SCFAs-p oducing bac e ia in he gu and plasma SCFAs le els is lac a e, which showed an inc eased p o- po ion o lac a e-p oducing bac e ia and educed plasma lac a e le els in SHR as compa ed o WKY. One possible explana ion migh be ela ed o he educed exp ession o MCT1 in SHR, he majo SCFA anspo e exp essed in he gu o he up ake o lac a e.[31] In e es ingly, a dec eased memb ane anspo e spe- cific o bu y a e (MCT4) was obse ed in colonic samples om SHR, which was significan ly up- egula ed by bu y a e, simila o ha p e iously desc ibed.[15] In e es ingly, LC40 and BFM, ha inc eased bu y a e-p oducing bac e ia, and bu y a e consump- ion inc ease plasma le els o bu y a e in SHR. This SCFA in- hibi s he diffe en ia ion o Th17 cells.[32,33] In ac , only hese ea men s change T cells pola iza ion in spleen, being wi h- ou effec s ace a e consump ion. In addi ion, sys emic bu y a e migh ac di ec ly a he ascula wall educing oxida i e s ess and imp o ing endo helial dys unc ion, as p e iously desc ibed in ApoE−/−mice.[34] Hype ension is associa ed wi h he al e ed exp ession o gu igh junc ion p o eins, inc eased pe meabili y, and gu pa hology.[35] SCFAs ha e been demons a ed o exe many ben- eficial effec s on in es inal epi helium, including inhibi ion o inflamma ion,[36] and modula ion o oxida i e s ess.[37] Fu he - mo e, an imp o ed ba ie unc ion by SCFAs has been epo ed in i o,[38–41] ex i o,[42] andinanimals udies. [15,43] In ag ee- men wi h hese da a, we also ound ha p obio ics, and ace a e and bu y a e consump ion inc eased mRNA le els o igh junc- ion p o ein occludin and ZO-1 in he colon. Howe e , no di- ec measu es o gu pe meabili y we e pe o med in he p esen s udy. Inc eased in es inal pe meabili y in adul hype ensi e SHR has been ela ed o educed goble cells.[35] These cells p o- duce mucins, which p o ec he gu om pa hogen in asion, he eby egula ing he gu immune esponse.[44] Howe e , MUC- 2, he main s uc u al componen o he mucus laye , and MUC-3 ansc ip s in SHR, we e only inc eased by bo h SCFAs. In ad- di ion, IL-18, a cy okine impo an o issue epai ,[38] was also inc eased in colon om SHR- ea ed g oups as compa ed o un- ea ed SHR. O e all, ou esul s sugges ha p obio ics migh Mol. Nu . Food Res. 2020,64, 1900616 © 2020 WILEY-VCH Ve lag GmbH & Co. KGaA, Weinheim 1900616 (9 o 13) 16134133, 2020, 6, Downloaded om h ps://onlinelib a y.wiley.com/doi/10.1002/mn .201900616 by Uni e sidad De G anada, Wiley Online Lib a y on [08/01/2024]. See he Te ms and Condi ions (h ps://onlinelib a y.wiley.com/ e ms-and-condi ions) on Wiley Online Lib a y o ules o use; OA a icles a e go e ned by he applicable C ea i e Commons License