RESEARCH ARTICLE
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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
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1900616 (1 o 13)
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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
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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
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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).
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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).
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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
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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
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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
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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
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