Antiulcer activity of proanthocyanidins is mediated via suppression of oxidative, inflammatory, and apoptotic machineries

Abstract

Gastric ulcer (GU) is a lesion in the gastric mucosa associated with excessive oxidative damage, inflammatory response, apoptotic events, and irritation which may develop into cancer. However, medications commonly used in GU treatment cannot normalize gastric mucosa, while causing several adverse effects. Proanthocyanidins (PAs) are dietary flavonoids with numerous biological and pharmacological activities. In the current investigation, we studied the potential anti‐ulcerative activity of PAs against acidified ethanol (HCl/ethanol)‐caused gastric ulceration. Fifty male albino Wistar rats were allocated into five equal groups: control, HCl/ethanol (3 mL/kg), lansoprazole (LPZ, 30 mg/kg) + HCl/ethanol, and PAs (100 and 250 mg/kg) + HCl/ethanol. LPZ and PAs were applied one week before gastric ulcer induction. PAs pretreatment notably reduced gastric mucosal macroscopic and microscopic pathological changes in a dose‐dependent manner. Additionally, PAs activated the innate antioxidant molecules including glutathione and its derived antioxidants (glutathione peroxidase and glutathione reductase), along with superoxide dismutase and catalase, while attenuating pro‐oxidant formation, including malondialdehyde and nitric oxide. Interestingly, PAs supplementation at a higher dose suppressed gastric inflammatory and apoptotic responses, as demonstrated by the reduced levels of interleukin‐1β, interleukin‐6, tumor necrosis factor alpha, high‐mobility group box 1, cyclooxygenase 2, prostaglandin E2, nuclear factor kappa‐B, Bcl‐2‐associated X protein, and caspase‐3, while B cell lymphoma 2 was elevated. Hence, PAs could exhibit antiulcer activity by protecting gastric tissue from the development of oxidative damage, inflammatory responses, and apoptosis events associated with ulceration.

Practical implications
Gastric ulcer is a lesion in the gastric mucosal layer associated with excessive inflammatory response, apoptotic events, oxidative damage, and irritation, and may develop into cancer with about 5%–10% morbidity rate. However, medications commonly used in GU treatment cannot normalize gastric mucosa, while causing several adverse effects. Therefore, new therapeutic approaches are needed to treat or prevent gastric ulceration. Proanthocyanidins (PAs, condensed tannins) are dietary flavonoids found in abundance in different plant species, including their fruits, bark, and seeds. Due to their potent antioxidative activity, PAs have been applied to prevent or treat oxidative stress‐related diseases, including cancer, as well as metabolic, neurodegenerative, cardiovascular, and inflammatory disorders. Here, we examine the potential therapeutic role of proanthocyanidins (PAs) against acidified ethanol‐induced gastric ulcer in rats through evaluating oxidative challenge, inflammatory response, apoptotic events, and histopathological changes in the gastric tissue.

Full text

J Food Biochem. 2022;00:e14070. wileyonlinelibrary.com/journal/jfbc 
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https://doi.org/10.1111/jc.14070
© 2022 Wiley Periodicals LLC.
Received:18September2021
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Revised:8D ecemb er2021
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Accepted :9Decemb er2021
DOI:10.1111/jfbc.14070
ORIGINAL ARTICLE
Antiulcer activity of proanthocyanidins is mediated via
suppression of oxidative, inflammatory, and apoptotic
machineries
Maha S. Lokman1,2 | Dalia Zaafar3 | Hussam A. Althagafi4 | Mohamed M. Abdel Daim5 |
Abdulrahman Theyab6 | Ahmad Hasan Mufti7 | Mohammad Algahtani8 | Ola A. Habotta9 |
Abdullah A. A. Alghamdi10 | Khalaf F. Alsharif11 | Ashraf Albrakati12 |
Atif Abdulwahab A. Oyouni13,14 | Amira A. Bauomy15 | Roua S. Baty16 |
Ahmed S. Zhery17 | Khalid E. Hassan18 | Ahmed E. Abdel Moneim2 | Rami B. Kassab2,4
1Biolog yDepartment,CollegeofScienceandHumanities,PrinceSat tambinAbdulA zizUniversit y,Alkhar j,SaudiArabia
2Depar tmentofZoolog yandEntomolog y,FacultyofS cience,HelwanUniversity,Cairo,Egy pt
3Depar tmentofPharma colog yandToxicology,Facult yofPharmacy,ModernUniversityforTechnolo gyandInformat ion,Cairo,Eg ypt
4Depar tmentofBiolog y,FacultyofSciencea ndArts,Al-BahaUniversit y,Almakhwah,SaudiArabia
5Depar tmentofPharma colog y,FacultyofVeterinar yMedicine,SuezCanalUniver sity,Ismailia,Egypt
6Depar tmentofLabor atoryMedici ne,SecurityForcesHospital,Mecca,SaudiArabia
7MedicalGeneticsDe part ment,FacultyofMedicine,UmmAl-QuraUniversity,SaudiArabia
8Depar tmentofClinic alLaboratoriesScie nces,CollegeofA ppliedMedicalScien ces,TaifUniversit y,Taif,SaudiArabia
9Depar tmentofForensicMedicineandToxicology,Facult yofVeterinaryMe dicine,Mansou raUnive rsit y,Mansoura,Egypt
10Depa rtme ntofBiology,Facult yofScience,Al-BahaUnive rsity,SaudiArabia
11Depar tmentofClinic alLab orator yScie nces,CollegeofA ppliedMedicalScien ces,TaifUniversit y,Taif,SaudiArabia
12Depar tmentofHumanAn atomy,CollegeofMedicine,TaifUnive rsit y,Taif,SaudiArabia
13Depar tmentofBiology,Facult yofSciences,Uni versit yofTabuk,Tabuk,SaudiArabia
14GenomeandBiotechnologyU nit,FacultyofSciences,UniversityofTabuk,Tabuk,SaudiAra bia
15Depar tmentofScienceLabor atorie s,Coll egeofScienceandArts,QassimUniversity,SaudiArabia
16Depar tmentofBiotechnolog y,CollegeofScience,TaifUniversity,Taif,SaudiArabia
17KasrAl-EiniSch oolofMed icine,C airoUniversity,Cairo,Egypt
18Depar tmentofPathology,CollegeofMedicine,TaifUniversit y,Taif,SaudiAra bia
Correspondence
AshrafAlbra kati,DepartmentofHuman
Anatomy,CollegeofMedicine,Taif
University,P.O.Box11099,Taif21944,
SaudiArabia.
Email:a.albrakati@tu.edu.sa
Funding information
Thisworkw assupportedbyTaif
UniversityResearchersSup port ing
Program(projectnumb er:
TURSP-2020/153),TaifUniversit y,Saudi
Arabia.
Abstract
Gastriculcer(GU)isalesioninthegastricmucosaassociatedwithexcessiveoxidative
damage,inflammatoryresponse,apoptoticevents, and irritationwhichmaydevelop
intocancer.However,medicationscommonly usedin GU treatmentcannot normal-
ize gastric mu cosa, while causin g several adverse ef fects. Proa nthocyanidins (PAs)
aredietaryflavonoidswithnumerousbiologicalandpharmacologicalactivities.Inthe
currentinvestigation,westudiedthepotentialanti-ulcerativeactivityofPAsagainst
acidified ethanol (HCl/ethanol)-caused gastr ic ulceration. Fift y male albino Wistar
ratswereallocatedintofiveequalgroups: control,HCl/ethanol(3mL/kg),lansopra-
zole(LPZ, 30 mg/kg) + HCl/ethanol, and PAs(100and 250 mg/kg) +HCl/ethanol.
LPZandPAswereappliedoneweekbeforegastriculcerinduction.PAspretreatment

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1 | INTRODUCTION
Gastriculcer(GU)isalesion inthegastric mucosallayer associated
with excessive inflammator y response, apoptotic events, oxidative
damage, and irritation, and may develop into cancer with about
5%–10% morbidity rate (Lanas & Chan, 2017). Findings yielded
by emerging studies suggest that GU pathogenesis is linked to
modern d ietary habi ts, as well as long-term consu mption of anti-
inflammatory drugs and Helicobacter pyloriinfection.Thesecaus-
ative factors activate neutrophils in the gastric tissue to produce
excessive am ounts of reac tive oxygen s pecies (ROS) and n itrogen
reactive species (RNS) resulting in the depletion of endogenous
antioxidantsystemand development of mucosal oxidative damage
(Al-Quraishy et al., 2017; Lanas & Chan, 2017). ROS initiates an
inflamma tory resp onse throu gh activat ion of redox-sensitive t ran-
scriptionfactors,includingnuclear factor kappa-B,that upregulate
thevarietyofpro-inflammatorycytokines,adhesionmolecules,and
othermediators,therebyenhancinggastriculceration.However,the
exactmechanismsimplicatedinthepathogenesisofGUremainun-
clear(Zhouetal.,2020).
Hydrochloric acid and ethanol (acidified ethanol) model has
been wid ely used to ulce rate gast ric mucosal e xperime ntally, as it
is known to over produce c ytotoxic fre e radical s which trig ger the
development of oxidative stress and inflammation (Abdelfattah
et al., 2019). This mo del is associat ed with morp hological a nd ne-
crotic alte rations in th e gastric m ucosa, alon g with distur bance of
gastricsecretion,neutrophilactivation,hemorrhagiclesions,edema,
and epithelial exfoliation, which mimic GU pathogenesis in human
(Aziz et al., 2019;Shinetal.,2020).Thecurrent interventionssuch
asprotonpumpinhibitors,H2blockers,andantacidsareassociated
withadversereactions,includinghypoacidity,gynecomastia,impo-
tence,osteoporosis,bonefracture,andcardiovascularimpairments
(Kulikova et al., 2020; Zhang et al., 2019). Therefore, alternative
drugsthatdonotinducesuchharmfulsideeffects,whilehavinghigh
therapeutic efficiency are required.
notably reduced gastric mucosal macroscopic and microscopic pathological changes
inadose-dependentmanner.Additionally,PAsactivatedtheinnateantioxidantmol-
eculesincludingglutathioneanditsderivedantioxidants(glutathioneperoxidaseand
glutat hi onereductase),alongwit hsup eroxidedismut aseandcat alase,whileat tenuat-
ingpro-oxidantformation,includingmalondialdehydeandnitricoxide.Interestingly,
PAssupplementationatahigherdosesuppressedgastricinflammatoryandapoptotic
responses, as demons trated by the reduced levels of interleukin-1β, interleukin-6,
tumornecrosisfactoralpha,high-mobilitygroupbox1,cyclooxygenase2,prostaglan-
dinE2,nuclearfactorkappa-B,Bcl-2-associatedXprotein,andcaspase-3,whileBcell
lymphoma2 was elevated.Hence,PAscouldexhibitantiulceractivitybyprotecting
gastric t issue from the devel opment of oxidative dam age, inflammator y responses ,
and apoptosis events associated with ulceration.
Practical implications: Gastriculcerisalesioninthegastricmucosallayerassociated
with excessive inf lammatory resp onse, apoptotic event s, oxidative damage, an d ir-
ritation,andmaydevelopintocancer withabout5%–10%morbidityrate.However,
medicationscommonlyusedinGUtreatmentcannotnormalizegastricmucosa,while
causingseveral adverseeffects.Therefore, newtherapeuticapproachesareneeded
to treat or prevent g astric ulceration . Proanthocyanid ins (PAs, conde nsed tannins)
aredietaryflavonoids foundinabundanceindifferentplant species,includingtheir
fruits,bark,andseeds.Duetotheir potentantioxidativeactivity,PAshavebeenap-
plied to prevent ortreat oxidative stress-related diseases, including cancer, as well
asmetabolic, neurodegenerative,cardiovascular,and inflammatorydisorders.Here,
weexamine the potential therapeuticroleofproanthocyanidins(PAs)against acidi-
fiedethanol-inducedgastric ulcer in ratsthrough evaluatingoxidative challenge, in-
flammatoryresponse,apoptoticevents,andhistopathologicalchangesinthegastric
tissue.
KEYWORDS
apoptosis,gastriculcer,inflammation,oxidativedamage,proanthocyanidins

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LOKMAN e t AL.
Naturaldietaryantioxidantshavebeenproveneffectiveindis-
eas eprevent ionan dtreatment(Lieta l.,20 21),andare,t he refor e,
thenatural choice in this domain. Proanthocyanidins (PAs, con-
densedtannins)aredietaryflavonoidsfoundinabundanceindif-
ferentplantspecies,includingtheirfruits,bark,andseeds(Wang
etal.,2020).Dueto theirpotentantioxidativeactivit y,PAshave
beenappliedtopreventortreatoxidativestress-relateddiseases,
including cancer, as well as metabolic, neurodegenerative, car-
diovascular, and inflammatory disorders (Beecher, 2004; Wang
etal.,2020).
Hence, theaimofthecurrentstudy wasto investigatethepo-
tentialantiulcerroleofPAsadministeredintwodosesagainstacid-
ified ethanol-induced gastric ulcerationinrats by evaluating redox
homeostasis,andinflammatoryandapoptoticchallenges,as wellas
conducting microscopic and macroscopic analyses.
2 | MATERIALS AND METHODS
2.1 | Chemicals and reagents
Purifiedgrapeseedsoligomericproanthocyanidinsandlansoprazole
wereobtainedfrom Sigma ChemicalCompany,St.Louis,MO,USA .
Ethanolwas purchasedfromAl-GomhoriaCompanyfor medicines
andmedicalsupplies,Cairo,Egypt.Allotherusedchemicalswereof
high analytical grade.
2.2 | Animal grouping and study design
FiftymalealbinoWistarrats,10-week-oldweighing170–180gwere
purchasedfromVACSERA(Cairo,Egypt).Priortostar tingtheexperi-
mental protocol, rats were acclimatized to thecontrolledlaboratory
conditions for seven days by housing them in polypropylene cages
(at25°Cunder normal light/darkcycle) and werefedastandard ro-
dent diet with freeaccess towater. The experimental protocol was
approved by t he Instituti onal Animal Ethi cs Committ ee and was in
accordan ce with the guide lines for animal c are and use at Helw an
University (approval no: HU2020/Z/RKA1020-01). After the 7-day
acclimatization period,ratswereallocatedrandomlyintothe follow-
ingfivegroups(n =10):
Controlgroup:receiveddailyoraladministrationofnormalsaline
solution(3mL/kg)foroneweek.
Acidifiedethanol(HCl/ethanol)group:receiveddailyoraladmin-
istrationof normalsalinesolution(3mL/kg)dailyforoneweekbe-
fore induction of gastritis.
Lansoprazole+HCl/ethanol (LPZ+HCl/ethanol) group: received
dailyoraladministrationofLPZ(30mg/kg)foroneweekbeforein-
ducinggastritisbyapplyingacidifiedethanol(Zhouetal.,2020).
Proanthocyanidins+HCl/ethanol(PAs-100+HCl/ethanol)group:
receiveddailyoraladministrationofPAsatadoseof100mg/kgfor
oneweekbeforeinducinggastritisbyapplyingacidifiedethanol.
Proanthocyanidins+HCl/ethanol(PAs-250+HCl/ethanol)group:
receiveddailyor ala dministrationofPAsatadoseof2 50mg/kgfor
oneweekbeforeinducinggastritisbyapplyingacidifiedethanol.
TheexperimentedPAsdoseswereselectedbasedonaprelimi-
narystudyusingdifferentPAsdoses(50,100, and250mg/kg)and
the highest doses showed potent antiulcer effec ts than the lower
dose.
2.3 | Gastric ulcer induction
Priortoulcerinduction,foodwaswithheldfor 24hrwithoutrestrict-
ingwater intake.Gastriculcerwas inducedinallgroups except con-
trol(whichreceivedonlythevehicle)byoraladministrationof0.15M
HCland ethanol(60% inwater) solution (Sigma,St.Louis, MO, USA)
at3mL/kgdoseviaanorogastrictubeaccordingtoSonetal.(2015).
2.4 | Gastric ulcer lesion
Ratsweresacrificedbycervicaldecapitation,afterwhichthestom-
achtissueswereseparatedandfixedin4%formalinsolutionforone
hour.The collected tissues werethen opened by an incision along
the greater curvature and were photographed using a Samsung cam-
era(WB30F,Samsung,Japan).Thetotalerosivemucosallesionarea
(mm2) was determined by a ImageJ sof twareversion1.5 (National
InstitutesofHealth,USA).
The followingexpression was usedtocalculatethe percentage
oflesioninhibitionbyPAspretreatment:
where%Larearepresentsthepercentageofulceratedareasorhemor-
rhages in the gastric mucosa.
2.5 | Gastric ulcer index (GUI) determination
Thed egr eeofinjuryint hegastricmucosawase stimateda ccordingtoa
0–5sco rin gm e tho df org ros sp ath o log yde ve l ope dbyAr a be tal .( 2015) ,
as following: 0 = no lesions; 1 = small hemorrhagic lesions; 2 = lesions
smaller than 2 mm; 3 = lesions 2– 3 mm; 4 = lesions 3– 4 mm; 5 = lesion
greaterthan4mm.Themeanscorewasdetermined ,calculateda ndt he
GUIwasexpressedbyatechnicianblindedtothestudyprotocol.
2.6 | Gastric mucus adherence measurement
Alcianblue(Sigma,USA),whichisanacidmucopolysaccharidestain-
ingdye,wasused to estimatethe adherence ofgastricmucosa ac-
cordingtotheprotocolproposedbySonetal.(2015).Theamountof
Percent of lesioninhibition (%)=
%
Larea untreated lesion rats −%Larea PAs treated lesion rats
%Larea untreated lesion rats ,

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thealcian blue (µg/gtissue)reflectedthedegree ofgastricmucosa
adherence.
2.7 | Histopathological and immunohistochemical
examinations
Collectedtissue samples werefixed in 10% formalin, embedded in
paraffin, and sectioned into 4-mm-thick specimens. Sections were
then stained with H&E st ain and alcian bluestainand were inves-
tigatedunderaNikon EclipseE200-LED(Tokyo,Japan)microscope
(400×magnification).
2.8 | Estimation of reactive oxygen species (ROS)
Intracellular generation of ROSingastrictissue was measured using
greenfluorescencestain(2,7-dichlorofluoresceindiacetate,DCF-DA)
according t o the method desc ribed by Wang & Roper (2014) with 
some mod ification. In b rief, gastric tis sue homogenate s were incu-
bated in DCF-DAdissolvedin the medium at 37°C for 30 min. The
meanfluorescence intensity ofDCF-DAwas evaluatedbya fluores-
cenceplatereader(excit ationʎ4 85nmandemissionʎ525nm).
2.9 | Examination of oxidative stress indices in the
gastric tissue
Glandularpartsofthegastrictissuewereweighedandthenho-
mogenized i n a PH 7.4 , 50 mM Tris–HCl ice -col d medium. The
gastric homogenate was centrifuged for 10 min at 1000 g and
4°Cbeforebeingstoredat−80°C.Thedeterminationofthesu-
pe rna tan tpr ot ein co nte nt was per f or m eda cc ord ingto theLo wry
method (L owry et al. , 1951). Li pid peroxida tion levels were e x-
amined in t he form of malon dialdehyde amo unt (MDA) via the
thiobarbituricacidreactionmethod(Ohkawaetal.,1979).Inad-
dition,nitricoxide(NO)levelwasdeterminedbytheGriesssolu-
tion method(Green et al.,1982)andglutathione (GSH) content
wasassayed basedontheSedlakandLindsay method(Sedlak &
Lindsay,1968).
2.10 | Examination of antioxidative enzymes in the
gastric tissue
SOD act ivity was teste d colorimetric ally at λ = 560 nm in the
gastric supernatants by applying the Nishikimi et al. method
(Nishikimi et al., 1972). CAT activity was determined using the
methoddescribedbyAebi(1984),whichisbasedontheH2O2 de-
composition rate at λ =240nm.Glutathionereductase(GR)and
glutathione peroxidase(GPx)were estimated based on the pro-
cedures describedbyPaglia & Valentine(1967)and Factor et al.
(1998),respectively.
2.11 | Examination of pro- inflammatory mediators
in the gastric tissue
For the determination of the pro-inflammatory cytokines in the
gastric tissue, commercial ELISA kits were used for IL-1β (cata-
log no. RAB0278), IL-6 (catalog no. EZRIL6), and TNF-α (catalog
no. EZRTNFA) obtained from Merck Millipore (Toronto, Ontario,
Canada)andSigma-Aldrich(St.Louis,MO,USA),respectively.
2.12 | Real- time PCR (molecular assay)
Toextracttotal RNA from gastrictissue,RNeasyPlus Minikit was
used(Qiagen,Valencia,CA).Forfirst-strandcDNAsynthesis,oneµg
of RNAa n dhe x a m ersw e rep r o ces s e dus i n gth e S cri p tTM c DNAs y n-
thesiskit(Bio-Rad,CA).TheDNAsamplesforreal-timePCRanalysis
wererun in triplicate.PowerSYBR1 Green (Life Technologies, CA)
was used to am plify cDNA in an A pplied Biosys tems Instrum ent.
ThePCRreac tionwasperformedforfourminutesat95°C,followed
by4 0cycles of 94°C for60s,55°Cfor60 s, and a finalextension
for10minat72°C.The∆Ct wasc alculatedafterPCRamplification
by subtracting the GapdhCt from eachsample Ct. AllPCRprimers
used for Hmgb1, Nos2, Ptgs2, Nrf2I2, and Hmox1were plotted using
theNCBIPrimer-Blast(JenaBioscienceGmbH,Jena,Germany).The
examinedgeneprimersequencesandaccessionnumbersaregiven
inTable1.
2.13 | Statistical analyses
All results were expressedas mean ± SD. One-wayANOVAwas
carried out, and Tukey's test was conducted for comparisons
between groups. All analyses were conducted using st atistical
TAB LE 1 Primersequencesofgenesanalyzedinrealtime
polymerase chain reaction
Name Forward primer (5′−3′) Reverse primer (5′−3′)
Gapdh AGTGCCAGCCTCG
TCTCATA
GATGGTGATGGG
TTTCCCGT
Nfe2l2 TTGTAGATGACCAT
GAGTCGC
ACTTCCAGGGGCA
CTGTC TA
Hmox1 TTAAGC TGGTG ATG
GCCTCC
GTGGGGCATAGA
CTGGGTTC
Nos2 GT TCCTC AGGC TTG
GGTCTT
TGGGGGAACACA
GTAATGGC
Hm gb1 G TGCCTCGCGGAG
GAAAATC
AGTTG ACAGA AGC
ATCCGGG
Ptgs2 CTCAGCCATGCAGC
AAATCC
GGGTGGGCTTCAG
CA GTA AT
Abbreviations:Gapdh,Glyceraldehyde-3-phosphatedehydrogenase;
Hm gb1 ,Highmobilitygroupprotein1;Hmox1,Hemeoxygenase1;
Nfe2l2,Nuclearfac torer ythroid2-relatedfactor2;Nos2,Inducible
nitricoxidesynthase;Ptgs2,prostaglandin-endoperoxidesynthase2.

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package S PSS version 23 .0 and p < .05 was considered statisti-
cally significant.
3 | RESULTS
3.1 | Antiulcer effect of proanthocyanidins
MacroscopicobservationsinHCL/ethanol-injectedratsshowedob-
vioustransmuralinflammationwithhyperemia,mucosaledema,and
severeulcerations.However,administrationofPAsatlow(100mg/
kg)andhigh(250mg/kg)dosesdecreasedmarkedlytheulcerations
anditsassociatedinflammatorysignsascomparedtoHCL/ethanol-
injected group. Additionally, PAs decreased the development of
ulcerareaandthegastriculcerindexaswellincomparisonwiththe
experimentalgastritisgroup(Figure1a–c).
Additionally,gastric mucosal adherence was assessedusing alcian
blue staining, the results revealed that the binding capacity of alcian
blue to the g astric mucus w as markedly redu ced in the HCl/ethanol 
injectedgroup,whereasfindingsofLPZ-orPAs-pretreatedgroupswere
similartocontrols,indicatingthattheysuccessfullypreservedthebind-
ingabilit yofalcianbluetothegastricmucosallayer(Figure1d).
3.2 | Proanthocyanidins administration protects
gastric tissue following HCl/ethanol administration
Histological analyses revealedthat, while healthy controlsandPAs
administered groups had intact stomach wall architecture and com-
pletemucosal layer,ratstreatedwithHCl/ethanolexhibitedsevere
gastricdamage,with extensively swollen submucosal layer,gastric
mucos ad esquamation,thin ni ng ,p al eness,ande ro si on ,aswellasse-
verely disruptedgastricglands. Theselesionswereassociatedwith
infiltration of inflammatorycells to all stomach layers.However,in
rats pr etreated with PAs, g astric lesion s were alleviated an d gas-
tric mucosal injur y was lessened and was associated with low des-
quamation of the gastric epithelial lining and low inflammatory cell
infiltration.
FIGURE 1 EffectsofPAson(a)themorphologyofgastriculceration,(b)gastriculcerarea(mm2),(c)gastriculcerindex,and(d)alcianblue
bindingtogastricmucusinratsexposedtoacidifiedethanol.Dataareexpressedasmean± SDandwereanalyzedusingone-wayANOVA,
followedbyTukey'sposthoctestatp <.05.Barsthatdonotsharethesameletters(superscripts)aresignificantlydifferentfromeach
other.EtOH/HCL:acidifiedethanol,LPZ:lansoprazole(30mg/kg),PAs-100:proanthocyanidines(100mg/kg),PAs-250:proanthocyanidines
(250mg/k g),n = 10
(a)
(b) (c) (d)

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Additionally, the gastroprotective activity of PAs administra-
tion was also confirmed via PAS-alcian blue staining, which was
pronounced in control and PAs-pretreated groups, but was of low
intensityinthe HCl/ethanolgroup. Conversely, PAsadministration
likeLPZwasfoundtoincreasestain intensity,reflectingtheabilit y
ofPA stoincr easeglyco pr ot ei nc ontentinthega str ic mu cosaand re -
ducegastricerosionfollowingHCl/ethanoladministration(Figure2).
3.3 | Effect of proanthocyanidins on redox status
following HCl/ethanol administration
Comparedtothecontrolgroup,ratsintheHCl/ethanol-administered
groupexhibitedsignificantlyhigherROS(Figure3a),MDA(Figure3b),
andNOlevels(Figure3c),alongwith lower GSH levels(Figure 3d),
andasignificantdecreaseintheantioxidantenzymesactivity[(SOD,
Figure 4a), (C AT, Figure 4b), (GR , Figure 4 c), and (GPx , Figure 4a)]
activ ity. Notably, PAs supp lementatio n (in both 100 and 2 50 mg/
kg dose) re stored the bal ance betwee n oxidants and an tioxidants
in the gas tric tissu e following HC l/ethanol injec tion by decre asing
ROS, MDA,and NO productionand enhancing theexaminedanti-
oxidantproteins.Atthemolecular level, HCl/ethanol-treatedgroup
showedasignificantdownregulationinmRNAexpressionofHmox1
(Figure 5a) an d Nfe2I2 (Figure 5b). Similar to LPZ, PAs administra-
tion at both doses significantly upregulated the transcriptional level
oftheseantioxidantpromoters,thusexhibitingitsantioxidativepo-
tencyagainstoxidativeinjuryassociatedwithgastriculceration.
3.4 | Effect of proanthocyanidins on inflammatory
markers following HCl/ethanol administration
HCl/ethanoladministration induced gastric tissue inflammation, as
reflectedinasignificantelevation(p <.05)inthepro-inflammatory
cy t oki n el e ve ls[(TN F-α, Fig u re6 a) ,( I L-1β,Figure6b),(IL-6,Figure6c),
and(Hmgb1,Figure6d)].Thisinflammatorycascadeextendedtothe
upregulation of Nos2(Fig ure7a)andNF-κBlevel(Figure7b),inaddi-
tion to Pt gs2(Fi gur e7c )a nditsp ro duc t ,PG E2(Fi gur e7d )wh encom -
pared to th e control group. How ever,th is inflammator y response
was signif icantly supp ressed follow ing PAs administ ration at both
doses(100and250mg/k g),reflectingtheprotectiveimpact ofPAs
against inflammatory events that are triggered during the develop-
ment of gastric ulceration.
3.5 | Effect of proanthocyanidins on apoptotic
alterations following HCl/ethanol administration
Toinvestigate theapoptoticevents associated with gastric ulcera-
tion, Bax, caspase-3, and Bcl2 levels inthe gastric tissue homoge-
nate were assessed. Our findings revealed that rats that were
adminis tered acidif ied ethanol s howed higher B ax (Figure 8b) an d
caspase-3levels(Figure8c),whereasBcl2(Figure8a)wasdecreased
significantly (p < .05) co mpared to the cont rols. On the con trary,
bothPAs(100and250mg)andLPZ(30mg/kg)administrationwere
foundtoprotectgastriccelllossbyinhibitingpro-apoptoticproteins,
Baxandcaspase-3,andincreasinganti-apoptoticproteinBcl2.
4 | DISCUSSION
Antioxid ants regulate red ox homeostasis in t he biological sys tem
and provid e protection aga inst oxidative infl ammatory and ap op-
totic challenges that are associated with gastrointestinal disorders
(Akandaet al., 2018; Linetal., 2016). Findings yielded by thepre-
sent study demonstrated the gastroprotective effect of proantho-
cyanidinsagainstacidifiedethanol-inducedgastritisinrats.Acidified
ethanolinjectionisassociatedwithinflammation,oxidativedamage,
andhistopathologicalalteration,which resultsin gastric ulceration
(Abdelfattahetal.,2019;Al-Quraishyetal.,2017).Interestingly,the
macroandmicroscopicobservationsshowedthatPAspretreatment
waseffec tiveinalleviatinggastriculcersinrats,asdemonstratedby
adose-dependentreductionof gastric lesions, gastric ulcerindex,
andgastricwallmucouslevels.Previousstudieshavedemonstrated
thegastroprotectiveroleofantioxidants,thussuppor ting our find-
ings(Abdelfattahetal.,2019;Zhouetal.,2020).
Thecorrelationbetweeninflammationandtheacidifiedethanol-
induced gastriculcer model is well established (Raish et al.,2021).
Determination ofthelevelsofpro-inflammatorymediators reflects
thegastricvisceralinjury.Ourinvestigationshowedmarkedelevation
ofpro-inflammatorymarkersincludingTNF-α,  I L - 1 β,IL-6,andNF-κB
p65 accompa nied by upreg ulation of mRN A expressi on of Hm gb1 ,
Nos2, and P tgs2following HCl/ethanol injection, which was coun-
teracted following the administration of PAs.Duringinflammatory
conditions, theinflamed gastric cells and the activated leukocytes
and macrophages produce excess amount s of pro-inflammatory
cytokines and enzymes, thus help to initiate the innate immune
respons e (Abdelf attah et al ., 2019).T NF-α,  I L - 1 β, and I L-6are pro-
inflammatorycytokinesthathaveintenseimpactsoninflammation
andimmunityandhavebeenfoundtobeincreasedfollowingH.py-
loriinfection-inducedgastriculcerandcancer(Azadegan-Dehkordi
etal.,2015;El-Omaretal.,2001;Tuetal.,2008).Meanwhile,Cox 2
isknownasinflammatorymediatorthatplayaroleasanimmediate-
early response in inflammation and has been found to be activated
indif ferentexperimentalgastritismodels (Abdelfattahet al., 2019;
Santosetal., 2018).Cox2is therate-limitingenzymeinprostaglan-
dins(PGE2)productionfromthearachidonicacid.Theupregulated
mRNAexpressionofCox2mayexplaintheincreasedPGE2levelfol-
lowingHCl/ethanolinjec tionexpression.
High-mobilit y group box 1 (Hmgb1) is a pro-inflammatory cy-
tokine th at can create a s tate of chroni c inflammati on and could in-
crease th e risk of epithelial ma lignancies develo pment. Hmgb1 is a
nuclear protein that could initiate several sequences of inflammatory
reactions strongly involved in gastriculcer pathogenesis. It isone of
the knowninjury markers that could depend on the NF-κB pathway
(Ghweiletal.,2020),whichinturnmayelevatetheexpressionofIL-1β

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and TNF- α(Kangetal.,2014)andthusdelayulcerhealing.Ourre-
sults are suppor ted by the findings yielded by several recent studies
whichdemonstratedthatHMGB1levelsareincreasedingastriculcers
or even gast ric cancer s, and may inhibit r ecovery (Al zokaky, A. A.m,
etal.,2020).GastriculcerisassociatedwithROSoverproductionthat
triggers the accumulation of innate immune cells at the site of injury
resultingintranslocationandactivationofNF-κB which enhances the
secretionandexpressionofpro-inflammatorymediators,asconfirmed
byourfindings(Chenetal.,2017;Kangetal.,2014).
Interestingly, PAs administration inhibited the developed
inflammatory reactions following HCl/ethanol injection in a
dose-dependent manner, as evidenced by the decreased pro-
inflammatorycytokinesandNos2,P tgs2,Hm gb1mRNAexpressions
alon gw it hPG E2 an dN F- κBlevels.Downregulationofinflammatory
mediatorswasshowntoprotectgastrictissue(Akandaetal.,2018).
PAswas foundto exertanti-inflammatory ac tivit y through deacti-
vationofNF-κB,modulatingarachidonicacidpathway,suppressing
eicosanoidproducingenzymes, downregulating mRNA expression,
andreleaseof pro-inflammatorycytokines, and deactivating Cox-2
aswellasregulatingmitogen-activatedproteinkinasesignaling(Chu
et al., 2016;Limtrakul et al., 2016; Martinez-Micaelo et al., 2012).
Inparallelwith previousrepor ts, ourresultsindicate thatPAsmay
FIGURE 2 EffectsofPAsonhistopathologicalchangesfollowingHCl/ethanol-inducedgastriculcerinrats.Scalebar= 80 µm
FIGURE 3 EffectsofPAsonlevelsofoxidativestressmarkers:(a)ROS,(b)MDA(nmol/mgprotein),(c)NO(µmol/mgprotein)and(d)GSH
(mmol/mgprotein)inratsexposedtoEtOH/HCl.Dataareexpressedasmean± SDandwereanalyzedusingone-wayANOVAfollowedby
Tukey'sposthoctestatp <.05.Barsthatdonotsharethesamelet ters(superscripts)aresignificantlydifferentfromeachother.EtOH/HCL,
acidifiedethanol;GSH,glutathione;LPZ,lansoprazole(30mg/kg);MDA,malondialdehyde;NO,nitricoxide;PAs-100,proanthocyanidines
(100mg/kg);PAs-250,proanthocyanidines(250mg/kg);ROS,reactiveoxygenspecies,n = 10
(a) (b)
(c) (d)

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suppress early steps of inflammation and are able to modulate the
production of pro-inflammatory mediators through inhibition of
iNOS,Cox2,PGE-2,andNF-κB activity.
Oxidativestressplaysafundamentalroleinthepathophysiology
ofinflammatory disorders includinggastriculceration(Abdelfattah
etal., 2019). Our findingsindicatethe developmentofgastric oxi-
dativedamage, ascharacterizedby theupregulation ofROS,MDA,
andNO,aswellasbydepletionof antioxidant molecules andtheir
regulators,Nrf2andHO-1.Anamplebodyofevidenceindicatesthat
ROS produc ed in gastri c mucosa by neu trophils pl ays an essenti al
FIGURE 4 EffectsofPAsontheactivityofenzymaticantioxidativemarkers:(a)SOD(U/mgprotein),(b)CAT(U/mgprotein),(c)GR(µmol/
mgprotein),and(d)GPx(U/mgprotein)inratsexposedtoEtOH/HCl.Dataareexpressedasmean± SDandwereanalyzedusingone-way
ANOVAfollowedbyTukey'sposthoctestatp <.05.Barsthatdonotsharethesameletters(superscripts)aresignificantlydifferentfrom
eachother.CAT,catalase;EtOH/HCL,acidifiedethanol;GPx,glutathioneperoxidase;GR,glutathionereductase;LPZ,lansoprazole(30mg/
kg);PAs-100,proanthocyanidines(100mg/kg);PAs-250,proanthocyanidines(250mg/kg);SOD,superoxidedismutase,n = 10
(a) (b)
(c) (d)
FIGURE 5 EffectsofPAsonmRNAexpressionof(a)Hmox1and(b)Nfe2l2inratsexposedtoEtOH/HCl.PCRfindingsareexpressedas
the mean ± SD of triplicate assays referenced to Gapdhandexpressedasfoldchanges(log2scale)comparedtothemRNAexpressionin
thecontrolsandEtOH/HCLinjectedgroup.Datawereanalyzedusingone-wayANOVAfollowedbyTukey'sposthoctestatp < .05. Bars
thatdonotsharethesameletters(superscripts)aresignificantlydifferentfromeachother.EtOH/HCL,acidifiedethanol;LPZ,lansoprazole
(30mg/kg);PAs-100,proanthocyanidines(100mg/k g);PAs-250,proanthocyanidines(250mg/kg);Hmox1,hemeoxygenase-1;Nfe2l2,
nuclearfactorerythroid2-relatedfactor2,alsoknownasnuclearfactorerythroid-derived2-like2,n = 10
(a) (b)

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role in the d amage cause d to the gastr ic mucosal ep ithelium. T he
imbalancebetweenincreased ROS levels anddepletedantioxidant
levels is one of the pathophysiological mechanisms of gastric ulcer
progression(Yeoetal.,2021).RecentstudiesfurtherrevealthatHCl
and/orethanolcauseshypoxiabydisturbingthemicrocirculationin
gastricmucosa,generatingROS,andinitiatinglipidperoxidation(Suo
etal.,2016).TheincreasedMDAisassociatedwithROSproduction
andusedasanindicator for lipidperoxidationandconfirms the in-
cidenceofoxidativedamage.Additionally,upregulationofiNOSex-
pressionincreasesNOproduction,whereby excess NOreact swith
O2toproduceperoxynitriteresultinginseverecellularimpairments
(Sunet al., 2021).Indeed,the depletedcellular antioxidativecapac-
ityduringthedevelopmentof gastriculcerationislinkedwith ROS
overproduction and the downregulation of Nrf2/HO-1 signaling
(Yeoetal.,2018),asobtainedinthecurrentstudy.
The curr ent study reveale d that PAs administr ation reduced
ROS generation, gastric lipid peroxidation, and NO formation,
while inc reased antioxidat ion markers, inc luding GSH, GP x, GR,
SOD, and CAT, and upregulated mRN A expression of Nrf2 and
HO -1, dem o nst r ati ngt hatit s ant i- ulc ero gen icp rop e r tie sar ep a r tly 
dependentonitsantioxidant function. The antioxidativecapacity
of PAs has been at tribute d to their abil ity to scaven ge free rad-
icals through donating an electron to free radicals - OH groups
connectedtothephenolicring,resultingintheterminationofthe
oxidative c hain reactions ( Yang et al., 2018. 2018.). In ad dition,
PAsinhibitedROSgenerationandlipoperoxidationinerythrocy tes
and lymphocytes following cadmium exposure in rat s (Nazima
etal.,2016).Moreover,Rajput et al.(2019)reported thatPAs in-
hibited oxidative indults mediated by AflatoxinB1inthe bursaof
Fabriciusofbroilersbydecreasinglipoperoxidationandenhancing
thelevelandexpressionofcellularantioxidants.Baket al. (2016)
showedthatPAsinhibitedROSgenerationandincreasedthelevel
andexpressionofantioxidantenzymesinbothHepG2cellsandrat
liver.InadditiontoitsabilitytoinhibitROSproductionassociated
withgastriculceration,PAswasfoundtoenhancetheexpression
ofNrf 2andH O-1.N rf2isat ra nscriptio nalfactorth atcontrols and
regulate s the activit y and expressi on of antioxidant en zymes. It
was also found to maintain the integrity of the mucosal membrane
inresponsetoethanolexposure(Badretal., 2019).HO-1isanin-
ducible enzymethatcatalyzeshemeintoFe2+,CO,andbiliverdin.
HO-1 wasfound to protect thecellthroughits antioxidant,anti-
inflammatory,andanti-apoptoticproperties.TheupregulatedNrf2
FIGURE 6 EffectsofPAsonlevelsofpro-inflammator ycytokines(a)TNF-αlevels(pg/mgprotein),(b)IL-1β(pg/mgprotein),(c)IL-6
levels(pg/mgprotein),and(d)H m gb1 mRNAexpressioninratsexposedtoEtOH/HCl.PCRfindingsareexpressedfromtriplicateassays
referenced to Gapdhandexpressedasfoldchanges(log2scale)comparedtothemRNAexpressioninthecontrolsandEtOH/HCLinjected
group.Alldataareexpressedasmean± SDandwereanaly zedusingone-wayANOVAfollowedbyTukey'sposthoctestatp < .05. Bars that
donotsharethesameletters(superscripts)aresignificantlydifferentfromeachother.EtOH/HCL ,acidifiedethanol;H mgb1,Highmobility
groupbox1;IL-1β,interleukin1β;IL-6,interleukin6;LPZ,lansoprazole(30mg /kg);PAs-100,proanthocyanidines(100mg/kg);PAs-250,
proanthocyanidines(250mg/kg);TNF-α,tumornecrosisfactorα,n = 10
(a) (b)
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andHO-1followingPAsadministrationprovideagastroprotective
effect against HCl/ethanol-induced oxidative insults. Previous
studiesdemonstrated thatPAsinhibitedthedevelopmentofhe-
patic oxidative injur y in response to lead intoxication through
activ ating Nrf2 /AREsi gnaling (Lon g et al., 2016). In the diab etic
model,PAsexhibitedantioxidativecapacitythroughinducingNrf2
andHO-1expression(Dingetal.,2020).
Accumulative studies link between oxidative stress, inflamma-
tion,andthedevelopmentofapoptosisduringgastriculceration.In
thecurrentstudy,apoptoticcascadewasobservedingastrictissue
FIGURE 7 EffectsofPAsonmRNAexpressionandlevelof(a)Nos2,(b)NF-κB,(c)Ptgs2,and(d)PGE2(ng/mgprotein)inratsexposedto
EtOH/HCl.PCRfindingsareexpressedfromtriplicateassaysreferencedtoGapdhandexpressedasfoldchanges(log2scale)comparedto
themRNAexpressioninthecontrolsandEtOH/HCLinjectedgroup.Alldataareexpressedasmean± SDandwereanalyzedusingone-way
ANOVAfollowedbyTukey'sposthoctestatp <.05.Barsthatdonotsharethesameletters(superscripts)aresignificantlydifferentfrom
eachother.EtOH/HCL,acidifiedethanol;LPZ,lansoprazole(30mg/kg);NF-κB,nuclearfactorkappa-light-chain-enhancer;Nos2,inducible
nitricoxidesynthase;PAs-100,proanthocyanidines(100mg/kg);PAs-250,proanthocyanidines(250mg/kg);PGE2,prostaglandinE2;Ptgs2,
prostaglandin-endoperoxidesynthase2(cyclooxygenase-2,Cox2),n = 10
(a) (b)
(c) (d)
FIGURE 8 EffectsofPAsonlevelsofapoptoticmarkers:(a)Bcl-2(ng/mgprotein),(b)Bax(ng/mgprotein)and(c)caspase-3(µmolpNA/
min/mgprotein)inratsexposedtoEtOH/HCl.Dataareexpressedasmean± SDandwereanalyzedusingone-wayANOVAfollowedby
Tukey'sposthoctestatp <.05.Barsthatdonotsharethesamelet ters(superscripts)aresignificantlydifferentfromeachother.Bax,Bcl-
2-associatedXprotein;Bcl2,B-celllymphoma2;EtOH/HCL,acidifiedethanol;LPZ,lansoprazole(30mg/kg);PAs-100,proanthocyanidines
(100mg/kg);PAs-250,proanthocyanidines(250mg/kg),n = 10
(a) (b) (c)

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upon HCl/ethan ol injecti on as demons trated by the el evated pro-
apoptoticmarkers, Bax andcaspase3,andthe decreased Bcl2,the
anti-apoptoticmarker.Abdelfattahetal.(2019)reportedthatgastric
epithelium loss was pronounced following HCl/ethanol injection.
Theauthors recordeda significantincreaseinthepro-apoptogenic
proteins anda decrease in theanti-apoptotic protein. Theseapop-
totic eventsingastriculceration have beenexplained by the over-
produc tion of ROS. Interestingly, PAs administration blocked the
apoptoti c machinery a ssociated with g astric erosi ons. It has been
suggestedthatPAsfromdifferentnaturalresourcesisabletoinhibit
cell death via an intrinsic pathway through enhancing Bcl2 proteins
and inhibiting mitochondrial dysfunction (Puiggròs et al., 2014).
Additionally,theanti-apoptoticactivity of PAshas been attributed
toscavengetheaccumulatedfreeradicals(Bouazizetal.,20 07).
5 | CONCLUSION
Theresults reportedinthisworkindicate that PAs administration
iscapable of protecting gastric tissuefollowingHCL/ethanol injec-
tion, as evidenced by the improved macroscopic and microscopic
struc tures, the decr eased gastric le sions, and gastr ic ulcer index.
PAs were also sh own to restore the b alance betwe en the gastric
pro-oxidants and antioxidants while inhibiting inflammatory reac-
tions and suppressing apoptotic events. All these findings indicate
thatPAsarepromisinggastroprotectiveagentsandcouldbeusedto
treatorpreventgastriculcerationanditsassociatedoxidativedam-
age,inflammation,apoptosis,andhistologicalchanges.
CONFLICT OF INTEREST
Theauthordeclaresthatthereisnoconflictofinterest.
AUTHOR CONTRIBUTIONS
Maha Lokman: Conceptualization. Dalia Zaafar: Conceptualization.
Mohamed M. Abdel- Daim: Formal analysis . Abdulrahman Theyab:
Investigation. Hasan Mufti:Formal analysis. Mohammad Algahtani:
Investigation. Ola Habotta: Project administration. Abdullah A.
Alghamdi: Resources. Khalaf F. Alsharif: Software. Ashraf Albrakati:
Writing—review&editing.Atif A. Oyouni:Validation.Amira Bauomy:
Writing—original draft. Roua S. Bat y: Writing—review & editing.
Ahmed Zehry: Funding acquisition. Rami Kassab: Methodology.
Hussam A. Althagafi: Supervision. Khalid E. Hassan: Formal
analysis. Ahmed E. Abdel Moneim:Fundingacquisition;Supervision;
Validation;Visualization.
ETHICAL APPROVAL
TheexperimentalprotocolwasapprovedbytheInstitutionalAnimal
Ethics Committe e and was in accordance w ith the guideline s for
animalcareanduseatHelwanUniversity(approvalno:HU2020/Z/
RKA1020-01).
DATA AVAIL AB ILI T Y STAT EME N T
Thedataareavailableuponrequestfromthecorrespondingauthor.
ORCID
Maha S. Lokman https://orcid.org/0000-0003-2830-3479
Dalia Zaafar https://orcid.org/0000-0002-7201-3658
Hussam A. Althagafi https://orcid.org/0000-0003-0311-2975
Ashraf Albrakati https://orcid.org/0000-0002-4116-7865
Atif Abdulwahab A. Oyouni https://orcid.org/0000-0001-8762-4999
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How to cite this article:Lokman,M.S.,Zaafar,D.,Althagafi,
H.A.,AbdelDaim,M.M.,Theyab,A .,HasanMufti,A.,
Algahtani,M.,Habotta,O.A.,Alghamdi,A.A .A.,Alsharif,K.
F.,Albrakati,A.,Oyouni,A.A.A.,Bauomy,A.A.,Baty,R.S.,
Zhery,A.S.,Hassan,K.E.,AbdelMoneim,A.E.,&Kassab,R .
B.(2022).Antiulceractivityofproanthocyanidinsismediated
viasuppressionofoxidative,inflammator y,andapoptotic
machineries. Journal of Food Biochemistry,00,e14070.
https://doi.org/10.1111/jfbc.14070