GPETAFLR, a biopeptide from Lupinus angustifolius L., protects against oxidative and inflammatory damage in retinal pigment epithelium cells

J Food Biochem. 2019;43:e12995. wileyonlinelibrary.com/journal/jfbc  | 1 of 7 https://doi.org/10.1111/jfbc.12995 © 2019 Wiley Periodicals, Inc. 1 | INTRODUCTION Proteins from plants have raised as an option for the isolation of bioactive peptides (Lee & Hur, 2017; Pihlanto, Mattila, Makinen, & Pajari, 2017). These biopeptides are short amino acid sequences, inactive in the native protein, that are released by digestive enzymes and then reach systemic circulation as bioactive molecules (Millan‐ Linares, Millan, Pedroche, & Yust, 2015). Recently, our laboratory has demonstrated the presence of a novel octapeptide isolated Received:8February2019 | Revised:22June2019 | Accepted:26June2019 DOI: 10.1111/jfbc.12995 SHORT COMMUNICATION GPETAFLR, a biopeptide from Lupinus angustifolius L., protects against oxidative and inflammatory damage in retinal pigment epithelium cells Maria C. Millan‐Linares1 | Rocio Toscano2,3 | Ana Lemus‐Conejo2,3 | Maria E. Martin4 | Justo Pedroche2 | Francisco Millan2 | Sergio Montserrat‐de la Paz3 Abbreviations:AMD,age‐relatedmaculardisease;BRB,blood–retinalbarrier;CNV,choroidneovascularization;GSH,glutathione;IFN,interferon;IL,interleukin;LPS,lipopolysaccharide;NO,nitricoxide;PBS,phosphate‐bufferedsaline;ROS,reactiveoxygenspecies;RPE,retinalpigmentepithelium;TNF,tumornecrosisfactor;VEGF,vascularendothelialgrowth factor. 1CellBiologyUnit,InstitutodelaGrasa, CSIC,Seville,Spain 2Department of Food & Health, Instituto de laGrasa,CSIC,Seville,Spain 3DepartmentofMedicalBiochemistry, MolecularBiology,andImmunology,School ofMedicine,UniversidaddeSevilla,Seville, Spain 4DepartmentofCellBiology,Facultyof Biology,UniversidaddeSevilla,Seville,Spain Correspondence SergioMontserrat‐delaPaz,Departmentof MedicalBiochemistry,MolecularBiology, andImmunology,SchoolofMedicine, UniversidaddeSevilla,Av.Dr.Fedriani3, 41071Seville,Spain. Email:[email protected] Abstract GPETAFLR,anoctapeptidereleasedfromtheenzymatichydrolysisoflupine(Lupinus angustifolius L.) protein, has demonstrated anti‐inflammatory effect in myeloid lineage. Thisworkaimstoevaluateinretinalpigmentepithelium(RPE)cellstheprotectiverole ofGPETAFLRonbothoxidativeandinflammatorymarkersknowntobeinvolvedin age‐relatedmaculardegeneration(AMD).Incomparisonwithstimulatedcontrolcells, GPETAFLRincreasedglutathioneproductionanddiminishedthesecretionandgene expressionofVEFG,IL‐1β,IL‐6,IFNγ,andTNF‐α,aswellasreactiveoxygenspecies, andnitriteoutput.OurfindingsrevealthatGPETAFLR,anovelplantpeptide,isable toprotectagainstRPEoxidativestressandinflammation.Takentogether,theseresults strongly support innovative nutritional strategies considering Lupinus angustifolius L. as sourceofproteinstopreventtheonsetandprogressionofAMD. Practical applications WerevealanovelnutraceuticalimpactofGPETAFLRpeptideinhumanRPEcellsto preventoxidativeandinflammatorymediators.Ourresultssupportthattheintake of Lupine angustifoliusL.,proposedtobeareservoirofGPETAFLR,couldlessenthe functionaldecayofRPEcells,leadingthereforetoaslowdownoftheprogressof AMDduringage.Notonlythiswork,butalsofuturesimpleclinicalstudiesshould raise new nutritional strategies focused on understanding the etiological role of the foods, nutrition, and metabolism in the pathogenesis of ocular disorders. KEYWORDS age‐related macular degeneration, biopeptide, lupine seeds, Lupinus angustifolius, retinal pigment epithelium 2 of 7 | MILLAN‐LINARES Et AL. from Lupinus angustifolius L. protein hydrolysates. As depicted inFigure1,theaminoacidicsequencewasdescribedasglycine– proline–glutamate–threonine–alanine–phenylalanine–leucine–arginine(GPETAFLR)withα‐helixthree‐dimensionalstructure(protein structuremodelwasgeneratedbytheautomatedSWISS‐MODEL homology modelingpipeline,Bienertetal.,2017). Previously,antioxidative and anti‐inflammatory properties of GPETAFLR have been tested in a human myeloid lineage (Millan‐Linares et al., 2018). GPETAFLR showed decrease the expression of pro‐inflammatory cytokinesasTNF‐α or IL‐1β and increase those that participate in the anti‐inflammatory response as IL‐10 or IL‐4 in THP‐1‐derived macrophages (Millan‐Linares et al., 2015), monocyte‐derived osteoclasts (Millan‐Linares et al., 2018), and primary human monocytes (Montserrat‐de la Paz et al., 2019). Nutrition,age,oxidativestress,orastateofchronicinflammation, among other causes, may induce dysfunction and degenerationofretinalpigmentepithelium(RPE)(Levyetal.,2015).RPEisa monolayer of pigmented epithelial cells located between the retina and choroid that plays a critical role in maintaining visual function and thereby in the pathogenesis of retinal degenerative diseases (Pang,Zhou,&Kuang,2018).RPEregulatesthevolumeandchemical composition of the subretinal space and nutrient transport between theretinaandchoroid.Therefore,RPE functionsasblood–retinal barrier that protects the health and integrity of the retina and choroid(Qinetal.,2017;Sonodaetal.,2009).Recentstudiesarehelping toclarifytheroleofRPEcellsintheimmuneandinflammatoryresponseoftheretina(Montserrat‐delaPazetal.,2016).Thelossof integrityandfunctionalityofRPEcellsinvolvesirreversibledamage to the photoreceptors and consequently a loss of central vision. This isknownasage‐relatedmaculardegeneration(AMD)(MontserratdelaPaz,Naranjo,Bermudez,etal.,2017),whichistheleadingcause of blindness and its prevalence continues to increase in parallel with theincreaseinlifeexpectancyagingandchangeinlifestyleofthe worldpopulation(Montserrat‐delaPazetal.,2016). Therefore,thisstudyaimsforthefirsttimetheroleofGPETAFLR, isolated from lupine (Lupinus angustifoliusL.),onoxidativestressand inflammationinRPEcells. 2 | MATERIAL AND METHODS 2.1 | Chemicals Thefollowing ELISA kitswere purchased fromDiacloneResearch (Besancon, France): VEGF, IFNγ, TNF‐α, IL‐6, and Human IL‐1β. Hydrogenperoxide(H2O2)waspurchasedfromPanreac(Barcelona, Spain).OtherchemicalswerepurchasedfromSigma‐AldrichChem. (St.Louis,MO,USA). 2.2 | GPETAFLR synthesis GPETAFLR,originallyisolatedfromlupine(Lupinus angustifolius L.) protein hydrolysates (Millan‐Linares et al., 2015; Millan‐Linares, Yust,Alcaide‐Hidalgo, Millan,& Pedroche,2014), wasfinallysynthesized at 95% purity, measured by HPLC‐UV at 220 nm, followingFmoc solid‐phase method by theBarcelona ScientificPark Foundation(Barcelona,Spain). 2.3 | ARPE‐19 cell culture Dr. Edilberto Ojeda (UPV/EHU) kindly provided ARPE‐19 (human RPEcells).Cellswerecultureday37°C/5%CO2 in a medium resulting from the mixture of DMEM and F12 (1:1) (Life Technologies) completedwith100U/mLpenicillin,100μg/ml streptomycin, and 2 mM L‐glutamine (Lonza) and 10% fetal bovine serum (Hyclone). Every3–4days,subculturesweremadeusing0.25%trypsin‐EDTA (Montserrat‐delaPazetal.,2016).Cellswerepassagedbeforecoming to confluence, in order to keep them in an undifferentiated state. FortheexperimentsweusedARPE‐19cellsat5–10passages. FIGURE 1 Chemical(a)andsecondarythree‐dimensional(b)structureofGPETAFLRpeptide,anoctapeptideisolatedfromLupinus angustifoliusL.,whichaminoacidsequenceisidentifiedas:Glycine(G),Proline(P),Glutamate(E),Threonine(T),Alanine(A),Phenylalanine (F),Leucine(L),andArginine(R).Yellowcolorwasusedtosidechain,greencolorwasusedtoaminogroup,andbluecolorwasusedto carboxylgroup | 3 of 7 MILLAN‐LINARES Et AL. 2.4 | Cell viability assay (MTT) The chemical reduction to formazan of MTT (3‐(4,5‐dimethylthi‐ azol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) is the basis of the colorimetric assay used to determine ARPE‐19 cells viability. Briefly, afterseedingin96‐wellplates105ARPE‐19cells/well,theculture was incubated during 24 hr in the presence of concentrations of the peptideGPETAFLR,between50μg/ml and 100 μg/ml, then 0.5 mg/ ml of MTT in aqueous solution was added to continue incubation 2hrat37°C(Montserrat‐delaPaz,Naranjo,Lopez,etal.,2017).In ordertoestimatecell viability,aMultiskan Spectrumplatereader (ThermoLab systems) was used to quantify the % of absorbance at 570nmwavelengthcomparedtoa620nmreference. 2.5 | Intracellular reactive oxygen species (ROS) The intracellular ROS was determined using the CellROX Green Reagent (ThermoFisher Scientific, Madrid, Spain). After in vitro stimulation with H2O2 at 100 μM,ARPE‐19cellswereexposedto 50 μg/ml and 100 μg/mlofthepeptideGPETAFLRfor24hrandthen withCellROXGreenReagent(5μM)for30min.Cellswerewashed withphosphate‐bufferedsalineandfixedwith3.7%formaldehyde, and the fluorescence signal was analyzed in a Fluoroskan Microplate Fluorometer(ThermoFisherScientific)equippedwitha485/555excitation/emission filter set. The autofluorescence of cells was measuredunderthesameconditionsbutwithoutaddingCellROXGreen Reagent (Lopez et al., 2017). Data shown refer to the % of intracellularROSproductionandtothecomparisonwithapositivecontrol (100%ROSproduction)aftercelltreatmentinthepresenceofH2O2. 2.6 | Glutathione (GSH) assay AfterinvitrostimulationwithH2O2 at 100 μM,ARPE‐19cellswere exposed to 50 μg/ml and 100 μg/ml of the peptide GPETAFLR for 24hr.Cellextractswereobtainedin5%sulfosalicylicacidfollowed bytwofreeze/thawcycles(Yan,Liang,Li,&Zheng,2015).GSHwas determinedinsamplesofcellextractsbymeasuringtheformationof p‐nitrophenolfrom5,5′‐dithiobis(2‐nitrobenzoicacid)inthepresence ofGSHreductaseandthereducedformofnicotinamideadeninedinucleotidephosphateaccordingtotheGSHAssayKit(CS0260;Sigma‐ Aldrich).Datashownrefertothe%ofintracellularGSHconcentration and to the comparison with a negative control (untreated cells, 100% GSHconcentration). 2.7 | Nitrite production Briefly,afterbeingstimulatedwithH2O2 at 100 μM,anARPE‐19cell culture (105 cells/well in 24‐well plates), was incubated for 24 hr in the presence of 50 μg/ml and 100 μg/mlofthepeptideGPETAFLR. TheGriessreagent(Sigma‐Aldrich)wasusedtocalculatetheproduction of nitrite, considered an indication of nitric oxide (NO) generation. Once transferred 100 μl of the culture supernatant to a 96‐well plate, a volume of 100 μl of Griess reagent was added (Quilez, Montserrat‐de la Paz, De la Puerta, Fernandez‐Arche, & Garcia‐Gimenez,2015).ABioTekplatereadermeasuredabsorbance at 540 nm wavelength, using a sodium nitrite standard curve to estimate concentration. 2.8 | Cytokine release After stimulation treatment with 100 ng/ml of lipopolysaccharide (LPS)invitro,ARPE‐19cellswereculturedin24‐wellplatesata105 cells/well density and then treated 24 hr with two doses of the peptideGPETAFLR,50and100μg/ml.ELISAkitswereusedtocalculate in cell culture supernatants several concentrations in pg/ml, from every standard curve: vascular endothelial growth factor (VEGF), IL‐1β, IFNγ, IL‐6, and TNF‐α. A Multiskan Spectrum plate reader measured absorbance at 450 nm wavelength. 2.9 | RNA isolation and quantitative real‐time PCR analysis AfterinvitrostimulationwithLPSat100ng/ml,theARPE‐19cells were cultured in 24‐well plates at 105 cells/well density, and incubated during 24 hr in the presence of 50 μg/ml and 100 μg/ml of thepeptideGPETAFLR.TrisureReagent(Bioline)wasusedtoextract total RNA. In a NanoDrop ND‐1000 Spectrophotometer (Thermo Scientific), RNA qualitywas evaluated byA260/A280 ratio. Reverse transcriptionwasperformedusing1,000ngRNA(iScript,BioRad). TwentynanogramsofthecDNAobtainedwereusedastemplatefor real‐timepolymerasechainreactionamplifications.Amplificationof each specificgeneproductwasperformedusingaCFX96system (BioRad).EveryPCRreactioncontainedbrilliantSYBRgreenQPCR Supermix(BioRad),theprimerpairsforthecorrespondinggeneand cDNA template. As housekeeping genes glyceraldehyde 3‐phos‐ phate dehydrogenase (GAPDH) and 18‐S ribosomal RNA (18‐S) were used Table 1 shows both the sequence and the information for theprimers.Everyreactionwasdoneintriplicate.Toestimatethe relativemRNAexpressionofanalyzedgenes,theaveragethreshold cycle(Ct)valuesofthetriplicateswereused.Withthestandard2‐ (ΔΔCt)method,themagnitudeofchangeofmRNAexpressionforcandidate genes was assessed (Montserrat‐de la Paz, Rodriguez, et al., 2017;Naranjoetal.,2016).Todeterminetherelativeexpressionof thestudiedgenes,weusedtheaverageoftheCtdataofhousekeepingsamples.Resultswerenormalizedusinghousekeepinggenesexpression and showed as percentage of control samples. 2.10 | Statistical evaluation Data in figures and text are expressed as arithmetic mean ± SD (standarddeviations).Everyexperimentwasperformedintriplicate. ForevaluationoftheresultsweusedGraphPadPrismVersion6.01 software(SanDiego,CA,USA).Significanceofparametervariations within treated groups was evaluated by one‐way analysis of variance, following Tukey’s multiple comparisons test as post hoc test. Those p values fewer .05 were determined statistically significant. 4 of 7 | MILLAN‐LINARES Et AL. Target No. GenBank Direction Secuence (5′ ‐‐> 3′) GAPDH NM_001289746 Forward CACATGGCCTCCAAGGAGTAAG Reverse CCAGCAGTGAGGGTCTCTCT 18‐S NR_003286.2 Forward GGCCCTGTAATTGGAATGAGTC Reverse CCAAGATCCAACTACGAGCTT iNOS NM_000625 Forward ACCCAGACTTACCCCTTTGG Reverse GCCTGGGGTCTAGGAGAGAC IL‐1βNM_000576 Forward GGGCCTCAAGGAAAAGAATC Reverse TTCTGCTTGAGAGGTGCTGA IL‐6 NM_000600 Forward TACCCCCAGGAGAAGATTCC Reverse TTTTCTGCCAGTGCCTCTTT TNFαNM_000594 Forward TCCTTCAGACACCCTCAACC Reverse AGGCCCCAGTTTGAATTCTT IFNγNM_000619 Forward CAGGCAGGACAACCATTACTGGGATGCTC Reverse TGAACTCATCCAAGTGATGGCTGAACTGTCG VEGF NM_001171623.1 Forward CCCACTGAGGAGTCCAACAT Reverse TTTCTTGCGCTTTCGTTTTT TABLE 1 SequencesofRT‐qPCR primersforgeneexpressionanalysis FIGURE 2 Cellviability(a),IntracellularROS(b),GSH(c),andnitrite(d)production,expressedaspercentageoffluorescence/absorbance, andiNOSmRNAlevels(e)relativetocellstreatedwithH2O2,afterthetreatmentofARPE‐19cellswithGPETAFLRat50and100μg/ml for 24hr.Valuesarepresentedasmeans±SD (n = 3). Different letters denote statistical differences (p < .05) | 5 of 7 MILLAN‐LINARES Et AL. 3 | RESULTS AND DISCUSSION Treatment during 24 hr with peptide GPETAFLR up to 200 μg/ ml doses, had no cytotoxic effect in ARPE‐19 cells (Figure 2a). ConsideringtheMTTassay,50and100μg/ml of peptide showed morethan98%viabilityofthecells.Inpreviousreports,GPETAFLR were not cytotoxic to primary human monocytes (Montserrat‐de la Paz et al., 2019), human monocyte‐derived osteoclasts (Millan‐ Linares et al., 2018), and THP‐1 cells (Millan‐Linares et al., 2015) at the same concentrations. Progressofmany eyediseases,includingAMD,glaucoma,and cataracts, are related to oxidative damage, resulted from excess productionofNOorROS(Yonekawa,Miller,&Kim,2015).Forthat reason,quantificationofbothROSandNOconcentrationsinARPE cells,letusanalyzethepreventiveroleofpeptideGPETAFLRonoxidative conditions. H2O2remarkablyincreasedbothintracellularROS (Figure 2b) and nitrites (Figure 2d), compared to nonstimulated cells. GPETAFLR decreased ROS production in H2O2‐stimulated ARPE cells. The relative increase caused by H2O2 in nitrite production was higherthanthatofROS.Inaddition,LPSstimulatedtheiNOSmRNA transcriptional activity which was downregulated by GPETAFLR (Figure2e).GSHprotectsagainstoxidativedamageinmanytissues, includingRPE(Sun,Zheng,Wang,&Liu,2018).GPETAFLRincreased GSHlevelagainstoxidativedamageinARPE‐19cells(Figure2c).We have no evidence of previous studies regarding the effect of a plant peptideonthebalanceofROS,NO,andGSHinhumanRPEcells. In order to determine possible anti‐inflammatory effects on humanRPEcellsofthepeptideGPETAFLR,westudiedinARPE‐19 bothreleaseandgeneexpressionofIFNγ, IL‐1β,TNF‐α,andIL‐6. AsshowninFigure3,LPSstimulatedthetranscriptionalactivity of genes IL‐1β(Figure3a),IL‐6(Figure3b),TNF‐α (Figure 3c), and IFNγ (Figure 3d). GPETAFLR, particularly at 100 μg/ml, showed toproducelessinflammatorymediatorsthanLPS‐stimulatedcells (Figure4).PresentfindingsinARPE‐19cellsconfirmanti‐inflam‐ matory effects of peptide GPETAFLR, emphasizing a possible function on acute inflammation. In the literature, it possible to find large number of examples where plant‐derived biopeptides are usedasanti‐inflammatoryorantioxidantcompounds.Oneofthem is1,2,3,4,6penta‐O‐galloyl‐β‐D‐glucose, a naturally polyphenolic compound present in some medicinal herbs as Rhuschinensis Mill. Fagopyrum tataricum, commonly known as buckwheat, is another example of bioactive plant. Researchers found that buckwheat extracts may inhibit adipogenesis and inflammatory response duringadipocytedifferentiationof3T3‐L1cells.22Brazilianred propolis (Apis mellifera), Copaifera oleoresins, flavonoid fraction of Bergamot Juice (Citrus bergamia), effusanin C (Isodon japonicus), oligomeric proanthocyanidins (crataegus oxyacantha) are others isolated compounds with anti‐inflammatory actions in activated monocytes and macrophages (Montserrat‐de la Paz et al., 2019).Aswellaspriorstudies(Millan‐Linaresetal.,2018,2015), showingGPETAFLRasoneofthemajorbioactivepeptideswith anti‐inflammatory activity, our observations suggest that this octapeptide isolated from Lupinus angustifolius L. is associated with a remarkablepreventionofoxidativeandinflammatoryinRPEcells. Therefore, dietary lupine protein intake could play a key role in maintaining ocular homeostasis. VEGF,animportantinducerofvascularpermeabilityandacentral regulator of new blood vessel growth, is remarkably involved inchoroidneovascularizationformationinwetAMD(Grisantietal., 2015).Inthisprocess,ithasbeenproposedthatbothoxidativeand FIGURE 3 GeneexpressionofIL‐1β(a),IL‐6(b),TNF‐α(c),andIFNγ (d) relative to untreated cells (control) after the treatment of ARPE‐19cellswithGPETAFLRat50and100μg/mlfor24hr.Valuesarepresentedasmeans±SD (n = 3). Different letters denote statistical differences (p < .05) 6 of 7 | MILLAN‐LINARES Et AL. inflammatory state of RPE may be also implicated (Fang, Yang, & Yang,2014).Therefore,weinvestigatedinARPE‐19cellstheeffects of GPETAFLR on VEGF secretion and gene expression (Figure 5). Interestingly, the gene expression and protein secretion of VEGF wassignificantlyreducedinhumanRPEcellsexposedtoGPETAFLR. 4 | CONCLUSIONS We reveal a novel nutraceutical impact of GPETAFLR peptide in humanRPEcellstopreventinflammatorycytokines,NO,GSH,and ROS.Briefly,ourresultssupportthattheintakeofLupine angustifoliusL.,proposedtobeareservoirofGPETAFLR,couldlessenthe functionaldecayofRPEcells,leadingthereforetoaslowdownofthe progressofAMDduringage.Notonlythiswork,butalsofuturesimple clinical studies should raise new nutritional strategies focused on understanding the etiological role of the foods, nutrition, and metabolism in the pathogenesis of ocular disorders. ACKNOWLEDGMENTS ThisworkwassupportedbygrantAGL2012‐40247‐C02‐01fromthe SpanishMinistryofEconomyandCompetitiveness.Authorsthank Cell BiologyUnitofInstitutodelaGrasa forits assistanceduring thefulfilmentofthisworkandResearchScientistEnriqueMartínez Forceforhishelpwiththeproteinstructuremodels.SMPacknowledgesfinancialsupportfrom“VOwnResearchPlan”(Universityof Seville). CONFLICTS OF INTEREST The authors state no conflict of interest. ORCID Sergio Montserrat‐de la Paz https://orcid. org/0000‐0001‐5400‐3192 FIGURE 4 CytokinereleaseofIL‐1β(a),IL‐6(b),TNF‐α(c),andIFNγ(d)afterthetreatmentofARPE‐19cellswithGPETAFLRat50and 100 μg/mlfor24hr.Valuesarepresentedasmeans±SD (n = 3). Different letters denote statistical differences (p < .05) FIGURE 5 VEGFgeneexpression(a)andsecretion(b)relativetountreatedcells(control)afterthetreatmentofARPE‐19cellswith GPETAFLRat50and100μg/mlfor24hr.Valuesarepresentedasmeans±SD (n = 3). Different letters denote statistical differences (p < .05) | 7 of 7 MILLAN‐LINARES Et AL. REFERENCES Bienert, S., Waterhouse, A., de Beer, T.A. P., Tauriello, G.,Studer, G., Bordoli,L.,&Schwede,T.(2017).TheSWISS‐MODELRepository— New features and functionality. Nucleic Acids Research, 45, D313–D319.https://doi.org/10.1093/nar/gkw1132 Fang,I.M.,Yang,C.H.,&Yang,C.M.(2014).Docosahexaenoicacid reduces linoleic acid induced monocyte chemoattractantprotein‐1 expression via PPARγ and nuclear factor‐κB pathway in retinal pigment epithelial cells. Molecular Nutrition & Food Research, 58, 2053–2065. 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