(−)-Methyl-Oleocanthal, a New Oleocanthal Metabolite Reduces LPS-Induced Inflammatory and Oxidative Response: Molecular Signaling Pathways and Histones Epigenetic Modulation

  Ci a ion: Mon oya, T.; Ala cón-de-la-Las a, C.; Cas ejón, M.L.; O ega-Vidal, J.; Al a ejos, J.; Sánchez-Hidalgo, M. (−)-Me hyl- Oleocan hal, a New Oleocan hal Me aboli e Reduces LPS-Induced In lamma o y and Oxida i e Response: Molecula Signaling Pa hways and His ones Epigene ic Modula ion. An ioxidan s 2022,11, 56. h ps://doi.o g/10.3390/an iox 11010056 Academic Edi o : S anley Omaye Recei ed: 3 Decembe 2021 Accep ed: 24 Decembe 2021 Published: 27 Decembe 2021 Publishe ’s No e: MDPI s ays neu al wi h ega d o ju isdic ional claims in published maps and ins i u ional a il- ia ions. Copy igh : © 2021 by he au ho s. Licensee MDPI, Basel, Swi ze land. This a icle is an open access a icle dis ibu ed unde he e ms and condi ions o he C ea i e Commons A ibu ion (CC BY) license (h ps:// c ea i ecommons.o g/licenses/by/ 4.0/). an ioxidan s A icle (−)-Me hyl-Oleocan hal, a New Oleocan hal Me aboli e Reduces LPS-Induced In lamma o y and Oxida i e Response: Molecula Signaling Pa hways and His ones Epigene ic Modula ion Ta iana Mon oya 1, Ca alina Ala cón-de-la-Las a 1, Ma ía Luisa Cas ejón1, Juan O ega-Vidal 2, Joaquín Al a ejos 2and Ma ina Sánchez-Hidalgo 1,* 1Depa men o Pha macology, Facul y o Pha macy, Uni e sidad de Se illa, 41012 Se illa, Spain; [email p o ec ed] (T.M.); cala [email p o ec ed] (C.A.-d.-l.-L.); [email p o ec ed] (M.L.C.) 2Depa men o Ino ganic and O ganic Chemis y, Facul y o Expe imen al Sciences, Campus de Excelencia In e nacional Ag oalimen a io (ceiA3), Uni e si y o Jaén, 23071 Jaén, Spain; jo 00007@ ed.ujaen.es (J.O.-V.); [email p o ec ed] (J.A.) *Co espondence: [email p o ec ed] Abs ac : The an ioxidan and an i-in lamma o y esponses o ( − )-me hyl-oleocan hal (me -OLE), a new me aboli e o he ex a i gin oli e oil (EVOO) phenolic oleocan hal (OLE), we e explo ed in lipopolysaccha ide (LPS)-induced mu ine pe i oneal mac ophages. Possible signaling pa hways and epigene ic modula ion o his ones we e s udied. Me -OLE inhibi ed LPS-induced in acellula eac i e oxygen species (ROS) and ni i e (NO) p oduc ion and dec eased he o e exp ession o he p o-in lamma o y enzymes COX-2, mPGES-1 and iNOS in mu ine mac ophages. In addi ion, me - OLE was able o signi ican ly dec ease he ac i a ion o p38, JNK, and ERK mi ogen-ac i a ed p o ein kinases (MAPKs) and blocked canonical and non-canonical in lammasome signaling pa hways. On he con a y, me -OLE up egula ed haem oxigenase 1 (HO-1) and nuclea ac o (e y h oid-de i ed 2)-like 2 (N -2) exp ession in ea ed cells. Finally, me -OLE p e ea ed spleen cells coun e ac ed LPS induc ion, p e en ing H3K18 ace yla ion o H3K9 and H3K27 deme hyla ion. O e all, hese esul s p o ide no el mechanis ic insigh s in o he bene icial e ec s o me -OLE ega ding he egula ion o he immune–in lamma o y esponse h ough epigene ic changes in his one ma ke s. This e ealing e idence sugges s ha he me hyla ed me aboli e o OLE may con ibu e signi ican ly o he bene icial e ec s ha a e associa ed wi h he secoi idoid- ela ed compound and he usual consump ion o EVOO. Keywo ds: an ioxidan ; his ones; in lamma ion; mac ophages; me aboli e; me hyla ion; oleocan hal; oli e oil 1. In oduc ion Mac ophages a e majo componen s o he inna e immune sys em and play a c i i- cal ole in modula ing in lamma o y and immune esponses [ 1 ]. Ex acellula bac e ial lipopolysaccha ide (LPS) ac s as pa hogen-associa ed molecula pa e n and is ecognized by he Toll-like ecep o (TLR)-4, inducing mac ophages o an ac i a ed s a e, p oducing p o- in lamma o y cy okines and chemokines and enhancing he exp ession o in lamma o y- ela ed enzymes, such as inducible ni ic oxide syn hase (iNOS), cyclooxygenase (COX)-2 and mic osomal p os aglandin E syn hase (mPGES)-1, which syn hesize ni ic oxide (NO) and p os aglandin (PG)E 2 , espec i ely [ 2 ]. Addi ionally, LPS-s imula ed mac ophages dis up he balance o he in acellula educ ion–oxida ion s a e, leading o oxida i e s ess, usually accompanied by damage ha is media ed by eac i e oxygen species (ROS) [ 3 ]. The p ocess o gene exp ession o hese p o-in lamma o y media o s in ol es mul iple signal ansduc ion pa hways, which a e mainly h ough mi ogen-ac i a ed p o ein kinases An ioxidan s 2022,11, 56. h ps://doi.o g/10.3390/an iox11010056 h ps://www.mdpi.com/jou nal/an ioxidan s An ioxidan s 2022,11, 56 2 o 18 (MAPKs), nuclea ansc ip ion ac o -kappa B (NF- κ B), janus kinase/signal ansduce and ansc ip ion ac i a o o ansc ip ion (JAK/STAT) o in lammasome ac i a ion. Fu - he mo e, he nuclea ac o (e y h oid-de i ed 2)-like 2 (N -2)/haem oxygenase-1 (HO-1) an ioxida i e axis, which exe s a egula i e unc ion on he ac i a ion o ROS, MAPKs, and in lammasome signaling pa hways, is ep essed in he e en o he induc ion o he ac i a ed mac ophages s a e [2,3]. Eme ging e idence sugges s ha epigene ic p ocesses ha a ec gene exp ession wi hou causing changes in he nucleo ide sequence occu a e ex e nal s imuli exposu e, and may con ibu e o he pa hophysiology o in lamma o y p ocesses [ 4 ]. In pa icula , his one H3 me hyla ion a lysine 9 (H3K9), one o he mos conse ed epigene ic ma ke s, is co ela ed wi h gene silencing and he modula ion o immune cell di e en ia ion and immune esponses, and he e o e, in luences he ou come o in lamma ion. Simila ly, H3 ace yla ion on lysine 18 (H3K18ac) is a pe missi e ma ke on genes encoding cy okines ha co ela e o in lamma ion, such as in e leukin (IL)-1 β , IL-6 o IL-17 [ 5 – 7 ]. Fu he mo e, pos - ansla ional his one modi ica ions ha e eme ged as p ospec i e he apeu ic a ge s. Unde s anding he c oss-link o hese mechanisms could be c ucial in designing new immune–in lamma o y app oaches ha a e e ec i ely ela ed o se e al in lamma o y diseases [8]. In his con ex , die a y nu ien s could modi y physiological and pa hological p o- cesses h ough c i ical epigene ic mechanisms, p omo ing modi ica ions o gene exp ession wi hou al e a ion o he gene ic code. In pa icula , speci ic, unc ional oods such as ex a i gin oli e oil (EVOO) ha e displayed an i-in lamma o y ac i i ies in human mac ophages h ough epigene ic mechanisms [9]. The heal h-p omo ing p ope ies o EVOO ha e been co ela ed wi h i s peculia chemical composi ion. EVOO polyphenols a e mino seconda y me aboli es ha ha e been widely s udied due o hei wide unc ional e sa ili y, including hei an i-in lamma o y, an ioxidan , ca diop o ec i e, chemop e en i e, and neu op o ec i e p ope ies [ 10 ]. Es- sen ially, he an i-in lamma o y ac i i ies o oli e oil, especially phenolic compounds, ha e ecen ly been linked o hei po en ial o induce epigene ic modi ica ions such as gene exp ession, DNA me hyla ion and his one modi ica ion [10,11]. Examples o he main oli e polyphenols a e y osol, hyd oxy y osol, oleocan hal (OLE), oleacein, oli e ligus oside and oleu opein. OLE ep esen s up o 10% o he o- al polyphenol con en in EVOO (0.2–498 mg/kg) [ 12 ] and has ecei ed mo e scien i ic a en ion, due o i s in e es ing biological ac i i ies, bo h wi hin in i o and in i o sys- ems, including an i-in lamma o y, an ioxidan , ca diop o ec i e, chemop e en i e and neu op o ec i e p ope ies [ 10 , 13 ]. In ac , ecen ly, we ha e epo ed he p e en i e ole o die a y OLE-supplemen ed e ec s in a collagen-induced a h i is (CIA) mu ine model and he abili y o OLE o diminish he acu e in lamma o y esponse in LPS-induced mu ine pe i oneal mac ophages [14,15]. Me abolic ans o ma ions o he p esence o me aboli es can a ec he pha macologi- cal ac i i y o he pa e n compounds. I has been epo ed ha OLE can emain in ac in he s omach o up o 4 h and en e he small in es ine unhyd olyzed. Then, non-hyd olyzed OLE ollows u he me abolic eac ions ela ed o phase I and II in he li e , namely, hyd oxyla ion o hyd a ion and me hyla ion, espec i ely. Consequen ly, López-Ye ena e al. (2021) p oposed wo plausible me aboli es o OLE, howe e , me -OLE was he main ci cula ing conjuga e o OLE de ec ed in all issues analyzed om a s a e he acu e in ake o a e ined oli e oil con aining 0.3 mg/mL o OLE [ 16 ]. In ela ion o bio ans o ma ion, he me hyla ed me aboli es a e mainly me abolized by glucu onida ion and sulpha ion, as hey a e no subs a es o me hyl ans e ases. Pe haps, he majo disad an age o he EVOO phenols is hei empe a u e ins abili y, pho olabili y, and inadequa e pha macokine ic p o ile. To educe hese handicaps, me hyla- ion o he phenolic hyd oxyl g oups (O-me hyla ion) may inc ease he chemical s abili y o hei s uc u es, while con e ing g ea e lipophilici y, inc easing hei me abolic s abili y and memb ane anspo and acili a ing abso p ion and g ea e o al bioa ailabili y. Recen An ioxidan s 2022,11, 56 3 o 18 s udies also indica e ha he me hyla ion p ocess inc eases biological ac i i y wi hou al e ing he apeu ic indices [17]. Taking his backg ound in o accoun , he aim o he p esen wo k was o in es i- ga e he po en ial an ioxidan and an i-in lamma o y e ec s o a new OLE me aboli e, ( − )-me hyl-oleocan hal (me -OLE), in LPS-induced mu ine pe i oneal mac ophages. Speci - ically, in acellula ROS, NO, p o-in lamma o y cy okines p oduc ion (IL-1 β , IL-6, IL-17, IL-18, umo nec osis ac o (TNF)- α and in e e on (IFN)- γ ), and he p o ein exp essions o p o-in lamma o y enzymes (COX-2, iNOS, and mPGES-1) we e e alua ed. In addi- ion, he possible molecula signaling pa hways in ol ed in hei bene icial e ec s, such as N -2/HO-1, MAPKs, and he canonical and noncanonical in lammasome, we e also s udied. Finally, o s udy he ole o he epigene ic mechanisms unde lying me -OLE an i-in lamma o y e ec s, we explo ed me -OLE induced epigene ics changes in his one ma ke s (H3K9me3, H3K27me3 and H3K18ac) and cy okines-co ela ed p oduc ion com- pa ed o OLE in spleen cells a e LPS induc ion. 2. Ma e ials and Me hods 2.1. Reagen s Sol en s used o ex ac ion, analy ical hin-laye ch oma og aphy (TLC), column ch o- ma og aphy (CC) and as cen i ugal pa i ion ch oma og aphy (FCPC), such as die hyl e he (E 2 O), e hyl ace a e (E OAc), n-hexane (Hex), dichlo ome hane (DCM) and e hanol (E OH) we e o analy ical g ade and we e pu chased om VWR Chemicals (P olabo ® , Fon enay-sous-Bois, F ance). Wa e (H 2 O), used o ch oma og aphic sepa a ions, was o ul apu e g ade and was p oduced by Milli-Q wa e (1.8 M Ω ) equipmen (Me ck ® , KGaA, Da ms ad , Ge many). Ace oni ile (ACN) and me hanol (MeOH), used o high- pe o mance liquid ch oma og aphy (HPLC), and chlo o o m, used o de e mine op ical o a ion alues, we e o HPLC g ade and we e pu chased om VWR ® (Mad id, Spain). Deu e a ed chlo o o m (CDCl 3 ) was used o p epa e solu ions o isola ed compounds o nuclea magne ic esonance (NMR) analysis and we e pu chased om VWR ® (Mad id, Spain). Ace ic acid (AcOH), used o HPLC, was pu chased om VWR ® (Mad id, Spain). Silica gel 60 F240 p ecoa ed aluminum shee s we e pu chased om Me ck ® (Da ms ad , Ge many). N-me hylu ea, used o p epa e N-me hyl-N-ni osou ea (see Supplemen a y Ma e ials), was pu chased om Sigma-Ald ich ® (Mad id, Spain). Sodium hyd oxide was pu chased om VWR Chemicals®(P olabo, Fon enay-sous-Bois, F ance). 2.2. Ins umen s HPLC analyses we e pe o med on a Wa e s HPLC ins umen , equipped wi h a C18 e e sed-phase Sphe iso b ODS-2 column, 250 × 3 mm i.d, 5 µ m (Wa e s Ch oma og a-phy Di ision®, Mild o d, MA, USA), and a pho odiode a ay de ec o . Reac ions unde mic owa e i adia ion we e achie ed on a CEM Disco e monomodal mic owa e eac o wi h empe a u e and p essu e in e nal p obes. A sealed essel was used o pe o m he eac ion. Pu i ica ion o syn hesized compounds was ca ied ou on a FCPC-200 ® ins umen (K oma on Technologies ® , Ange s, F ance) ha was i ed wi h a o o and wi h a o al column capaci y o 200 mL. Ro a ion could be adjus ed om 0 o 2000 pm. Sol en was pumped by an All ech 627 isoc a ic pump (All ech Associa es ® , Dee ield, IL, USA). The sample was injec ed in o he FCPC column wi h a 3725i-038 manual injec o (Rheodyne ® , Co a i, CA, USA) equipped wi h a 10 mL sample loop. The ins umen was equipped wi h a UV-Vis Linea UVIS 200 de ec o (Linea Ins umen Co ® , Reno, NV, USA) se a 280 nm. Mass spec a (ESIMS) we e eco ded on an Esqui e 6000 ion mass spec ome e (B uke Dal onics, B emen, Ge many) i ed wi h an elec osp ay ioniza ion (ESI) in e ace, ope a - ing in posi i e mode. High- esolu ion mass spec a (HRMS) we e pe o med on an Agilen 6520B spec ome e (Agilen echnologies, Waldb onn, Ge many) i ed wi h a quad upole ime-o - ligh (Q-TOF) mass spec ome e . An ioxidan s 2022,11, 56 4 o 18 P o on nuclea magne ic esonance ( 1 H NMR) and ca bon nuclea magne ic esonance ( 13 C NMR) spec a o he isola ed and syn hesized compounds we e eco ded on a B uke A ance DPX 400 spec ome e (B uke Dal onic GmbH ® , Rheins e en, Ge many) a 400 and 100 MHz, espec i ely. Deu e a ed chlo o o m, wi h e ame hylsilane (TMS) as in e nal e e ence, was used o dissol e he samples. Coupling cons an s (J) a e p o ided in he z (Hz) and he mul iplici ies o signals a e epo ed using he ollowing abb e ia ions: single (s), b oad single (b s), double (d), double o double s (dd), double o double o double s (ddd), iple ( ), double o iple (d ), quad uple (q) and mul iple (m). Speci ic o a ions ([ α ] D ) o chi al compounds we e calcula ed by measu ing he co e- sponding op ical o a ion ( α ) in chlo o o m on a Jasco P-200 au oma ic pola ime e (Jasco Analy ical Ins umen ® , Eas on, MD, USA) using cells o qua z wi h a pa h leng h o 1 dm. 2.3. Isola ion o (−)-Oleocan hal (OLE) om Oli e Oil A sample o OLE was pu i ied om oli e oil phenolic ex ac by a combina ion o wo echniques ( as cen i ugal pa i ion ch oma og aphy (FCPC) and semi-p epa a i e high-pe o mance liquid ch oma og aphy (HPLC)), ollowing he p ocedu e desc ibed in Diez-Bello e al., (2019) [ 18 ]. In sho , an oli e oil sample (300 g) was ex ac ed wi h a mix u e o MeOH/H 2 O 8:2, acco ding o a no malized p ocedu e (IOC, 2017), o a o d a phenolic ex ac (1.50 g), which was ac iona ed wi h a FCPC-200 ® ins umen (K oma on Technologies, Ange s, F ance) using a qua e na y biphasic sol en sys em composed o hexane/e hyl ace a e/e hanol/wa e (2:3:2:3, / / / ). A pooling ac ion (180 mg), mainly con aining OLE, was u he pu i ied by semi-p epa a i e HPLC using he sol en s ace oni ile/ace ic acid (99.8:0.2, / , sol en A) and wa e /ace ic acid (99.8:0.2, / , sol en B) unde a linea g adien om 20 o 25% o sol en A. As a esul , pu e OLE (25 mg) was ob ained, in which nuclea magne ic esonance (NMR) da a (Figu es S1 and S2) ag eed wi h hose epo ed in he li e a u e [18]. 2.4. Syn hesis o (−)-Me hyl-Oleocan hal (Me -OLE) om (−)-Ligus oside A wood sample o Olea eu opaea L., ob ained om p uning a ee g owing in he p o ince o Jaén, Spain, was used o isola e ( − )-ligus oside. A sample o wood chips (300 g) was ex ac ed wi h E OAc (4 L) o 2 h a e lux. Sol en was e apo a ed unde educed p essu e a 40 ◦ C o gi e he co esponding d y ex ac (15 g), which was column ch oma og aphed o gi e ligus oside (0.34 g), as epo ed be o e by he au ho s [ 19 ]. The pu i y o ligus oside (84%) was de e mined by an ex e nal s anda d me hod using he HPLC peak a ea a 280 nm. P e iously, a calib a ion cu e (y = 1734.5 x + 1656; R 2 = 0.9981) (G aphic S1) was cons uc ed wi h six s anda ds (0.1–1 mg/mL in MeOH) (Table S1) o pu e ( − )-ligus oside. Fo analy ical HPLC analyses, sepa a ion was ca ied ou by a s ep g adien wi h mix u es o MeOH/AcOH (99.8:0.2, / , sol en A) and H 2 O/AcOH (99.8:0.2, / , sol en B). The g adien p og am consis ed o a linea g adien om 20 o 80% A in 55 min, a linea g adien om 80 o 100% A in 5 min and ano he 10 min o e u n o ini ial condi ions. Nex , in o de o syn he ize ( − )-me hyl-ligus oside, a solu ion o diazome hane in E 2 O (35 mL), eshly p epa ed om N-me hyl-N-ni osou ea (3.5 g) and aq. KOH (50%), was added d opwise o a solu ion o ligus oside (200 mg) in MeOH (10 mL) and he eac ion was le o s i o 60–90 min. The eac ion was moni o ed by TLC and by HPLC using he g adien elu ion desc ibed abo e. Finally, sol en was emo ed unde educed p essu e o gi e a b own solid (205 mg). The c ude was pu i ied by as cen i ugal pa i ion ch oma og aphy (FCPC) using a qua e na y biphasic sol en sys em composed o Hex/E OAc/E OH/H 2 O (2:5:2:5, / / / ) a a low o 7 mL/min and a o a ion speed o 1200 pm. As a esul , 125 mg (71% yield) o ( − )-me hyl-ligus oside as a sligh yellow– whi e solid was ob ained: ESIMS: m/z 561 [M + Na] + (Figu e S11); 1 H NMR (400 MHz, CDCl 3 ) (Figu e S9): δ (ppm) 7.51 (s, 1H, H-9), 7.15 (m, 2H, H-4 0 , H-8 0 ), 6.85 (m, 2H, H-5 0 , 7 0 ), 6.07 (m, 1H, H-5), 5.92 (b s, 1H, H-7), 4.81 (d, 1H, J 1”,2” = 7.8 Hz, H-1”), 4.25 (d , 1H, J 10a,20 = 6.9 Hz, J 10a,10b = 10.7 Hz, H-1 0 a), 4.12 (d , 1H, J 10b,20 = 6.9 Hz, J 10b,10a = 10.7 Hz, H-1 0 b), An ioxidan s 2022,11, 56 5 o 18 3.96 (dd, 1H, J 3,2a = 4.5 Hz, J 3,2b = 9.2 Hz, H-3), 3.89 (dd, 1H, J 6”a,5” = 1.8 Hz, J 6”a,6”b = 11.9 Hz, H-6”a), 3.76 (s, 3H, C6 0 -OCH 3 ), 3.71 (s, 3H, C11-OCH 3 ), 3.67 (dd, 1H, J 6”b,5” = 5.7 Hz, J 6”b,6”a = 11.9 Hz, H-6”b), 3.37 (m, 4H, H-2”, H-3”, H-4”, H-5”), 2.85 ( , 2H, J 20,10 = 6.9 Hz, H-2 0 ), 2.70 (dd, 1H, J 2a,3 = 4.5 Hz, J 2a,2b = 14.2 Hz, H-2a), 2.44 (dd, 1H, J 2b,3 = 9.2 Hz, J 2b,2a = 14.2 Hz, H-2b), 1.64 (dd, 3H, J 6,7 = 1.5 Hz, J 6,5 = 7.0 Hz, H-6). 13 C NMR (100 MHz, CDCl 3 ) (Figu e S10): δ (ppm) 173.1 (C-1), 168.6 (C-11), 159.9 (C-6 0 ), 155.1 (C-9), 131.3 (C-4), 131.0 (C-4 0 , C-8 0 ), 130.5 (C-3 0 ), 124.8 (C-5), 115.0 (C-5 0 , C-7 0 ), 109.4 (C-10), 100.8 (C-1”), 95.1 (C-7), 78.5 (C-5”), 77.9 (C-3”), 74.8 (C-2”), 71.5 (C-4”), 66.8 (C-1 0 a,C-1 0 b), 62.8 (C-6”a, C-6”b), 55.7 (C6 0 - O C H 3 ), 51.9 (C11-O C H 3 ), 41.2 (C-2a, C-2b), 35.1 (C-2 0 ), 31.8 (C-3), 13.5 (C-6). [α]25 D : − 154.0 (c0.7, MeOH). NMR da a o his compound ag ee wi h hose epo ed in he li e a u e [ 20 ]. The pu i y o ( − )-me hyl-ligus oside (90%) was de e mined using he ligus oside’s calib a ion cu e desc ibed abo e. Finally, o ob ain me -OLE, a mix u e o me hyl-ligus oside (100 mg), wa e (5 mL) and DMSO (0.4 mL) was subjec ed o mic owa e i adia ion o 9 min a 180 ◦ C. The eac ion was moni o ed by TLC and HPLC. Then, he c ude was ex ac ed wi h E 2 O and he sol en was emo ed unde educed p essu e o gi e a b own oil (58 mg). The pu i ica ion was achie ed by FCPC using Hex:E OAc:E OH:H 2 O (1:1:1:1, / / / ) as a biphasic sol en sys em. As a esul , 39 mg (64% yield) o me -OLE as a yellow oil was ob ained: ESIMS: m/z341 [M + Na] + (Figu e S7): HRMS (ESI/Q-TOF) m/z319.1540 [M + H] + co esponding o C 18 H 22 O 5 (Figu e S8); 1 H NMR (400 MHz, CDCl 3 ) (Figu e S3): δ (ppm) 9.63 (b s, 1H, H-8), 9.23 (d, 1H, J 9,3 = 2.0 Hz, H-9), 7.10 (m, 2H, H-4 0 ,H-8 0 ), 6.83 (m, 2H, H-5 0 ,H-7 0 ), 6.62 (q, 1H, J 5,6 = 7.1 Hz, H-5), 4.19 (m, 2H, H-1 0 ), 3.78 (s, 3H, C6 0 -OC H3 ), 3.61 (m, 1H, H-3), 2.95 (ddd, 1H, J 7b,8 = 1.2 Hz, J 7b,3 = 8.7 Hz, J 7b,7a = 18.3 Hz, H-7b), 2.83 ( , 2H, J 20,10 = 7.0 Hz, H-2 0 ), 2.73 (ddd, 1H, J 7a,8 = 1.0 Hz, J 7a,3 = 5.5 Hz, J 7a,7b = 18.3 Hz, H-7a), 2.68 (dd, 1H, J 2b,3 = 8.2 Hz, J 2b,2a = 15.9 Hz, H-2b), 2.61 (dd, 1H, J 2a,3 = 6.7 Hz, J 2a,2b = 15.9 Hz, H-2a), 2.07 (d, 3H, J 6,5 = 7.1 Hz, H-6). 13 C NMR (100 MHz, CDCl 3 ): δ (ppm) 200.5 (C-8), 195.2 (C-9), 172.0 (C-1), 158.5 (C-6 0 ), 154.3 (C-5), 143.4 (C-4), 130.0 (C-4 0 ,C-8 0 ), 129.8 (C-3 0 ), 114.1 (C-5 0 ,C-7 0 ), 65.2 (C-1 0 ), 55.4 (C6 0 -O C H 3 ), 46.3 (C-7), 37.0 (C-2), 34.3 (C-2 0 ), 27.4 (C-3), 15.4 (C-6). The ull assignmen o 1H and 13C NMR esonances was suppo ed by DEPT (Figu e S4), HSQC (Figu e S5) and HMBC (Figu e S6) spec al analyses. [α]25 D : − 1.7 (c1.0, CHCl 3 ). The pu i y o me -OLE (97%) was de e mined by an ex e nal s anda d me hod using he HPLC peak a ea a 280 nm. P e iously, a calib a ion cu e (y = 3121.8x − 6706.6; R 2 = 0.9974) (G aphic S2) was cons uc ed wi h six s anda ds (0.01–0.5 mg/mL in MeOH) (Table S2) o pu e OLE a ailable in au ho s’ lab. 2.5. Animals Swiss mice we e p o ided by Ha lan In e auna Ibé ica ® (Ba celona, Spain) and main ained in a empe a u e-, humidi y- and ligh -con olled oom, and we e allowed ee access o wa e and ood. All animal p ocedu es ollowed we e in acco dance wi h he ecommenda ions o he Eu opean Union on animal expe imen a ion (Di ec i e o he Eu opean Counsel 2012/707/EU) and we e app o ed by he Animal E hics Commi ee o he Uni e si y o Se illa (23 July 2018/119). 2.6. Mu ine Mac ophages and Spleen Cells Isola ion and Cul u e Cells we e collec ed 72 h a e in ape i oneal s e ile hioglycola e injec ion (3.8% w/ ) as p e iously desc ibed by Mon oya e al. (2019) [ 14 ]. The collec ed mac ophages we e cul u ed wi h me -OLE (50, 25, 12.5 µ M) p e- ea men s o 30 min (min) and hen s imula ed wi h LPS om Esche ichia coli (5 µ g/mL) (Sigma-Ald ich ® , S . Louis, MO, USA) incuba ing hem o 18 h (h) a 5% CO 2 37 ◦ C. The supe na an s and cells samples we e collec ed and s o ed a − 80 ◦ C un il cy okine measu emen and Wes e n blo ing, espec i ely. The mice spleens we e ha es ed and passed h ough a nylon cell s aine (BD ® Biosciences, F anklin Lakes, NJ, USA) wi h supplemen ed RPMI1640 medium (10% e al cal se um, 2 mM glu amine, 1 mM sodium py u a e, 50 µ M 2-me cap oe hanol and 1% An ioxidan s 2022,11, 56 6 o 18 penicillin and s ep omycin), in o de o ob ain a cell suspension. Pelle ed cells we e esuspended in ed blood lysis bu e (BD ® Biosciences, F anklin Lakes, NJ, USA) and hen washed wi h phospha e bu e solu ion (PBS). Cells (1 × 10 6 cells/mL) we e le un ea ed o ea ed wi h OLE (50 µ M) o , me -OLE (50, 25, 12.5 µ M) o 30 min and we e LPS-s imula ed (5 µ g/mL) du ing an 18 h pe iod a 5% CO 2 , a 37 ◦ C. A e incuba ion, cell pelle s and supe na an s we e collec ed and s o ed a − 80 ◦ C un il he quan i ica ion o cy okine le els and his one ex ac ion, espec i ely. 2.7. Cell Viabili y To e alua e me -OLE cy o oxici y, a sul o hodamine B (SRB) assay was pe o med [ 21 ]. An amoun o 1 × 10 5 cells/mL we e cul u ed in he p esence/absence o me -OLE (200–1.6 µ M) o 18 h. The abso bance was ead a 510 nm wi h a mic opla e eade (Bio ad ® , Mad id, Spain). Abso bance is exp essed as he pe cen age o iabili y when compa ed o un ea ed con ol cells (100% cell su i al). 2.8. Ni ic Oxide P oduc ion Ni i es le els, exp essed as he NO gene a ion index, we e quan i ied using G iess eagen in cul u e supe na an s (Sigma-Ald ich ® , S . Louis, MO, USA), ollowing he p o ocol epo ed by Mon oya e al. (2018) [ 22 ]. A sodium ni a e cu e was used as a s anda d o ex apola e he ni i e amoun , and he esul s we e exp essed as a pe cen age compa ed wi h DMSO-LPS ea ed cells (100% ni i es p oduc ion). 2.9. In acellula ROS P oduc ion The DCDFA assay ki was pe o med acco ding o he manu ac u e ’s ins uc ions (Abcam ® , Camb idge, UK). Cells (2.5 × 10 5 cells/mL) we e seeded on a black pla e and hen DCDFA (25 µ M) was added o each well ei he p e iously un ea ed o ea ed wi h me -OLE (50, 25, 12.5 µ M) and we e LPS-s imula ed du ing 18 h. The esul s we e exp essed using H 2 O 2 (Sigma-Ald ich ® , Ba celona, Spain) as a posi i e p o-oxidan con ol (100% in acellula ROS p oduc ion), compa ing luo escence in ensi y [14]. 2.10. His one Ex ac ion Acid ex ac ion was pe o med as p e iously epo ed by Hajji e al. (2010) [ 5 ] wi h b ie modi ica ions. The collec ed spleen cells we e washed wi h PBS, suspended in lysis bu e (10mM T is pH 6.5, 50 mM sodium bisul a e, 10 nM MgCl 2 , 8.6% suc ose, 1% T i on X-100) and incuba ed 15 min a 4 ◦ C. A e ha , he samples we e cen i uged a 3500 pm o 10 min a 4 ◦ C and ewashed. The supe na an was hen emo ed and disca ded and T is–EDTA bu e (10 mM T is pH 7.4 and 13 mM EDTA) was added o he samples. The p ecipi a ed nucleus was esuspended wi h acid sul u ic 0.2M. A e 1 h incuba ion ime, samples we e cen i uged a 15,000 pm o 1 h a 4C, sal ing supe na an s o incuba ion wi h ace one o e nigh a − 20 ◦ C. Cen i uged samples we e dilu ed wi h H 2 O and p o ein con en was measu ed. 2.11. Enzyme-Linked Immunoso ben Assay IL-6, IFN- γ (Diaclone ® , Besancon Cedex, F ance), IL-1 β (R&D Sys em ® , Minneapolis, Cánada, USA), TNF- α and IL-17 (Pep o ech ® , London, UK) concen a ions in cul u e media we e measu ed using speci ic enzyme-linked immunoso ben assay (ELISA) ki s. 2.12. Wes e n Blo ing Whole cell lysa es, p epa ed as desc ibed by Mon oya e al. (2019) [ 14 ], and ex ac ed his ones we e p o ided in o 25 µ g p o ein aliquo s. P o ein samples we e sepa a ed by SDS-PAGE (15 o 10%) and elec oblo ed on o ni ocellulose memb anes. Speci ic p ima y an ibodies we e used. The memb anes we e hen incuba ed wi h he co esponding seconda y an ibody o 2 h. The esul s we e ob ained om a leas six independen expe imen s. A chemiluminescence ligh de ec ion ki (Pie ce ® , Rock o d, IL, USA) and An ioxidan s 2022,11, 56 7 o 18 Ame sham Image 600 equipmen (GE Heal hca e ® , Chicago, IL, USA) we e used o he de ec ion o immunosignals. Da a we e no malized wi h a housekeeping con ol and quan i ied by Image P ocessing and Analysis in Ja a (Image J®, Be hesda, MD, USA). 2.13. S a is ical Analysis Da a in igu es and ex a e epo ed as a i hme ic means ± s anda d e o (SEM) om a leas six independen expe imen s ca ied ou in iplica e. Resul s we e e alua ed using G aph Pad P ism e sion 5.01 so wa e (San Diego, CA, USA), analyzing he s a is ical signi icance by a one-way analysis o a iance (ANOVA), ollowed by he Tukey’s mul iple compa isons es . The p- alues < 0.05 we e conside ed s a is ically signi ican . Figu es om densi ome y expe imen s a e ep esen a i es o di e en expe imen s pe o med on a di e en day. 3. Resul s 3.1. Chemis y The syn hesis o ( − )-me -OLE was ca ied ou he ein om ( − )-ligus oside (Figu e 1), inspi ed by he eac ion epo ed by Skal sounis’ g oup o he con e sion o oleu opein (same s uc u e as ligus oside bu wi h an addi ional OH a C-5 0 ) in o oleacein (same s uc u e as OLE bu wi h an addi ional OH a C-5 0 ) [ 23 ]. These au ho s achie ed he semi- syn hesis o oleacein om oleu opein unde K apcho deca bome hoxyla ion condi ions in one single s ep. They e luxed oleu opein, dissol ed in we DMSO, wi h wo equi alen s o an ino ganic sal (NaCl) o 10 h o ob ain oleacein wi h a 21% yield, a e pu i ica ion by silica gel column ch oma og aphy. P ocopio’s g oup la e imp o ed his a ac i e semi-syn hesis h ough changing DMSO by wa e and hea ing in a mic owa e eac o o ge oleacein in jus 20 min wi h a 48% yield [ 24 ]. Ou g oup was also wo king o imp o e he semi-syn hesis o oleacein and OLE om oleu opein and ligus oside, espec i ely, ollowing he ecommenda ions o a p e ious wo k aimed a adap ing he classical K apcho deca boxyla ion expe imen al condi ions o an aqueous mic owa e scena io [ 25 ], when P ocopio’s wo k came o ligh (unpublished esul s). An ioxidan s 2022, 11, x FOR PEER REVIEW 8 o 19 In o de o pu i y he inal compound and aking in o accoun he known sensi i i y o ela ed OLE and oleacein o decomposi ion when in con ac wi h solid s a iona y phases, such as silica gel [27], and upon exposu e o oxygen and ligh [12], we used he FCPC echnique o he pu i ica ion o me -OLE. This allowed us o ob ain pu e me -OLE wi h he same success as when we p e iously pu i ied OLE om an oli e oil phenolic ex ac [18]. This silica- ee ch oma og aphic echnique has shown o be a good op ion o isola e hese compounds [28] and consequen ly imp o e he yield o he eac ion s ep. Thus, we ha e pe o med he con e sion o me hyl-ligus oside in o me -OLE in a 64% yield, which is he bes yield epo ed o da e o he K apcho con e sion o ligus o- side/oleu opein o hei de i a i es in o he co esponding dialdehydes. Me -OLE has been syn hesized in his wo k o he i s ime. Figu e 1. Syn hesis o (−)-me hyl-oleocan hal om (−)-ligus oside. Reagen s and condi ions: (a) CH 2 N 2 /E 2 O; (b) H 2 O-DMSO, MW, 180 °C, 9 min. 3.2. E ec s o Me -OLE on Cell Viabili y Fi s , we e alua ed he cell iabili y a e me -OLE ea men s using a SRB assay. Da a show ha me -OLE was no cy o oxic a e 18 h o ea men wi h concen a ions o 1.6 up o 200 μM and did no comp omise he cell iabili y signi ican ly (≥80%) (Figu e 2). The e o e, based on ou p e ious wo k using OLE [14], we selec ed 12.5, 25 and 50 μM concen a ions o me -OLE o be s udied in he ollowing assays. Figu e 2. E ec o me -OLE on cell iabili y. Mac ophages we e p e ea ed wi h me -OLE (200–1.6 μM) o 18 h. Cell su i als we e exp essed as he pe cen age o iabili y wi h espec o 100% om con ol, un ea ed cells. Resul s a e p esen ed as mean ± SEM o a leas six independen expe i- men . 3.3. E ec s o Me -OLE on IL-1β, IL-6, IL-17, IFN-γ and TNF-α P oduc ion To explo e he e ec s o me -OLE on p o-in lamma o y cy okine p oduc ion, we e alua ed IL-1β, IL-6, IL-17, IFN-γ and TNF-α le els. As shown in Figu e 3, a e 18 h o               μ    Figu e 1. Syn hesis o ( − )-me hyl-oleocan hal om ( − )-ligus oside. Reagen s and condi ions: (a) CH2N2/E 2O; (b) H2O-DMSO, MW, 180 ◦C, 9 min. Thus, ou syn hesis o me -OLE (Figu e 1) s a ed wi h he isola ion o ligus oside om oli e wood ollowing a p ocedu e p e iously epo ed by us [ 19 ]. Then, ligus oside was me hyla ed using diazome hane, p epa ed in si u om N-me hyl-N-ni osou ea. The con e sion o ligus oside in o me hyl-ligus oside was quan i a i ely pe o med a oom empe a u e o a ound 1 h. I he s a ing ma e ial (ligus oside) had been pu e enough, me hyl-ligus oside would ha e been su icien ly pu e o be used di ec ly in he nex s ep. Howe e , ligus oside was isola ed wi h a pu i y o 84% om he oli e wood ex ac and i was con enien o submi c ude me hyl-ligus oside o pu i ica ion, in o de o elimina e he mino componen s ha accompanied he s a ing ma e ial (ligus oside). On his occasion, we used as cen i ugal pa i ion ch oma og aphy (FCPC) o ge adequa ely pu e me hyl-ligus oside in one single un. Then, pu e me hyl-ligus oside was submi ed o he K apcho eac ion unde mic owa e i adia ion. Se e al a emp s we e ca ied ou , wi h a ying empe a u es, eac ions ime and sol en a ios, un il he bes condi ions An ioxidan s 2022,11, 56 8 o 18 we e ound. Thus, he eac ion o me hyl-ligus oside in wa e , wi h he minimum amoun o DMSO o dissol e he subs a e, was pe o med in a mic owa e eac o o 9 min a 180 ◦ C, eaching he comple e con e sion o me hyl-ligus oside in o me -OLE. I is wo h no ing ha his K apcho eac ion ook place in he absence o ino ganic sal , as Mu ph ee’s g oup [ 25 ] came o obse e in some cases and as Fe nández-Bolaños’ g oup has ecen ly claimed [26]. In o de o pu i y he inal compound and aking in o accoun he known sensi i i y o ela ed OLE and oleacein o decomposi ion when in con ac wi h solid s a iona y phases, such as silica gel [ 27 ], and upon exposu e o oxygen and ligh [ 12 ], we used he FCPC echnique o he pu i ica ion o me -OLE. This allowed us o ob ain pu e me -OLE wi h he same success as when we p e iously pu i ied OLE om an oli e oil phenolic ex ac [ 18 ]. This silica- ee ch oma og aphic echnique has shown o be a good op ion o isola e hese compounds [ 28 ] and consequen ly imp o e he yield o he eac ion s ep. Thus, we ha e pe o med he con e sion o me hyl-ligus oside in o me -OLE in a 64% yield, which is he bes yield epo ed o da e o he K apcho con e sion o ligus oside/oleu opein o hei de i a i es in o he co esponding dialdehydes. Me -OLE has been syn hesized in his wo k o he i s ime. 3.2. E ec s o Me -OLE on Cell Viabili y Fi s , we e alua ed he cell iabili y a e me -OLE ea men s using a SRB assay. Da a show ha me -OLE was no cy o oxic a e 18 h o ea men wi h concen a ions o 1.6 up o 200 µ M and did no comp omise he cell iabili y signi ican ly ( ≥ 80%) (Figu e 2). The e o e, based on ou p e ious wo k using OLE [ 14 ], we selec ed 12.5, 25 and 50 µ M concen a ions o me -OLE o be s udied in he ollowing assays. An ioxidan s 2022, 11, x. h ps://doi.o g/10.3390/xxxxx www.mdpi.com/jou nal/an ioxidan s Figu e 2. E ec o me -OLE on cell iabili y. Mac ophages we e p e ea ed wi h me -OLE (200–1.6 µ M) o 18 h. Cell su i als we e exp essed as he pe cen age o iabili y wi h espec o 100% om con ol, un ea ed cells. Resul s a e p esen ed as mean ±SEM o a leas six independen expe imen . 3.3. E ec s o Me -OLE on IL-1β, IL-6, IL-17, IFN-γand TNF-αP oduc ion To explo e he e ec s o me -OLE on p o-in lamma o y cy okine p oduc ion, we e alua ed IL-1 β , IL-6, IL-17, IFN- γ and TNF- α le els. As shown in Figu e 3, a e 18 h o exposu e o LPS, mu ine cells exhibi ed highe le els o p o-in lamma o y cy okines han uns imula ed con ol cells (++ p< 0.01; +++ p< 0.001 s. uns imula ed cells). On he con a y, when cells we e ea ed wi h me -OLE, we obse ed a signi ican ly down- egula ion o IL-1 β , IL-6, IL-17, IFN- γ and TNF- α sec e ions when compa ed o he LPS- DMSO g oup (** p< 0.01; *** p< 0.001 s. LPS-DMSO s imula ed cells). An ioxidan s 2022,11, 56 9 o 18 3.4. E ec s o Me -OLE on In acellula ROS and NO P oduc ions In e ms o elucida ing he ole o me -OLE in he oxida i e and in lamma o y esponse media ed by LPS, we measu ed in acellula ROS and NO le els using DCFDA and G iess assays, espec i ely, in LPS-induced mu ine pe i oneal mac ophages. Da a e ealed ema kable o e p oduc ions o ROS and NO induced by LPS in mu ine mac ophages when compa ed o uns imula ed cells (+ p< 0.05; ++ p<0.01; +++ p< 0.001 s. uns imula ed con ol cells) (Figu e 4A,B). Meanwhile, le els o bo h media o s we e signi ican ly educed a e me -OLE ea men s (** p< 0.01; *** p< 0.001 s. cells s imula ed cells). An ioxidan s 2022, 11, x FOR PEER REVIEW 9 o 19 exposu e o LPS, mu ine cells exhibi ed highe le els o p o-in lamma o y cy okines han uns imula ed con ol cells (++ p < 0.01; +++ p < 0.001 s. uns imula ed cells). On he con- a y, when cells we e ea ed wi h me -OLE, we obse ed a signi ican ly down- egula ion o IL-1β, IL-6, IL-17, IFN-γ and TNF-α sec e ions when compa ed o he LPS-DMSO g oup (** p < 0.01; *** p < 0.001 s. LPS-DMSO s imula ed cells). Figu e 3. P o-in lamma o y cy okine le els we e down- egula ed in me -OLE ea ed cells. Mac- ophages we e p e ea ed wi h me -OLE (50, 25 o 12.5 μM) o 30 min and hen, LPS-s imula ed du ing 18 h. (A) IL-1β, (B) IL-6, (C) IL-17, (D) IFN-γ and (E) TNF-α le els we e measu ed by ELISA on cell supe na an s. Resul s a e p esen ed as he mean ± SEM o a leas six independen expe i- men s. ++ p < 0.01; +++ p < 0.001 s. uns imula ed con ol cells; ** p < 0.01; *** p < 0.001 s. LPS-DMSO s imula ed cells. 3.4. E ec s o Me -OLE on In acellula ROS and NO P oduc ions In e ms o elucida ing he ole o me -OLE in he oxida i e and in lamma o y e- sponse media ed by LPS, we measu ed in acellula ROS and NO le els using DCFDA and G iess assays, espec i ely, in LPS-induced mu ine pe i oneal mac ophages. Da a e ealed ema kable o e p oduc ions o ROS and NO induced by LPS in mu- ine mac ophages when compa ed o uns imula ed cells (+ p < 0.05; ++ p <0.01; +++ p < 0.001 s. uns imula ed con ol cells) (Figu e 4A,B). Meanwhile, le els o bo h media o s we e signi ican ly educed a e me -OLE ea men s (** p < 0.01; *** p < 0.001 s. cells s imu- la ed cells). B CD A E Figu e 3. P o-in lamma o y cy okine le els we e down- egula ed in me -OLE ea ed cells. Mac ophages we e p e ea ed wi h me -OLE (50, 25 o 12.5 µ M) o 30 min and hen, LPS-s imula ed du ing 18 h. ( A ) IL-1 β , ( B ) IL-6, ( C ) IL-17, ( D ) IFN- γ and ( E ) TNF- α le els we e measu ed by ELISA on cell supe na an s. Resul s a e p esen ed as he mean ± SEM o a leas six independen expe i- men s. ++ p< 0.01; +++ p< 0.001 s. uns imula ed con ol cells; ** p< 0.01; *** p< 0.001 s. LPS-DMSO s imula ed cells. 3.5. Me -OLE Down-Regula ed iNOS, COX-2 and mPGES-1 P o ein O e exp ession Induced by LPS in Mu ine Mac ophages To in es iga e whe he he e ec s o me -OLE on NO accumula ion we e associa ed wi h he exp ession o he iNOS p o ein, we pe o med an immunoblo ing assay wi h cell lysa es. Consis en ly, cells om secoi idoid- ela ed phenolic g oups showed dec eased iNOS p o ein o e exp ession when compa ed o hose o he LPS-DMSO exposed g oup (*** p< 0.001 s. LPS-DMSO s imula ed cells) (Figu e 4C). In addi ion, o gain u he insigh in o he an i-in lamma o y po en ial o me -OLE, we s udied i s e ec s on COX-2 and PGE 2 ela ed bioma ke s. As expec ed, a e LPS exposu e, COX-2 and mPGES-1 exp essions inc eased signi ican ly (+++ p< 0.001 s. uns imula ed con ol cells). On he con a y, me -OLE p e ea men s e ec i ely coun e ac ed induc ion o An ioxidan s 2022,11, 56 16 o 18 5. Conclusions Collec i ely, his s udy epo s, o he i s ime, he semi-syn hesis o a new me hy- la ed OLE me aboli e and i s an ioxidan and an i-in lamma o y e ec s in he esponse o mu ine mac ophages o LPS exposu e. Gene ally, me -OLE inhibi ed he exp ession o p o-in lamma o y enzymes, (iNOS, COX-2 and mPGES-1), cy okines (IL-6, IL-17, IL-1 β , TNF- α , IFN- γ and IL-18) and egula ed in acellula ROS and p oduc s ha a e ela ed o oxida i e damage, such as NO le els, ia MAPKs, N -2/HO-1 and bo h canonical and non-canonical in lammasome signal pa hway modula ion. Fu he mo e, bo h OLE and me -OLE we e able o egula e epigene ic mechanisms by modula ing his one me hyla- ion (H3K9me3 and adnH3K27me) and ace yla ion (H3K18ac) and by down- egula ing cy okine- ela ed p oduc ion in LPS-exposed spleen immune cells. This e ealing e idence sugges s ha he me hyla ed me aboli e o OLE may con- ibu e signi ican ly o he bene icial e ec s ha a e associa ed wi h he secoi idoid- ela ed compound and he usual consump ion o EVOO. Thus, me -OLE could be a p omising he apeu ic agen used in a ious immune—in lamma o y pa hologies, and he elucida ion o hei s uc u e—ac i i y ela ionship migh be a ele an goal o u u e di ec ions. Supplemen a y Ma e ials: Supplemen a y ma e ials a e a ailable online h ps://www.mdpi.com/ a icle/10.3390/an iox11010056/s1, syn hesis o diazome hane’s p ecu so ; Wes e n Blo ing; Cali- b a ion cu es: G aphics S1 and S2, Tables S1 and S2; Spec oscopy da a o OLE (Figu es S1 and S2), me -OLE (Figu es S3–S8) and me hyl-ligus oside (Figu es S9–S11). Au ho Con ibu ions: Concep ualiza ion, M.S.-H. me hodology, T.M.; expe imen a ion con ibu ion, M.L.C.; p oduc ex ac ions and chemical syn hesis, J.O.-V. and J.A.; so wa e, alida ion and o mal analysis, T.M.; in es iga ion, T.M.; da a cu a ion, T.M.; w i ing—o iginal d a p epa a ion, T.M. and M.S.-H.; w i ing— e iew and edi ing, C.A.-d.-l.-L., T.M., J.A. and M.S.-H.; isualiza ion and supe ision, C.A.-d.-l.-L. and M.S.-H. All au ho s ha e ead and ag eed o he published e sion o he manusc ip . Funding: This esea ch was suppo ed by Minis e io de Economía y Compe i i idad o Spain, g an numbe AG-2017-89342-P and, Jun a de Andalucía unded by CTS-259, FQM-182. Ins i u ional Re iew Boa d S a emen : The s udy was conduc ed acco ding o he guidelines o he Eu opean Union ega ding animal expe imen a ion (Di ec i e o he Eu opean Counsel 2012/707/EU) and we e app o ed by he Animal E hics Commi ee o he Uni e si y o Se illa (23 July 2018/119). In o med Consen S a emen : No applicable. Da a A ailabili y S a emen : Da a is con ained wi hin he a icle. Acknowledgmen s: The au ho s g a e ully acknowledge he assis ance o Cen e o Technology and Inno a ion Resea ch, Uni e si y o Se ille (CITIUS) and, he Technical Scien i ic Ins umen a ion Cen e (CICT) o he Uni e si y o Jaen. T. Mon oya hanks suppo om a Pos g adua e P og am o PIF ellowship and inancial sponso ship om VI Plan P opio de In es igación y T ans e encia a Uni e sidad de Se illa. Con lic s o In e es : The au ho s decla e no con lic o in e es . Re e ences 1. Wynn, T.A.; Chawla, A.; Polla d, J.W. Mac ophage biology in de elopmen , homeos asis and disease. Na u e 2013 ,496, 445–455. [C ossRe ] [PubMed] 2. Yao, Y.; Liu, K.; Zhao, Y.; Hu, X.; Wang, M. P e os ilbene and 4 0 -Me hoxy es e a ol Inhibi ed Lipopolysaccha ide-Induced In lamma o y Response in RAW264.7 Mac ophages. Molecules 2018,23, 1148. [C ossRe ] [PubMed] 3. B üne, B.; Dehne, N.; G ossmann, N.; Jung, M.; Namgaladze, D.; Schmid, T.; Von Kne hen, A.; Weige , A. Redox Con ol o In lamma ion in Mac ophages. An ioxid. Redox Signal. 2013,19, 595–637. [C ossRe ] [PubMed] 4. Shanmugam, M.K.; Se hi, G. Role o Epigene ics in In lamma ion-Associa ed Diseases. Epigene ics De . Dis. 2013 ,61, 627–657. [C ossRe ] 5. Hajji, N.; Joseph, B. Epigene ic egula ion o cell li e and dea h decisions and de egula ion in cance . Essays Biochem. 2010 ,48, 121–146. [C ossRe ] An ioxidan s 2022,11, 56 17 o 18 6. Zhao, S.; Zhong, Y.; Fu, X.; Wang, Y.; Ye, P.; Cai, J.; Liu, Y.; Sun, J.; Mei, Z.; Jiang, Y.; e al. H3K4 Me hyla ion Regula es LPS-Induced P oin lamma o y Cy okine Exp ession and Release in Mac ophages. Shock 2019,51, 401–406. [C ossRe ] 7. Imu a, H.; Fuji a, D.; Oba, S.; Kiyosue, A.; Nishima su, H.; Yudo, K.; Suzuki, E. His one me hyla ion and deme hyla ion a e implica ed in he ansien and sus ained ac i a ion o he in e leukin-1 β gene in mu ine mac ophages. Hea . Vessel. 2020 ,35, 1746–1754. [C ossRe ] 8. Hachiya, R.; Shiihashi, T.; Shi akawa, I.; Iwasaki, Y. The H3K9 me hyl ans e ase Se db1 egula es TLR4-media ed in lam-ma o y esponses in mac ophages. Sci. Rep. 2016,28, 28845. [C ossRe ] 9. Bo doni, L.; Fedeli, D.; Fio ini, D.; Gabbianelli, R. Ex a Vi gin Oli e Oil and Nigella sa i a Oil P oduced in Cen al I aly: A Compa ison o he Nu igenomic E ec s o Two Medi e anean Oils in a Low-G ade In lamma ion Model. An ioxidan s 2019 ,9, 20. [C ossRe ] 10. Cas ejón, M.L.; Mon oya, T.; Ala cón-De-La-Las a, C.; Sánchez-Hidalgo, M. Po en ial P o ec i e Role Exe ed by Secoi idoids om Olea eu opaea L. in Cance , Ca dio ascula , Neu odegene a i e, Aging-Rela ed, and Immunoin lamma o y Diseases. An ioxidan s 2020,9, 149. [C ossRe ] 11. Fabiani, R.; Vella, N.; Rosignoli, P. Epigene ic Modi ica ions Induced by Oli e Oil and I s Phenolic Compounds: A Sys ema ic Re iew. Molecules 2021,26, 273. [C ossRe ] [PubMed] 12. Cice ale, S.; Conlan, X.; Ba ne , N.W.; Keas , R. S o age o ex a i gin oli e oil and i s e ec on he biological ac i i y and concen a ion o oleocan hal. Food Res. In . 2013,50, 597–602. [C ossRe ] 13. Segu a-Ca e e o, A.; Cu iel, J.A. Cu en Disease-Ta ge s o Oleocan hal as P omising Na u al The apeu ic Agen . In . J. Mol. Sci. 2018,19, 2899. [C ossRe ] 14. Mon oya, T.; Cas ejón, M.L.; Sánchez-Hidalgo, M.; González-Benjumea, A.; Fe nández-Bolaños, J.G.; De-La-Las a, C.A. Oleocan- hal Modula es LPS-Induced Mu ine Pe i oneal Mac ophages Ac i a ion ia Regula ion o In lammasome, N -2/HO-1, and MAPKs Signaling Pa hways. J. Ag ic. Food Chem. 2019,67, 5552–5559. [C ossRe ] 15. Mon oya, T.; Sánchez-Hidalgo, M.; Cas ejón, M.; Rosillo, M.; González-Benjumea, A.; Ala cón-De-La-Las a, C. Die a y Oleocan- hal Supplemen a ion P e en s In lamma ion and Oxida i e S ess in Collagen-Induced A h i is in Mice. An ioxidan s 2021,10, 650. [C ossRe ] [PubMed] 16. López-Ye ena, A.; Vall e dú-Que al , A.; Jáu egui, O.; Ga cia-Sala, X.; Lamuela-Ra en ós, R.; Esc ibano-Fe e , E. Tissue Dis ibu ion o Oleocan hal and I s Me aboli es a e O al Inges ion in Ra s. An ioxidan s 2021,10, 688. [C ossRe ] [PubMed] 17. Wen, L.; Jiang, Y.; Yang, J.; Zhao, Y.; Tian, M.; Yang, B. S uc u e, bioac i i y, and syn hesis o me hyla ed la onoids. Ann. N. Y. Acad. Sci. 2017,1398, 120–129. [C ossRe ] [PubMed] 18. Diez-Bello, R.; Ja din, I.; Lopez, J.; El Haoua i, M.; O ega-Vidal, J.; Al a ejos, J.; Salido, G.; Salido, S.; Rosado, J. ( − )-Oleocan hal inhibi s p oli e a ion and mig a ion by modula ing Ca 2+ en y h ough TRPC6 in b eas cance cells. Biochim. e Biophys. Ac a (BBA)-Bioene g. 2019,1866, 474–485. [C ossRe ] 19. Pé ez-Bonilla, M.; Salido, S.; an Beek, T.A.; Lina es-Palomino, P.J.; Al a ejos, J.; Nogue as, M.; Sánchez, A. Isola ion and iden i ica ion o adical sca enge s in oli e ee (Olea eu opaea) wood. J. Ch oma og . A 2006,1112, 311–318. [C ossRe ] 20. Chang, H.C.; Wang, S.W.; Chen, C.Y.; Hwang, T.L.; Cheng, M.J.; Sung, P.J.; Liao, K.W.; Chen, J.J. Secoi idoid glucosides and an- i-in lamma o y cons i uen s om he s em ba k o F axinus chinensis. Molecules 2020,25, 5911. [C ossRe ] 21. Skehan, P.; S o eng, R.; Scudie o, D.; Monks, A.; McMahon, J.M.; Vis ica, D.; Wa en, J.T.; Bokesch, H.; Kenney, S.; Boyd, M.R. New Colo ime ic Cy o oxici y Assay o An icance -D ug Sc eening. JNCI J. Na l. Cance Ins . 1990 ,82, 1107–1112. [C ossRe ] [PubMed] 22. Mon oya, T.; Apa icio-So o, M.; Cas ejón, M.L.; Rosillo, M.A.; Sánchez-Hidalgo, M.; Begines, P.; Fé nandez-Bolaños, J.G.; Ala cón- de-la-Las a, C. Pe ace yla ed-Hyd oxy y osol, a new hyd oxy y osol de i a e, a enua es LPS-induced in lamma o- y esponse in mu ine pe i oneal mac ophages ia egula ion o non-canonical in lammasome, N 2/HO1 and JAK/STAT signaling pa hways. J. Nu . Biochem. 2018,57, 110–120. [C ossRe ] [PubMed] 23. Vougogiannopoulou, K.; Lemus, C.; Halabalaki, M.; Pe gola, C.; We z, O.; Smi h, A.B., III; Michel, S.; Skal sounis, L.; Deguin, B. One-s ep semisyn hesis o olea-cein and he de e mina ion as a 5-lipoxygenase inhibi o . J. Na . P od. 2014 ,77, 441–445. [C ossRe ] [PubMed] 24. Cos anzo, P.; Bonacci, S.; Ca ia i, L.; Na di, M.; Oli e io, M.; P ocopio, A. Simple and e icien sus ainable semi-syn hesis o oleacein [2-(3,4-hyd oxyphenyl) e hyl (3S,4E)-4- o myl-3-(2-oxoe hyl)hex-4-enoa e] as po en ial addi i e o edible oils. Food Chem. 2018,245, 410–414. [C ossRe ] [PubMed] 25. Mason, J.D.; Mu ph ee, S.S. Mic owa e-Assis ed Aqueous K apcho Deca boxyla ion. Synle 2013,24, 1391–1394. [C ossRe ] 26. Fe nández-Bolaños, J.M.; Maya-Cas illa, I.; González-Benjumea, A. Use o DMSO o he Syn hesis o Oleacein and Oleocan hal. WO2018162769A2, 13 Sep embe 2018. 27. Ka koula, E.; Skan za i, A.; Melliou, E.; Magia is, P. Quan i a i e Measu emen o Majo Secoi idoid De i a i es in Oli e Oil Using qNMR. P oo o he A i icial Fo ma ion o Aldehydic Oleu opein and Ligs oside Aglycon Isome s. J. Ag ic. Food Chem. 2014,62, 600–607. [C ossRe ] [PubMed] 28. Angelis, A.; Michailidis, D.; An oniadi, L.; S a hopoulos, P.; Tsan ila, V.; Nuzilla d, J.-M.; Renaul , J.-H.; Skal sounis, L.A. Pilo con inuous cen i ugal liquid-liquid ex ac ion o ex a i gin oli e oil biophenols and g am-scale eco e y o pu e oleocan hal, oleacein, MFOA, MFLA and hyd oxy y osol. Sep. Pu i . Technol. 2021,255, 117692. [C ossRe ] An ioxidan s 2022,11, 56 18 o 18 29. Du ing, A.; La ondelle, Y. The O-me hyla ion o ch ysin ma kedly imp o es i s in es inal an i-in lamma o y p ope ies: S uc u e– ac i i y ela ionships o la ones. Biochem. Pha macol. 2013,86, 1739–1746. [C ossRe ] 30. Koi ala, N.; Thuan, N.H.; Ghimi e, G.P.; Van Thang, D.; Sohng, J.K. Me hyla ion o la onoids: Chemical s uc u es, bioac i i ies, p og ess and pe spec i es o bio echnological p oduc ion. Enzym. Mic ob. Technol. 2016,86, 103–116. [C ossRe ] 31. Cas ejón, M.L.; Mon oya, T.; Ala cón-De-La-Las a, C.; González-Benjumea, A.; Vázquez-Román, M.V.; Sánchez-Hidalgo, M. Die a y oleu opein and i s acyl de i a i e amelio a e in lamma o y esponse in pe i oneal mac ophages om p is ane-induced SLE mice ia canonical and noncanonical NLRP3 in lammasomes pa hway. Food Func . 2020,11, 6622–6631. [C ossRe ] 32. Sco ece, M.; Gomez, R.; Conde, J.; Lopez, V.; Gomez-Reino, J.; Lago, F.; Iii, A.S.; Gualillo, O. Oleocan hal Inhibi s P oli e a ion and MIP-1αExp ession in Human Mul iple Myeloma Cells. Cu . Med. Chem. 2013,20, 2467–2475. [C ossRe ] 33. Iacono, A.; Gómez, R.; Spe y, J.; Conde, J.; Bianco, G.; Meli, R.; Gómez-Reino, J.J.; Smi h, A.B.; Gualillo, O. E ec o oleocan hal and i s de i a i es on in lamma o y esponse induced by lipopolysaccha ide in a mu ine chond ocy e cell line. A h i is Rheum. 2010,62, 1675–1682. [C ossRe ] [PubMed] 34. Gallo, K.A.; Ellswo h, E.; S oub, H.; Con ad, S.E. The apeu ic po en ial o a ge ing mixed lineage kinases in cance and in lamma ion. Pha macol. The . 2020,207, 107457. [C ossRe ] [PubMed] 35. Yu, Y.; He, J.; Li, S.; Song, L.; Guo, X.; Yao, W.; Zou, D.; Gao, X.; Liu, Y.; Bai, F.; e al. Fib oblas g ow h ac o 21 (FGF21) inhibi s mac ophage-media ed in lamma ion by ac i a ing N 2 and supp essing he NF- κ B signaling pa hway. In . Immunopha macol. 2016,38, 144–152. [C ossRe ] 36. Sha ma, D.; Kannegan i, T.-D. The cell biology o in lammasomes: Mechanisms o in lammasome ac i a ion and egula ion. J. Cell Biol. 2016,213, 617–629. [C ossRe ] 37. Tschopp, J.; Sch ode , K. NLRP3 in lammasome ac i a ion: The con e gence o mul iple signaling pa hways on ROS p oduc- ion. Na . Re . Immunol. 2010,10, 210–215. [C ossRe ] 38. Kuma i, A.; Bhawal, S.; Kapila, S.; Yada , H.; Kapila, R. Heal h-p omo ing ole o die a y bioac i e compounds h ough epigene ic modula ions: A no el p ophylac ic and he apeu ic app oach. C i . Re . Food Sci. Nu . 2020,21, 1–21. [C ossRe ] [PubMed] 39. Ide aabdullah, F.; Zeisel, S.H. Die a y Modula ion o he Epigenome. Physiol. Re . 2018,98, 667–695. [C ossRe ] 40. Ca adonna, F.; Consiglio, O.; Lupa ello, C.; Gen ile, C. Science and Heal hy Meals in he Wo ld: Nu i ional Epigenomics and Nu igene ics o he Medi e anean Die . Nu ien s 2020,12, 1748. [C ossRe ] 41. Ál a ez-E ico, D.; Ven o-To mo, R.; Sieweke, M.; Balles a , E. Epigene ic con ol o myeloid cell di e en ia ion, iden i y and unc ion. Na . Re . Immunol. 2015,15, 7–17. [C ossRe ] 42. Roh a , D.M.; He, Y.; Fa kash, E.A.; Schon eld, M.; Tsou, P.; Sawalha, A.H. Inhibi ion o EZH2 Amelio a es Lupus—Like Disease in MRL/lp Mice. A h i is Rheuma ol. 2019,71, 1681–1690. [C ossRe ] [PubMed] 43. Huang, J.; Wan, D.; Li, J.; Chen, H.; Huang, K.; Zheng, L. His one ace yl ans e ase PCAF egula es in lamma o y molecules in he de elopmen o enal inju y. Epigene ics 2015,101, 62–72. [C ossRe ] 44. Yi, Y.-S. Func ional In e play be ween Me hyl ans e ases and In lammasomes in In lamma o y Responses and Diseases. In . J. Mol. Sci. 2021,22, 7580. [C ossRe ] [PubMed] 45. Jiang, L.; Wang, J.; Liu, Z.; Jiang, A.; Li, S.; Wu, D. Sodium bu y a e alle ia es lipopolysaccha ide-induced in lamma o y esponses by down-Regula ion o NF- κ B, NLRP3 Signaling Pa hway, and ac i a ing his one ace yla ion in bo ine mac o-phages. F on . Ve . Sci. 2020,7, 579674. [C ossRe ] [PubMed] 46. Sun, H.-J.; Ren, X.-S.; Xiong, X.-Q.; Chen, Y.-Z.; Zhao, M.-X.; Wang, J.-J.; Zhou, Y.-B.; Han, Y.; Chen, Q.; Li, Y.-H.; e al. NLRP3 in lammasome ac i a ion con ibu es o VSMC pheno ypic ans o ma ion and p oli e a ion in hype ension. Cell Dea h Dis. 2017 , 8, e3074. [C ossRe ] 47. Fang, P.; Chen, C.; Zheng, F.; Jia, J.; Chen, T.; Zhu, J.; Chang, J.; Zhang, Z. NLRP3 in lammasome inhibi ion by his one ace yla ion amelio- a es se o lu ane-induced cogni i e impai men in aged mice by ac i a ing he au ophagy pa hway. B ain Res. Bull. 2021 , 172, 79–88. [C ossRe ] 48. Liu, C.-C.; Huang, Z.-X.; Li, X.; Shen, K.-F.; Liu, M.; Ouyang, H.-D.; Zhang, S.-B.; Ruan, Y.-T.; Zhang, X.-L.; Wu, S.-L.; e al. Up egula ion o NLRP3 ia STAT3-dependen his one ace yla ion con ibu es o pain ul neu opa hy induced by bo ezomib. Exp. Neu ol. 2018,302, 104–111. [C ossRe ]