Effects of Plant Sphingolipids on Inflammatory Stress in Differentiated Caco-2 Cells

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1 Journl of Oleo Siene Copyright 2017 y Jpn Oil Chemists Soiety doi : /jos.ess17171 J. Oleo Si. 66, (12) (2017) NOTE Effets of Plnt Sphingolipids on Inflmmtory Stress in Differentited Co-2 Cells Shinji Ymshit 1, Tkuy Seino 1, Kzuhiko Aid 2 nd Mikio Kinoshit 1 1 Deprtment of Food Siene, Oihiro University of Agriulture nd Veterinry Mediine, Oihiro, Hokkido , JAPAN 2 Innovtion Center, Nippon Flour Mills Co., Ltd., Atsugi, Kngw , JAPAN Astrt: To determine the mehnism underlying the nti-inflmmtory effets of plnt sphingolipids, espeilly plnt gluosylermide (GlCer), the effets of plnt sphingolipids on inflmmtory stress in differentited Co-2 ells were ompred to those of sphingolipid of niml origin, gltosylermide (GlCer). Addition of GlCer or GlCer suppressed ell injury used lipopolyshride ()- nd TNFα-indued inflmmtory stress nd indution of poptosis in differentited Co-2 ells. There ws no differene in the suppressive effet etween GlCer nd GlCer. The inflmmtory ytokines nd hemokines indued y were suppressed y GlCer. GlCer remined on the ell surfe. The results of this study n e summrized s follows: 1) sphingolipids suh s GlCer hve potent nti-inflmmtory effets; 2) GlCer suppresses -indued prodution of ytokines nd poptosis; 3) sphingolipids my remin on the surfe of ells, nd 4) the hemil properties of sphingolipids my prevent the intertion etween nd its reeptor. Key words: sphingolipid, glyosylermide, ytokine, olon inflmmtion 1 INTRODUCTION Sphingolipids nd their metolites hve funtions s intrellulr meditors essentil for ell differentition, poptosis, prevention of melnin formtion, nd so on 1 4. Orlly dministered sphingolipids undergo hydrolysis, through the tion of intestinl enzymes, for susequent uptke y muosl ells 5. Following intke, sphingolipids exert nti-rinom effets, nd, therefore, dietry sphingolipids hve ttrted onsiderle ttention s funtionl onstituents of food. Although ovine rin hs een the sphingolipid resoure, outrek of ovine spongiform enephlopthy BSE mde its use to food nd osmetis for humn diffiult 6. The funtionlity of sphingolipids in food, minly gluosylermide GlCer of plnt nd yest origin, ws investigted in our lortory, fousing on the effets on digestive orgns. Our previous studies indited tht GlCer hs nti-ibd 7 nd nti-olon ACF effets in vivo 8. In ddition, sphingolipids of plnt origin hve een shown to hve n poptosis-induing effet on olon ner ells in vitro 2. At the eginning of tht study, we speulted tht the dministered GlCer ws deomposed in the digestive trt, nd its onstituents, ermides or sphingoid ses, exerted the oserved effets. Therefore, ermide or sphingoid ses derived from omplex sphingolipids were used s sphingolipid metolites in the ove-mentioned in vitro study 2. However, reent experiments on intestinl inflmmtion nd olon denom indued in our niml experiments 7, 8 showed tht portion of the dministered GlCer is exreted in fees nd tht suppression mehnism of inflmmtory relted ytokines in the olon indites similr tendeny even when different mehnisms re used to indue inflmmtion. Therefore, it ws suggested tht GlCer itself my hve n intestinl protetive effet. In this study, we exmined the effets of plnt sphingolipids on inflmmtory stress in differentited Co-2 ells, whih were used s n in vitro model of intestinl ells. The effets of vrious sphingolipids on these ells were lso exmined. Correspondene to: Mikio Kinoshit, Oihiro University of Agriulture nd Veterinry Mediine, Oihiro, Hokkido , JAPAN E-mil: kinosit@oihiro..jp Aepted August 14, 2017 (reeived for review July 28, 2017) Journl of Oleo Siene ISSN print / ISSN online

2 S. Ymshit, T. Seino, K. Aid et l. 2 EXPERIMENTAL PROCEDURES 2.1 Preprtion of GlCer A ommeril GlCer-rih preprtion from Nippon Flour Mills Atsugi, Jpn ws used for isoltion of mize GlCer. To isolte GlCer, the preprtion ws dissolved in hloroform nd pplied to sili gel olumn followed y preprtive thin-lyer hromtogrphy, s desried previously 2. The GlCer frtion ws srped off the pltes nd resuspended in 1:5:5 mixture of distilled wter D.W. / hloroform/methnol. The otined suspension ws sonited for 15 minutes. D.W. ws dded to ring the solution to 1:1:1 D.W./hloroform/methnol, nd the solution ws sonited gin for 15 minutes. The orgni phse solution ws pplied to n evportor to otin purified mize GlCer purity 90. Whet flour GlCer purity 99 nd ovine rin GlCer purity 97 were purhsed from Wko Pure Chemil Osk, Jpn nd Sigm Aldrih St. Louis, MO, respetively. 2.2 Cell study Cell ulture Co-2 ells, otined from Riken Gene Bnk Tsuku, Jpn, were mintined in DMEM supplemented with 10 FCS, 2 mm glutmine, nd 0.1 mm nonessentil mino ids. For routine mintenne, Co-2 ells were trypsinized nd pssged t 1:3 rtio, immeditely prior to rehing onfluene. For spontneous differentition 9, the time t whih the ells initilly rehed onfluene, s determined y light mirosopy, ws designted s dy 0, nd the ells were trnsfeted 21 dys lter Cell ount Co-2 ells were seeded t density of ells/ ml in 24-well pltes Nun, Rohester, NY in 1 ml of ulture medium, for 21 dys, t 37, under n tmosphere of 5 CO 2. The medium ws then repled with test medium ontining no FBS, inflmmtory stimultors 50 μg/ml or 50 ng/ml, nd sphingolipids GlCer or GlCer were dded for 0 48 hours. After stimultion, ells were rinsed with PBS Nissui, Tokyo, Jpn, trypsinized, nd ounted using ounting hmer EM-Teholor; Hirshmnn, Eerstdt, Germny Apoptosis ssy Co-2 ells were seeded t density of ells/ ml in L-Tek TM Chmer Slides Thermo Sientifi, Wlthm, MA in 0.5 ml of ulture medium, for 21 dys, t 37, under n tmosphere of 5 CO 2. The medium ws, then, repled y test medium ontining no FBS, inflmmtory stimultors 50 μg/ml or 50 ng/ml, nd sphingolipids GlCer or GlCer were dded for 48 hours. Apoptoti ells were quntified s reported previously 10, 11, y ounting ells with hrteristi frgmented nulei on DAPI stining, under fluoresene mirosope. The perentge of poptoti ells ws lulted s: numer of poptoti ells/totl numer of ells Cytokine rry ssy Cytokine seretion ws exmined using Cytokine Arry Kit Humn Cytokine Arry Pnel A; R&D Systems, Minnepolis, MN. The deteted ytokines were the following: CD-40 Lignd, Chemokine C-C motif lignd 1 I-309, Complement Component 5 C5/C5, Grnuloyte olonystimulting ftor G-CSF, Grnuloyte mrophge olony-stimulting ftor GM-CSF, Growth relted onogenelph GRO-α, Interferon-gmm IFN-γ, Interferon-γ-indued protein 10 IP-10, Interleukin-1lph IL-1α, IL-1β, IL-1r, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL- 12p70, IL-13, IL-16, IL-17A, IL-17E, IL-18, IL-21, IL-32, Monoyte hemotti nd tivting ftor MCAF ; Mrophge inflmmtory protein-1 lph MIP-1α : CCL3 / Mrophge inflmmtory protein-1 et MIP-1β : CCL4, Mrophge migrtion inhiitory ftor MIF, Interferoninduile T ell lph hemottrtnt I-TAC, Regulted on tivtion, norml T ell expressed nd sereted RANTES, Interellulr dhesion moleules-1 ICAM-1, Stroml ell-derived ftor 1 SDF-1, Plsminogen tivtor inhiitor -1 PAI-1 : Serpin E1, Triggering reeptor expressed on myeloid ells 1 TREM-1, nd Tumor nerosis ftor-lph. This ssy ws performed ording to the mnufturer s protool. Briefly, ells were seeded in 100-mm dishes ells for 21 dys t 37 under n tmosphere of 5 CO 2. The medium ws then repled with test medium ontining no FBS. Cells were inuted in this medium supplemented with nd/or GlCer, nd, 24 hours lter, ells were srped off the pltes nd lysed with hypotoni lysis uffer 1 M NCO 3, 10 μl/ml Protese Inhiitor Coktil for use with mmmlin ell nd tissue extrts Sigm, St. Louis, MO for 30 minutes on ie. Moreover, ommeril lysis uffer Lysis Buffer 17; R&D Systems ws dded to otin the ell lyste. Memrnes oted with 36 different nti-ytokine ntiodies were exposed to the lyste, nd ytokines were deteted y fluoresene. Dt were quired with ImgeJ softwre TLC nlysis To determine the movement of exogenous GlCer, TLC nlyses of ells, ell surfe, nd medium were onduted. Briefly, fter 48-hour inution of GlCer 50 μm with ells nd medium or only with medium, the totl lipids of ells, ell surfe 10, nd medium were extrted, nd the lkline-stle lipid frtion ws prepred nd sujeted to TLC with 95:12 v/v hloroform/methnol s the solvent Sttistil nlysis The results were nlyzed y nlysis of vrine nd Sheffé s test. In ll nlyses, differenes were onsidered sttistilly signifint when p J. Oleo Si. 66, (12) (2017)

3 Sphingolipids nd Inflmmtory Stress Fold Chnge (vs. Blnk = 1.0) Fold Chnge (vs. Blnk = 1.0) Fold Chnge (vs. Blnk = 1.0) A: Whet GlCer B: GlCer +10μM +50μM +10 GlCer µm +50 GlCer µm GlCer GlCer µM µm GlCer GlCer µm GlCer µM µm GlCer C: Mize GlCer 50 50μM µm GlCer 50 50µM GlCer μM µm GlCer µM µm GlCer μM µm GlCer µM µm GlCer GlCer µm GlCer µm GlCer +10 GlCer µm +50 GlCer µm GlCer GlCer GlCer Fig. 1 Effets of glyosylermide on the viility of differentited Co-2 ells under onditions of - or -indued stress. Differentited Co-2 ells were ultured for 48 hours using inflmmtory stimultors with/without sphingolipids. A: GlCer from whet. B: GlCer from ovine rin. C: GlCer from mize.,, Vlues with different supersript letters in the sme olumn differ signifintly p 5. Error rs indite stndrd derivtion SD. Bold indites Blnk RESULTS nd DISCUSSION Under the ulture onditions used herein, Co-2 re dherent ells. Therefore, to determine ell viility nd inflmmtory stress, the numers of dherent ells were ssessed. Addition of or, whih re induers of inflmmtory stress, deresed the numer of dherent ells y However, this effet ws suppressed y supplementtion with 10 μm GlCer. To determine the effets of different sugr omponents of glyosylermide GlCer nd GlCer, the effets of GlCer from ovine rin under these onditions of stress were lso exmined. Both GlCer nd GlCer suppressed the redution in vile ell numers upon exposure to or. The reovery levels otined with eh sphingolipid were essentilly the sme, ut slight differenes were oserved Fig. 1. To determine the effets of different sphingoid ses of plnt GlCer ginst nd tions, the effets of whet nd mize GlCer, whih hve different sphingoid ses, were tested. The levels of vile ells upon exposure to nd were nerly the sme for oth GlCer hving different omposition of sphingoid ses. These oservtions indited tht differenes in sugr nd sphingoid ses did not ffet ell numers under onditions of or stress. Thus, the GlCer from whet ws used in susequent experiments. The mehnisms underlying the suppression of ell prolifertion y nd were sed on poptoti ell deth Fig. 2. Therefore, the effets of GlCer on - nd -indued poptosis were investigted. DAPI stining of differentited Co-2 ells treted with or showed ggregted nd frgmented nulei Fig. 2. The levels of - nd -indued poptoti-like ells were pproximtely 13 nd 10, respetively. Addition of 10 μm GlCer suppressed phenotype, n effet tht ws dosedependent. Differenes in oth sugr nd sphingoid ses did not ffet the suppression of poptoti ell deth indued y inflmmtory stress in differentited Co-2 ells dt not shown. To determine the loliztion of GlCer in Co-2 ells, TLC nlyses of the ell surfe nd medium were onduted. After 48 hours of ultivtion with GlCer, the mjority of GlCer ws reovered from the medium nd ell surfe Fig. 3. The effets of 24-hour tretment with sphingolipids on the prodution of inflmmtion-relted ytokines in differentited Co-2 ells stimulted y were ssessed with ytokine rry kit tht n, simultneously, detet 36 ytokines Humn Cytokine Arry Pnel A; R&D Systems. The densities of positive nd negtive ontrol spots in this kit were lmost equivlent, inditing tht this method is suitle for the nlysis of ytokine ontent. First, ddition of GlCer 50 μm lone did not hve n effet on the prodution of severl ytokines Fig. 4. On the other hnd, ddition of inresed the levels of inflmmtory ytokines nd hemokines Fig. 4A nd B. Supplementtion with GlCer suppressed the prodution of most inflmmtory ytokines nd hemokines indued y. The levels of most nti-inflmmtory ytokines nd other ytokines did not hnge signifintly Fig. 4C nd D. Nevertheless, some nti-inflmmtory nd other ytokines were indued upon tretment. Addition of indued IL-6, IL-8, IL-12p70, nd RANTES, nd ws lso slightly indued. The ddition of GlCer together with signifintly suppressed the expression of these ytokines. IL-6, IL-8, IL-12p70, RANTES, nd re indued downstrem of the J. Oleo Si. 66, (12) (2017) 1339

4 S. Ymshit, T. Seino, K. Aid et l. A B C D (%) Cells Apoptoti signling pthwy whih is prt of the Toll-like reeptor signling pthwy 12. These results indited tht the mehnism of suppression of -medited inflmmtory stress in Co-2 ells ws sed on the regultion of the Toll-like reeptor signling pthwy 12 14, suggesting tht the suppressive effet of glyosylermide ourred upstrem of the Toll-like reeptor signling pthwy 14. On the other hnd, other reserhers reported tht sphingolipids, espeilly ermide, hve n nti-inflmmtory effet fter stimultion y inding to reeptors of the CD300 fmily, whih re regultors of the immune system 15, 16. The reltionship etween ell surfe reeptors nd sphingolipids should e investigted. In our previous study, we reported tht dietry GlCer ould inhiit the indution of ytokines, espeilly hemokines, promoted y DMH dministrtion 8. The mehnism ws similr to tht reported previously in DSS inflmmtion model 7. Therefore, we speulted tht dietry GlCer + 10μM + 50μM 50μM µm GlCer + + GlCer GlCer 10μM GlCer50μM GlCer +10 µm +50 µm GlCer +10 µm +50 µm GlCer GlCer GlCer GlCer Fig. 2 Effets of GlCer on poptosis indued y or in differentited Co-2 ells. Cells were stined with 4,6-dimidino-2- phenylindole DAPI fter ultivtion for 48 hours using with/without GlCer. Sle r = 50 μm. A: Blnk. B: +. C: +10 μm GlCer whet. D: Indution of poptosis in Co-2 ells y or.,, Vlues with different supersript letters in the sme olumn differ signifintly p 5. Error rs indite stndrd derivtion SD. Fig. 3 Loliztion of exogenous GlCer in differentited Co-2 ells inuted with GlCer determined y TLC. Moile phse: Chloroform:Methnol 95:12,v/v. Detetion: 50 H 2 SO 4. Cells were ultured for 48 hours using GlCer, nd then these were frtionted C, D, nd E. A: GlCer stndrd. B: Only medium inuted with GlCer. C: Medium. D: Cell surfe. E: Cells. my protet the surfe of the olon from the effets of vrious drugs. Moreover, dietry sphingolipids were shown to regulte intestinl sorption of severl lipids 17, 18. Milk sphingomyelin hs strong inhiitory effets on holesterol nd α toopherol sorption 17, whih re thought to e due to the higher degree of sturtion nd to the longer hin length of its ftty yl groups tht slow the rte of luminl lipolysis, mielle soluiliztion, nd trnsfer of mielle lipids to enteroyte. GlCer lso hve high degree of sturtion nd long-length hin ftty yl groups Therefore, GlCer my suppress the sorption of vrious drugs, inluding. Moreover, most GlCer ws reovered from the medium nd ell surfe, suggesting tht GlCer my exert its funtion on the surfe of Co-2 ells. Thus, sphingolipids my regulte the intertion etween nd its reeptors on the ell surfe s desried ove. The results of this study n e summrized s follows: 1 sphingolipids, suh s GlCer, hve potent nti-inflmmtory effet; 2 GlCer suppresses the -induted prodution of ytokines nd poptosis; 3 sphingolipids my remin on the ell surfe, nd 4 the hemil properties of sphingolipids my prevent the intertion etween nd its reeptor. Dietry GlCer my protet the olon surfe from the hrmful effets of vrious drugs. Further studies in ell model systems re required to determine the moleulr mehnism underlying these effets. Referenes 1 Sugwr, T.; Kinoshit, M.; Ohnishi, M.; Miyzw, T. Apoptosis indution y whet-flour sphingoid ses in DLD-1 humn olon ner ells. Biosi. Biotehnol J. Oleo Si. 66, (12) (2017)

5 Sphingolipids nd Inflmmtory Stress A Fold Chnge ( vs. Blnk = 1.0) Fold Chnge ( vs. Blnk = 1.0) B + 50 µm GlCer 50 µm GlCer C D Fold Chnge v s. Blnk = 1.0) Fold Chnge (vs. Blnk = 1.0) 1.01 ( Fig. 4 Effets of GlCer on ytokine levels in differentited Co-2 ells stimulted y fold hnge vs. Blnk. Cells were ultured for 24 hours using with/without GlCer, nd then were nlyzed for levels of vrious ytokines. A: Inflmmtory ytokines. B: Chemokines. C: Anti-inflmmtory ytokines. D: Other ytokines. p 5 vs. group. Error rs indite stndrd derivtion SD. Bold indites Blnk = 1.0. Biohem. 66, Aid, K.; Kinoshit, M.; Sugwr, T.; Ono, J.; Miyzw, T.; Ohnishi, M. Apoptosis induement y plnt nd fungus sphingoid ses in humn olon ner ells. J. Oleo Si. 53, Kinoshit, M.; Hori, N.; Aid, K.; Sugwr, T.; Ohnishi, M. Prevention of melnin formtion y yest ereroside in B16 mouse melnom ells. J. Oleo Si. 56, Hnnun, Y.A.; Linrdi, C.M. Sphingolipid rekdown produts: nti-prolifertive nd tumor-suppressor lipids. Biohim. Biophys. At 1154, Sugwr, T.; Kinoshit, M.; Ohnishi, M.; Ngt, J.; Sito, M. Digestion of mize sphingolipids in rts nd uptke of sphingdienine y Co-2 ells. J. Nutr. 133, Adm, D. Review lmes BSE outrek on lf feed. Nture 412, J. Oleo Si. 66, (12) (2017) 1341

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