Docosapentaenoic Acid (22:5n-3) Downregulates mrna Expression of Pro-inflammatory Factors in LPS-activated Murine Macrophage Like RAW264.

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1 Journl of Oleo Siene Copyright 217 y Jpn Oil Chemists Soiety oi : 1.565/jos.ess17111 Doospentenoi Ai (22:5n-3) Downregultes mrna Expression of Pro-inflmmtory Ftors in LPS-tivte Murine Mrophge Like RAW264.7 Cells Ynzhu Tin 1, Ami Ktsuki 1, Donto Romnzzi 2, Mtthew R. Miller 2, Seren L. Ams 2, Kzuo Miyshit 1 n Msshi Hosokw 1,* 1 Fulty of Fisheries Sienes, Hokkio University, Minto, Hkote, Hokkio , JAPAN 2 Cwthron Institute, 98 Hlifx Street Est Nelson 71, NEW ZEALAND Astrt: Doospentenoi i (22:5n-3, n-3 DPA) is n-3 polyunsturte ftty i (PUFA) foun in fish oil, n hs een reporte to hve helth enefits. This stuy investigte onversion of n-3 DPA, n exmine the nti-inflmmtory effets of n-3 DPA on tivte mrophges. Murine mrophge-like RAW264.7 ells were inute in ulture mei ontining n-3 DPA for 72 h. The level of n-3 DPA in the ftty i omposition of the totl lipi frtion inrese in ose-epenent mnner. Furthermore, the levels of ooshexenoi i (DHA) n eiospentenoi i (EPA) were higher in trete ells thn in ontrol ells. In RAW264.7 ells stimulte y lipopolyshrie (LPS), n-3 DPA signifintly ownregulte mrna expression of pro-inflmmtory ftors suh s IL-6, IL-1β, inos n COX-2. Proution of IL-6 ws lso reue y n-3 DPA in ose-epenent mnner. We foun tht n-3 DPA tretment resulte in greter IL-6 mrna own-regultion thn tht hieve with EPA tretment, n ws similr to tht of DHA tretment. Furthermore, expression levels of IL-6 n IL-1β mrnas were mesure in the presene of the elt-6 esturse inhiitor SC26196 in the ulture meium to inhiit the onversion of n-3 DPA to DHA. There ws no signifint ifferene in the own-regultion in the mrna expression of proinflmmtory ytokines in RAW264.7 ells y n-3 DPA with or without presene of SC These results emonstrte tht n-3 DPA exhiits nti-inflmmtory effets in tivte RAW264.7 ells, whih re inepenent of DHA onversion. Key wors: n-3 oospentenoi i, nti-inflmmtion, mrophge, n-3 polyunsturte ftty is, ytokine 1 INTRODUCTION Long hin n-3 polyunsturte ftty is PUFAs suh s ooshexenoi i DHA n eiospentenoi i EPA re well known for their enefiil helth effets. These n-3 PUFAs re mjor ftty is in fish oil. In prtiulr, DHA is enrihe in tun fish, n EPA in srines. Doospentenoi i 22:5n-3, n-3 DPA is the thir most prevlent n-3 PUFA in mrine oil. Sel met, slmon n lone ontin n-3 DPA t omprtively high levels, pproximtely 2-7 of totl ftty i omposition 1, 2. Furthermore, n-3 DPA levels in humn milk hve een reporte to e higher thn EPA levels, n re omprle to DHA levels 3. In previous stuies, n-3 DPA hs een reporte to inhiit ngiogenesis 4 n pltelet ggregtion 5, n to improve lipi metolism in oth in vitro n in vivo moels 6 8. In epiemiologil investigtions of oler ults, higher n-3 DPA levels in the loo hve een orrelte with lower totl mortlity, inluing eth from oronry hert isese 9. However, in omprison to the vst numer of stuies on EPA n DHA, informtion regring the helth enefits of n-3 DPA is very limite. Previous humn n roent stuies suggest tht ietry n-3 DPA inreses not only the onentrtion of n-3 DPA in plsm triglyeries, ut lso the levels of oth EPA n DHA 1, 11. Reently, we reporte tht the ioonversion profile of n-3 DPA is ifferent mong humn ell lines estlishe from ifferent tissues n loo. We foun tht n-3 DPA ws onverte to DHA in HepG2 ells heptolstom ell line, ut not in Co-2 olon rinom ell line or THP-1 monoyti leukemi ell line ells, even though retro-onversion of n-3 DPA to EPA ws oserve * Corresponene to: Msshi Hosokw, Fulty of Fisheries Sienes, Hokkio University, Minto, Hkote, Hokkio , JAPAN E-mil: hoso@fish.hokui..jp Aepte My 23, 217 (reeive for review My 7, 217) Journl of Oleo Siene ISSN print / ISSN online

2 Y. Tin, A. Ktsuki, D. Romnzzi et l. in ll three ell lines 12. This metoli informtion is importnt when onsiering the nutritionl roles of n-3 DPA. Chroni low-gre inflmmtion hs een ientifie s the primry trigger for the pthogenesis of metoli synrome, riovsulr iseses n ners During the evelopment of inflmmtion, proution of pro-inflmmtory ytokines n hemokines suh s interleukin IL -1β, IL-6, monoyte hemottrtnt protein-1 MCP-1 n regulte on the tivtion of norml T-ell expresse n serete RANTES inreses ue to immune ell tivtion 16, 17. Mrophges ply ruil role in the regultion n proution of ytokines n hemokines 18. Furthermore, ylooxygense-2 COX-2 n nitri oxie synthse NOS proue inflmmtory meitors suh s prostglnin E 2 n nitri oxie NO, respetively 19, 2. Therefore, suppression of pro-inflmmtory ftors proution in tivte mrophges is very importnt for the prevention of inflmmtory-relte iseses. Herein, we report the nti-inflmmtory effet of n-3 DPA on murine mrophge-like RAW264.7 ells tivte y lipopolyshrie LPS. Initilly, the onversion of n-3 DPA in RAW264.7 ells ws evlute. The nti-inflmmtory effet of n-3 DPA ws then investigte n ompre with those of EPA n DHA. In RAW264.7 ells, n-3 DPA n e onverte to oth DHA n EPA. We utilize the inhiitor SC26196 to stop the onversion of n-3 DPA to DHA n evlute the effet of n-3 DPA on IL-6 n IL-1β mrna expression in the presene of this elt-6 esturse inhiitor. The present results showe tht own-regultion of IL-6 mrna expression y n-3 DPA ws more potent thn tht y EPA, n similr s tht y DHA. Furthermore, n-3 DPA exhiite nti-inflmmtory effets in LPS-tivte RAW264.7 ells inepenent of its onversion to DHA. 2 EXPERIMENTAL PROCEDURES 2.1 Mterils The ftty i n-3 DPA, EPA n DHA were purhse from Cymn Chemil Ann Aror, MI, USA. Fetl ovine serum FBS, RPMI 164 meium, peniillin n streptomyin were otine from Gio, Thermo Fisher Sientifi In. Wlthm, MA, USA. LPS n the elt-6 esturse inhiitor SC26196 α,α-iphenyl-4-3-pyriinylmethylene mino -1-piperzinepentnenitrile were purhse from Sigm Chemil St. Louis, MO, USA. α-toopherol ws purhse from Wko Pure Chemil Inustries Osk, Jpn. 2.2 Cell ulture Murine mrophge-like ell line RAW264.7 ws purhse from DS Phrm Biomeil Suit, Osk, Jpn. RAW264.7 ells ells/well were pre-inute in 12-well pltes with 1 ml RPMI 164 ontining 1 FBS, 1 U/mL peniillin, n 1 μg/ml streptomyin. Cells were mintine t 37 in humiifie tmosphere ontining 5 CO 2 for 48 h. n-3 DPA, EPA, or DHA μm ws e into the ulture meium, n the ells were inute for 72 h. The elt-6 esturse inhiitor, SC μm ws e into the ulture meium 3 h prior to the ition of n-3 DPA t 45 h pre-inution. n-3 PUFAs n SC26196 were e s ethnol n imethylsulfoxie DMSO solutions, respetively. Finl onentrtions of ethnol n DMSO were juste to.1 in the ulture meium. Then, inflmmtion in ells ws inue y LPS.1 μg/ml of finl onentrtion for 6 h in the presene of n-3 PUFAs n/or SC To prevent n-3 PUFAs oxition, 1 μm α-toopherol ws e in the ulture meium uring n-3 PUFA tretment. 2.3 Extrtion of ellulr lipis n ftty i nlysis RAW264.7 ells ells/ ish were pre-inute in 1 mm ish with 1 ml RPMI 164 supplemente with 1 FBS, 1 U/mL peniillin, n 1 μg/ml streptomyin t 37 in humiifie tmosphere ontining 5 CO 2 for 48 h. Tretments with n-3 PUFAs n/or SC26196 were onute s esrie in setion 2.2 Cell ulture. Following inution for 72 h with n-3 PUFAs n/or SC26196, RAW264.7 ells were wshe three times with phosphteuffere sline PBS, n were entrifuge t 115 x g for reovery. Totl lipi ws extrte from the ells y the Bligh n Dyer metho 21. Ftty i ompositions of ellulr lipis were etermine y gs hromtogrphy GC ; methyl ester erivtives were prepre oring to proeures esrie in previous pper 22. GC nlysis ws performe using Shimzu GC-214 gs hromtogrph equippe with flme-ioniztion etetor FID n Omegwx-32 pillry olumn 3 m.32 mm i..: Supelo, Bellefonte, PA, USA. The tempertures of the etetor, the injetor, n the olumn were 26, 25 n 2, respetively. The rrier gs, helium, flow ws t 5 kp. The split rtio ws 1:49 v/v. Eh ftty i ws ientifie se on the retention times of uthenti stnrs. 2.4 Quntittive rel-time RT-PCR RAW264.7 ells ells/well were seee into 12-well pltes with 1 ml RPMI 164 supplemente with 1 FBS, 1 U/mL peniillin, n 1 μg/ml streptomyin. Cells were inute t 37 in humiifie tmosphere ontining 5 CO 2 for 48 h. Ftty is n-3 DPA, EPA or DHA t μm were e into the ulture meium together with 1 μm α-toopherol, n ells were further inute for 72 h. Totl RNA ws extrte from the ells with the RNesy Mini Kit QIAGEN GmH, Hilen, Germny oring to mnufturer s protool. Then, DNA ws synthesize from totl RNA using the High-C- 115

3 Anti-inflmmtory effet of n-3 oospentnoi i on RAW264.7 ells pity DNA Arhive Kit Applie Biosystems Jpn Lt, Tokyo, Jpn. Quntittive rel time RT-PCR ws performe on the ABI Prism 75 Applie Biosystems Jpn Lt, Tokyo, Jpn. Cyling onitions for PCR were s follows; 5 for 2 min, 95 for 1 min, n 4 yles of 95 for 15 s, followe y 6 for 1 min. PCR primers n TqMn proes were otine from TqMn Gene Expression Assys Applie Biosystems Jpn Lt, Tokyo, Jpn ; IL-6: Mm44619_m1, IL-1β: Mm434228_m1, COX-2: Mm478374_m1, inos: Mm4452_m1; MCP-1: Mm441242_m1, RANTES: Mm132427_m1, GAPDH: Mm _g Enzyme-linke immunosorent ssy The onentrtion of IL-6 in the ulture meium of RAW264.7 ells ws mesure y enzyme-linke immunosorent ssy ELISA using ommerilize kits Thermo Sientifi, Freerik, MD, USA oring to the mnufturer s protool. 2.6 Sttistil Anlysis All vlues re expresse s men SE n 3. Sttistil ifferenes were etermine y the Sheffe s test t p.5. 3 RESULTS 3.1 Ftty i omposition of totl lipi in RAW264.7 ells trete with n-3 DPA RAW264.7 ells were inute with μm n-3 DPA for 72 h, n ftty i omposition ws etermine y GC-FID. The perentge of n-3 DPA in the ells inrese in ose-epenent mnner, n rehe with 75 μm n-3 DPA Tle 1. Cytotoxiity ws not oserve y the tretment with 75 μm n-3 DPA for 72 h t not shown. Conversely, n-3 DPA in ontrol ells trete with ethnol ws foun to e The perentge of EPA n DHA lso signifintly inrese to n 13 7, respetively, with 75 μm n-3 DPA, lthough 2:4n-6 level ws reue. In ition, 16: n 18: inrese, n 16:1, 18:1n-9 n 18:1n-7 erese in the ells trete with n-3 DPA Tle Effet of n-3 PUFAs on the expression of pro-inflmmtory ytokines in RAW264.7 ells The effets of n-3 DPA on mrna expression of pro-inflmmtory ftors n protein proution in RAW264.7 ells were investigte. Expression of IL-6, IL-1β, MCP-1, n RANTES mrnas inue y LPS stimultion ws signifintly suppresse y n-3 DPA Fig. 1. Furthermore, n-3 DPA tretment in RAW264.7 ells mrkely erese mrna expressions of inos n COX-2, whih proue the inflmmtory ftors NO n PGE 2. Reution of IL-6 on- Tle 1 Ftty i omposition of totl lipi extrte from RAW264.7 ells trete with n-3 DPA. (%) Control n-3 DPA 25 μm 5 μm 75 μm 14: 6±.4 6± ± ±.2 16: 13.66± ± ± ±8 16:1 4.56± ± ± ±.12 18: 16.89± ± ± ±.7 18:1n ± ±5 1.33± ±5 18:1n-7 11± ± ± ±.3 18:2n-6 1± ± ± ±.3 2:1n-9 2±.3.39±.3 2±.2.15±.1 2:2n ± ±.17 2±.4.51±.7 2:3n-6.56±.3 5±.8.52±.5.58±.1 2:4n ± ± ±.9 5.9±.6 2:5n-3 (EPA).16±..93± ± ±.35 22:5n-3 (n-3 DPA) 1.99±.9 18± ± ±5 22:6n-3 (DHA) 2.43± ± ±5 13±7 RAW264.7 ells were trete with μm n-3 DPA for 72 h. Control ells were trete with ethnol. Dt re presente s mens±se, n=3. Different letters enote signifint ifferenes t p<.5 (Sheffe s F test). 1151

4 Y. Tin, A. Ktsuki, D. Romnzzi et l. IL-6 IL-1β IL-6/GAPDH IL-1β/GAPDH Fig. 1 MCP-1/GAPDH inos/gapdh LPS n-3dpa - - MCP-1 inos (μm) RANTES/GAPDH COX-2/GAPDH LPS n-3dpa RANTES COX (μm) Effet of n-3 oospentenoi i DPA on mrna expression of pro-inflmmtory ftors in lipopolyshrie LPS -stimulte RAW264.7 ells. Cells were inute in ulture meium ontining n-3 DPA for 72 h, n were then stimulte with.1 μg/ml LPS for 6 h in the presene of n-3 DPA. α-toopherol 1 μm ws e into the ulture meium together with n-3 DPA. The mrna expression level ws etermine y rel-time quntittive RT- PCR, n gene expression ws normlize to GAPDH mrna level. Dte re presente reltive to LPS+, n re expresse s mens SE, n 3. Different letters enote signifint ifferenes t p.5 Sheffe s test. entrtion ws lso oserve in the ulture meium of n-3 DPA-trete RAW264.7 ells Fig. 2. The reution ws ose-epenent. In the present stuy, we further ompre mrna expression n protein proution of IL-6 in RAW264.7 ells trete with EPA, n-3 DPA n DHA. All of the n-3 PUFAs 1152

5 Anti-inflmmtory effet of n-3 oospentnoi i on RAW264.7 ells IL-6 seretion(ng/ml) LPS n-3 DPA Fig. 2 - mrkely suppresse mrna expression of IL-6 s ompre to LPS+ ells without n-3 PUFA tretment Fig. 3. Tretment with n-3 DPA n DHA resulte in signifintly greter own-regulte IL-6 mrna expression s ompre to EPA tretment. In ition, IL-6 onentrtion in the ulture meium ws signifintly reue y EPA, n-3 DPA, n DHA to , n 8 4 ng/ml, respetively, s ompre with ng/ml oserve in ontrol RAW264.7 ells Tle 2. The suppressive effet of DHA on IL-6 seretion ws greter thn tht of EPA, n ws similr to tht of n-3 DPA. 3.3 Anti-inflmmtory effets of n-3 DPA uner elt-6 esturse inhiition Our results showe tht n-3 DPA ws inorporte into RAW26.47 ells, n ws prtly onverte to DHA n EPA Tle 1. Therefore, it is unler whether the suppressive effet of n-3 DPA on LPS-inue mrna expression of pro-inflmmtory ytokines is epenent on n-3 DPA or the onverte DHA. To exmine the nti-inflmmtory effet of n-3 DPA in etil, we mesure mrna expression levels of pro-inflmmtory ytokines in LPS-stimulte RAW264.7 ells trete with elt-6 esturse inhiitor. Aition of the elt-6 esturse inhiitor SC26196 in the ulture meium ompletely inhiite the ioonversion of n-3 DPA to DHA, n le to the umultion of n-3 DPA in RAW264.7 ells Fig. 4A. Tretment with n-3 DPA suppresse LPS-inue mrna expression of IL-6 n IL-1β even in the presene of SC26196 Fig. 4B n 4C. In ition, no signifint ifferene ws oserve in n-3 DPAmeite reution of IL-6 n IL-1β mrna expressions (μm) Effet of n-3 oospentenoi i DPA on IL-6 seretion from lipopolyshrie LPS -stimulte RAW264.7 ells. Cells were inute in ulture meium with n-3 DPA for 72 h, n were then stimulte with.1 μg/ml LPS for 6 h in the presene of n-3 DPA. α-toopherol 1 μm ws lso e into the ulture meium together with n-3 DPA. Conentrtion of IL-6 ws mesure y ELISA. Dte re presente s mens SE, n 3. Different letters enote signifint ifferenes t p.5 Sheffe s test. IL-6/GAPDH LPS EPA n-3 DPA DHA following LPS-stimultion in RAW264.7 ells trete with or without SC These finings inite tht the ntiinflmmtory effet of n-3 DPA is, t lest prtilly, ine- 75 µm Fig. 3 Down-regultion of IL-6 mrna expression y n-3 polyunsturte ftty is PUFAs i n lipopolyshrie LPS -stimulte RAW264.7 ells. Cells were inute in ulture meium with EPA, n-3 DPA or DHA for 72 h, n were then stimulte with.1 μg/ml LPS for 6 h in the presene of n-3 PUFAs. α-toopherol 1 μm ws lso e into the ulture meium together with n-3 PUFAs. The mrna expression ws etermine y rel-time quntittive RT-PCR, n gene expression ws normlize to the levels of GAPDH. Dte re presente reltive to LPS+, n re expresse s mens SE, n 3. Different letters enote signifint ifferenes t p.5 Sheffe s test. Tle 2 Effet of n-3 PUFAs on IL-6 seretion in LPSstimulte RAW264.7 ells. LPS- LPS+ LPS+75 μm EPA LPS+75 μm n-3 DPA LPS+75 μm DHA IL-6 onentrtion (ng/ml).± ±5 1.92± ±3 8±4 Cells were trete with n-3 DPA, EPA or DHA (75 μm) for 72 h in ulture meium, n then stimulte y.1 μg/ml LPS for 6 h. To prevent ftty is oxition, 1 μm α-toopherol ws e into the ulture meium. IL-6 onentrtion in the ulture meium ws mesure y ELISA. Dte re presente s the mens±se, n=3. Different letters enote signifint ifferenes t p<.5 (Sheffe s F test). 1153

6 Y. Tin, A. Ktsuki, D. Romnzzi et l. % (A) 2:4n-6 EPA n-3dpa DHA Control n-3 DPA n-3 DPA (75 μm) + SC26196 (1 μm) IL-6/GAPDH (B) LPS - SC n-3 DPA(75 μm) IL-1β/GAPDH LPS - SC n-3 DPA(75 μm) Fig. 4 Ftty i omposition n mrna expression of pro-inflmmtory ytokines in RAW264.7 ells trete with n-3 oospentenoi i DPA n the elt-6 esturse inhiitor SC Cells were trete with 75 μm n-3 DPA for 72 h, n were then stimulte with.1 μg/ml lipopolyshrie LPS for 6 h. The elt-6 esturse inhiitor SC μm ws e in the ulture meium 3 h efore ition of n-3 DPA. α-toopherol 1 μm ws e in the ulture meium together with n-3 DPA. A Ftty i omposition of totl lipi extrte from RAW264.7 ells efore LPS stimultion ws nlyze y GC. The mrna expressions of IL-6 B n IL-1β C in the LPS-stimulte RAW264.7 ells trete with or without n-3 DPA n SC26196 were etermine y rel-time quntittive RT-PCR. Dte re presente reltive to LPS+, n were expresse s the mens SE, n 3. Different letters enote signifint ifferenes t p.5 Sheffe s test. (C) penent of n-3 DPA to DHA onversion. 4 DISCUSSION The ftty i, n-3 DPA, is one of the intermeite ftty is proue uring the onversion of EPA to DHA vi elongtion, esturtion, n β-oxition of 24:6 n-3. In previous stuy, we hve reporte tht the metolism of n-3 DPA is ifferent in humn ell lines. In HepG2 ells, n-3 DPA ws onverte to DHA. However, it ws not onverte in Co-2 or THP-1 ells, lthough retro-onversion to EPA ws oserve in ll three ell lines 12. In the present stuy, we nlyze the onversion of n-3 DPA in murine mrophge-like RAW264.7 ells. RAW264.7 ells were inute in ulture mei ontining μm n-3 DPA for 72 h, n n-3 DPA level inrese in ose-epenent mnner. Furthermore, oth DHA n EPA levels lso inrese when ompre to those of ontrol ells. Chroni inflmmtion is well known to e involve in the onset of metoli synrome n riovsulr isese 13, 14. Mrophges ply ruil role in inflmmtion n immune funtions, inluing ytokine proution n inution of pro-inflmmtory-ssoite enzymes The n-3 ftty is, EPA n DHA, hve een reporte to exhiit nti-inflmmtory effets through suppression of inflmmtory ftors proue in mrophges 23, 24. As ompre with DHA n EPA, there is little known regring the nti-inflmmtory effets of n-3 DPA on mrophges. Therefore, in the present stuy, the regultion of 1154

7 Anti-inflmmtory effet of n-3 oospentnoi i on RAW264.7 ells mrna expression n protein seretion of pro-inflmmtory ftors y n-3 DPA ws investigte using LPS-stimulte RAW264.7 ells. In ition, we ompre the nti-inflmmtory effets of n-3 DPA with DHA n EPA. We emonstrte tht n-3 DPA reue mrna expression of pro-inflmmtory ftors suh s IL-6, IL-1β, MCP-1, RANTES, inos n COX-2 in LPS-stimulte RAW264.7 ells. Furthermore, n-3 DPA tretment suppresse LPS- inue IL-6 proution in RAW264.7 ells in ose-epenent mnner. These results inite tht n-3 DPA is n effetive ftty i for ttenuting the proution of inflmmtory ftors through own-regultion of mrna expression. We further ompre the nti-inflmmtory effets of n-3 DPA, EPA, n DHA on LPS-tivte RAW264.7 ells. All three ftty is signifintly suppresse IL-6 mrna expression n protein proution in LPS-stimulte RAW264.7 ells. It is noteworthy tht own-regultion of IL-6 mrna expression in ells trete with n-3 DPA n DHA ws signifintly greter thn tht in EPA-trete ells. Furthermore, IL-6 onentrtion in the ulture meium of ells inute with DHA ws signifintly lower thn tht of EPA n ws similr to n-3 DPA. These results suggest tht DHA n n-3 DPA re more effetive thn EPA in lleviting LPS-inue pro-inflmmtory ftor proution n mrna expression in RAW264.7 ells. Welon et l. reporte tht DHA exhiits greter nti-inflmmtory effet on humn THP-1 mrophges s ompre to tht of EPA 25. Norris n Dennis reporte tht EPA iniretly inhiits COX through elongtion to n-3 DPA 26. However, no informtion regring the ility of n-3 DPA to regulte mrna expression of pro-inflmmtory ftors in tivte mrophges ws esrie. The present results emonstrte tht n-3 DPA is highly potent nti-inflmmtory ftty i in LPS-stimulte RAW264.7 ells. In RAW264.7 ells trete with n-3 DPA, levels of DHA n EPA, s well s n-3 DPA, were signifintly inrese. However, it ws unler whether own-regultion of IL-6 mrna expression y n-3 DPA in LPS-inue RAW264.7 ells ws epenent on the onversion of n-3 DPA to DHA or ue to the n-3 DPA itself. Therefore, we exmine the nti-inflmmtory effet of n-3 DPA in the presene of the elt-6 esturse inhiitor SC , whih inhiits the n-3 DPA to DHA onversion. n-3 DPA signifintly ownregulte mrna expressions of IL-6 n IL-1β in the presene of SC26196, to the sme egree s tht oserve y n-3 DPA tretment lone. These results inite tht n-3 DPA n iretly exert helth enefits vi its nti-inflmmtory properties tht re inepenent of onversion to DHA. In previous stuies, it hs een reporte tht DHA regultes the proution of pro-inflmmtory meitors through inhiition of NF-κ B tivtion. In ition, n-3 DPA-erive 7, 8, 17-trihyroxy-9, 11, 13, 15E, 19Z-oospentenoi i RvD1 n-3 DPA n 7, 14-ihyroxy-8, 1, 12, 16Z, 19Z-oospentenoi i MR1 n-3 DPA hve een ientifie s speilize meitors tht t to resolve inflmmtion 28. Further reserh is require to lrify the moleulr mehnism unerlying the nti-inflmmtory effets of n-3 DPA. 5 CONCLUSION Tretment of RAW264.7 ells with n-3 DPA le to n umultion of n-3 DPA, s well s n inrese in DHA n EPA. n-3 DPA signifintly own-regulte mrna expression of pro-inflmmtory ftors n reue IL-6 proution. The own-regultion of IL-6 mrna y n-3 DPA ws similr s tht inue y DHA, n more effetive thn EPA. As the own-regultion of mrna expression of proinflmmtory ytokines y n-3 DPA ws unffete y the ition of elt-6 esturse inhiitor SC26196, we onlue tht the nti-inflmmtory effet of n-3 DPA is epenent on its originl form, rther thn on its onverte DHA in the ells. ACKNOWLEDGMENT This work ws supporte through funing from the JST Jpnese-New Zeln Strtegi Interntionl Collortive Reserh Progrm SICORP on Funtionl Foos n New Zeln Ministry for Business Innovtion n Employment CAWX1416 n CAWX1318. Referenes 1 Byelshov, O.A.; Sinlir, A.J.; Kur, G. Dietry soures, urrent intkes, n nutritionl role of omeg-3 oospentenoi i. Lipi Tehnol. 27, Mulvney, W.J.; Jhngr, S.; Ingrm, B.A.; Turhini, G.M.; Winerg, P.C. Reovery of omeg-3 profiles of ultivte lone y ietry mrolge supplementtion. J. Appl. Phyol. 27, Koletzko, B.; Mrotzek, M.; Bremer, H.J. Ftty i omposition of mture humn milk in Germny. Am. J. Clin. Nutr. 47, Tsuji, M.; Murot, S.I.; Morit, I. Doospentenoi i 22:5, n-3 suppresse tue-forming tivity in enothelil ells inue y vsulr enothelil growth ftor. Prostglnins Leukot. Essent. Ftty Ais 68, Aki, S.; Murt, T.; Kittni, K.; Sto, T. Involvement of lipoxygense pthwy in oospentenoi i-in- 1155

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