Torenia concolor Lindley var. formosana Yamazaki extracts improve inflammatory response and lipid accumulation via PPARs activation

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1 BioMeiine (ISSN ) Septemer 2017, Vol. 7, No. 3, Artile 18 Pges DOI: /mn/ Originl rtile Toreni onolor Linley vr. formosn Ymzki extrts improve inflmmtory response n lipi umultion vi PPARs tivtion Yu-Chi Ling 1, Jun-Cheng Hu 2, Pei-Ying Li 3, Gun-Jhong Hung 1, Yueh-hsiung Kuo 1,4, Che-Yi Cho 2,5, * 1 Deprtment of Chinese Phrmeutil Sienes n Chinese Meiine Resoures, College of Chinese Meiine, Chin Meil University, Tihung, 404, Tiwn 2 Deprtment of Helth n Nutrition Biotehnology, Asi University, Tihung, 413, Tiwn 3 Shool of Phrmy, College of Phrmy, Chin Meil University, Tihung, 404, Tiwn 4 Deprtment of Biotehnology, Asi University, Tihung, 413, Tiwn 5 Deprtment of Meil Reserh, Chin Meil University Hospitl, Chin Meil University, Tihung, 404, Tiwn Reeive 5 th of April, 2017 Aepte 2 n of My, 2017 Author(s) This rtile is pulishe with open ess y Chin Meil University Keywors: Toreni onolor Linley vr. formosn Ymzki; Anti-inflmmtion; Lipi metolism; PPARs ABSTRACT Bkgroun/Introution: At present, humn iet is replete with sugr n ft. Anorml metolism n hyperglyemi or hyperlipiemi in the oy inues the evelopment of n overtive n ontinuous inflmmtory response, resulting in oesity n metoli synromes, inluing hypertension, hyperlipiemi, n insulin resistne. Toreni onolor Linley vr. formosn Ymzki (TC), perennil reeping hereous plnt, is tritionl Chinese meiinl her wiely use for the tretment of het stroke, hing musles n ones, ol, ysentery, n mustion. Purpose: This stuy evlute the influene of TC on inflmmtion responses n lipi metolism. Methos: In this stuy, groun TC power ws extrte with 95% ethnol. The ethnol ws remove y vuum onentrtion, n the resulting extrt ws further extrte with numer of solvents of ifferent polrity to proue four finl extrts: n ethnol extrt (TCEE), n ethyl ette extrt (TCEAE), n n- utnol extrt (TCBUE), n wter extrt (TCWE). The nti-inflmmtory effiy of the extrts n their pility for lipi metolism regultion ws then explore. Results: TCEE, TCEAE, n TCBUE exhiite goo nti-inflmmtory effiy; TCEAE lso simultneously regulte lipi metolism. In RAW264.7 ells, these three extrts suppresse the expression of inos n vi the signling pthwy tivtion of the trnsription ftor peroxisome prolifertor tivte reeptor γ (PPARγ) n therey showe nti-inflmmtory effiy. In 3T3-L1 ells, these three extrts promote lipi metolism n reue lipi umultion through the tivtion of PPARα n the inrese expression of iponetin, thus emonstrting regultion of lipi metolism. Conlusion: These results inite tht TC possesses nti-inflmmtory effiy n n regulte lipi metolism through the tivtion of trnsription ftor PPARs. We speulte tht these nutreutil effets re ttriutle to etulin, n tive ingreient in this herl meiine.. 1. Introution Nowys, the humn iet ommonly ontins high ontent of rohyrtes, fts, n exess lories. Nturlly, too muh lori intke will result in oesity. Oesity is one of the most ommon glol metoli isorers. Dt from the WHO emonstrte tht more thn 1.3 illion ults worlwie re overweight (oy mss inex: kg/m 2 ) n further 600 million re oese (oy mss inex: 30 kg/m 2 ). Oesity is often onomitnt with other symptoms (e.g., hypertension, hyperlipiemi, n insulin resistne), whih re together terme metoli synrome. Suh metoli normlities n long-term hyperglyemi or hyperlipiemi re likely to inue overtive n ontinuous inflmmtory retions in the oy, whih my result in mny severe iseses, suh s riovsulr isese (Axen et l., 2003; Nel, 2003), rthritis, Alzheimer s isese, type 2 ietes mellitus, n ner (Hn n Yoshimur, 2002). Mny epiemiologi stuies hve emonstrte the high mortlity rte of oese iniviuls, espeilly ue to riovsulr isese. In morily oese iniviuls, their ipoytes re * Corresponing uthor. Deprtment of Fmily Meiine, Chin Meil University Hospitl, No. 2, Yuh-Der Ro, Tihung 404, Tiwn. E-mil ress: yho@si.eu.tw (C.-Y. Cho). 29

2 likely to e insulin-resistnt, whih mens tht they re unle to reeive insulin signls to metolize gluose, proteins, n ftty is. Furthermore, on the one hn, insulin-resistnt ipoytes my serete exessive hemokines suh s monoyte hemottrtnt protein-1 (MCP-1) to ttrt irulting mrophges to infiltrte ipose tissue. The mrophges re stimulte y ipoyte-serete ftty is, n this genertes inflmmtory retions. On the other hn, the mrophges my serete inrese mounts of tumor nerosis ftor-α (TNF-α) to inue the trnsformtion of norml ipoytes to new insulin-resistne ipoytes, n this genertes greter egree of severe hroni inflmmtory retions n metoli synrome in oese iniviuls. If the mrophge inflmmtory retions inue y ipose tissue seretions re meliorte, the insulin resistne of ipoytes will e reue, n hroni inflmmtory retions will e prevente. Inflmmtion retions re losely relte to the progression of tissue mge use y numerous iseses. Therefore, stuies proing the nti-inflmmtory effets of vrious meiines n ompouns re extremely ruil. Peroxisome prolifertor tivte reeptor γ (PPARγ) is lign-tivte trnsription ftor of the nuler reeptor superfmily tht ontrols the expression of vriety of genes involve in ftty i metolism, ipogenesis, inflmmtion, n insulin sensitivity. Serhing for nutreutil ingreients from nturl prouts tht n prevent inflmmtion n oesity is pivotl in preventing iseses n promoting helth. Consequently, the mrket for nutreutil foos is lrge. Toreni onolor Linley vr. formosn Ymzki (TC), whih elongs to the Srophulriee fmily, is plnt ntive to Tiwn. As stte in Pen-tso Kng-mu, TC is effiious in meliorting lung fire, resolving phlegm, issipting stsis, etoxifying, n relesing het. Moreover, some previous stuies hve inite tht TC hs nti-inflmmtory tive ingreients, e.g., etulin n mslini i (Lin et l., 2009). It is with this in min tht the present stuy explore the effiy of severl ifferent TC extrts in the improvement of inflmmtion n lipi metolism in two ell types n investigte the possile moleulr mehnisms of their tion. 2. Mterils n Methos 2.1. Chemils n Regents Oil re O, 3-isoutyl-1-methylxnthine (IBMX), insulin from ovine pnres n lipopolyshrie (LPS) were purhse from Sigm (St. Louis, MO, USA). Thizolyl lue tetrzolium romie (MTT) n imethyl sulfoxie (DMSO) were purhse from Amerso (Cohrn, Ohio, USA). Dexmethsone (DEX) ws purhse from Alf Aesr (Heyshm, Lnshire, UK). Primry ntioies ginst PPARα, PPARγ, n iponetin were purhse from Thermo (Grn Isln, NY, USA). Primry ntioies ginst β-tin n were purhse from Genetex (Alton Prkwy, Irvine, USA) n primry ntioy ginst inos ws purhse from Novus (Southprk Wy, Littleton, USA). Seonry ntioies ginst got nti-mouse IgG n got ntirit IgG were purhse from Jkson Immunoreserh (West Grove, PA, USA). Mmmlin Protein Extrtion Regent for protein extrtion ws purhse from Thermo (Grn Isln, NY, USA). The Brfor regent for protein quntifition ws purhse from Bio Bsi In. (Mrkhm, Ontrio, Cn). The Totl RNA Extrtion Miniprep System kit for RNA extrtion ws purhse from Viogene (Tipei, Tiwn) Extrtion methos The TC ws groun to power, whih ws extrte with 95% ethnol followe y prtition extrtion. For the ethnol extrtion, TC power ws first soke in 95% ethnol for 3 ys. The ethnol ws remove y vuum onentrtion to proue onentrte, whih ws weighe n then egsse with uling nitrogen gs to remove the remining ethnol resiue. This proess generte n ethnol-free, onentrte extrt. One liquot of the extrt ws re-issolve with H 2 O n ple into prtition-extrtion flsk into whih ethyl ette n n-utnol were sequentilly e, in the orer of inresing solvent polrity, for prtition extrtion. The two resulting orgni extrts were onentrte y vuum onentrtion n referre to respetively s TCEAE, ompose of TC n ethyl ette, n TCBUE, ompose of TC n n-utnol. The remining, onentrte queous phse fter the extrtion ws referre to s TCWE n ws ompose of TC n wter. The other liquot of the forementione ethnol-free, onentrte extrt, whih ws not use in the prtition extrtion, ws set sie n referre to s TCEE. The orgni extrts TCEAE, TCBUE, n TCEE were re-issolve in solute ethnol for the genertion of stok solutions, wheres the TCWE extrt ws mixe with PBS (Phosphte-uffere sline) to proue fourth stok solution. All of the four stok solutions were store t -20 C prior to further use Cell Culture The mouse mrophge ell line RAW (BCRC No ) ws otine from the Bioresoures Colletion n Reserh Center (BCRC) of the Foo Inustry Reserh n Development Institute (Hsinhu, Tiwn) n routinely ulture in Duleo s Moifie Egle s Meium (DMEM; GIBCO- BRL, Grn Isln, NY, USA) supplemente with 10% fetl ovine serum (FBS; GIBCO-BRL) n ntiiotis (100 U/ml peniillin n 100 μg/ml streptomyin; GIBCO-BRL) t 37 C in humiifie tmosphere with 5% CO 2. The ells were suulture every 2 3 ys, n the meium ws hnge every 2 ys. To ssess the effets of the extrts on ell ytotoxiity, RAW264.7 ells were seee in 96- well plte t ells/well. After 24 h, the ells were trete with ifferent onentrtions of the four extrts or vehile, n the ytotoxiity of the extrts ws nlyze. The mouse firolst 3T3-L1 ells (BCRC No ) were lso otine from BCRC n mintine in DMEM ontining 10% lf serum (CS; GIBCO-BRL) n ntiiotis (100 U/ml peniillin n 100 μg/ml streptomyin; GIBCO-BRL). The ells were seee in 6-well plte t ells/well n grown to 100% onfluene. To inue ipoyti ifferentition, 2 ys fter rehing onfluene (Dy 0), the ells were expose to ulture meium ontining 0.5 mm 3-isoutyl-methylxnthine (IBMX), 1 μm DEX, n 1 μg/ml insulin (Bovine) (the MDI hormonl oktil) for 2 ys (Dy 2). This meium ws reple with fresh omplete meium ontining insulin with 10% FBS, n the ells were inute for further 2 ys (Dy 4). Therefter, until the ells were fully ifferentite, fresh meium with 10% FBS ws supplie every other y Mesurement of nitrite proution Nitrite oxie (NO) hs short hlf-life n is stle oxie me- 30

3 tolite; hene, the mesurement of nitrite ontent woul provie informtion regring NO proution. In this stuy, the TC ells were trypsinize, ilute to onentrtion of ells/ml with ulture meium, n then 100 μl of ell solution ws inoulte into 96-well plte. The pltes were ple in n inutor fille with 5% CO 2 t 37 C for 18 h, fter whih the initil ulture meium ws remove from the pltes, n they were then trete jointly with the smples n LPS n ple in n inutor (5% CO 2, 37 C) for 18 h. Susequently, 100 μl of the superntnt ws remove from eh well y pipette n trnsferre to ifferent 96-well plte. After the ition of Griess regent (100 μl), the resulting solution ws llowe to stn in the rk for 10 min efore the sorne of the solution t wvelength of 540 nm ws mesure with n ELISA plte reer. The solution s sorne ws ompre with n sorne lirtion urve to etermine its nitrite ontent Oil re O stin Oil re O is izo ye, non-polr n liposolule tht stins the ipose roplets in ipoytes to form re olor tht n e employe s metri to evlute ipose roplet ontent in ipoytes. In this stuy, 0.35% solution of oil re O power in isopropnol ws mixe with eionize wter in wter-to-stin rtio of 2:3 for use. After the ompletion of ell ifferentition t ifferent smple onentrtions in 12-well pltes, the pltes were rinse with PBS until the smple olor ws wshe off, following whih 300 μl of formlehye (10% solution in PBS) ws e to eh of the wells, llowe to stn for 1 h, n rinse with eionize wter y using pipettes until the formlehye oor h ompletely issipte. Susequently, 1 ml of 60% isopropnol ws e to eh well n llowe to stn for 2 min to ensure the full evportion of the wter. The oil re O stin ws e to the wells, left to stn for 30 min, n then rinse off with eionize wter. Wter ws e to over the ells n the ells were photogrphe, fter whih the wells were ir-rie. Then, 1 ml of 60% isopropnol ws e to eh well, n eh plte ws shken horizontlly on shker until the re ipoytes were issolve to form solution. The sorne of the solution ws mesure t the wvelength of 510 nm y n ELISA plte reer Protein extrtion The ell solutions were ilute to onentrtion of ells/ ml, inoulte in 6-m 2 ulture ishes, n ple in n inutor fille with 5% CO 2 t 37 C for 18 h. After removl of the ulture meium, ulture meium ontining LPS n the smples ws e to the ishes, whih were inute in the inutor for further 24 h, rinse with PBS, n then the ells were ollete on ie with sptul. The ollete ells were entrifuge t 4,500 rpm for 5 min t 4 C. After removl of the superntnt, 100 μl of ell lysis gent (M-PER ) ws e to the entrifuge tue, remove, n re-e severl times with syringe. Susequently, the tue ws entrifuge t 1,000 rpm for 15 min t 4 C. The superntnt ontine the originl ellulr proteins n ws store t -20 C prior to use Protein quntifition A solution of ovine serum lumin ws use to generte lirtion urve n portion of the ove extrte protein solution ws ilute with H 2 O in orer to ensure the protein onentrtion ws within the ynmi rnge of the lirtion urve. A 10- μl liquot ws trnsferre to 96-well plte mixe Brfor regent. The resulting mixture ws llowe to stn for 2-3 min t room temperture. Susequently, the sorne of the solution ws mesure t wvelength of 595 nm n use to evlute the protein onentrtion se on the pre-etermine lirtion urve Western lotting Proteins were resolve y SDS-polyrylmie gel eletrophoresis n then trnsferre to polyvinyl ifluorie memrnes. The lot memrnes were loke with 4% non-ft milk for 1 h t room temperture, followe y inution with primry ntioies t 4 C overnight. After wshing three times, the lots were inute with nti-rit or nti-mouse HRP-onjugte seonry ntioies for 1 h t room temperture. Finlly, the lots were visulize y enhne hemiluminesene using Fujifilm LAS-3000 hemiluminesene etetion system (Fujifilm; Tokyo, Jpn) Quntifition softwre AlphEseFC 4.0 imging softwre ws use to nlyze the stining intensities of the western lots. The intensity of kgroun stining ws sutrte n the intensity of the trget protein n ws ivie y the intensity of the ontrol n to proue the quntittive t Sttistil nlysis Eh experimentl t set ws expresse s the men ± stnr evition (men ± SD). When the t were onfirme to follow norml istriution, the t sets were sujete to one-wy ANOVA n Dunn s Multiple Rnge Test to etermine whether there ws sttistilly signifint ifferene etween two inepenent smple groups (P < 0.05). All sttistil nlyses were onute with Sttistil Prout n Servie Solutions (SPSS) softwre version Results 3.1. The effet of the TC extrts on the survivl rte n nitrite proution in the RAW264.7 ells RAW264.7 mrophges were trete with 100 ng/ml of LPS to inue inflmmtory retions n then trete with TC extrts. As shown in Tle 1, uner onitions tht i not ffet the ellulr survivl rte, this stuy nlyze the proue ontent of nitrite, n inflmmtory meitor. It ws foun tht 150 μg/ml of TCEE, 150 μg/ml of TCEAE, 250 μg/ml of TCBUE, n 400 μg/ ml of TCWE ll showe signifint inhiitory effet on nitrite proution The effet of the TC extrts on the inflmmtion-relte protein expression in the RAW264.7 ells As shown in Fig. 2, TCEE n TCEAE signifintly reue the expression level of inos protein, showing mrkely greter inhiition thn the positive ontrol group (DEX) (P < 0.05). The 31

4 Tle 1 The effets of the TC extrts on the nitrite proution n ell survivl rte in the RAW264.7 ells. Tretment Cell viility (%) Nitrite proution (μm) Vehile LPS TCEE 150 μg/ml ± 0.41 h ± 416f ± ± ± ± 1.24 LPS Vehile Nitrite / Cell viility ± ± 0.26 e ± ± 0.26 LPS TCEAE 150 μg/ml ± ± 0.26 LPS TCBUE 250 μg/ml ± 1.21e 5.71 ± ± 4.12 e ± 3.92e ± 2.09 LPS TCWE 400 μg/ml LPS Dexmethsone 1 μm ± ± ± 0.46 e 5.41 ± 0.20 Vlues re the men ± SD (n = 5) n were nlyze using one-wy ANOVA followe y Dunn s new multiple rnge test. Dexmethsone (1 μm) is the positive ontrol n ells were trete with 100 ng/ml LPS to inue inflmmtion. inos v- v ex EE EAE BUE WE - PPARγ β-tin LPS 5.0 Relte protein expression inhiitory effiies of TCBUE n TCWE were lower thn those of TCEE or TCEAE, ut were similr to the positive ontrol group (P > 0.05). This stuy thus ientifie tht, of the ifferent TC extrts, TCEE n TCEAE exerte the strongest inhiitory effet on the expression of inos protein. TCEE, TCEAE, n TCBUE signifintly inrese the expression level of the PPARγ protein. These inreses were signifintly ifferent from wht ws oserve in the lnk group, the positive ontrol group, n the negtive ontrol group (P < 0.05), while TCWE h no signifint effet on the expression. This stuy foun tht, of the ifferent TC extrts, TCEAE n TCBUE were le to signifintly inrese PPARγ protein expression. TCEE n TCEAE oth signifintly reue the protein expression level of the pro-inflmmtory ytokine, with the oserve inhiition eing greter thn tht seen in the positive ontrol group. Of the TC extrts, TCEAE ws most effetive inhiitor while TCBUE n TCWE showe the lest effetive inhiition. Thus, this stuy foun tht, of the ifferent TC extrts, TCEE n TCEAE exerte the strongest inhiitory effet on the expression of protein. 4.0 vehilevehile ex methsone 1 μm TCEE 150 μg/ml TCEAE 150 μg/ml TCBUE 250 μg/ml TCWE 400 μg/ml inos PPARγ Fig. 2 - The effets of TCEE, TCEAE, TCBUE, n TCWE on the inflmmtory-relte protein expression in the RAW264.7 mrophges. Vlues re the men ± SD (n = 3) n were nlyze using one-wy ANOVA followe y Dunn s new multiple rnge test. Brs not shring ommon letter re signifintly ifferent from eh other (P < 0.05). ifferent TC extrts, TCEAE n TCBUE exerte the strongest effets on the promotion of iponetin. TCEE, TCEAE, n TCBUE signifintly inrese the expression level of the PPARα protein. TCEAE n TCBUE were the most effiient n signifintly ifferent from the ifferentition group n the vehile group (P < 0.05), wheres TCWE proue no signifint effet on the PPARα protein expression. This stuy foun tht, of the ifferent TC extrts, TCEAE h the most signifint effet on inresing the expression level of the PPARα protein. TCEE n TCEAE inhiite protein expression, ut this inhiition ws not signifintly ifferent from the ifferentition group (P > 0.05). Menwhile, the inhiitory effets of TCBUE n TCWE were even less signifint. TCEE n TCEAE h inhiition rtes of 16% n 23% ompre with those in the ifferentition group, respetively. The present stuy foun tht, of the ifferent TC extrts, TCEE n TCEAE ex The effet of the TC extrts on the expression of lipi metolism-relte proteins in the 3T3- L1 ells As shown in Fig. 3, TCEAE n TCBUE promote the expression of iponetin, n it==they were not signifintly ifferent from the level in the ifferentition group (P > 0.05). However, TCEE n TCWE reue the expression level of iponetin, with them not eing signifintly ifferent from tht of the vehile group (P > 0.05). TCEAE n TCBUE promote the level of iponetin protein expression to 128% n 141% of tht in the ifferentition group, respetively. This stuy foun tht of the 4.5 Fig. 1 - An experimentl smple of TC. 32

5 v ex EE EAE BUE WE Aiponetin PPARα β-tin Aiponetin TCEE 150 μg/ml TCEAE 200 μg/ml TCBUE 500 μg/ml TCWE 2000 μg/ml Differentite ontrol ifferentite vehile TCEE 150 μg/ml TCEAE 200 μg/ml TCBUE 500 μg/ml TCWE 2000 μg/ml PPARα Lipi eumultion (%) Relte protein expression 2.0 Unifferentite Fig. 3 - The effets of TCEE, TCEAE, TCBUE, n TCWE on the ipogeni-relte protein expression in the 3T3L1 ipoytes. Vlues re the men ± SD (n = 3) n were nlyze using one-wy ANOVA followe y Dunn s new multiple rnge test. Brs not shring ommon letter re signifintly ifferent from eh other (P < 0.05) e it t en fer if Un erte the strongest inhiitory effet on the protein expression of pro-inflmmtory ytokine. f Di te nti e fer ol ntr o EE TC l g/m μ 50 1 E T A CE l g/m μ 00 2 E T U CB l g/m μ 00 5 E T CW l g/m μ Fig. 4 - The effets of TCEE, TCEAE, TCBUE, n TCWE on the lipi umultion in the 3T3-L1 ipoytes. Vlues re the men ± SD (n = 3) n were nlyze using one-wy ANOVA followe y Dunn s new multiple rnge test. Brs not shring ommon letter re signifintly ifferent from eh other (P < 0.05) The effet of the TC extrts on the lipi genertion in the 3T3-L1 ells As shown in Fig. 4, 5 ys fter ell ifferentition, 3T3-L1 preipoytes were trete with TCEE, TCEAE, TCBUE, or TCWE t ifferent onentrtions for 72 h. The results of the MTT ssy suggeste tht no ytotoxiity ws oserve t 150 μg/ml TCEE, 200 μg/ml TCEAE, 500 μg/ml TCBUE, or 2000 μg/ml TCWE. Therefore, these onentrtions of TC extrts were use to tret the ells in the susequent experiments n oserve the effets on the genertion of ipose roplets. Lipi ifferentition ws inue y ifferentition gent in 3T3-L1 pre-ipoytes n then the pre- ipoytes were trete with the TC extrts or their tive ingreients. The genertion of ipose roplets ws oserve uner onitions tht i not ffet the ellulr survivl rte. The results showe tht 200 μg/ml TCEAE exerte signifint inhiitory effet on the proution of ipose roplets (P < 0.05), with n inhiition rte of 29.3%; no other TC extrts resulte in sttistilly signifint effet. Of the tive ingreients, 100 μm etulin signifintly inhiite the genertion of ipose roplets (P < 0.05), with n inhiitory rte of 35.3%. 4. Disussion mtory ytokines (Cooper et l., 2010), n this exertes inflmmtion n les to sustntil NO proution. Through metoli proesses, NO n e onverte to nitrite, whih plys pivotl role in the ourse of inflmmtion. Nitrite n inue the proution of other inflmmtory sustnes, ll of whih ooperte to inue inflmmtory retions in the oy. Stuies hve suggeste tht phenoli ompouns, flvonoi ompouns, n triterpenois my inhiit LPS-inue inflmmtory retions in RAW264.7 murine mrophges (Chen et l., 2011). The experimentl results of this stuy inite tht TCEE, TCEAE, n TCBUE h signifint inhiitory effets on nitrite proution; in prtiulr, TCEE h the most signifint effet. At onentrtion of 100 μg/ml, the inhiitory effet ws stisftory. However, when the onentrtion ws inrese to 150 μg/ml, the inhiitory effet on nitrite proution ws similr to tht of DEX, n estlishe nti-inflmmtory rug. These results suggest tht the nti-inflmmtory mehnism of TC might our through the L-rginine- NO pthwy The effet of the TC extrts on the inflmmtory meitor proution in the RAW264.7 ells 4.2. The effet of the TC extrts on the inflmmtion-relte protein expression in the RAW264.7 ells After the stimultion of mrophges y LPS, mrophgeinue inflmmtion will use the etyltion of pro-inflm- Both inos n re importnt proteins in the ourse of inflmmtion. NOS (Nitri oxie synthse) n e lssifie into 33

6 Mrophge Infiltrtion (Pn et l., 2009). The results of this present stuy inite tht TCEAE h the est inhiitory effet on lipi genertion, ft whih ompelle us to speulte tht the tive ingreients of triterpenois n sterois, suh s etulin n mslini i, enle TCEAE to regulte the lipi metolism n erese lipi proution. However, stuy involving further purifition, isoltion, n nlysis is neee in orer to ientify n onfirm these tive ingreients. inos Nitrite PPARα PPARγ Tretment LPS-inue inflmmtion Aiponetin Aipoytes Leptin COX-2 NF-κB 4.4. The effets of the TC extrts on the expression of lipi metolism-relte proteins in the 3T3- L1 ells Prrine loop Aiponetin, n ipoytokine tht promotes insulin sensitivity, is serete only y ifferentite ipose tissues, n three types of seretions n our: high, meium, n low moleulr weight (Wki et l., 2003; Tso et l., 2003). Of the three types, high-moleulr-weight iponetin hs the highest metoli tivity, promoting insulin sensitivity n therey lleviting the symptoms of ietes mellitus (Pjvni et l., 2004; Wng et l., 2008; Ymuhi et l., 2013). Also, its loo onentrtion is lower in oese iniviuls thn in other iniviuls (Arit et l., 1999). is pro-inflmmtory protein, ut s 30% of in the oy is serete y ipoytes, it is lso n ipokine. The mehnism y whih influenes insulin sensitivity is thought to inlue the following steps: is proue y peritonel ft n n iretly enter n stimulte the liver to use triglyerie seretion (Nonogki et l., 1995), ffet insulin signl trnsution, n influene insulin-inue glyogen proution in heptoytes (Senn et l., 2002), the ltter of whih therey reues insulin sensitivity n les to hyperglyemi (Tsigos et l., 1997). Mny isruptions in the regultion of lipi metolism elong to the ownstrem genes of PPARα, e.g., lipoproteins on n in the vsulr enothelium, n polipoprotein A-I (ApoA-I) n polipoprotein A- II (ApoA-II) on the lipoprotein lipse. PPARα n promote lipoprotein lipse expression, using lipoproteins to relese ftty is, helping them to enter tissues, n therey filitting their entrne into heptoytes. Furthermore, PPARα ws shown to regulte the expression of ipogeni-relte genes n promote lipolysis in heptoytes, reuing the ftty is use for triglyerie proution in the liver n thus eresing the lipi onentrtion in loo (Shoonjns et l., 1996). In the present stuy, TCEAE n TCBUE inrese the levels of iponetin, wheres TCEE n TCEAE inhiite the expression of the protein; furthermore, TCEAE h signifint effet on the tivtion of the PPARα protein expression. Therefore, we speulte tht the nti-lipi umultion effet of TCEAE my hve een hieve vi the tivtion of PPARα n the susequent inrese in iponetin expression, whih in turn promote lipi metolism to reue triglyerie proution n enhne insulin sensitivity. Thus, the expression of ws inhiite n the regultion of lipi metolism ws hieve. Oesity-relte hroni inflmmtion prtilly rises from inrese mrophge infiltrtion to ipose tissue. Those mrophges n relese pro-inflmmtory ftors tht influene jent ipoytes vi prrine n inue insulin resistne (Grimle, 2002, Cinti et l., 2005, Hirsk et l., 2007). The present stuy foun tht the TCEE n TCEAE extrts h the strongest inhiitory effet on lipi proution, n we speulte tht otulin, mslini i, n stigmsterol, the tive ingreients of triterpenois n sterois in TC, enle TC to regulte insulin resistne. Pro-inflmmtory ytokines n inrese proution, reue iponetin seretion, n influene insulin Mrophges Toreni onolor Linley vr. formosn Ymzki Fig. 5 - A suggeste mehnism of TC for nti-inflmmtion n nti-lipi umultion. three types: neuronl nitri oxie synthse (nnos), enothelil nitri oxie (enos), n inuile nitri oxie synthse (inos). inos exists mostly in mrophges, lymphoytes, n vsulr smooth musle ells. When those ells re stimulte, high level of inos ours, leing to high proution of NO (Nthn n Xie, 1994; Thiemermnn, 1994)., menwhile, exerts pivotl role in the regultion of mny immune funtions, suh s mrophge tivtion, B-ell evelopment, inflmmtory retions, hemtopoieti funtions, n ute retions. Therefore, n exess of is usully ssoite with some iseses tht rise from immune overretion, suh s rheumtoi rthritis n Crohn s isese. is type of pro-inflmmtory ytokines, whih plys pivotl role in ute inflmmtory retions n inreses vsulr permeility to eliit inflmmtory retions with symptoms suh s re swelling, het, n pin. It is the min regultor of ontinul stimultion n tivtion of mrophges for the seretion of more pro-inflmmtory ytokines (Nthn, 1987). Ativte PPARγ n inhiit the expression of the nuler trnsription ftor NF-κB in the mrophges, reue the tivity of inos, n inhiit the proution of (Peng et l., 2005; Jing et l., 1998). The TC extrts (TCEE, TCEAE, n TCBUE) ll signifintly inhiite the expression of the inos protein n susequently inhiite the proution of nitrite; similrly, they h signifint inhiitory effet on the expression of n ll signifintly tivte PPARγ protein expression. Given ll the ove finings n reports, we n speulte tht TCEE, TCEAE, n TCBUE showe nti-inflmmtory effiy y inhiiting the expression of inos n the protein through the signling pthwys of tivte trnsription ftor PPARγ The effet of the TC extrts on the lipi genertion in the 3T3-L1 ells 3T3-L1 pre-ipoytes egin to umulte lipis fter ifferentition inue y IBMX. Stuies hve suggeste tht mslini i, n tive triterpenoi in TC, oul tivte the Akt/PKB signling pthwy to promote insulin sensitivity n reue lipi umultion, s shown in experiments with 3T3- L1 ipoytes (Cstellno et l., 2013). However, some stuies hve foun tht stigmsterol, n tive ingreient in sterois, oul prevent ovrin, prostte, rest, n olon ners, n lso exert the potent nutreutil effets of ntioxition n loo gluose erese 34

7 sensitivity, n these ftors ll rete onitions suite to the evelopment of metoli synrome. The regultion of inflmmtory ftors (e.g., NF-κB, TNF-α,, COX-2, n inos) n ipogeni relte ftors (e.g., IL-1β, MCP- 1,, CRP, PAI-1, PPARα n iponetin) n le to the suppression of inflmmtory retions (Hsu et l., 2013). In ition to the inution of inflmmtory ftors in RAW264.7 mrophges, LPS lso inues inflmmtory ftors in 3T3-L1 ipoytes, e.g., the tivtion of the NF-κB trnsution pthwy n the proinflmmtory ytokines (e.g.,, TNF-α n IL-10) to ffet insulin sensitivity (Chirumolo et l., 2014). TCEAE inhiite proution, ut it lso sustntilly tivte the expression of the PPARα protein. PPARs re regre s therpeuti trgets for riovsulr isese: the tivtion of these not only iretly regultes the genes of vsulr n inflmmtory ells involve in theroslerosis, ut lso iniretly promotes gluose utiliztion n serum lipi profiles. Prior reserh in our lortory foun tht TC extrts oul tivte PPARα n PPARγ (t not shown). Therefore, we speulte tht TCEAE oul improve mrophge infiltrtion-inue insulin resistne through the tivtion of PPARα to reue the proution of pro-inflmmtory ftors in the mrophges, inhiit the genertion of pro-inflmmtory ytokine, tivte PPARα n inrese the expression of iponetin, whih ll oul le to the regultion of lipi metolism n the meliortion of insulin sensitivity. 5. Conlusions TCEE, TCEAE, n TCBUE inhiite the proution of nitrite (n inflmmtory meitor) vi the inhiition of inos protein expression in RAW264.7 ells n inhiite the expression of proinflmmtory ytokine vi the tivtion of trnsription ftor PPARγ n the inrese in its expression, these three extrts emonstrte goo nti-inflmmtion effiy. TCEAE reue the proution of ipose roplets vi n inrese in iponetin n n tivtion of PPARα protein expression in 3T3-L1 ells, therey emonstrting goo pity to llevite lipi umultion. This stuy emonstrte the potentil of TC in the evelopment of nutreutil foos to prevent inflmmtion n oesity. In the future, olumn hromtogrphy my e employe to ientify the omposition of TCEAE; from this, the tive ingreients responsile for the nti-inflmmtory n nti-lipi umulting effets my e isolte n purifie for further niml experiments in whih the nutreutil effets of the tive ingreients n their moleulr mehnism oul e even further explore. Aknowlegments This work ws supporte y reserh grnt (MOST B ) from the Ministry of Siene n Tehnology, Tipei, Tiwn n Chin Meil University uner the Aim for the Top University Pln of the Ministry of Eution, Tiwn, n the Deprtment of Helth Clinil Tril n Reserh Center for Exellene (DOH102-TD-C ), Tiwn. Conflits of Interest Sttement The uthors elre no onflits of interest for this work. Open Aess This rtile is istriute uner terms of the Cretive Commons Attriution Liense whih permits ny use, istriution, n reproution in ny meium, provie originl uthor(s) n soure re reite. REFERENCES [1] Arit Y, Kihr S, Ouhi N, Tkhshi M, Me K, Miygw J, et l. 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