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Anti-Inflammatory Anti-oxidative Stress Activities

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Anti-inflammatory Anti-oxidative Stress Activities
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  Research Article Anti-in 󿬂 ammatory/Anti-oxidative Stress Activities and Differential Regulationof Nrf2-Mediated Genes by Non-Polar Fractions of Tea  Chrysanthemum zawadskii   and Licorice  Glycyrrhiza uralensis Tien-Yuan Wu, 1 Tin Oo Khor, 2 Constance Lay Lay Saw, 2 Stephanie C. Loh, 3 Alvin I. Chen, 3 Soon Sung Lim, 4 Jung Han Yoon Park, 4 Li Cai, 3 and Ah-Ng Tony Kong 1,2,5 Received 28 June 2010; accepted 11 October 2010; published online 22 October 2010  Abstract.  Accumulating evidence from epidemiological studies indicates that chronic in 󿬂 ammation andoxidative stress play critical roles in neoplastic development. The aim of this study was to investigate theanti-in 󿬂 ammatory, anti-oxidative stress activities, and differential regulation of Nrf2-mediated genes bytea  Chrysanthemum zawadskii  (CZ) and licorice  Glycyrrhiza uralensis  (LE) extracts. The anti-in 󿬂 ammatory and anti-oxidative stress activities of hexane/ethanol extracts of CZ and LE wereinvestigated using  in vitro  and  in vivo  approaches, including quantitative real-time PCR (qPCR) andmicroarray. Additionally, the role of the transcriptional factor Nrf2 (nuclear erythroid-related factor 2)signaling pathways was examined. Our results show that CZ and LE extracts exhibited potent anti-in 󿬂 ammatory activities by suppressing the mRNA and protein expression levels of pro-in 󿬂 ammatorybiomarkers IL-1 β , IL-6, COX-2 and iNOS in LPS-stimulated murine RAW 264.7 macrophage cells. CZand LE also signi 󿬁 cantly suppressed the NO production of LPS-stimulated RAW 264.7 cells. Addition-ally, CZ and LE suppressed the NF- κ B luciferase activity in human HT-29 colon cancer cells. Bothextracts also showed strong Nrf2-mediated antioxidant/Phase II detoxifying enzymes induction. CZ andLE induced NQO1, Nrf2, and UGT and antioxidant response element (ARE)-luciferase activity inhuman hepatoma HepG2 C8 cells. Using Nrf2 knockout [Nrf2 ( − / − )] and Nrf2 wild-type (+/+) mice, LEand CZ showed Nrf2-dependent transactivation of Nrf2-mediated antioxidant and phase II detoxifyinggenes. In summary, CZ and LE possess strong inhibitory effects against NF- κ B-mediated in 󿬂 ammatory aswell as strong activation of the Nrf2-ARE-anti-oxidative stress signaling pathways, which wouldcontribute to their overall health promoting pharmacological effects against diseases including cancer. KEY WORDS:  anti-in 󿬂 ammatory; anti-oxidative stress; chrysanthemum; licorice; Nrf2; phase II drugmetabolizing/detoxifying enzymes. INTRODUCTION Normal in 󿬂 ammation in general is a process involvinginteractions between pro-in 󿬂 ammatory and anti-in 󿬂 ammatorysignaling pathways. In 󿬂 ammation occurs when the tissue isinjured or infected by external challenges (1). In this context,generally acute in 󿬂 ammation is self-limiting and recovered byitself(1).However,chronicin 󿬂 ammationcouldincreasetheriskofdevelopingdiseasessuchascancerinthein 󿬂 amedtissues(2).In Eastern Asia, the use of plants including roots andfruits as herbal medicines is common. Licorice and teachrysanthemum are two popular herbal medicines used totreat various in 󿬂 ammatory diseases. Licorice ( Glycyrrhiza )species have been used in Europe as herbal medicines forcenturies as well. Licorice root is used for the treatment of gastric or duodenal ulcers, hepatitis, sore throats, coughs,bronchitis, arthritis, allergies, and cardiovascular disease(1,3). There are numerous dietary supplements stomach ulcers, bronchitis, and sore throat, as well as infections causedby viruses, such as hepatitis but have not been approved bythe Food and Drug Administration (FDA; National Centerfor Complementary and Alternative Medicines (NCCAM;http://nccam.nih.gov/health/licoriceroot/)). Licorice, includingglycyrrhizin, isoliquiritigenin, liquiritigenin, licochalcone Aand B, and  β -glycyhrritinic acid (4 – 6), have been shown topossess anti-in 󿬂 ammatory activities and glycyrrhizin caninhibit reactive oxygen species (ROS). Licorice has also beenshown to inhibit the expression of COX-2 and other pro-in 󿬂 ammatory proteins (3,7,8). There are currently (as of 09/ 29/2010) 12 on-going, completed or terminated clinical trials 1 Graduate Program in Pharmaceutical Science, Department of Pharmaceutics, Ernest Mario School of Pharmacy, Rutgers, TheState University of New Jersey, Piscataway, New Jersey, USA. 2 Department of Pharmaceutics, Ernest Mario School of Pharmacy,Rutgers, The State University of New Jersey, Room 228, 160Frelinghuysen Road, Piscataway, New Jersey 08854, USA. 3 Department of Biomedical Engineering, Rutgers, The State Uni-versity of New Jersey, Piscataway, New Jersey, USA. 4 Department of Food Science and Nutrition, Hallym University,Chuncheon, South Korea. 5 To whom correspondence should be addressed. (e-mail: kongt@rci.rutgers.edu) The AAPS Journal, Vol. 13, No. 1, March 2011 (  #  2010) DOI: 10.1208/s12248-010-9239-4 1  1550-7416/11/0100-0001/0 # 2010 American Association of Pharmaceutical Scientists  on  “ licorice or licorice-related ”  dietary supplement listed onClinicaltrial.gov (http://clinicaltrials.gov). Chrysanthemumwhich includes  󿬁 ve germacrane-type sesquiterpenes, kikka-nols D, D monoacetate, E, F, and F monoacetate, wereisolated from the ethyl acetate-soluble portion and two 󿬂 avanone glycosides, (2 S )- and (2 R )-eriodictyol 7- O -beta- D -glucopyranosiduronic acids, and a phenylbutanoid glycoside,(2 S , 3 S )-1-phenyl-2,3-butanediol 3- O -beta- D -glucopyranoside,isolated from the  󿬂 owers of   Chrysanthemum , has been usedto treat vertigo, hypertension, bacterial and viral infectiousdiseases (9 – 12). Botanical chrysanthemum tea or extract areavailable as dietary supplements and promoted as healthenhancing products. Extract of chrysanthemum has beenshown to possess strong anti-oxidative stress, anti-in 󿬂 amma-tory effects and previous studies revealed that differentextraction methods yielding different soluble fraction of extracts would possess different effects of anti-in 󿬂 ammationand immunomodulation (9,13). A completed clinical trial of  chrysanthemum extract as dietary supplement to lower serumLDL cholesterol and raising HDL cholesterol is listed onClinicaltrial.gov (http://clinicaltrials.gov).Despite these diverse potential health bene 󿬁 cial andpharmacological effects of licorice and chrysanthemum, themolecular and the signaling mechanisms leading to thesebiological effects are still unclear. We theorized that the non-polar fractions of licorice and chrysanthemum, may possessanti-in 󿬂 ammatory and anti-oxidative stress properties thatcould be further developed for diseases prevention includingcancer chemoprevention. In this study, we aim to investigatethe transcription regulation of LE and CZ on the transcrip-tional factor nuclear erythroid-related factor 2 (Nrf2) signal-ing pathway that controls the expression of many anti-oxidative stress and phase II drug metabolizing (DM)/detoxifying enzymes, which are typically elicited by manychemopreventive compounds (14 – 16).Nrf2 plays an important role to mediate phase II detoxify-ing/antioxidant enzymes expression. Under normal conditions,Nrf2 appears to be associated with actin-binding Keap 1 thatforms Nrf2-Keap1 complex preventing Nrf2 from entering intothe nuclear and promoting its proteasomal degradation. Typi-cally, the half-life of Nrf2 in un-stimulated mammalian cells is15 – 45 min. Upon treatments of the cells with oxidants such asH 2 O 2 , oxidative stress or cancer chemopreventive compounds,conformationalchangesoccurduetooxidationofthiol-sensitiveamino acids present in the Nrf2-Keap 1 complex and woulddrivethedissociationofNrf2from Keap1,therebyallowingthetranslocation of Nrf2 into the nucleus, Nrf2 binds to theantioxidant response element (ARE) of ARE-target genesandleadstoenhancedphaseIIdetoxifying/antioxidantenzymesexpression (17,18). Therefore, in the current study, we utilized theNrf2-de 󿬁 cientmice(Nrf2 − / − ;KO)andNrf2wild-type(Nrf2+/+; WT) mice to examine whether the  in vivo  phase II DM/detoxifying/anti-oxidative/properties elicited by the extractswould be mediated by Nrf2. MATERIALS AND METHODSPlant Extracts Whole plants of   Chrysanthemum zawadskii  (CZ) Her-bich var.  latilobum  (Maxim.) Kitamura and licorice roots,derived from  Glycyrrhiza uralensis  (LE) Fisch. were pur-chased from a local drug store (Dea Guang Medical,Chunchon, South Korea) and identi 󿬁 ed by Emeritus Profes-sor Hyung Jun Ji (Seoul National University, Seoul, Korea).Dried and ground  C. zawadskii  (5 kg) (CZ) and roots of   G.uralensis  (5 kg) (LE) were dip-extracted with hexane:ethanol(70 L) at a ratio 9:1 ( v / v ) at room temperature for 24 h. Theslurry was then  󿬁 ltered through  󿬁 lter paper and the residuewas re-extracted twice. The combined extracts were  󿬁 ltered,and the  󿬁 ltrates were concentrated under reduced pressure at40°C to yield the hexane/ethanol extract of CZ (412 g, 0.82%yield) and LE (455 g, 0.91% yield). Cell Culture and Treatment The murine RAW 264.7 macrophage cells, a well-established model system for many in 󿬂 ammatory studies aswell as the luciferase reporter assay of nuclear factor kappa-light chain enhancer of B cells (NF κ B) stabilized in humancolon cancer cells HT-29 (HT-29-N9) were used to investigatethe anti-in 󿬂 ammatory effects of licorice and chrysanthemumextracts (19). Similarly, the Nrf2-mediated ARE luciferaseassay stabilized in human hepatoma HepG2 cell (HepG2-C8)was used to investigate the potential of the extracts inactivating the Nrf2/ARE signaling pathway (20). Mousemacrophage cell line RAW 246.7, was obtained fromAmerican Type Culture Collection. HepG2-C8 and HT-29-N9 cells were generated in our laboratory as describedpreviously (21 – 24). All cells were cultured in Dulbecco ’ sModi 󿬁 ed Eagle ’ s Medium (Invitrogen Corp., Carlsbad, CA,USA) supplemented with 10% ( V  / V  ) FBS (Lifeblood Med-ical, Inc), penicillin 100 U/ml, and streptomycin 100  μ g/ml.Cells were maintained in a humidi 󿬁 ed incubator with 5%CO 2  at 37°C. The cells were treated with LPS (1  μ g/ml,Sigma, St. Louis, MO, USA) alone or pretreated 1 h with LE25  μ g/ml, CZ 25  μ g/ml, or curcumin (CUR) 10  μ g/ml (aspositive control) dissolved in DMSO before they werechallenged with LPS (19,23) Nitrite Assay The culture medium of the cells treated with differentcompounds was mixed with a Griess reagent in an equalvolume of 0.1% (1 mg/ml)  N  -(1-naphthyl)ethylenediaminedihydrochloride in deionized water and 1.0% (10 mg/ml)sulfanilic acid in 5% phosphoric acid solution. The mixedsample was incubated at 37°C for 30 min. Absorbance at548 nm was measured and concentrations were calculatedusing a sodium nitrite standard curve. RNA Isolation and Reverse Transcription Polymerase ChainReaction Analysis The RAW 246.7 cells were cultured in six-well plates andwere challenged by LPS 1  μ g/ml with or without pretreatmentwith LE, CZ, or CUR for 8 h at the 37°C incubator. TotalRNA was isolated by TRIZOL® according to the manufac-turer ’ s protocol (Invitrogen Corp., Carlsbad, CA, USA).First-strand cDNA was synthesized from 5  μ g of total RNAusing SuperScript III First-strand Reverse Transcriptase(Invitrogen Corp. Carlsbad, CA, USA) and oligo dT primers 2 Wu  et al  .  according to the manufacturer ’ s instructions. After reversetranscription, the polymerase chain reaction (PCR) reactionswere performed by using 1  μ l of reverse transcriptionproduct, 1  μ l of primer mixture ( 󿬁 nal concentration,10  μ mol/L), and 8  μ l of Platinum®  Taq  DNA Polymerasekit (Invitrogen Corp. Carlsbad, CA, USA), and performedwith initial denaturation at 94°C for 2 min, 25 cycles of ampli 󿬁 cation, and extension at 72°C for 10 min. PCRproducts were fractionated on 1.5% agarose gel. The primersused in this experiment are shown in Table I. Western Blotting The RAW 246.7 cells were challenged by LPS 1  μ g/mlwith or without pretreatment with LE, CZ, or CUR. After24 h, the cells were washed with ice-cold phosphate buffersaline (PBS) (pH 7.4), and scraped into microcentrifugetubes and pelleted. Cells were resuspended and lysed inRIPA buffer (Sigma, St. Louis, MO). 20  μ g protein perlane was loaded onto 4 – 15% SDS-PAGE (Bio-Rad Labo-ratories, Hercules, CA). After separation by SDS-PAGE,the protein was transferred onto nitrocellulose membrane(Millipore Corp., Billerica, MA, USA), and then wasblocked in 5% bovine serum albumin (BSA; FisherScienti 󿬁 c, Fair Law, NJ, USA) in tris – buffer saline tween-20 (TBST) solution for 1 h. Membranes were probed byrespective antibodies including  β -actin, COX2, cPLA 2 , andiNOS (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA)overnight at 4°C. Blots were washed with TBST solution15 min for four times and incubated with respectivesecondary antibodies for 1 h. After washing 15 min forfour times with TBST solution, the immunoreactive bandswere determined by adding SuperSignal West Femto mix(1:1 mix of stable peroxide buffer and luminol/enhancersolution, Thermo Scienti 󿬁 c, Rockford, IL) to detectimmunoreactive bands which were then visualized andquanti 󿬁 ed by Bio-Rad ChemiDoc XRS system (Hercules, CA). Enzyme-Linked Immunosorbent Assay The RAW 264.7 cells were cultured in 96-well plate with200  μ l medium. IL-6 and IL-1 β  enzyme-linked immunosorb-ent assay (ELISA) assay kits were purchased from InvitrogenCorporation, Carlsbad, CA, USAThe assays were performedaccording to the manufacturer ’ s instructions. For the ELISAassay, 50  μ l of incubation buffer was  󿬁 rst added to all thewells. After adding incubation buffer, 50  μ l standard diluentbuffer and 50  μ l of standards, controls, or samples were addedto each well in a stepwise fashion. Luciferase Reporter Assay The NF- κ B- and ARE luciferase activities were measuredusing a luciferase reporter assay system according to themanufacturer ’ s instructions (Promega, Madison, WI, USA).Brie 󿬂 y, after treatments, the cells were washed with ice-coldPBS andharvested in reporter lysis buffer. After centrifugation,10  μ l of the supernatants were mixed with 50  μ l of luciferaseassay substrate and measured for luciferase activity by using aSirius Luminometer (Berthold Detection Systems GmbH D-75173 Pforzheim, Germany). The luciferase activity was nor-malized against known protein concentrations and expressed asfold induction of luciferase activity over the control cells, whichwere treated with 0.1% DMSO. The protein level wasdetermined by Bio-Rad protein assay according to the manu-facturer ’ s instructions as we have described previously (22,25). Quantitative Real-Time PCR Assays The HepG2-C8 cells were cultured in six-well platesand were treated with respective extracts for 8 h at 37°C Table I.  Murine Primers for PCRGene Forward ReverseGAPDH 5 ′ -TGC TCG AGA TGT CAT GAA GG-3 ′  5 ′ -TTG CGC TCA TCG TAG GCT TT-3 ′ IL-1 β  5 ′ -GAG TGT GGATCC CAA GCA AT-3 ′  5 ′ -CTC AGT GCA GGC TAT GAC CA-3 ′ IL-6 5 ′ -AGT TGC CTT CTT GGG ACT GA-3 ′  5 ′ -GCC ACT CCT TCT GTG ACT CC-3 ′ TNF- α  5 ′ -ACG GCA TGG ATC TCA AAG AC-3 ′  5 ′ -GGT CAC TGT CCC AGC TT-3 ′ iNOS 5 ′ -GTG GTG ACA AGC ACA TTT GG-3 ′  5 ′ -GGC TGG ACT TTT CAC TCT GC-3 ′ COX-2 5 ′ -TCC TCC TGG AAC ATG GAC TC-3 ′  5 ′ -TGA TGG TGG CTG TTT TGG TA-3 ′ Nrf-2 5 ′ -AGC AGG ACA TGG AGC AAG TT-3 ′  5 ′ -TTC TTT TTC CAG CGA GGA GA-3 ′ UGT1A1 5 ′ -GTG GCC CAG TAC CTG ACT GT-3 ′  5 ′ -CGA TGG TCT AGT TCC GGT GT-3 ′ NQO-1 5 ′ -CAG ATC CTG GAA GGATGG AA-3 ′  5 ′ -AAG TTA GTC CCT CGG CCATT-3 ′ Table II.  Human Primers for Quantitative Real-Time PCRGene Forward ReverseGAPDH 5 ′ -TCG ACA GTC AGC CGC ATC TTC TTT-3 ′  5 ′ -ACC AAATCC GTT GAC TCC GAC CTT-3 ′ UGT1A1 5 ′ -TAA GTG GCT ACC CCA AAA CG-3 ′  5 ′ -TCT TGG ATT TGT GGG CTT TC-3 ′ NQO-1 5 ′ -CTG GAG TGT GCC CAATGC TA-3 ′  5 ′ -CAT GAA TGT CAT TCT CTG GCC A-3 ′ Nrf-2 5 ′ -TGC TTT ATA GCG TGC AAA CCT CGC-3 ′  5 ′ -ATC CAT GTC CCT TGA CAG CAC AGA-3 ′ 3Anti-Oxidative Effects of Chrysanthemum and Licorice  and the total RNA collected respectively. The primers forqPCR are listed in Table II (Integrated DNA Technologies,Coralville, IA, USA). The qPCR reactions were carriedout using 1  μ l cDNA product, 50 nM of each primer, andPower SYBR Green master mix (Applied Biosystems,Foster City, CA, USA) in 10  μ l reactions. The reactionswere performed using an ABI Prism 7900HT sequencedetection system ampli 󿬁 ed speci 󿬁 city was veri 󿬁 ed by  󿬁 rst-derivative melting curve analysis using the ABI software(SDS2.3, Applied Biosystems, Foster City, CA, USA).Relative quanti 󿬁 cation of each gene expression pro 󿬁 lewas calculated using a  ΔΔ Ct method and presented asrelative quantitative value (RQ value)=2 − ΔΔ Ct (RQmanager, Applied Biosystems, Foster City, CA, USA)(26,27). Animal and  In Vivo  Study The second generation (F2) Nrf2 ( − / − ) mice (C57BL/SV129) and the C57BL/6J wild-type mice (The JacksonLaboratory, Bar Harbor, ME) were used for the  in vivo study to investigate if the induction of phase II detoxify-ing/antioxidizing enzymes by the extracts was Nrf2-dependent (28). Five animals were used in each groupof Nrf2 ( − / − ) mice (Nrf2 KO) and wild-type mice (Nrf2WT). The mice were treated with vehicle (as a negativecontrol; cremophor: tween 80: ethyl alcohol: deionizedwater=2:1:1:6), LE 150 mg/kg, LE 300 mg/kg, CZ150 mg/kg, and CZ 300 mg/kg by oral gavage in a  󿬁 nalvolume of 100 – 110  μ l (13). After 12 h, livers were collected for the RNA extraction, and total RNA wereused for PCR, qPCR, and microarray analyses. Housingand care of the animals were in accordance with theguidelines established by the University ’ s AnimalResearch Committee consistent with the NIH Guidelinesfor the Care and Use of Laboratory Animals (Table III). Microarray Gene Expression Analysis Affymetrix MOE_430 microarrays (containing 45,101probes) were used to probe the global gene expression pro 󿬁 leof pooled RNA from Nrf2 WT or KO mice after oraladministration of CZ and LE of 150 mg/kg. These micro-arrays were conducted as published previously (27). The  “ .CEL ”  󿬁 les containing intensity values were created from thescanned image by using Microarray Suite 5 (Affymetrix). The.CEL  󿬁 les and the .CDF  󿬁 le (information on the location andidentity of different probe cells) were then analyzed using thedChip analysis software to identify genes that were differ-entially expressed in Nrf2-associated pathways in the liversamples of both treated and untreated controls (29 – 31).Normalization against the median using default settings indChip (median chip) was used and the expression values of each probe of all arrays (median intensities range 110 – 280)were calculated using default dChip model-based algorithmswith perfect match only for  󿬂 uorescence intensities. A trans-formed normalized data was generated and saved in an Excel 󿬁 le, which was later being imported into the IngenuityPathway Analysis Program (www.ingenuity.com, IPA 8.0)for further data characterization (29,32). Over 2,000 highly differentially expressed genes were  󿬁 ltered using a cut-off intensity at 1,750 and a subset of Nrf2-mediated oxidativestress response genes was performed using the canonicalpathway analysis function. Comparative analysis was per- Table III.  Con 󿬁 rmation of Genotype of the AnimalsGene Primers3 ′ -primer 5 ′ -GGA ATG GAA AAT AGC TCC TGC C-3 ′ 5 ′ -primer 5 ′ -GCC TGA GAG CTG TAG GCC C-3 ′ lacZ primer 5 ′ -GGG TTT TCC CAG TCA CGA C-3 ′ Fig. 1.  Effect of LE and CZ extracts on the mRNA expression of pro-in 󿬂 ammatory biomarkers (mRNA)in LPS-stimulated RAW 246.7 cells. LE 25  μ g/ml; CZ 25  μ g/ml; Curcumin 10  μ M. The gene bands werequanti 󿬁 ed by densitometry and normalized by GAPDH, ratio ( ***  compare with LPS-induced,  p <0.001;  ** compare with LPS-induced,  p <0.01;  *  compare with LPS-induced,  p <0.05) 4 Wu  et al  .
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