JOURNAL OF MEDICINAL FOODJ Med Food 9 (2) 2006, 145–153© Mary Ann Liebert, Inc. and Korean Society of Food Science and Nutrition

Baicalein Inhibits Adipocyte Differentiation by Enhancing COX-2 Expression

Min-Ho Cha,1,2 Il-Chul Kim,3 Bong-Hee Lee,2 and Yoosik Yoon1

1Department of Medical Research, Korea Institute of Oriental Medicine, Daejeon; 2Graduate School of Life Sciences and Biotechnology, Korea University, Seoul; and 3Department of Biology,

Chonnam National University, Gwangju, Korea

ABSTRACT Baicalein, one of the major flavonoids in Scutellaria baicalensis (Chinese Skullcap), is well known for its ef-fects on cell proliferation, apoptosis, and inflammation. Here we show that baicalein also inhibits the adipogenesis of 3T3-L1preadipocytes. Baicalein inhibited triglyceride accumulation during adipogenesis and significantly decreased the mRNA ex-pression of fatty acid-binding protein (FABP), a marker of adipogenesis. Microarray analysis revealed that several genes,which are differentially expressed during adipogenesis, were modulated by baicalein treatment in 3T-L1 cells. The expressionof FABP, apolipoprotein D, and insulin-like growth factor 2, which was markedly up-regulated during adipogenesis, wasdown-regulated by baicalein. Cyclooxygenase (COX)-2 mRNA expression, which was decreased during adipogenesis, wasup-regulated by baicalein. These COX-2 mRNA expression patterns were mirrored by the expression of COX-2 protein andits enzymatic activity. NS-398, a COX-2 inhibitor, partially abrogated the baicalein-induced inhibition of adipogenensis. Thus,the anti-adipogenic effect of baicalein may be mediated by its ability to enhance the expression of COX-2, which is normallydown-regulated during adipogenesis.

KEY WORDS: • adipogenesis • baicalein • cyclooxygenase-2 • microarray • Scutellaria baicalensis • 3T3-L1 preadipocytes

145

INTRODUCTION

BAICALEIN (FIG. 1), one of the major flavonoids in Scutel-laria baicalensis (Chinese Skullcap), is known to affect

a variety of biological processes. It has been shown thatbaicalein arrests the cell cycle in Hep G2 cells at the G1/Sphase by decreasing the expression of cyclin A, cyclin D,and cyclin-dependent kinases.1–3 Baicalein was also re-ported to inhibit cell proliferation in human T-lymphocy-toleukemia cells by suppressing platelet-derived growth fac-tor-A mRNA expression.4 In addition, it has been found toinduce apoptosis in Hep G2 cells by elevating caspase-3 andby decreasing Bcl-2 expression.5,6 Baicalein also has an-tioxidant and anti-inflammatory properties. Shao et al.7

showed with an ischemia-reperfusion model that the pres-ence of baicalein decreases the formation of reactive oxy-gen species in hypoxic conditions. Baicalein was also foundto reduce nitric oxide formation in microglial cells in re-sponse to endotoxin/cytokine treatment by repressing in-hibitory nitric oxide synthase mRNA expression.8 In addi-tion, baicalein decreased the production of pro-inflammatory

interleukins and eotaxin,9–11 and suppresses the synthesis ofprostaglandin E by inhibiting the mitogen-activated proteinkinase cascade.12,13 Miyamoto et al.14 reported thatbaicalein also blocked the synthesis and secretion ofleukotrienes C and D in the guinea pig lung by inhibitinglipoxygenase.

Adipogenesis, which is critical in controlling the body fatmass, is known to progress via two stages. The first is thedifferentiation of preadipocytes to adipocytes, while the sec-ond involves the accumulation of fat in adipocytes.15 Manygene products, including fatty acid binding protein (FABP),fatty acid synthase, glucose transporter 4, and apolipopro-teins, are involved in adipocyte differentiation and fat ac-cumulation.16–19 The expressions of these genes are regu-lated by transcription factors including peroxisomalproliferation-activating receptor-� (PPAR�) and theCCAAT enhancer binding proteins (C/EBPs).19,20 Adipo-genesis has been studied extensively in vitro usingpreadipocyte cell lines, including 3T3-L1.21 When culturedin defined media, 3T3-L1 cells deposit triglycerides in cy-toplasmic lipid droplets and express genes that are expressedin adipocytes in vivo.22–26 In this study, we showed, by us-ing the 3T3–L1 preadipocyte in vitro system, that baicaleineffectively inhibits adipogenesis, possibly by altering the ex-pression of adipogenic genes, including cyclooxygenase(COX)-2.

Manuscript received 21 July 2005. Revision accepted 23 January 2006.

Address reprint requests to: Yoosik Yoon, Department of Medical Research, Korea Insti-tute of Oriental Medicine, 461-24, Jeonmin-dong, Yuseong-gu, Daejeon, Korea, 305-811,E-mail: ysyoon66@naver.com

MATERIALS AND METHODS

Cell culture and reagents

3T3-L1 cells obtained from the Korean Cell Line Bank(Seoul, Korea) were maintained in Dulbecco’s Modified Ea-gle’s Medium supplemented with 10% fetal bovine serum,100 �g/mL streptomycin, and 100 units/mL penicillin. Thecells were fed with fresh medium every 2 days and subcul-tured when they reached a confluence of 80%. Baicalein andNS-398 were purchased from Sigma Chemical Co. (St.Louis, MO).

Induction of adipogenesis in 3T3-L1 cells

Confluent cells were cultured with differentiation induc-tion medium (1 �g/mL insulin, 0.25 �M dexamethasone, and0.5 mM 3-isobutyl-1-methylxanthine in Dulbecco’s Modi-fied Eagle’s Medium containing 10% fetal bovine serum) for2 days, after which they were maintained in differentiationmaintenance medium (1 �g/mL insulin in Dulbecco’s Mod-ified Eagle’s Medium containing 10% fetal bovine serum).The medium was changed every 2 days until the cells wereharvested. To test the effect of baicalein, it was added to thedifferentiation induction medium and the differentiationmaintenance medium until the cells were harvested. Cell vi-ability was measured by the 3-(4,5-dimethylthiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) method as de-scribed.27

Oil Red O staining

Cells were washed with phosphate-buffered saline (PBS),fixed in 4% (wt/vol) paraformaldehyde for 1 hour, andstained with Oil Red O as described by Kasturi and Joshi.28

After two washes with PBS, the fat droplets in the cells wereobserved under an inverted light microscope.

Triglyceride content assay

Differentiated cells in six-well plates were harvested in500 �L of PBS, frozen, and then sonicated. The triglyceridelevels in the sonicates were measured by using theTRIGlYZYME-V reaction kit (Shinyang Chemical Co.,Seoul, Korea) according to the manufacturer’s instructions.

Microarray assay

Total RNA was extracted from 3T3-L1 cells by the acid-phenol method using Trizol (GibcoBRL, Burlington, ON,Canada), and double-stranded cDNA was synthesized by us-ing the SuperScript Choice system (LifeTechnologies,Gaithersburg, MD). After cDNA synthesis, unincorporated de-oxyribonucleotide triphosphates were removed, and the cDNAwas labeled with biotin by using a GeneChip IVT labeling kit(Affymetrix Inc., Santa Clara, CA). Mouse oligonucleotidechips were obtained from Affymetrix, Inc., and assays wereconducted according to the manufacturer’s instructions. Thearray was scanned by using an Agilent GeneArray Scannerfrom Affymetrix, Inc., and the data were analyzed by usingImaGene software (BioDiscovery Co., Boston, MA).

Real-time polymerase chain reaction (PCR)

Total RNA was extracted by the acid-phenol method us-ing Trizol (GibcoBRL), and 1 �g was reverse-transcribedfor 60 minutes at 42°C using reverse transcription premix(Bioneer, Daejeon, Korea). PCR was performed in 25-�Lreaction volume containing SYBR Green PCR Mastermix(Qiagen, Hilden, Germany), 2 �L of cDNA, and 1 �M eachprimer. The reaction mixtures were preheated at 95°C for15 minutes and then subjected to 40 cycles of (melting at95°C for 30 seconds, annealing at 60°C for 30 seconds, elon-gation at 72°C for 30 seconds) and measurements at 72°Cfor 20 seconds using a Roter-gene 2000 real-time cycler(Corbett, Mortlake, NSW, Australia). The upstream anddownstream primers used were, respectively, as follows:COX-2, 5�-AAGACTTGCCAGGCTGAACT-3� and 5�-C-TTCTGCAGTCCAGGTTCAA-3�; FABP, 5�CCTCGAA-GGTTTACAAAATG-3� and 5�GAAGTCACGCCTTTC-ATAAC-3�; insulin-like growth factor-2 (IGF-2), 5�-T-TGGTGCTTCTCATCTCTTT-3� and 5�-GTCTCCAGG-TGTCATATTGG-3�; apolipoprotein D (ApoD), 5�-GAAA-GGAAACTGCATTCAAG-3� and 5�-CTGGAGGGAGAT-AAGGATTT-3�; and glyceraldehyde 3-phosphate dehy-drogenase (GAPDH) (used as an internal standard), 5�-CTA-CATGGTCTACATGTTCC-3� and 5�-CTGACAATCTTG-AGTGAGTT-3�.

Protein extraction and western blot

Cells were harvested using a cell scraper and lysed in alysis buffer [20 mM Tris (pH 7.4), 5 mM EDTA, 0.1% Tri-ton X-100, and 0.01% 2-mercaptoethanol]. The lysates werethen sonicated and centrifuged (13,000 g for 20 minutes at4°C) to remove insoluble material. The supernatant wastransferred to a new tube and stored at �20°C until analy-sis. The protein content was determined by using the Bio-Rad DC Protein Assay (Bio-Rad Laboratories Ltd., Her-cules, CA). Protein extracts (20 �g) were heated at 95°C for3 minutes, resolved by 10% sodium dodecyl sulfate–poly-acrylamide gel electrophoresis, and electrotransferred to ni-trocellulose membranes at 80 V for 2 h by using a semidrytransfer (Bio-Rad Laboratories). The membranes were then

146 CHA ET AL.

HO

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FIG. 1. Chemical structure of baicalein.

blocked for 2 hours at room temperature with Tris-bufferedsaline containing 5% albumin and 0.1% Tween 20, and thenincubated with anti-COX-2 antibody (diluted 1:1,000)(TAKARA, Shiga, Japan) for 3 hours at room temperatureand subsequently with horseradish peroxidase-conjugatedanti-rabbit secondary antibody (diluted 1:3,000) (AmershamBiosciences UK, Buckinghamshire, UK) for 1 hour at roomtemperature. Peroxidase activity was visualized by using anECL kit (Amersham Biosciences). Actin was used as anequal loading control.

COX-2 activity assay

Cells were harvested and sonicated in PBS. COX-2 ac-tivity was measured by using a COX activity assay kit (Cay-man Chemical Co., Ann Arbor, MI) according to the man-ufacturer’s instructions.

Statistical analysis

The data were expressed as means � SD from at leastthree independent experiments. Statistical significance was

BAICALEIN-INDUCED RECOVERY OF COX-2 147

FIG. 2. Inhibitory effect of baicalein on fat accumulation and FABP mRNA expression during adipogenesis. (A) Time course of triglycerideaccumulation during adipogenesis and inhibitory effect of 100 �M baicalein treatment. (B) Inhibition of triglyceride accumulation according tothe baicalein concentration. The preadipocytes were differentiated for 7 days with various concentrations of baicalein ranging from 0 to 200 �M.(C) Microscopic observation of Oil Red O-stained 3T3-L1 cells after 7 days of adipocyte differentiation with or without 100 �M baicalein. (D)Inhibition of FABP mRNA expression by baicalein. The preadipocytes were differentiated for 2 days with various concentrations of baicalein,and their total RNAs were subjected to real-time PCR. These data were normalized on the basis of the untreated adipocyte value. DMSO, di-methyl sulfoxide. (E) Effects of baicalein on cell viability during adipogenesis. The preadipocytes were differentiated for 2 days with variousconcentrations of baicalein, and cell viability was detected by MTT assay. The data were analyzed statistically by t tests and one-way ANOVA.*P � .05, **P � .01, ***P � .001 compared with untreated adipocytes.

analyzed by Student’s t test and one-way analysis of vari-ance (ANOVA) using SPSS Version 10.0 (SPSS Inc.,Chicago, IL).

RESULTS

Baicalein blocks triglyceride accumulation during adipogenesis

As adipocytes differentiate, their intracellular levels oftriglyceride increase continuously. We found thatbaicalein effectively prevented this triglyceride accumu-lation in differentiating 3T3-L1 preadipocytes (Fig. 2A).Baicalein at concentrations of more than 100 �M signif-icantly decreased triglyceride accumulation (Fig. 2B). Mi-croscopic observation of the Oil Red O-stained adipocytesrevealed that baicalein significantly decreased the forma-tion of intracellular fat deposits (Fig. 2C). The mRNA ex-pression of the free fatty acid transporter (FABP), a well-known marker of adipogenesis,19 was also decreased bybaicalein (Fig. 2D). When cell viability was measured byusing the MTT method, we found that baicalein had noeffect on cell viability at 100 �M (Fig. 2E). Thus, the in-

hibitory effect of baicalein on fat accumulation is not dueto its cytotoxicity.

Effect of baicalein on gene expression during adipogenesis

To elucidate the molecular mechanism behind the anti-adipogenic effect of baicalein, we sought to identify thegenes whose expressions were altered by baicalein. To dothis, preadipocytes were differentiated for 7 days with orwithout baicalein, and their total RNAs were subjected tomicroarray analyses. Of the 14,110 genes on the microar-rays, about 462 genes were up- or down-regulated by overthreefold during adipocyte differentiation. Some that werealso up- or down-regulated by over threefold during adipo-genesis and showed altered expression patterns due tobaicalein are listed in Tables 1 and 2, respectively.

The microarray assay showed that, among genes involvedin lipid metabolism, ApoD and FABP mRNA expressionswere increased by over ninefold in differentiated adipocytescompared with preadipocytes, which confirms previous re-ports.19,29,30 The expression of phospholipid scramblase 2was also significantly elevated. IGF-2, which regulates cell

148 CHA ET AL.

TABLE 1. GENES THAT ARE UP-REGULATED BY MORE THAN THREEFOLD DURING ADIPOGENESIS BUT ARE DOWN-REGULATED BY BAICALEIN TREATMENT

Gene Biological function Adipocyte Baicalein

ApoD Lipid transport 21.1 3.0FABP Lipid transport 9.2 1.3Phospholipid scramblase 2 Lipid metabolism 9.8 1.4Growth arrest-specific 6 Regulation of cell cycle 9.8 1.6IGF-2 Regulation of cell cycle 19.7 2.5C/EBP, � Regulation of transcription 21.3 2.3cut-like (Drosophila) Regulation of transcription 14.9 1.5Neurogenin 3 Regulation of transcription 14.9 2.1Matrix metalloproteinase 2 Collagen catabolism 4.9 1.1Complement component 4-binding protein Complement activation 17.1 2.5Olfactomedin 1 Development 10.6 1.1Pyruvate dehydrogenase E1 �2 Glycolysis 10.6 1.7Glycine receptor, �3 subunit Ion transport 12.1 2.0t-complex-associated testis expressed 2 Movement 22.6 1.7Apolipoprotein B-editing complex 2 mRNA editing 16.0 2.1Granzyme C Protein metabolism 14.9 2.3Rabphilin 3A Protein transport 11.3 1.7Cholinergic receptor, muscarinic 4 Signal transduction 18.4 2.6G protein-coupled receptor 34 Signal transduction 14.9 1.6G protein-coupled receptor kinase 2, Signal transduction 22.6 2.6

Grouchogene-related (Drosophila)Thyroid-stimulating hormone receptor Signal transduction 13.9 2.1wingless-related MMTV integration site 4 Signal transduction 17.1 2.1Amiloride-sensitive sodium channel Sodium ion transport 34.3 2.0Allograft inflammatory factor 1 — 24.3 1.1B lymphocyte-activation antigen — 22.6 2.6Bone sialoprotein — 24.3 2.1H19 fetal liver mRNA — 13.9 1.7Myelin and lymphocyte protein — 9.8 1.6Trypsinogen 16 — 13.9 1.5

Shown are the ratios of the microarray signals of untreated and baicalein-treated adipocytes relative to the preadipocytesignal. The biological functions of the genes were obtained from https://www.affymetrix.com/analysis/netaffx/index.affx.

cycle, and matrix metalloproteinase 2, which cleaves extra-cellular matrix, were also highly up-regulated during adi-pogenesis. Baicalein effectively blocked the increased ex-pressions of these adipogenesis-related genes (Table 1). Inaddition, the mRNA levels of COX-2, which regulatesprostaglandin synthesis, were decreased by more than three-fold during adipogenesis, as previously reported,31,32 whilethe expression of �-protocadherin, plakophilin 1, andtrophinin, which are components of cell adhesion molecules,were also down-regulated during adipogenesis. Baicalein re-versed these patterns (Table 2).

To verify the microarray results, we examined COX-2,IGF-2, FABP, and ApoD gene expression during adipo-genesis and the effects of baicalein on this by real-time PCRexperiments. It was confirmed that COX-2 mRNA expres-sion was significantly decreased during adipogenesis andthat baicalein restored it to preadipocyte levels (Fig. 3A).The expressions of IGF-2, FABP, and ApoD were also con-firmed to be up-regulated during adipogenesis and reducedto preadipocyte levels by baicalein treatment (Fig. 3B–D).

Baicalein restoration of mRNA expression and proteinlevel of COX-2 during adipogenesis

The expression of COX-2 mRNA during adipogenesiswith and without baicalein was analyzed by a time coursereal-time PCR experiment. This revealed that the effect of

baicalein was most evident at day 2, namely, at the earlyphase of adipogenesis (Fig. 4A). To confirm that thebaicalein-induced increase in COX-2 mRNA expression re-flects an increase in COX-2 protein levels, COX-2 proteinlevels were quantified by western blotting. A time courseexperiment revealed that baicalein had its strongest effect atday 2 (Fig. 4B), similar to what was observed for the mRNAlevels.

Baicalein induces recovery of COX-2 activity during adipogenesis

We performed a COX-2 activity assay and found that dur-ing adipogenesis, COX-2 activity steadily decreased duringadipogenesis, but it was reversed by baicalein (Fig. 5A). Theconcentration of baicalein able to increase COX-2 activitywas more than 100 �M (Fig. 5B).

COX-2 inhibitor induces recovery of baicalein-inhibited adipogenesis

To confirm that the inhibitory effects of baicalein on adi-pogenesis are mediated by the restoration of COX-2 activ-ity, a COX-2 inhibitor, NS-398, used to treat the cells alongwith baicalein during adipogenesis. Triglyceride content,which was decreased in adipocytes by baicalein, was sig-nificantly restored by the COX-2 inhibitor in a dose-depen-

BAICALEIN-INDUCED RECOVERY OF COX-2 149

TABLE 2. GENES THAT ARE DOWN-REGULATED BY MORE THAN THREEFOLD DURING ADIPOGENESIS

BUT ARE UP-REGULATED BY BAICALEIN TREATMENT

Gene Biological function Adipocyte Baicalein

COX-2 Prostaglandin synthesis 0.3 1.4Calsyntenin-3 Cell adhesion 0.1 2.0Trophinin Cell adhesion 0.1 1.7�-Protocadherin Cell adhesion 0.2 1.3plakophilin 1 Cell adhesion 0.3 1.5Myosin heavy chain IIX Cytoskeleton organization 0.2 1.4Receptor tyrosine kinase-like orphan receptor 2 Skeletal development 0.1 1.4Carbonic anhydrase 15 Carbohydrate metabolism 0.2 1.4Chromodomain helicase DNA binding protein 1 Chromatin assembly 0.3 1.3�4 Ion transport 0.3 1.2Potassium voltage-gated channel, subfamily Q, member 2 Ion transport 0.3 1.3Sodium iodide symporter Ion transport 0.3 1.3Alkaline phosphatase 5 Metabolism 0.2 1.2Peroxisomal biogenesis factor 13 Peroxisome organization 0.1 1.5Vacuolar protein sorting 39 (yeast) Protein transport 0.2 1.5Adaptor-related protein complex 3, �2 subunit Protein transport 0.3 1.3Nuclear receptor subfamily 4, group A, member 2 Regulation of transcription 0.1 1.5cut-like 1 (Drosophila) Regulation of transcription 0.2 1.3Ankyrin repeat domain 3 Regulation of transcription 0.2 1.7PDZ protein interacting specifically with TC10 Signaling cascade 0.2 1.6Keratin-associated protein 13 — 0.2 1.6Dihydropyrimidinase-like 2 — 0.2 1.2Small proline rich-like 3 — 0.2 1.2t-complex-associated testis expressed 2 — 0.2 1.1Son cell proliferation protein — 0.3 1.1

Shown are the ratios of the microarray signals of untreated and baicalein-treated adipocytes relative to the preadipocyte signal. Thebiological functions of the genes were obtained from https://www.affymetrix.com/analysis/netaffx/index.affx.

150 CHA ET AL.

FIG. 4. Baicalein-induced restoration of COX-2mRNA and protein expression during adipogenesis. Thetime course is shown of COX-2 mRNA expression (A)and COX-2 protein level (B) during adipogenesis andthe effect of baicalein. The preadipocytes were differ-entiated for 2, 4, and 7 days with or without 100 �Mbaicalein. COX-2 mRNA expressions were obtained byreal-time PCR experiments, and protein levels werequantified by western blot. The data were analyzed sta-tistically by t test. *P � .05, **P � .01 compared withthe control value. Actin was used as an equal loadingcontrol.

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FIG. 3. Effects of baicalein on the mRNA levels of COX-2, IGF-2, FABP, and ApoD during adipogenesis. 3T3-L1 preadipocytes were differ-entiated for 7 days with or without 100 �M baicalein and then subjected to real-time PCR to measure the mRNA levels of COX-2 (A), IGF-2(B), FABP (C), and ApoD (D). The data were normalized on the basis of preadipocyte value and analyzed statistically by t test. *P � .05, **P �.01 compared with the preadipocyte value.

dent manner up to 77% of the untreated adipocyte level (Fig.6), showing that the inhibitory effect of baicalein is medi-ated, at least partially, by COX-2 regulation.

DISCUSSION

Baicalein is one of the major flavonoids in S. baicalen-sis. It is well known for its ability to affect cell prolifera-tion, apoptosis, and inflammation.1,5,8,9 Here, we show thatit also inhibits adipocyte differentiation. It was found thatbaicalein significantly inhibited triglyceride accumulation inadipocytes during adipogenesis and decreased FABP mRNAexpression, which is a well-known marker of adipogenesis(Fig. 2B and D).19 To elucidate the mechanism behind theeffect of baicalein, we used a microarray assay to search forthe genes that are up- or down-regulated during adipogene-sis and then found genes whose expression patterns are al-tered by baicalein. We found that 462 of the 14,100 geneson the microarray were up- or down-regulated by over three-

fold during adipogenesis. Of these, FABP, ApoD, andC/EBP�, which have been reported to induce adipogenesisand triglyceride accumulation in adipocytes,19,20,29 and IGF-2, a growth factor that activates cell proliferation, wereup-regulated by over ninefold in differentiated adipocytescompared with preadipocytes, but were significantly down-regulated by baicalein treatment (Table 1). The expression ofmetalloproteinase 2, which is involved in cleaving the extra-cellular matrix, was also increased by about fivefold duringadipogenesis, but was decreased to the level in preadipocytesby baicalein treatment. The latter observation is in agreementwith the report of Liu et al.,33 who showed that baicaleindown-regulates metalloproteinase 2 mRNA expression andinhibits cell proliferation. Cell adhesion molecules, including�-protocadherin, plakophilin 1, and trophinin, were down-regulated during adipogenesis, and this action was reversedby baicalein (Table 2). In addition, adipogenesis was accom-panied by a significant decrease of COX-2 expression, whichis in agreement with other studies,31,32 but COX-2 mRNA

BAICALEIN-INDUCED RECOVERY OF COX-2 151

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FIG. 5. Effect of baicalein on the activity of COX-2. (A) Time course experiment showing the change in COX-2 activity during adipogenesisand the effect of baicalein. The preadipocytes were differentiated for 2, 4, and 7 days with or without 100 �M baicalein. (B) Changes in COX-2 activity according to the baicalein concentration. The preadipocytes were differentiated for 7 days with various concentrations of baicalein. Thedata were normalized according to the preadipocyte value of each experiment and were analyzed statistically by t test and one-way ANOVA.*P � .05, **P � .01 compared with untreated adipocytes.

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FIG. 6. Restoration of triglyceride accumulation byCOX-2 inhibitor. The preadipocytes were differentiatedfor 7 days with 100 �M baicalein and various concen-trations of the COX-2 inhibitor, NS-398. The data werenormalized according to the untreated adipocyte valueand were analyzed statistically by t test. ***P � .001compared with adipocytes treated with baicalein butwithout NS-398.

expression, protein levels, and enzymatic activity were allreversed by baicalein (Figs. 4 and 5).

The COXs catalyze the rate-limiting and first committedstep of prostanoid biosynthesis. The availability of this en-zyme is directly related to the level of prostaglandin pro-duction. Two COX isoforms have been identified, namely,COX-1, a constitutive form, and COX-2, an inducible form.While the former is believed to be responsible for home-ostasis, the latter is thought to be mainly involved in patho-logical conditions.34,35 A recent study suggested that COX-2 may be involved in body fat regulation and may in-hibit adipogenesis. Mice that are heterozygous for the COX-2 gene (Cox-2 �/�) showed a 3.5-fold increase in their fatpad compared with wild-type mice (Cox-2 �/�).36 More-over, Yan et al.31 reported that COX-2 mRNA expressionin 3T3-L1 cells was decreased during adipogenesis but thattumor necrosis factor-� (TNF-�) reversed this effect in adose-dependent manner. TNF-� is known to induce COX-2expression. When 3T3-L1 cells were treated simultaneouslywith TNF-� and NS-398, an inhibitor of COX-2, the adi-pogenesis that had been inhibited by TNF-� started toprogress again. This means that the inhibition of adipogen-esis by TNF-� is mediated by COX-2. Arachidonic acid isalso known to inhibit adipogenesis, and it was reported thatthis effect is also mediated by COX-2.31,32,37–39 In addition,Banerjee et al.40 showed that nimesulide, a COX-2 inhibitor,induces the expression of PPAR�, which is a transcriptionfactor that plays a major role in adipogenesis. In this study,a COX-2 inhibitor, NS-398, restored triglyceride accumula-tion in adipocytes, which was inhibited by baicalein (Fig.6). The products of COX-2, prostaglandins, have also beenreported to inhibit adipogenesis. Prostaglandin F2� isknown to activate p42/p44 mitogen-activated protein kinase,which decreases PPAR� mRNA expression and thereby in-hibits adipogenesis.41 Moreover, prostaglandin E2 is alsoknown to have an anti-adipogenic effect.36 Thus, the resultsof this study agree with previous observations regarding theinhibitory role of COX-2 in adipogenesis, and suggest thatthe anti-adipogenic effect of baicalein is mediated by therestoration of COX-2 expression.

ACKNOWLEDGMENTS

This research was supported in part by grantM1052701000005N270100000 from the Ministry of Sci-ence and Technology of Korea, and in part by a grant fromKorea Institute of Oriental Medicine.

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