Effects of Atractylodes macrocephala Koidzumi[PMIDb21669278]

Journal of Ethnopharmacology 137 (2011) 396– 402 Contents lists available at ScienceDirect Journal of Ethnopharmacology jo ur nal homep age : www.elsev ier .com/ Effects of Atractylodes macrocephala Koidzumi rhi and an Chang Ke -Mi Gi Soon C ang a Department o b Department o c Department o , Yong d Acupuncture e Division of Ho f Department o ea g Department of Physiology, College of Medicine, Kyung Hee University, Seoul, Republic of Korea a r t i c l e i n f o Article history: Received 4 Ma Received in re Accepted 30 M Available onlin Keywords: Atractylodes m Obesity 3T3-L1 adipoc Oil Red O stain Phospho-Akt Perilipin a b s t r a c t 1. Introdu Obesity eases throu as overweig to adverse m and cholest unfavorable bers and ad ∗ Correspon Hee Universit Korea. Tel.: +8 E-mail add 1 These auth 0378-8741/$ – doi:10.1016/j. rch 2011 vised form 6 May 2011 ay 2011 e 6 June 2011 acrocephala Koidzumi yte ing Ethnopharmacological relevance: Atractylodes macrocephala Koidzumi (AMK) is an herbal medicine tradi- tionally used for treatment of abdominal pain, gastrointestinal disease, obesity, and related complications. Aim of the study: We investigated the effects and molecular mechanism of AMK rhizome water extract on 3T3-L1 adipogenesis and an animal model of obesity. Materials and methods: To study the effect of AMK on adipogenesis in vitro, differentiating 3T3-L1 cells were treated every two days with AMK at various concentrations (1–25 �g/ml) for eight days. Oil Red O staining was performed to determine the lipid accumulation in 3T3-L1 cells. To elucidate the inhibitory mechanism of AMK on adipogenesis, phosphorylation levels of Akt and expression of perilipin, were analyzed by Western blotting. AMK was administered orally to high fat diet (HFD)-induced obese rats to confirm its effect in vivo. Results: AMK inhibited 3T3-L1 adipocyte differentiation in a dose-dependent manner without cellular toxicity. Phospho-Akt expression was highly decreased by AMK treatment, whereas there was no signifi- cant change in perilipin expression. AMK administration significantly reduced the body weight of rats fed a HFD. Plasma triglyceride levels were significantly lower in the AMK-treated HFD group than those in the HFD control group or normal diet (ND) group, although serum total, HDL- and LDL-cholesterol levels did not differ between the groups. Conclusion: These results demonstrate an inhibitory effect of AMK on adipogenesis through reduction of an adipogenic factor, phospho-Akt. AMK had a beneficial effect, reducing body weight gain in a HFD-induced animal model of obesity. © 2011 Elsevier Ireland Ltd. All rights reserved. ction has become one of the most common metabolic dis- ghout the world. More than 1 billion adults are classified ht. Obesity and being overweight are known to lead etabolic effects on insulin resistance, blood pressure, erol and triglyceride concentrations in plasma. These consequences are caused by increased adipocyte num- ipocyte mass, which results from a massive adipocyte ding author at: Department of Physiology, College of Medicine, Kyung y, #1 Hoegi-Dong, Dongdaemoon-Gu, Seoul 130-701, Republic of 2 2 961 0286; fax: +82 2 964 2195. resses: mbi@khu.ac.kr, ewmed@hanmail.net (B.-I. Min). ors contributed equally to this work. differentiation process which generates mature adipocytes from preadipocytes. Excess fat is accumulated in adipocytes as triglyc- eride and massive amounts of lipids can be released from adipocytes, resulting in elevated triglyceride content in plasma and tissues including muscle and liver, which can lead to physiologic dysfunction in tissues (Tang et al., 1999; Frühbeck et al., 2001). Adipocyte differentiation is a complex process that involves expression of several adipocyte-specific genes including peroxi- some proliferator-activated receptor-gamma (PPAR�), sterol reg- ulatory element binding protein-1c (SREBP-1c), CCAAT/enhancer binding protein-alpha (C/EBP�) and fatty acid synthase (FAS), which lead to morphological changes and lipid accumulation within the cells (Christy et al., 1991; Chawla et al., 1994). It is well established that activation of the serine/threonine kinase Akt (PKB) (PI3K-Akt/PKB) pathway is the major signaling mechanism in adipocyte differentiation by which insulin and certain growth see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. jep.2011.05.036 animal model of obesity un Kima,b,1, Mihyun Kimc,1, Sang Deog Oha, Sang hoia, Sun-Kwang Kimd,e, Hyunsu Baef, Chulhun K f East-West Medicine, Graduate School, Kyung Hee University, Seoul, Republic of Korea f Alternative Medicine, Graduate School, CHA University, Seoul, Republic of Korea f Medical Science, Graduate School of East-West Medical Science, Kyung Hee University and Meridian Science Research Center, Kyung Hee University, Seoul, Republic of Korea meostatic Development, National Institute for Physiological Sciences, Okazaki, Japan f Physiology, College of Oriental Medicine, Kyung Hee University, Seoul, Republic of Kor locate / je thpharm zome on 3T3-L1 adipogenesis n Leec, Boram Suna, c, Byung-Il Mina,g,∗ in, Republic of Korea Administrator 文本框 A C.K. Kim et al. / Journal of Ethnopharmacology 137 (2011) 396– 402 397 factors stimulate adipogenesis. Treatment with PI3K inhibitors has been shown to completely block the differentiation process of 3T3- L1 preadipocytes, indicating that PI3K is necessary for adipocyte differentiation (Tomiyama et al., 1995; Magun et al., 1996; Christoffers downstream L1 cells res involvemen (Kohn et a dependent degradation terified fatt lipases, lipi perilipin (T to hormone sis via phos Most work perilipin A ciation wit increased d (Souza et al Despite medication effects from when they Recently, m from plants in the mana mortality. F to be one o diabetic eff signaling pa over, severa medicines s (Momordica 2008), and g to significa diet (HFD)- Atractylo Compositae Asia for trea sity, and rel 2005). It ha pharmacolo 1989; Tsun (Kiso et al., Matsuda et 1979; Li et these anti-o In this s AMK, 3T3-L Lipid accum ing. Among suppressed phosphoryl administere activities in induced bo blood. 2. Materia 2.1. Prepara Ten herb were purch cal Center, Kyung Hee University (Seoul, Korea), and the voucher specimen was deposited at the laboratory of Medical Science, Grad- uate school of East-West Medical Science, Kyung Hee University. The dried herbs were soaked in 10 volumes of cold distilled water rnight and then were extracted after being boiled for 3 h ◦C. T and c nes he ex erim 3-L1 rine ined BRL, I, D ified ate p y of 2 hang % [v /BRL nd 1 ocyt ] an on d type two agn n Axi eatm b ext thr of h ere ion m il Red 3T3 (PBS . Aft solu ratur and ol. F e-lin ll via l viab ,5-di MO, of M s in a the m s we , abs , USA re to en et al., 1998; Xia and Serrero, 1999). Moreover, Akt is a effector of the PI3K pathway and its activation in 3T3- ults in differentiation into adipocytes, suggesting the t of Akt-mediated signaling in adipocyte differentiation l., 1996). On the other hand, activation of cAMP- kinase (PKA) stimulates lipolysis which results from of accumulated triglycerides into glycerol and nones- y acids (NEAF) in adipocytes. Lipolysis involves several d hydrolase, and lipid droplet-containing proteins like ansey et al., 2001, 2004). Perilipin in particular binds -sensitive lipase (HSL) and promotes adipocyte lipoly- phorylation-dependent and independent mechanisms. regarding lipolysis has showed that overexpression of increases storage of triglycerides in fibroblasts in asso- h a decrease in lipolysis, and its expression is highly uring the differentiation of preadipocytes to adipocytes ., 1998, 2002). short-term benefits of drug treatment for obesity, -induced weight loss is often associated with side these remedies as well as rebound weight gain are discontinued (Abdollahi and Afshar-Imani, 2003). any researchers have shown that natural compounds like herbal medicines and their derivatives are effective gement of obesity without significant adverse effects or or example, Ginsenoside Rh2 was successfully shown f the active components in Ginseng that exerts anti- ects and prevents obesity in association with the AMPK thway in 3T3-L1 adipocytes (Hwang et al., 2007). More- l animal studies demonstrated that a variety of herbal uch as Zingiber officinale (Han et al., 2005), bitter melon charantia) (Huang et al., 2008), ephedra (Jeong et al., inseng (Kim et al., 2005) have beneficial effects leading nt weight loss or inhibition of weight gain in high fat induced obesity models. des macrocephala Koidzumi (AMK) belonging to the family, is an herbal medicine traditionally used in East tment of abdominal pain, gastrointestinal disease, obe- ated complications (China Pharmacopoeia Commission, s been reported that extracts from AMK have various gical activities associated with anti-tumor (Mori et al., eki et al., 2005; Kimura, 2006), anti-lipid-peroxidation 1985), antiulcer (Kubo et al., 1983; Nogami et al., 1986; al., 1991) and anti-inflammation activities (Endo et al., al., 2007a,b). However, the mechanism responsible for besity effects has not been discovered. tudy, to evaluate the potential anti-obesity effects of 1 preadipocytes were differentiated by inducing agents. ulation in the cells was measured by Oil-Red O stain- 10 herbal medicines, AMK water extract significantly lipid accumulation with a concomitant decrease in AKT ation. In addition to this in vitro study, AMK was orally d to rats fed a HFD to demonstrate its anti-obesity vivo. Our results showed that AMK can decrease HFD- dy weight gain as well as triglyceride concentration in ls and methods tion of herbal extracts s including Atractylodes macrocephala Koidzumi (AMK) ased from the Oriental Medicine of Kyung Hee Medi- for ove at 100 gauze medici until t the exp 2.2. 3T Mu mainta Gibco/ BCS (JB humid ferenti densit was c ing 10 [Gibco sone, a to adip 1% [v/v Finally pheno every 200× m with a 2.3. Tr Her filtered effects cells w induct days. 2.4. O The saline for 1 h Red O tempe times propan enzym (n = 4). 2.5. Ce Cel 2-yl)-2 Louis, 100 �l L1 cell 37 ◦C, crystal Finally (Emax compa he aqueous extracts were filtered through a distilled oncentrated using an evaporator. The extracted herbal were dried with a freeze dryer and stored at −20 ◦C periment was performed. The concentration used in ent was based on the dry weight of the extract. cell culture and stimulation 3T3-L1 preadipocytes (ATCC, Manassas, VA, USA) were in Dulbecco’s Modified Eagle’s Medium (DMEM; Grand Island, NY, USA) supplemented with 10% (v/v) aegu, Korea) and 1% (v/v) antibiotics (Gibco/BRL) in a atmosphere of 95% air and 5% CO2 at 37 ◦C. To dif- readipocytes into adipocytes, cells were seeded at a .4 × 105 cells/cm2. After four days of growth the media ed to adipocyte-induction media I (DMEM contain- /v] fetal bovine serum [FBS; JBI], 1% [v/v] antibiotics ], 0.5 mM isobutylmethylxanthine, 1 �M dexametha- 0 �g/ml insulin). After three days, cells were changed e-induction media II (DMEM containing 10% [v/v] FBS, tibiotics and 10 �g/ml insulin) for five additional days. ay eight, 3T3-L1 preadipocytes showed the adipocyte with accumulation of lipid droplets. Media was changed days. Appearance was recorded by microscopic using ification with an Axiovert S 100 (Carl Zeiss) equipped oCam. ent of herb extracts on 3T3-L1 cells racts were dissolved in adipocyte-induction media and ough 0.2 �m-pore syringe filters. To investigate the erb extracts on adipogenesis, differentiating 3T3-L1 treated every two days with an herb in adipocyte- edia at various concentrations (1–25 �g/ml) for eight O staining -L1 adipocytes were washed with phosphate-buffered , pH 7.4) and fixed with 4% paraformaldehyde (PFA) er washing with PBS, the cells were stained with Oil tion (0.2% [w/v] in isopropanol) for 1.5 h at room e. The cells were washed in distilled water three Oil Red O dye in lipid droplets was eluted into iso- inally, absorbance was measured at 490 nm using an ked immunosorbent assay (ELISA) reader (Emax, USA) bility assay ility was determined by MTT (3-(4,5-dimethylthiazol- phenyltetrazolium bromide) assay (Sigma Aldrich, St. USA). At the end of the experimental period (day 8), TT solution (5 mg/ml) was added to differentiated 3T3- 24-well plate. After the well was incubated for 2 h at edium was removed, and the synthesized formazan re dissolved in dimethyl sulfoxide (DMSO) (Sigma). orbance was measured at 570 nm using an ELISA reader ) (n = 4). Data was calculated as a percentage of MTT control cells. Administrator 文本框 B Administrator 线条 Administrator 线条 Administrator 文本框 C Administrator 文本框 D 398 C.K. Kim et al. / Journal of Ethnopharmacology 137 (2011) 396– 402 Table 1 Composition of experimental diets. Ingredient (%) Normal diet (ND) High fat diet (HFD) Casein, 80 Mesh 19.0 25.8 l-cystine Corn starch Maltodextrin Sucrose Cellulose, BW Soybean oil Lard Mineral mix Dicalcium ph Calcium carb Potassium ci Vitamin mix Choline bita Percent ener Protein Carbohydr Lipid 2.6. Wester Cells we buffer (20 m Nonidet P-4 pyrophosph deoxychola dithiothreit incubated o 20 min at 1 was measu 10, or 20 �g 0.2-�m po Hercules, CA SD; Bio-Rad antibody di actin [1:100 were probe body (anti-g The immun using West purchased f 2.7. Image All imag software, v signal inten subtraction actin (for pe 2.8. In vivo Six-wee (Samtaco, S flow room m humidity of ratory diet f diet group ( fat diet + AM compositio once a day istered 0.5 g were allow mental peri internation ffect o ells w 8) in a were s calc t the K (Atr ternat TG (So lygala Rafin foun ). Fo . The ken is (AR atisti a are wit ing cally ults ti-ad ytes valu onflu erbal medicine every two days while the preadipocytes dif- ated into adipocytes. Morphological changes were observed accumulation of lipids in the preadipocytes. Oil Red O stain- ealed that the lipid accumulation in AMK-treated cells was antly lower than the lipid accumulation in control cells and 0.3 0.4 29.9 0.0 10 3.3 16.2 33.2 8.9 200 4.7 6.5 2.4 3.2 1.9 31.7 S10026 0.9 1.3 osphate 1.2 1.7 onate 0.5 0.7 trate 1.6 2.1 V10001 0.9 1.3 rtrate 0.2 0.3 gy 20.0 20.0 ate 70.0 20.0 10.0 60.0 n blot analysis re washed three times with cold PBS and scraped in lysis M Tris–HCl [pH 7.4], 150 mM NaCl, 10% glycerol, 2% 0, 1 mM EDTA, 20 mM sodium fluoride, 30 mM sodium ate, 0.2% sodium dodecyl sulfate (SDS), 0.5% sodium te, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mM ol (DTT), 1 mM sodium vanadate). The cell lysate was n ice while vortexing for 15 min and centrifuged for 2,000 × g. Protein quantification of each supernatant red by Bradford assay (Ref). Each protein extract (5, ) was separated on 10% SDS-polyacrylamide gel onto lyvinylidene difluoride (PVDF) membranes (Bio-Rad, , USA) using a semi-dry transfer apparatus (Trans-Blot ). The membranes were then incubated with primary luted (Akt-p [1:1000], Akt [1:2000], perilipin [1:2000] 0]) in blocking solution at 4 ◦C overnight. Membranes d with horseradish peroxidase-labeled secondary anti- oat IgG or anti-rabbit IgG) for 1 h at room temperature. oreactive bands were detected by chemiluminescence -oneTM (iNtRON Biotechnology). All antibodies were rom Santa Cruz Biotechnology and Abcam. analysis e analysis was performed with ImageMaster 2DTM Elite ersion 3.1 (Amersham Pharmacia Biotechnology). The sity of each band was analyzed by lowest background and normalized by band intensity of Akt (for Akt-p) and rilipin). Fig. 1. E 3T3-L1 c 0 to day the cells Data wa represen trol), AM (Pinellia Nakai), S PTW (Po trifoliata care as in 1985 weeks was ta analys 2.9. St Dat ANOVA test, us statisti 3. Res 3.1. An adipoc To e post-c of an h ferenti due to ing rev signific anti-obesity activity k-old male Sprague-Dawley rats weighing 170–190 g eoul, Korea) were housed four per cage in a laminar air- aintained at a temperature of 22 ± 1 ◦C and a relative 55 ± 1%. After the animals were given a standard labo- or one week, they were randomly divided into a normal ND) (n = 6), high-fat diet group (HFD) (n = 6), and high- K group (HFD + AMK) (n = 6). Table 1 shows the HFD n. Rats in the HFD group were orally administered saline while rats in the HFD + AMK group were orally admin- AMK/kg body weight in saline. All experimental rats ed free access to the diet and water during the experi- od. The research was conducted in accordance with the ally accepted principles for laboratory animal use and other herb- cells showe adipocytes analysis to adipocytes. 3.2. AMK in To exam adipocyte were maint ious doses lipid drops Fig. 2(B), A dose-depen intracellula f various herbal medicines on cellular lipid droplets. Differentiating ere treated every 2 days with 10 �g/ml of herbal medicines (from day dipocyte-induction media. To determine lipid content accumulation, stained with Oil Red O at day 8 and optical density detected at 490 nm. ulated as a percentage of Oil Red O stained lipid in controls. Results mean ± SEM of four independent experiments (*p < 0.001). CTL (con- actylodes macrocephala Koidzumi), KSS (Kochia scoparia Schrader), PTB a Breitenbach), ACT (Artemisia capillaris Thunberg), AGN (Angelica gigas phora tonkinensis Gagnep), RGL (Rehmannia glutinosa Liboschitz var.), tenuifolia Willdenow), PFV (Perilla frutescens var. acuta), PTR (Poncirus esqul). d in the US guidelines (NIH publication #85-23, revised od intake and body weights were recorded daily for six rats were euthanized by anesthetic overdose and blood for triglyceride, total cholesterol, and HDL cholesterol C Laboratory, South Korea). cal analysis presented as mean ± SEM. Analysis was performed by h Bonferroni’s test for multiple comparisons, and by t- SPSS 11.0 software. A p-value of <0.05 was considered significant. ipogenic effects of 10 herbal medicines on 3T3-L1 ate the anti-adipogenic effects of 10 herbal medicines, ent 3T3-L1 preadipocytes were treated with 10 �g/ml treated cells. Of the 10 herbal treatments, AMK-treated d a 33% inhibitory effect on adipogenesis of 3T3-L1 (Fig. 1). Therefore, AMK was subjected to additional better understand its anti-adipogenic effect on 3T3-L1 hibits 3T3-L1 adipocyte differentiation ine the anti-adipogenic effect of AMK on 3T3-L1 differentiation, post-confluent 3T3-L1 preadipocytes ained in adipocyte-induction media and exposed to var- of AMK (1, 5, 10, 25 �g/ml). Subsequently, intracellular were measured by Oil Red O staining. As shown in MK effectively inhibited adipocyte differentiation in a dent manner in 3T3-L1 adipocytes. We next examined r toxicity by MTT assay. Fig. 2(C) shows that viability of C.K. Kim et al. / Journal of Ethnopharmacology 137 (2011) 396– 402 399 Fig. 2. Effect o (from 1 to 25 � content, Oil Re Results repres adipocyte-ind represent the AMK-treate up to 25 �g of lipid acc lar toxicity. O staining leading to a 3.3. Effects adipocytes Serine/th vated by ins 3-kinase (P To reveal th anti-adipog level of pho 3T3-L1 cell 5, 10, 25 �g adipocytes. level of pho the inhibiti can also be of the lipid 2002), we n AMK-induc entiation. A f AMK on cellular lipid droplets and cell viability of 3T3-L1 adipocytes. Differentiating 3T3- g/ml) for 8 days in adipocyte-induction media. Intracellular lipids were stained with Oil d O dye was dissolved in isopropanol and optical density detected at 490 nm (B). Data ent the mean ± SEM of four independent experiments (*p < 0.01, **p < 0.001 and ***p < 0 uction media for 8 days and treated with AMK every 2 days (see Section