Artemisia Scoparia Inhibits Adipogenesis in 3T3-L1 Pre-Adipocytes by Downregulating the MAPK Pathway
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ISSN (Print) 1225-9918 ISSN (Online) 2287-3406 Journal of Life Science 2018 Vol. 28. No. 9. 999~1006 DOI : https://doi.org/10.5352/JLS.2018.28.9.999 Artemisia scoparia Inhibits Adipogenesis in 3T3-L1 Pre-adipocytes by Downregulating the MAPK Pathway Jung Hwan Oh1, Fatih Karadeniz1,2, Youngwan Seo3,4 and Chang-Suk Kong1,2* 1Department of Food and Nutrition, College of Medical and Life Sciences, Silla University, Busan 46958, Korea 2Marine Biotechnology Center for Pharmaceuticals and Foods, College of Medical and Life Sciences, Silla University, Busan 46958, Korea 3Division of Marine Bioscience, College of Ocean Science and Technology, Korea Maritime and Ocean University, Busan 49112, Korea 4Department of Convergence Study on the Ocean Science and Technology, Ocean Science and Technology School, Korea Maritime and Ocean University, Busan 49112, Korea Received April 30, 2018 /Revised June 29, 2018 /Accepted July 4, 2018 Obesity is epidemic worldwide and has reportedly been linked to the progression of several metabolic and cardiovascular diseases. The natural products are decreasing the side effects of medicines used for obesity and also have health benefits dut to their numerous bioactive compounds. In this context, Artemisia scoparia is a widespread plant that has been suggested as possessing various types of bioactivity. In this study, the crude extract from A. scoparia (ASE) was tested for its ability to suppress adipogenesis in mouse 3T3-L1 pre-adipocytes. The molecular pathway by which ASE affects differ- entiation of 3T3-L1 cells was also investigated. The introduction of ASE to differentiating 3T3-L1 pre-adipocytes resulted in suppressed adipogenesis, as confirmed by decreased intracellular lipid accumulation. The differentiating cells treated with 10 and 100 μg/ml of ASE showed 21.9 and 29.0% less lipid accumulation, respectively, than untreated adipocytes. In addition, the results indicated that ASE treatment lowered the expression of the adipogenesis-related factors PPARγ, C/EBPα, and SREBP-1c. Furthermore, treating with ASE notably decreased levels of phosphorylated p38, ERK, and JNK in 3T3-L1 adipocytes. These results indicate that ASE exhibits significant anti-adipogenesis activ- ity by downregulating the MAPK and PPARγ pathways during the differentiation of 3T3-L1 pre- adipocytes. Therefore, A. scoparia may be a potential source of natural products against obesity. Key words : Adipogenesis, Artemisia scoparia, MAPK, PPARγ, 3T3-L1 Introduction pocytes, which are capable of storing excessive energy as fat and secreting adipose tissue-specific hormones that affect Obesity is characterized as irregular fat metabolism of the almost all metabolic pathways of the body [5]. During the body expressed as persistent fat accumulation. Obesity is the onset of obesity, the number of adipocytes rises redundantly main risk factor for most of major diseases such as Type as the adipose tissue expanses. The role of adipocytes, there- II diabetes [17], heart diseases [15], hypertension [31] and fore, is gaining increasing interest towards the efforts to pre- cancer [1]. In metabolic phenotype of obesity, the adipose vent and treat obesity along other metabolic diseases linked tissue function is abnormal and affected by several genetic with deteriorated adipocyte function [8]. Adipocyte cells are and environmental factors [18, 23]. Triacylglycerols are high- developed from pre-adipocytes through adipogenesis in- ly efficient sources of energy in the body, and mammals volving the conversion of mesenchymal stem cells (MSCs) have developed intricate mechanisms to store triacylglycer- to the pre-adipocytes, and differentiation of pre-adipocytes ols as fats in adipocytes to minimize the loss of energy [30]. into mature adipocytes. Adipose tissue is formed by specialized cell types called adi- Differentiation of the pre-adipocytes is strictly regulated by transcription factors and enzymes, mainly peroxisome *Corresponding author proliferator-activated receptor γ (PPARγ) and mitogen acti- *Tel : +82-51-999-5429, Fax : +82-51-999-5457 vated protein kinase (MAPK) pathways [6, 24]. PPARγ is *E-mail : [email protected] a member of the nuclear-receptor superfamily and has been This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License considered as the master regulator in adipogenesis along (http://creativecommons.org/licenses/by-nc/3.0) which permits CCAAT-enhancer-binding protein α (C/EBPα). Their se- unrestricted non-commercial use, distribution, and reproduction quential activation induces the expression of important pro- in any medium, provided the original work is properly cited. 1000 생명과학회지 2018, Vol. 28. No. 9 teins and enzymes in order to attain and maintain adipocyte pre-adipocytes was induced with a differentiation mixture characteristics. Several on the market obesity drugs, acting containing methylisobutylxanthine (0.5 mM), dexameth- as PPARγ ligands, target and inhibit this pathway of PPARγ asone (0.25 μM) and insulin (5 μg/ml) in culture medium through preventing the upregulation [4]. Recently, consid- after 2 days following the confluence as described earlier erable attention has been directed to development of active [11]. This was replaced with DMEM containing 10% FBS ingredients from natural sources with minor side effects and supplemented with insulin only (5 μg/ml) after 2 days of high biocompatibility for preventing and alleviating obesity. incubation. Initial culture medium was introduced after 2 Artemisia scoparia is a widespread plant that belongs to days incubation and replaced with fresh one every 2 days a very large flowering plant family of Asteraceae and grow- until the maturation of adipocytes. Successful adipocyte dif- ing natively across Eurasia. Leaves and flowers of A. scoparia ferentiation was confirmed by intracellular lipid droplets ob- are referred in traditional medicine sources with activities served under a light microscope (Nikon Instruments, Tokyo, such as diuretic, antiphlogistic and for treatment of hepatitis Japan). Sample was administered to the cell culture medium [25]. In addition, several studies reported antioxidant, in- starting with introduction of differentiation medium and in- secticidal, phytotoxic and anti-inflammatory properties of A. cluded in all medium changes. Cytotoxicity level of sample scoparia as well as chemical constituents derived from it such in 3T3-L1 cells was evaluated by MTT assay as previously as essential oils, flavonoids and coumarins [3, 7, 22, 28]. In described [11]. the present study, therefore, A. scoparia was analyzed to eval- uate its effect on the differentiation of 3T3-L1 adipocytes and Oil-Red O staining possible mechanism of action during adipogenesis. Accumulation of triglycerides as lipid droplets in 3T3-L1 adipocytes were confirmed by Oil-Red O staining of the tri- Materials and Methods glycerides with common staining procedures reported in our previous study [11]. Following the confirmation of adipo- Reagents genesis in cultured cells (6-well plate), culture medium was Fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s removed, and cells were washed with PBS. Cells were then medium (DMEM) were purchased from Gibco BRL (Grand fixed on wells with 3.7% fresh formaldehyde in PBS at room Island, NY). Antibodies for Western blotting were procured temperature for 1 hr. Oil-Red O (0.5% w/v) solution (60% from Cell Signaling Technology (Danvers, MA, USA). isopropanol and 40% water) was filtered and 1 ml of solution Primers for reverse transcription polymerase chain reaction was transferred to the wells. After staining incubation of 1 (RT-PCR) were obtained from Bio-RAD (Hercules, CA, hr, the Oil-Red O solution was aspired from the plates and USA). Remaining reagents were purchased from Sigma- the plates were washed with distilled water prior to ob- Aldrich (St. Tropez, MI, USA) unless otherwise specified. servation. Images of lipid droplets in 3T3-L1 adipocytes were collected by a Nikon Instruments microscope (Tokyo, Crude extract Japan). Finally, Oil-Red O stain retained in the cells was elut- The sample (3 kg) of A. scoparia was air dried and cut ed with 100% isopropanol and quantified by optical absorb- into small pieces prior to maceration. Ground sample was ance value at 500 nm using a microplate reader (Tecan extracted twice with methylene chloride (CH2Cl2) for 24 hr Austria GmbH, Austria). at room temperature. The extraction solution was dried in vacuo. The remains were then subjected to extraction again, RT-PCR twice with methanol (MeOH), using the same procedure as Analysis of mRNA levels was carried out by RT-PCR fol- above. Lastly, both extracts were combined and used for fur- lowing the method from previous reports [14]. Briefly, Total ther experiments. RNA was isolated from 3T3-L1 adipocytes using Trizol re- agent (Invitrogen Co., CA, USA) following the manu- Cell culture and adipocyte differentiation facturer’s instructions. For synthesis of cDNA from total Murine 3T3-L1 pre-adipocytes (ATCC® CL-173TM) were RNA isolates, RNA (1 μg) was added to RNase-free water cultured in DMEM supplemented with 10% FBS at 37℃ in and oligo (dT), denaturated at 70°C for 5 min and cooled a humidified atmosphere of 5% CO2. Differentiation of the immediately. RNA was reverse transcribed in a master mix Journal of Life Science 2018, Vol. 28. No. 9 1001 containing 1X RT buffer, 1 mM dNTPs, 500 ng oligo (dT), Results and Discussion 140 U M-MLV reserve transcriptase and 40 U RNase in- hibitor at 42°C for 60 min and at 72°C for 5 min using an Crude extract of A. scoparia (ASE) was used for evaluation automatic T100 Thermo Cycler (Bio-Rad, UK). The target of the anti-adipogenesis effect. Prior to in vitro experiments gene in synthesized cDNA was amplified using specific fol- using mouse pre-adipocyte 3T3-L1 cells, any cytotoxic prop- lowing sense and antisense primers as following: forward erties of ASE were analyzed with MTT formazan assay. The 5'-TTT-TCA-AGG-GTG-CCA-GTT-TC-3' and reverse 5'-AAT- cells were treated with or without ASE in various concen- CCT-TGG-CCC-TCT-GAG-AT-3' for PPARγ; forward 5‘- trations (10, 50, 100, 200 μg/ml).