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/lp/multidisciplinary-digital-publishing-institute/erigeron-annuus-l-pers-extract-inhibits-reactive-oxygen-species-ros-Bp3qCkc2kk
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- Multidisciplinary Digital Publishing Institute
- Copyright
- © 1996-2019 MDPI (Basel, Switzerland) unless otherwise stated
- ISSN
- 2076-3921
- DOI
- 10.3390/antiox8050139
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Abstract
antioxidants Article Erigeron annuus (L.) Pers. Extract Inhibits Reactive Oxygen Species (ROS) Production and Fat Accumulation in 3T3-L1 Cells by Activating an AMP-Dependent Kinase Signaling Pathway 1 2 3 3 , Yoon-Hee Choi , Ok-Hwan Lee , Yulong Zheng and Il-Jun Kang * Department of Food and Nutrition, Hallym Polytechnic University, Chuncheon 24210, Korea; cyh@hsc.ac.kr Department of Food Science and Biotechnology, Kangwon National University, Chuncheon 24341, Korea; loh99@kangwon.ac.kr Department of Food Science and Nutrition, Hallym University, Chuncheon 24252, Korea; zyl1994@naver.com * Correspondence: ijkang@hallym.ac.kr; Tel.: +82-33-248-2135; Fax: +82-33-256-3420 Received: 18 April 2019; Accepted: 20 May 2019; Published: 20 May 2019 Abstract: Obesity is one of the major public health problems in the world because it is implicated in metabolic syndromes, such as type 2 diabetes, hypertension, and cardiovascular diseases. The objective of this study was to investigate whether Erigeron annuus (L.) Pers. (EAP) extract suppresses reactive oxygen species (ROS) production and fat accumulation in 3T3-L1 cells by activating an AMP-dependent kinase (AMPK) signaling pathway. Our results showed that EAP water extract significantly inhibits ROS production, adipogenesis, and lipogenesis during di erentiation of 3T3-L1 preadipocytes. In addition, EAP decreased mRNA and protein levels of proliferator-activated receptor (PPAR ) and CCAAT/enhancer-binding protein alpha (C/EBP ). Moreover, EAP suppressed mRNA expressions of fatty acid synthase (FAS), lipoprotein lipase (LPL), adipocyte protein 2 (aP2) in a dose-dependent manner. Whereas, EAP upregulated adiponectin expression, phosphorylation levels of AMPK and carnitine palmitoyltransferase 1 (CPT-1) protein level during di erentiation of 3T3-L1 preadipocytes. These results suggest that EAP water extract can exert ROS-linked anti-obesity e ect through the mechanism that might involve inhibition of ROS production, adipogenesis and lipogenesis via an activating AMPK signaling pathway. Keywords: Erigeron annuus (L.) Pers; 3T3-L1 adipocyte; ROS production; fat accumulation; AMPK signaling pathway 1. Introduction Obesity is a major public health problem around the globe because it is implicated in metabolic syndromes, including type 2 diabetes, and cardiovascular diseases. In 2005, the World Health Organization (WHO) reported that 1.6 billion adults are overweight and 0.4 billion are obese among adults worldwide [1]. High-calories diet and deskbound lifestyle are the most dominant factors contributing to obesity [2]. Due to the rapid increase of obesity-related diseases, cellular and molecular mechanisms underlying fat metabolism need to be clarified. Obesity is affected by both the number and size of adipose tissue that is accelerated by adipogenesis and lipogenesis progression [3,4]. Regulating adipogenesis, therefore, provides a promising therapeutic approach for preventing obesity. Moreover, recent reports have explored the mechanism study of adipocyte life cycles. such as adipogenesis, lipogenesis, and lipolysis using 3T3-L1 cells [5,6]. Expression of cell-specific adipogenic transcription factors in addition to regulation of cell phase together with proliferator-activated receptor (PPAR ) and CCAAT/enhancer-binding protein alpha Antioxidants 2019, 8, 139; doi:10.3390/antiox8050139 www.mdpi.com/journal/antioxidants Antioxidants 2019, 8, 139 2 of 12 (C/EBP ) can facilitate the expression of genes. Adipocytes di erentiation is a convoluted procedure exaggerated through numerous hormones and transcript aspects. Adipogenesis is accurately and forcefully controlled by transcriptional flow where transcript aspects can trigger or suppress each other ’s expression in a consecutive method. Transcriptional cascade strategic actors include PPAR , a nuclear receptor, and C/EBP family members, such as C/EBP , C/EBP , and C/EBP, known to be dominant in adipocyte di erentiation by both in vitro and in vivo studies [7]. Adipocyte di erentiation, C/EBP , PPAR , and sterol regulatory element binding protein-1c (SREBP-1c), are reflected strategic controllers in adipogenesis, comprising adiponectin, leptin, lipoprotein lipase (LPL), and adipocyte-specific fatty acid binding protein (FABP) [8–11]. SREBP-1c expression is known to be controlled by insulin and AMP-dependent kinase (AMPK) signaling pathway [12,13]. AMPK is one of the most important targets to inhibit and control obesity [14,15]. AMPK can control cellular metabolism and glucose and lipid metabolism, thus controlling energy. AMPK can rapidly activate the uptake, storage, and metabolism of numerous substrates after it is activated in adipose tissue. Moreover, AMPK can activate fatty acid -oxidation by inactivating acetyl-CoA carboxylase (ACC) and upregulating the expression of carnitine palmitoyltransferase 1 (CPT1) [16]. Oxidative stress reasons many responses, as well as degradation of numerous fragments and alteration of intracellular indicator transduction. In the course of the adipogenesis, adipocytes as well as produce variation adipogenic cytokines, ROS and fatty acids [17]. Recent studies suggest that fat cell-derivative ROS roles as a key regulator of metabolic syndrome, comprising insulin conflict and obesity [18,19]. Furthermore, ROS productions plays a serious starring role in the instruction of adipocyte di erentiation related to the improved adipogenic transcription factors gene expressions [20]. The current study proposes that the escalation of cellular ROS level is a common sign in the pathological complaint, such as lipid accumulation [18]. Thus, lipid homeostasis is sustained by the fine-turning of lipogenesis and lipolysis, it is controlled by accommodating feat of numerous enzymes in the metabolic organs, principally adipose tissues, the liver, and muscles. Erigeron annuus (L.) Pers. (EAP) is a biennial plant belonging to the daisy family and is native to North America. It also grows widely in Korea near mountains and lakes as a naturalized plant. It has been used to treat multiple disorders, e.g., cyanotic detoxification, acute gastroenteritis, acute infectious hepatitis, schizophrenia, hypoglycemia, and anti-inflammation [21,22]. Recent studies have revealed that EAP possesses functions of antimicrobial, antitumor, anti-inflammatory, antioxidant, and cellular imperviousness-improving e ects [23,24]. However, the anti-obesity e ect of EAP has not been studied yet. Thus, the objective of this study was to elucidate the e ect of EAP on ROS production and lipid accumulation in the di erentiation of 3T3-L1 preadipocytes by measuring levels of adipocyte-specific transcription factors such as C/EBP , PPAR , and phosphorylated AMPK and ACC. 2. Materials and Methods 2.1. Reagents Dulbecco’s modified Eagle’s medium (DMEM) was purchased from Lonza (Cologne, Germany). Fetal bovine serum (FBS), bovine calf serum (BCS), insulin, and penicillin-streptomycin (P/S) were purchased from Gibco (Gaithersburg, MD, USA). Nitro blue tetrazolium (NBT), dexamethasone and 3-isobutyl-1-methylxanthine (IBMX) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other reagents of the highest grade available were obtained from commercial sources. WST-1 was purchased from Roche Molecular Biochemicals (Mannheim, Germany). 2.2. Preparation EAP Extracts Erigeron annuus (L.) Pers. (EAP) sample was collected locally in Chuncheon, Kangwon-Do, Korea. Leaves and stems of EAP were subjected to natural drying at room temperature. These dried leaves and stems of EAP (488.73 g) were crushed to pieces. Crushed leaves and stems were extracted three times with various extraction solvents (water, 30%-ethanol, 50%-ethanol, 70%-ethanol, and 100%-ethanol) Antioxidants 2019, 8, 139 3 of 12 for 3 h. The extract was concentrated with a vacuum evaporator (Rotavapor R-220, Buchi, Flawil, Switzerland), followed by freeze-drying with a freeze-drier (Il Shin Lab Co., Seoul, Korea) to obtain powder (water extract: 21.8 g, 30%-ethanol extract: 20.4 g, 50%-ethanol extract: 25.7 g, 70%-ethanol extract: 23.3 g, 100%-ethanol extract: 10.2 g). Extraction yields of water extract, 30%-ethanol extract, 50%-ethanol extract, 70%-ethanol extract, and 100%-ethanol extract of EAP were 27.3%, 25.6%, 32.2%, 29.2%, and 12.8%, respectively. 2.3. Cell Culture and Di erentiation 3T3-L1 cells were purchased from the ATCC. Cells were seeded into 24-well plates and were cultured in DMEM containing 10% BCS and 1% P/S at 37 C with 5% CO . Two days after complete confluence was reached (D0), cells were cultured in MDI di erentiation medium (DMEM containing 0.5 mM/L IBMX, 1 M/L dexamethasone, and 10 g/mL insulin), 1% PS, and 10% FBS for three days (D3). To evaluate the e ects of EAP extract on adipocyte di erentiation of 3T3-L1 preadipocytes, cells were cultured in MDI in the presence of various concentrations of EAP extract. Cells were then maintained in regular medium containing 1% PS, 10% FBS, and 10 g/mL insulin (D5). After five days of induction, the medium was changed to DMEM containing 1% PS and 10% FBS (D7). On day seven, cells were harvested for further experiment. 2.4. Cell Viability Assay Cell viability of 3T3-L1 preadipocytes and adipocytes was assessed using WST-1 assay. 3T3-L1 preadipocyte and adipocytes cells were incubated with EAP extracts (0–300 g/mL) for 24 h and seven days, respectively. WST-1 was added to the cultured medium, followed by incubation for 2 h. Cell viability was measured in absorbance at a wavelength of 570 nm. 2.5. NBT Assay and Oil Red O (ORO) Staining The e ect of the EAP on ROS production was determined by NBT assay during the di erentiation of 3T3-L1 cells. NBT is reduced by ROS to a dark-blue, insoluble form of NBT called formazan. On day seven after induction, the cells were incubated for 90 min in PBS containing 0.2% NBT. Formazan was dissolved in 50% acetic acid, and the absorbance was determined at 570 nm. The e ect of the EAP on fat accumulation in 3T3-L1 cells was evaluated by ORO staining. 3T3-L1 cells were fixed with 4% formaldehyde in PBS for 1 h at room temperature and washed twice with 60% isopropanol. After performing ORO staining for 1 h at room temperature, cells were washed with water to remove the excess stain. Stained cells were allowed to air dry and ORO stained cells were eluted with DMSO for quantitative analysis. The absorbance was measured at a wavelength of 490 nm on a spectrophotometer. 2.6. Real-Time Polymerase Chain Reaction (RT-PCR) Total RNA was isolated from cells after seven days of maturation using high pure RNA isolation kit (Roche Applied Science, Penzberg, Germany). NanoDrop (NanoDrop 2000c, Thermo Scientific, Waltham, MA, USA) was used to quantify total RNA. Then 1 g of total RNA was converted into cDNA using a cDNA synthesis kit (Roche Applied Science, Penzberg, Germany). Real-time quantitation was performed using Light Cycler 480 (Roche Diagnostics, Manneim, Germany). PCR reaction mix contained Light Cycler 480 SYBR Green I Master (Roche, Germany). The real-time PCR conditions were as follows: 95 C for 10 min followed by forty-five cycles at 95 C for 15 s, 60 C for 5 s, 72 C for 15 s. All experiments were performed three or more times. Expression levels of target genes were normalized against glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or -actin as internal controls. Primers used in the experiment are shown in Table 1. Antioxidants 2019, 8, 139 4 of 12 Table 1. Description of target genes and their primer sequences used for real-time polymerase chain reaction (PCR). Gene Name Accession No. Forward Primer Reverse Primer NM_011146 CGCTGATGCACTGCCTATGA AGAGGTCCACAGAGCTGATTCC PPAR C/EBP BC058161 CGCAAGAGCCGAGATAAAGC CACGGCTCAGCTGTTCCA aP2 NM_024406 CATGGCCAAGCCCAACAT CGCCCAGTTTGAAGGAAATC ACC NM_133360 GAATCTCCTGGTGACAATGCTTATT GGTCTTGCTGAGTTGGGTTAGCT FAS NM_007988 CTGAGATCCCAGCACTTCTTGA GCCTCCGAAGCCAAATGAG LPL NM_008509 ATCGGAGAACTGCTCATGATGA CGGATCCTCTCGATGACGAA Adiponectin NM_009605 ATCCACACGTGTACTCAC AGCATGGTCTACTTCCAG NM_011480.3 GGACGAGCTGGCCTTCGGTGA ATAGGGGGCGTCAAACAGGCCC SPEBP-1c BC083080 GTATGACTCCACTCACGGCAAA GGTCTCGCTCCTGGAAGATG GAPDH 1 2 3 PPAR , peroxisome proliferator-activated receptor . C/EBP , CCAAT/enhancer binding protein . aP2, adipocyte 4 5 6 7 protein 2. ACC, acetyl-CoA carboxylase. FAS, fatty acid synthase. LPL, lipoprotein lipase. SREBP-1c, sterol regulatory element binding protein-1c. GADPH, glyceraldehydes-3-phosphate dehydrogenase. 2.7. Analysis of Protein Level Cells were harvested using a cell scraper and lysed to obtain whole cell lysate. Cells lysates were clarified by centrifugation at 12,000 g for 30 min. Protein concentrations were measured with bicinchoninic acid (BCA) protein assay kit (Pierce Biotechnology, Waltham, MA, USA). Then 20 g of the protein extract was mixed with 2 sample bu er, heated at 95 C for 5 min, separated by 10% SDS-PAGE, and transferred to PVDF membrane at 100 V for 90 min. Membranes were blocked with 1 TBST comprising 5% skim milk at room temperature for 1 h, incubated with primary antibody overnight at 4 C, washed five times with 1 TBST (10 min each wash), incubated with secondary antibody at room temperature for 1 h, and washed five times with 1 TBST (10 min each wash). PPAR , C/EBP , SREBP-1c, phospho-AMPK (p-AMPK), total-AMPK, and phospho-ACC (p-ACC), CPT1 antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). The target protein was detected with Luminata — Forte Western HRP substrate (Millipore, Tokyo, Japan). The density of a specific band was analyzed using image J software (NIH, Bethesda, MD). 2.8. Statistical Analysis Experimental results are presented as mean standard deviation (SD) of three experiments. All results were statistically analyzed by Duncan’s multivariate analysis. Di erence between averages was considered statistically significant when the p-value was less than 0.05. 3. Results 3.1. EAP Water Extract Inhibits Lipid Accumulation and Ros Production in 3t3-l1 Adipocytes To observe e ects of EAP extracts on adipocytes di erentiation, 3T3-L1 cells were treated with MDI in the presence or absence of various EAP extracts (water, 30%-ethanol, 50%-ethanol, 70%-ethanol, and 100%-ethanol) at a concentration of 100 g/mL. Among the various EAP extracts, water extract has the greatest inhibitory e ect on adipogenesis (Figure 1). Thus, it was further analyzed for its e ect on adipocyte di erentiation and molecular mechanisms involved in its e ect. To evaluate the e ect of EAP water extract on the cell viability of preadipocyte and adipocytes, cultured 3T3-L1 cells were treated with various concentrations of EAP water extract and cultured for 24 h or seven days followed by cell viability using WST-1. Treatment with EAP water extract did not significantly a ect cell viability (Figure 2). Thus, we used this extract at up to 200 g/mL in subsequent experiments. We further determined the e ect of EAP water extract on lipid accumulation and ROS production in 3T3-L1 adipocytes by ORO staining and NBT assay. EAP water extract treatment dose-dependently inhibits lipid accumulation during adipogenesis (Figure 3). Moreover, Antioxidants 2019, 8, x FOR PEER REVIEW 5 of 12 Antioxidants 2019, 8, x FOR PEER REVIEW 5 of 12 Antioxidants 2019, 8, 139 5 of 12 treatment dose-dependently inhibits lipid accumulation during adipogenesis (Figure 3). Moreover, treatment dose-dependently inhibits lipid accumulation during adipogenesis (Figure 3). Moreover, the production of dark-blue formazan, which represents ROS production, was decreased in the production of dark-blue formazan, which represents ROS production, was decreased in the production of dark-blue formazan, which represents ROS production, was decreased in adipocytes adipocytes treated with EAP water extract when compared with MDI treated cells (Figure 3). adipocytes treated with EAP water extract when compared with MDI treated cells (Figure 3). treated with EAP water extract when compared with MDI treated cells (Figure 3). Figure 1. Effects of various extracts of EAP on lipid accumulation in 3T3-L1 adipocytes. EAP: Figure 1. E ects of various extracts of EAP on lipid accumulation in 3T3-L1 adipocytes. EAP: Erigeron Figure 1. Effects of various extracts of EAP on lipid accumulation in 3T3-L1 adipocytes. EAP: Erigeron annuus (L.) Pers. water extract, Post-confluent 3T3-L1 preadipocytes were differentiated in annuus (L.) Pers. water extract, Post-confluent 3T3-L1 preadipocytes were di erentiated in the presence Erigeron annuus (L.) Pers. water extract, Post-confluent 3T3-L1 preadipocytes were differentiated in the presence of various EAP extracts (water, 30% ethanol, 50% ethanol, 70% ethanol and 100% of various EAP extracts (water, 30% ethanol, 50% ethanol, 70% ethanol and 100% ethanol) for seven the presence of various EAP extracts (water, 30% ethanol, 50% ethanol, 70% ethanol and 100% ethanol) for seven days. Lipid accumulation of cells was determined by Oil Red O (ORO) staining days. Lipid accumulation of cells was determined by Oil Red O (ORO) staining followed by reading ethanol) for seven days. Lipid accumulation of cells was determined by Oil Red O (ORO) staining followed by reading absorbance at 490 nm. Values are presented as mean ± standard deviation. absorbance at 490 nm. Values are presented as mean standard deviation. Di erences between means followed by reading absorbance at 490 nm. Values are presented as mean ± standard deviation. Differences between means were considered statistically significant at P < 0.05. were considered statistically significant at p < 0.05. Differences between means were considered statistically significant at P < 0.05. (A) (B) (A) (B) Figure 2. Effects of EAP water extract on cell viability of 3T3-L1 preadipocytes (A) and adipocytes (B). Figure 2. Effects of EAP water extract on cell viability of 3T3-L1 preadipocytes (A) and adipocytes 3T3-L1 cells were treated with EAP water extract at various concentrations (50, 100, 200, and 300 g/mL) Figure 2. Effects of EAP water extract on cell viability of 3T3-L1 preadipocytes (A) and adipocytes (B). 3T3-L1 cells were treated with EAP water extract at various concentrations (50, 100, 200, and 300 for 24 h (A) and seven days (B). Cell viability was measured by WST-1 assay. Values are presented as mean (B). 3T3-L1 cells were treated with EAP water extract at various concentrations (50, 100, 200, and 300 μg/mL) for 24 h (A) and seven days (B). Cell viability was measured by WST-1 assay. Values are standard deviation. Differences between means were considered statistically significant at p < 0.05. μg/mL) for 24 h (A) and seven days (B). Cell viability was measured by WST-1 assay. Values are presented as mean ± standard deviation. Differences between means were considered statistically presented as mean ± standard deviation. Differences between means were considered statistically significant at P < 0.05. significant at P < 0.05. Antioxidants 2019, 8, x FOR PEER REVIEW 6 of 12 Antioxidants 2019, 8, 139 6 of 12 Antioxidants 2019, 8, x FOR PEER REVIEW 6 of 12 (A) (B) (A) (B) Figure 3. Effects of EAP water extract on lipid accumulation (A) and ROS production (B) in 3T3-L1 Figure 3. E ects of EAP water extract on lipid accumulation (A) and ROS production (B) in 3T3-L1 adipocytes. EAP: Erigeron annuus (L.) Pers. water extract, 3T3-L1 preadipocytes were induced to adipocytes. Figure 3. Effe EAP: cts of Eriger EAP on water annuus extract (L.) on Pers. lipid water accum extract, ulation3T3-L1 (A) and ROS preadipocytes production wer (e B) in induced 3T3-L1 differentiate in the presence of EAP (added on D0 of differentiation) for seven days. (A) Lipid to adipocytes. EAP: di erentiate in theEr pr igero esence n annu of us EAP (L.) Per (added s. water on D0 extract, 3T3-L1 of di erentiation) prea for dipocytes wer seven days.e induced t (A) Lipid o accumulation was determined by measuring absorbance at 490 nm. (B) ROS production was accumulation differentiate was in the presence determined by measuring of EAP (adde absorbance d on D0 at of d 490 nm. ifferentiation) f (B) ROS production or seven was day determined s. (A) Lipid determined by using nitro tetrazolium blue chloride and absorbance at 570 nm. Values are byaccumulation was determin using nitro tetrazolium blueed by chloridemeasuring absorbance at 490 nm. ( and absorbance at 570 nm. Values arB e) prROS production was esented as mean presented as mean ± standard deviation. Differences between means were considered statistically standar determ d deviation. ined by uDi sing er nitro tetrazoliu ences between means m blu wer e chlo e consider ride and absorbance at 570 ed statistically significant nm. Values are at p < 0.05. significant at P < 0.05. presented as mean ± standard deviation. Differences between means were considered statistically 3.2. E ect of EAP Water Extract on Adipogenic and Lipogenic Gene Expressions significant at P < 0.05. 3.2. Effect of EAP Water Extract on Adipogenic and Lipogenic Gene Expressions We evaluated the e ect of EAP water extract on mRNA and protein expression of adipogenic We evaluated the effect of EAP water extract on mRNA and protein expression of adipogenic 3.2. Effect of EAP Water Extract on Adipogenic and Lipogenic Gene Expressions target gene during adipogenesis of 3T3-L1 cells using real-time RT-PCR and western blot. Our results target gene during adipogenesis of 3T3-L1 cells using real-time RT-PCR and western blot. Our results revealed that EAP water extract at a concentration of 200 g/mL significantly downregulated the We evaluated the effect of EAP water extract on mRNA and protein expression of adipogenic revealed that EAP water extract at a concentration of 200 μg/mL significantly downregulated the mRNA expression of PPAR and C/EBP (Figure 4). Moreover, EAP dose-dependently suppressed target gene during adipogenesis of 3T3-L1 cells using real-time RT-PCR and western blot. Our results mRNA expression of PPARγ and C/EBPα (Figure 4). Moreover, EAP dose-dependently suppressed protein levels of PPAR , C/EBP and SREBP-1c in 3T3-L1 cell parallel with mRNA expression of revealed that EAP water extract at a concentration of 200 μg/mL significantly downregulated the protein levels of PPARγ, C/EBPα and SREBP-1c in 3T3-L1 cell parallel with mRNA expression of PPAR and C/EBP gene. Garcinia cambogia at a concentration of 100 g/mL, used as the positive mRNA expression of PPARγ and C/EBPα (Figure 4). Moreover, EAP dose-dependently suppressed PPARγ and C/EBPα gene. Garcinia cambogia at a concentration of 100 μg/mL, used as the positive control, decreased protein levels of PPAR , C/EBP and SREBP-1c in 3T3-L1 cell (Figure 5). These protein levels of PPARγ, C/EBPα and SREBP-1c in 3T3-L1 cell parallel with mRNA expression of control, decreased protein levels of PPARγ, C/EBPα and SREBP-1c in 3T3-L1 cell (Figure 5). These results indicate that EAP water extract exerts the anti-adipogenic e ect by blocking PPAR and CEBP PPARγ and C/EBPα gene. Garcinia cambogia at a concentration of 100 μg/mL, used as the positive results indicate that EAP water extract exerts the anti-adipogenic effect by blocking PPARγ and expression, which might have implications in anti-obesity e ects. control, decreased protein levels of PPARγ, C/EBPα and SREBP-1c in 3T3-L1 cell (Figure 5). These CEBPα expression, which might have implications in anti-obesity effects. results indicate that EAP water extract exerts the anti-adipogenic effect by blocking PPARγ and CEBPα expression, which might have implications in anti-obesity effects. (B) (A) Figure 4. E ects of EAP on mRNA expression of PPAR (A) and C/EBP (B) in 3T3-L1 adipocytes. (B) (A) Figure 4. Effects of EAP on mRNA expression of PPARγ (A) and C/EBPα (B) in 3T3-L1 adipocytes. EAP: Erigeron annuus (L.) Pers. water extract, 3T3-L1 preadipocytes were di erentiated in the presence EAP: Erigeron annuus (L.) Pers. water extract, 3T3-L1 preadipocytes were differentiated in the of EAP at di erent concentrations (50 to 200 g/mL) for seven days. The mRNA expression level was Figure 4. Effects of EAP on mRNA expression of PPARγ (A) and C/EBPα (B) in 3T3-L1 adipocytes. presence of EAP at different concentrations (50 to 200 μg/mL) for seven days. The mRNA expression measured by real time-PCR and normalized using housekeeping gene GAPDH. Values are presented EAP: Erigeron annuus (L.) Pers. water extract, 3T3-L1 preadipocytes were differentiated in the level was measured by real time-PCR and normalized using housekeeping gene GAPDH. Values as mean standard deviation. Di erences between means were considered statistically significant at presence of EAP at different concentrations (50 to 200 μg/mL) for seven days. The mRNA expression are presented as mean ± standard deviation. Differences between means were considered p < 0.05. level was measured by real time-PCR and normalized using housekeeping gene GAPDH. Values statistically significant at P < 0.05. are presented as mean ± standard deviation. Differences between means were considered statistically significant at P < 0.05. Antioxidants 2019, 8, x FOR PEER REVIEW 7 of 12 Antioxidants 2019, 8, 139 7 of 12 Figure 5. E ects of EAP on protein levels of PPAR , C/EBP and SREBP-1c in 3T3-L1 adipocytes. EAP: Erigeron annuus (L.) Pers. water extract. These cells were treated with EAP (from 50 to 200 g/mL) for seven days during di erentiation. (a) Western blot analysis of PPAR , C/EBP , and SREBP-1c. At day Figure 5. Effects of EAP on protein levels of PPARγ, C/EBPα and SREBP-1c in 3T3-L1 adipocytes. seven, total protein was harvested. (b–d) Protein levels were determined by western blotting. Values EAP: Erigeron annuus (L.) Pers. water extract. These cells were treated with EAP (from 50 to 200 are presented as mean standard deviation. Di erences between means were considered statistically μg/mL) for seven days during differentiation. (a) Western blot analysis of PPARγ, C/EBPα, and significant at p < 0.05. SREBP-1c. At day seven, total protein was harvested. (b–d) Protein levels were determined by western blott In order to ing. Values are confirm whether presented as this inhibitory mean ± standa e ectrd deviation. of EAP water Differences extract on betlipid ween means accumulation is were considere mediated thrd ough statist inhibition ically significant at of adipogenesis P < 0.05. and lipogenesis involving PPAR , C/EBP , and SREBP-1c, we further checked the FAS, LPL, aP2 and adiponectin gene expressions using RT-PCR. In order to confirm whether this inhibitory effect of EAP water extract on lipid accumulation is As shown in Figure 6, EAP water extract markedly suppressed mRNA expression of fatty acid mediated through inhibition of adipogenesis and lipogenesis involving PPARγ, C/EBPα, and synthase (FAS), LPL, and adipocyte protein 2 (aP2) in a dose-dependent manner. However, EAP water SREBP-1c, we further checked the FAS, LPL, aP2 and adiponectin gene expressions using RT-PCR. As extract dose-dependently increased adiponectin gene expression. These results indicated that EAP shown in Figure 6, EAP water extract markedly suppressed mRNA expression of fatty acid synthase water extract inhibits lipid accumulation and ROS production in 3T3-L1 adipocytes via inhibition of (FAS), LPL, and adipocyte protein 2 (aP2) in a dose-dependent manner. However, EAP water extract adipogenesis and lipogenesis. dose-dependently increased adiponectin gene expression. These results indicated that EAP water extract inhibits lipid accumulation and ROS production in 3T3-L1 adipocytes via inhibition of adipogenesis and lipogenesis. Antioxidants 2019, 8, x FOR PEER REVIEW 8 of 12 Antioxidants 2019, 8, 139 8 of 12 Figure 6. Effects of EAP on mRNA expressions of FAS, LPL, aP2, ACO, ACC and adiponectin genes in 3T3-L1 adipocytes. EAP: Erigeron annuus (L.) Pers. water extract. Cells were treated with EAP (50, 100, and 200 g/mL) for seven days during differentiation. At day seven, mRNA expression levels of FAS, LPL, Figure 6. Effects of EAP on mRNA expressions of FAS, LPL, aP2, ACO, ACC and adiponectin genes aP2 and adiponectin were determined by real-time PCR. Results are expressed as fold increase compared to in 3T3-L1 adipocytes. EAP: Erigeron annuus (L.) Pers. water extract. Cells were treated with EAP (50, the control after normalizing against GAPDH expression level. Values are presented as mean standard 100, and 200 μg/mL) for seven days during differentiation. At day seven, mRNA expression levels deviation. Differences between means were considered statistically significant at p < 0.05. of FAS, LPL, aP2 and adiponectin were determined by real-time PCR. Results are expressed as fold 3.3. EAP Water Extract Enhances AMPK Phosphorylation and Its Downregulation ACC increase compared to the control after normalizing against GAPDH expression level. Values are presented as mean ± standard deviation. Differences between means were considered statistically To elucidate how EAP water extract inhibited lipid accumulation and ROS production during significant at P < 0.05. the adipocyte di erentiation, AMPK phosphorylated protein level and ACC were measured. We determined whether EAP (200 g/mL) controlled the di erentiation of adipocytes and vitality 3.3. EAP Water Extract Enhances AMPK Phosphorylation and Its Downregulation ACC metabolism via the AMPK pathway in 3T3-L1 cells. The results show that EAP enhances AMPK To elucidate how EAP water extract inhibited lipid accumulation and ROS production during phosphorylation and ACC in MDI-induced adipocyte di erentiation. In addition, EAP upregulated CPT1 the adip protein ocyte differen level (Figur tiateion 7)., AMPK Garcinia pcambogia hosphorylat alsoed upr protei egulated n level both and ACC were measured phosphorylation AMPK. We and determined whether EAP (200 μg/mL) controlled the differentiation of adipocytes and vitality ACC as well as CPT1 protein levels in 3T3-L1 cells. These results indicated that EAP water extract may inhibit metabolism via t adipogenesis he AMPK pat and lipogenesis hway in via an 3T3-L1 cells activating. AMPK The resul signaling ts show pathway that EA . P enhances AMPK phosphorylation and ACC in MDI-induced adipocyte differentiation. In addition, EAP upregulated CPT1 protein level (Figure 7). Garcinia cambogia also upregulated both phosphorylation AMPK and ACC as well as CPT1 protein levels in 3T3-L1 cells. These results indicated that EAP water extract may inhibit adipogenesis and lipogenesis via an activating AMPK signaling pathway. Antioxidants Antioxidants 2019 2019 , , 88 , , x FOR 139 PEER REVIEW 9 of 9 of 12 12 Figure 7. E ects of EAP on the activation of AMPK signaling in 3T3-L1 adipocytes. EAP: Erigeron Figure 7. Effects of EAP on the activation of AMPK signaling in 3T3-L1 adipocytes. EAP: Erigeron annuus (L.) Pers. water extract. Cells were treated with EAP at 200 g/mL during MDI-induced annuus (L.) Pers. water extract. Cells were treated with EAP at 200 μg/mL during MDI-induced adipocytes. p-AMPK, AMPK, p-ACC, ACC, and CPT1 were determined by Western blot analysis. adipocytes. p-AMPK, AMPK, p-ACC, ACC, and CPT1 were determined by Western blot analysis. Band densities of p-AMPK and p-ACC were quantified and normalized with those of AMPK and Band densities of p-AMPK and p-ACC were quantified and normalized with those of AMPK and ACC expression levels, respectively. Values are presented as mean standard deviation. Di erences ACC expression levels, respectively. Values are presented as mean ± standard deviation. Differences between means were considered statistically significant at p < 0.05. between means were considered statistically significant at P < 0.05. 4. Discussion 4. Discussion Erigeron annuus (L.) Pers. (EAP) is a naturalized plant belonging to the daisy family. EAP has Erigeron annuus (L.) Pers. (EAP) is a naturalized plant belonging to the daisy family. EAP has been used to treat a variety of diseases in Korea, Japan, and China, including bronchitis, cough, been used to treat a variety of diseases in Korea, Japan, and China, including bronchitis, cough, and and convulsions [17,18]. However, the antiobesity activity of EAP has not been reported yet. This study convulsions [17,18]. However, the antiobesity activity of EAP has not been reported yet. This study evaluated the e ect of EAP water extract on lipid accumulation and ROS production in 3T3-L1 cells. evaluated the effect of EAP water extract on lipid accumulation and ROS production in 3T3-L1 cells. 3T3-L1 preadipocyte cells were treated with EAP water extract at varying concentrations. To further 3T3-L1 preadipocyte cells were treated with EAP water extract at varying concentrations. To further clarify the mechanisms involved in this e ect, we investigated the e ects of EAP on expression levels clarify the mechanisms involved in this effect, we investigated the effects of EAP on expression of adipogenic and lipogenic target gene as well as the AMPK signaling pathway. levels of adipogenic and lipogenic target gene as well as the AMPK signaling pathway. Our data demonstrated that EAP water extract attenuated the lipid accumulation by up to Our data demonstrated that EAP water extract attenuated the lipid accumulation by up to 67.4% 67.4% compared to MDI-treated control cells (Figure 3). Treatment with EAP water extract also compared to MDI-treated control cells (Figure 3). Treatment with EAP water extract also dose-dependently inhibits mRNA and protein expression of PPAR and C/EBP paralleled to reduced dose-dependently inhibits mRNA and protein expression of PPARγ and C/EBPα paralleled to lipid accumulation in adipocytes (Figure 5). During di erentiation of preadipocyte, adipogenesis reduced lipid accumulation in adipocytes (Figure 5). During differentiation of preadipocyte, is activated through the action of transcription factors, such as PPAR , C/EBP , and SREBP-1c [25]. adipogenesis is activated through the action of transcription factors, such as PPARγ, C/EBPα, and SREBP-1c is one of the earliest lipogenic genes besides LPL and FAS [26,27]. In the present study, EAP SREBP-1c [25]. SREBP-1c is one of the earliest lipogenic genes besides LPL and FAS [26,27]. In the water extract can inhibit adipogenesis and lipogenesis by downregulating adipogenic and lipogenic present study, EAP water extract can inhibit adipogenesis and lipogenesis by downregulating markers. Recent studies have shown that C/EBP , C/EBP , and C/EBP can also regulate adipocyte adipogenic and lipogenic markers. Recent studies have shown that C/EBPα, C/EBPβ, and C/EBPδ gene expression. In the early stage of adipocyte di erentiation, C/EBP and C/EBP activation can also regulate adipocyte gene expression. In the early stage of adipocyte differentiation, C/EBPβ and C/EBPδ activation can increase the expression of C/EBPα, PPARγ, and other lipogenic agents Antioxidants 2019, 8, 139 10 of 12 can increase the expression of C/EBP , PPAR , and other lipogenic agents [28,29]. We found that mRNA expression levels of FAS, LPL, and aP2 in 3T3-L1 cells after EAP treatment were significantly downregulated compared to those in DW-treated cells (Figure 6). LPL is linked to ACC and the hydrolysis of plasma triglycerides that contributes to fatty acid synthesis [30]. FAS is a lipogenic enzyme which is an important enzyme catalyzing the last step of fatty acid synthesis [31]. aP2 can stimulate adipogenesis which is controlled by C/EBP and PPAR transcriptional levels [32]. aP2 gene is vital to pathways linking obesity to insulin resistance. These results indicate that EAP water extract can prevent MDI-induced expression of a gene linked to adipogenesis and lipogenesis. The major regulator of energy metabolism is AMPK. Phosphorylated AMPK plays a role in the regulation of adipocyte differentiation [33]. AMPK has evolved to become a motivational target, especially for the treatment of obesity. It is possible to regenerate a key to inhibit lipogenesis of 3T3-L1 cells by natural complexes [34]. For this reason, AMPK modulation has been predicted to be a key to controlling obesity based on scientific research. We elucidated that EAP water extract could inhibit adipogenesis and phosphorylation level of AMPK and substrate ACC. AMPK can lead to fatty acid -oxidation through inactivation of ACC and aggregate CPT1 expression gene in adipose tissue [16]. We found that EAP (200 g/mL) could control the differentiation of adipocytes and vitality metabolism via the AMPK pathway in 3T3-L1 cells. EAP could enhance AMPK phosphorylation and ACC through MDI-induced adipocytes differentiation. EAP also upregulated CPT1 gene expression (Figure 7). These results suggest that EAP water extract inhibits lipid accumulation via activating AMPK signaling pathway. In conclusion, our data demonstrate that EAP water extract inhibits lipid accumulation and ROS production in 3T3-L1 cells. Moreover, EAP water extract is capable of inhibiting adipocytes di erentiation via downregulation of expression levels of C/EBP and PPAR . Furthermore, EAP water extract promotes AMPK phosphorylation and its downstream ACC. Phosphorylated AMPK also increases the expression of the fatty acid oxidase CPT1 gene. Based on these findings, EAP water extract may be useful as an e ective natural product to prevent obesity and obesity-related metabolic syndrome. Future identifying studies on bioactive compounds in EAP water extract and in vivo tests using animal HFD-induced obesity models are needed to examine whether EAP water extract could be used as a therapeutic agent and developed as an anti-obesity material, such as a functional food. 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Journal
Antioxidants – Multidisciplinary Digital Publishing Institute
Published: May 20, 2019