August 20141341 Regular Article Biol. Pharm. Bull. 37(8) 1341–1351 (2014)
Eugenol Ameliorates Hepatic Steatosis and Fibrosis by Down-Regulating SREBP1 Gene Expression via AMPK-mTOR-p70S6K Signaling Pathway Hee Kyung Jo, Go Woon Kim, Kyung Ju Jeong, Do Yeon Kim, and Sung Hyun Chung* Department of Pharmacology and Clinical Pharmacy, College of Pharmacy, Kyung Hee University; Seoul 130–701, Republic of Korea. Received April 2, 2014; accepted May 22, 2014 Beneficial effect of eugenol on fatty liver was examined in hepatocytes and liver tissue of high fat diet (HFD)-fed C57BL/6J mice. To induce a fatty liver, palmitic acid or isolated hepatocytes from HFD-fed Sprague-Dawley (SD) rats were used in vitro studies, and C57BL/6J mice were fed HFD for 10 weeks. Lipid contents were markedly decreased when hepatocytes were treated with eugenol for up to 24 h. Gene expressions of sterol regulatory element binding protein 1 (SREBP1) and its target enzymes were suppressed but those of lipolysis-related proteins were increased. As a regulatory kinase for lipogenic transcriptional factors, the AMP-activated protein kinase (AMPK) signaling pathway was examined. Protein expressions of phosphorylated Ca2+-calmodulin dependent protein kinase kinase (CAMKK), AMPK and acetyl-CoA carboxylase (ACC) were significantly increased and those of phosphorylated mammalian target of rapamycin (mTOR) and p70S6K were suppressed when the hepatocytes were treated with eugenol at up to 100 µM. These effects were all reversed in the presence of specific inhibitors of CAMKK, AMPK or mTOR. In vivo studies, hepatic triglyceride (TG) levels and steatosis score were decreased by 45% and 72%, respectively, in eugenoltreated mice. Gene expressions of fibrosis marker protein such as α-smooth muscle actin (α-SMA), collagen type I (Col-I) and plasminogen activator inhibitor-1 (PAI-1) were also significantly reduced by 36%, 63% and 40% in eugenol-treated mice. In summary, eugenol may represent a potential intervention in populations at high risk for fatty liver. Key words eugenol; fatty liver; fibrosis; AMP-activated protein kinase; sterol regulatory element binding protein
Non-alcoholic fatty liver disease (NAFLD) is one of the most common liver ailment worldwide.1) While hepatic steatosis is often asymptomatic, it can progress to non-alcoholic steatohepatitis (NASH). If untreated, NASH can progress to cirrhosis and increased risk of early mortality.2) Obesity and insulin resistance, as seen in type 2 diabetes mellitus (T2DM), and hypertriglyceridemia are well-documented risk factors for NAFLD.3) These factors are key targets for prevention and therapy of NAFLD tends to focus on addressing the obesity or insulin resistance rather than NAFLD itself. NAFLD in the hepatic steatosis (HS) phase can be reversed by lifestyle modification, while NASH is more difficult to treat. Thus, preventing the progression of HS to NASH is of primary importance. Lifestyle recommendations for NAFLD are generally limited to losing weight through energy restriction and/or increasing physical activity, which is often to fail to hold on to them. With this notion, phytochemicals obtained from medicinal plants or foods are attracting alternative options for the treatment of NAFLD and prevention to progress to NASH. Eugenol (4-allyl-2-methoxyphenol) has been identified in several aromatic plants such as cloves, cinnamon, basil and nutmeg as a supplement or a therapeutic ingredient in various medications and foods. In addition, it is widely used in agricultural applications to protect foods from microorganisms during storage and as a pesticide and fumigant. As a functional ingredient, it is included in many dental preparations and it has also been shown to enhance skin permeation of various drugs.4) In recent years, eugenol has been reported to have antioxidant,5) anti-inflammatory,6) anti-viral7) and anticancer8–10) activities. Although beneficial effects of eugenol The authors declare no conflict of interest. * To whom correspondence should be addressed. e-mail:
[email protected]
on oxidative stress and inflammation have been well-studied, pharmacological effects on metabolic diseases are largely unknown. Here, we examined whether eugenol has a beneficial effect on NAFLD and mechanism(s) of action using human hepatoma HepG2 cells, rat primary hepatocytes isolated from high fat diet (HFD)-fed Sprague-Dawley (SD) rats and HFDfed C57BL/6J mice.
MATERIALS AND METHODS Materials Eugenol, metformin, Oil Red O and Masson’s trichrome stain kit were purchased from Sigma (St. Louis, MO, U.S.A.). Eugenol was dissolved in 0.1% dimethyl sulfoxide (DMSO). Palmitate (Sigma) was bound to bovine serum albumin (BSA) at a 2 : 1 M ratio.11) Antibodies against AMPactivated protein kinase (AMPK), phospho-AMPK, acetylCoA carboxylase (ACC), phospho-ACC, mammalian target of rapamycin (mTOR), phospho-mTOR, p70S6K and phosphop70S6K were from Cell Signaling Technology (Beverly, MA, U.S.A.) and anti-actin was from Santa Cruz Biotechnology (Santa Cruz, CA, U.S.A.). Ca2+-calmodulin dependent protein kinase kinase (CAMKK) and phospho-Ser/Thr antibodies were from BD Biosciences (San Jose, CA, U.S.A.). Reverse transcriptase, cell proliferation assay kits and Dual luciferase assay system were supplied by Promega (Madison, WI, U.S.A.), and compound C, STO-609 and rapamycin were from Calbiochem (Darmstadt, Germany). Protein extraction, EASYBLUE total RNA extraction, ECL-reagent, Taq polymerase, SYBR-green and lipofectamin were from Intron Biotechnology Inc. (Beverly, MA, U.S.A.), and protein assay kit was from Bio-Rad (Hercules, CA, U.S.A.). Regular diet (RD) and HFD (Table 1) were purchased from Research Diets, Inc. (New © 2014 The Pharmaceutical Society of Japan
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Brunswick, NJ, U.S.A.). The other reagents and chemicals were of analytical grade commercially available. Cell Culture and Viability Assay The human hepatoma cell line HepG2 was purchased from Korean Cell Line Bank (Seoul, Korea), and rat hepatocytes were isolated by collagenase perfusion of the liver of SD rat. HepG2 cells and primary rat hepatocytes were grown in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco BRL, Grand Island, NY, U.S.A.) supplemented with 10% fetal bovine serum (FBS), 100 unit/ mL penicillin and 100 µg/mL streptomycin. Cells were maintained at subconfluent conditions in a humidified incubator at 37°C with ambient oxygen and 5% CO2. The cytotoxicity of eugenol was determined by a Cell Titer 96 AQueous One solution Cell Proliferation Assay kit (Promega). In brief, cells were seeded at 1.5×104 cells/well in a 96-well plate and treated with eugenol as indicated. After 48 h of treatment, 20 µL of 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)2-(4-sulfophenyl)-2H-tetrazolium (MTS) solution was added and incubated at 37°C for 30 min and then the absorbance was recorded at 490 nm using an enzyme-linked immunosorbent assay (ELISA) plate reader (Thermo LabSystem, Helsinki, Finland). Isolation of Primary Rat Hepatocytes For hepatic lipid accumulation, male SD rats were fed a HFD, starting at 3 weeks of age for the next 4 weeks. Primary hepatocytes were isolated by a two-step collagenase perfusion in situ.12) Briefly, under anesthesia with pentobarbital (intraperitoneal, 30 mg/kg body weight), livers were perfused with a Ca2+-free Hanks’ balanced solution (Invitrogen, MA, U.S.A.) at 10 mL/ min for 20 min, followed by a continuous perfusion with
serum-free DMEM containing collagenase H (Roche, Indianapolis, IN, U.S.A.), 10 m M N-(2-hydroxyethyl) perazine-N′-2ethanesulfonic acid (HEPES) and 0.004 N NaOH at 10 mL/min for 20 min. Hepatocytes were harvested and then centrifuged for 5 min at 350 rpm. The supernatant was discarded and the pellet, representing the hepatocytes, was gently resuspended in 50 mL DMEM media containing 10% FBS, 10−7 M dexametasone, 10−8 M insulin, 100 unit/mL penicillin and 100 µg/mL streptomycin. After 4 h incubation in a humidified atmosphere of 95% air 5% CO2, the medium was changed to remove unattached hepatocytes, and after 16–18 h cells were suspended in DMEM media containing 10% FBS, 100 unit/mL penicillin and 100 µg/mL streptomycin. Immunoprecipitation and Western Blot Analysis Protein extracts from HepG2 cells were prepared by addition of protein extraction reagent to cells after washing with ice-cold phosphate buffered saline (PBS). To harvest proteins in liver, liver was removed and homogenized for 30 s, and then insoluble protein was removed by centrifugation at 13000 rpm for 20 min. The protein concentration of the cell lysates was measured using a Bio-Rad protein assay kit. For immunoprecipitation, 300 µg of cell lysate was cleared with 20 µL of protein G-sepharose beads (Santa Cruz, CA, U.S.A.) and 1 µg of anti-CAMKK antibody was used. After the addition of 20 µL of G-sepharose beads, incubation was continued for an additional 2 h at 4°C. The beads were then collected by centrifugation and washed three times with PBS, the supernatant was removed, 30 µL of 2× loading buffer was added, and samples were run on a 8% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). For Western blot analysis, the
Table 1. Composition of the Experimental Diets RD 10% kcal g% Protein Carbohydrate Fat
19.2 67.3 4.3
Total (kcal/gm)
3.85
Ingredient
g
Casein, 80 mesh L-Cystine Corn starch Maltodextrin 10 Sucrose Cellulose, BW 200 Soybean oil Lard Mineral mix S10026 Dicalcium phosphate Calcium carbonate Potassium citrate Vitamin mix v10001 Choline bitartrate FD&C Yellow dye #5 (RD) Red dye #40 (HFD) Total
HFD 45% kcal kcal% 20 70 10
g% 24 41 24
100
kcal% 20 35 45 100
4.73 kcal
g
kcal
200 3 315 35 350 50 25 20 10 13 5.5 16.5 10 2 0.05
800 12 1260 140 1400 0 225 180 0 0 0 0 40 0 0
200 3 72.8 100 172.8 50 25 177.5 10 13 5.5 16.5 10 2 0.05
800 12 291 400 691 0 225 1598 0 0 0 0 40 0 0
1055.05
4057
858.15
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August 20141343 Table 2. RT-PCR and Real-Time PCR Primer Sequence Gene (Forward/reverse)
PCR (5′→3′)
Real-time PCR (5′→3′)
hSREBP1
GTGGCGGCTGCATTGAGAGTGAAG AGGTACCCGAGGGCATCCGAGAAT CAAGAACTGCACGGAGGTGT AGCTGCCAGAGTCGGAGAAC TGCCAGCTCTAGCCTTTAAATTC GTACCGCTGGCACATCAACTT TGCAGCACTCACCACCTTC TAGGCATCCATGACAACTA CCAGCCTGTGCTACCTTCTC GAAGCTTCTTGTCCCACTG GAAGCTTCTTGTCCCACTG TCTTGCTGCCTGAATGTGAGTTGG GGGCTATAGGGATCCATTTTTG CCTTTCAGATTAACGTCGGATTC TCCACCACCCTGTTGCTGTA ACCACAGTCCATGCCATCAC ACGACGGAGCCATGGATTG TTTGATTGGAGGCCCAGGGG GCAGTCGCTCATCAAGCTCT GGCTCATTTTCCAGGCTACC GTGTTGAACCTTCCCCGACT TGGAGGTAGGGAGGATCTGG GGCCAACTATGGTGGACATCA TACCAATCTGGCTGCACGAA TGATCAGCCAGGAGCAGCTG AGACAGTATGTGGCACTCTC TATGTGAGGATGCTGCTTCC CTCGGAGAGCTAAGCTTGTC GAAGCATCGAAGAATCTGAAGAG TCCAACACCAAGTAAGACCATC ATGGTCAACCCACCGTG CTTAGAGGGACAAGTGGCG TCCTACACGAGGATCAAGCG AGTCGCAATGCAAAGACCTG GATCCTGGAACGAGAACAC TGCTGCCAAAAGACAAGGG GATCCTGGAACGAGAACAC AGACTGTGGAACACGGTGGT CGAGGGTTGGTTGTTGATCTG ATAGCACTGTTGGCCCTGGA GGTAGTGGATACTCTGTCGTCCA CAGCAACATCATTCGGT TCCTCTGACATTTGCAGGTCTATC GTGAATCCAGTTATGGGTTCCC ATCATGTATCGCCGCAAACT GGGATGCGTGTAGTGTTGAAC ACTATATTTGGCCAATTTTGTG TGTGGCAGTGGTTTCCAAGCC CTGACAGAGGCACCACTGAA CAGAGGCATAGAGGGACAGC GTGGACCTCCTGGACCTCAG AGGAGCTCCGTTTTCACCAG GACACCCTCAGCATGTTCATC AGGGTTGCACTAAACATGTCAG GGACTCCTATGGTGGGTGACGAGG GGGAGAGCATAGCCCTCGTAGAT
ATACCACCAGCGTCTACC CACCAACAGCCCATTGAG CGGCTCGCCCACCT CGGGCCGCAAAGC TCCTGGTAGCATTATTCAGTAGTT TTGGAGACTTTCTCCGGTCAT AACAGACGGAAGCCCAAGC TCGGTGAGTGACCATTGCTC AACCCCAGTATCCCGTCTTT CAGTCACATTGGTGGCAAC ACAGTCGGTGAGGCCTCTTATGAA TCTGCTGCCTGAATGTGAGTTGG TTGATGTGCAAAATCCACAGG TGTGTTGTCCTCAGCGTCCT CGACGACCCATTCAAAAATC AACCCTGATTCCCCATCAC CAGGTCCTTGAGCTCCACAATC GCCCACAATGCCATTGAGA CAACCTGCATTTCCACAACCCCAA ACCTCCGAAGCCAAACGAGTTGAT CAGAGCCAGGTGCCACTTTT TGCTAGAGGGTGTACCAAGCTTT GCCCTCAGCTATGGTATTAC AGGAACTGCTCTCACAATGC TGATCAGCCAGGAGCAGCTG AGACAGTATGTGGCACTCTC TATGTGAGGATGCTGCTTCC CTCGGAGAGCTAAGCTTGTC GACCATCGGCGGCGATGAGAAA CCAGGCCCAGGAGCTTTATT TTAGAGTTGTCCACAGTTCGGAGA GGACATCTAAGGGCATCACA TCGCAAATGCCGCCA TCAAGCGGATCTGTTCTTCTGA GCAGTCTGCTTTGGAACCTC CCTCCTGTGTACTTGCCCAT CCCTTGATGAAGAGGGATCA ACTCCACAGGTGGGAACAAG GCGATACACTCTGGTGCTCA CTGGCAGAGTCGAAGGG CCAGCCTGTGCTACCTTCTC GAAGCTTCTTGTCCCACTGC TACCTGGGAGTTGGCGAGAA TTGCCACGTCATCTGGGTTT GTGACTGGTGGGAGGAATAC GAGCATCTCCATGGCGTAG GTGCAGCTCAGAGTCTGTCCAA TACTGCTGCGTCTGAAAATCCA CTGCTCCAGCTATGTGTGA TTACAGAGCCCAGAGCCATT CCAAGGGTAACAGCGGTGAA CCTCGTTTTCCTTCTTCTCCG TCATCAATGACTGGGTGGAA GCCAGGGTTGCACTAAACAT TGACAGGATGCAGAAGGAGA CGCTCAGGAGGAGCAATG
hFAS hSCD1 hACO hGPAT hCPT-1 hCD36 hGAPDH rSREBP1 rFAS rSCD1 rACO rGPAT rCPT-1 rCD36 rCPN/18S mLXRαα mSREBP1 mFAS mSCD1 mGPAT mCD36 mCPT-1 mACO mα-SMA mCol-I mPAI-1 mActin
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Fig. 1. Effects of Eugenol on Lipid Accumulation in HepG2 Cells and Primary Rat Hepatocytes (A, E) HepG2 cells and primary rat hepatocytes were treated with different concentrations (50, 75, 100, 200 µM) of eugenol for 48 h, and cell viability was determined by MTS assay. (B, F) HepG2 cells were pretreated with eugenol as indicated for 2 h and incubated with 200 µM PA for 48 h. In rat primary hepatocytes, eugenol was treated for 24 h. To determine lipid levels, cells were stained with 0.2% Oil Red O and examined by light microscopy at a magnification of 200×. Relative intracellular lipid levels were determined by quantifying each lipid droplet using spectrophotometer at 490 nm. The mRNA levels of lipogenesis- and lipolysis-related genes were measured using RT-PCR (C, G) and real-time PCR (D, H). Data are mean±S.E. of three independent experiments. # p
