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Effect of Capsosiphon fulvescens on Ethanol-induced Liver Damage in HepG2 Cells over Expressing CYP2E1

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Journal of ood and Nutrition esearch, 2017, Vol. 5, No. 7, Available online at Science and Education Publishing DOI: /jfnr Effect of Capsosiphon
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Journal of ood and Nutrition esearch, 2017, Vol. 5, No. 7, Available online at Science and Education Publishing DOI: /jfnr Effect of Capsosiphon fulvescens on Ethanol-induced Liver Damage in HepG2 Cells over Expressing CYP2E1 Haneul Jo 1,#, Ok-Kyung Kim 1,2,#, Ho-Geun Yoon 3, Eungpil Kim 4, Kyungmi Kim 5, Yoo-Hyun Lee 6, Kyung-Chul Choi 7, Jeongmin Lee 8, Jeongjin Park 1,2,*, Woojin Jun 1,2,* 1 Division of ood and Nutrition, Chonnam National University, Gwangju, South Korea 2 esearch Institute for Human Ecology, Chonnam National University, Gwangju, South Korea 3 Department of Biochemistry and Molecular Biology, Brain Korea 21 PLUS Project for Medical Sciences, Yonsei University College of Medicine, Seoul, South Korea 4 Marine Biotechnology esearch Center, Wando, South Korea 5 Department of Biofood Analysis, Korea Bio Polytechnic, Ganggyung, South Korea 6 Department of ood and Nutrition, The University of Suwon, Suwon, South Korea 7 Department of Biomedical Sciences and Department of Pharmacology, University of Ulsan College of Medicine, Seoul, South Korea 8 Department of Medical Nutrition, Kyung Hee University, Yongin, South Korea # Equally contributed to this work. *Corresponding author: Abstract In the present study, the protective effects of 10% ethanol extract of Capsosiphon fulvescens (CE10) against alcoholic liver damage were investigated in vitro using CYP2E1-overexpressing hepatocytes (HepG2/2E1). To determine whether CE10 attenuated ethanol-induced cell death, we compared the viability of HepG2/2E1 cells treated with 250 mm ethanol in the presence or absence of CE10. Cell viability significantly increased after treatment with CE10 and ethanol compared with that of cells treated with only ethanol. Additionally, CE10 inhibited ethanol-induced OS formation and lipid peroxidation. We also found that CE10 attenuated the mna expression of CYP2E1, as well as decreased ethanol-induced lipid droplets, through stimulation of the AMPK pathway. Based on these results, the protective effect of CE10 extract from C. fulvescens against liver damage and fatty liver induced by ethanol may occur via the alleviation of oxidative stress. Keywords: capsosiphon fulvescens, alcohol, CYP2E1, reactive oxygen species, liver damage Cite This Article: Haneul Jo, Ok-Kyung Kim, Ho-Geun Yoon, Eungpil Kim, Kyungmi Kim, Yoo-Hyun Lee, Kyung-Chul Choi, Jeongmin Lee, Jeongjin Park, and Woojin Jun, Effect of Capsosiphon fulvescens on Ethanol-induced Liver Damage in HepG2 Cells over Expressing CYP2E1. Journal of ood and Nutrition esearch, vol. 5, no. 7 (2017): doi: /jfnr Introduction Ingested alcohol (ethanol) is absorbed from the stomach and small intestine. As it cannot be stored in the body, it is subsequently oxidized in the liver [1]. In the liver, alcohol is converted to acetaldehyde by alcohol dehydrogenase (ADH), a cytosolic enzyme, or cytochrome P-450 2E1 (CYP2E1), a membrane-bound protein. Acetaldehyde is oxidized to acetate by the mitochondrial enzyme acetaldehyde dehydrogenase (ALDH), which ultimately produces CO2 and water [2]. The metabolism of alcohol in the liver induces the increase of NADH and reactive oxygen species (OS), which can cause several liver diseases including alcoholic fatty liver, alcoholic hepatitis, alcoholic fibrosis, and alcoholic cirrhosis [3]. CYP2E1 has been shown to be upregulated by a chronic or excessive alcohol intake and is a major factor in oxidative stress and liver injury via the generation of OS. Thus, chronic or excessive alcohol consumption can induce the overproduction of OS, which destroys the antioxidant defense systems and leads to oxidative stress in the liver [2,3,4]. Although the body has several antioxidant defense systems for the elimination of OS, including antioxidant nutrients and enzymes such as superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (G), and glutathione (GSH), when OS levels reach a certain threshold, the antioxidant defense systems are aggravated and subsequently induce lipid peroxidation (LPO) and cell damage in the liver [5]. In the liver, an increase in the redox potential (NADH/NAD) by alcohol oxidation promotes fatty liver and cell damage through inhibition of fatty acid oxidation and the activation of the TCA cycle [6]. In addition, several recent reports have shown that OS production by CYP2E1 activation caused hepatic lipogenesis through the inhibition of AMP-activated protein kinase (AMPK) expression and an increase in the expression of sterol regulatory element-binding protein (SEBP)-1 [7,8,9]. Because AMPK is necessary for cellular energy homeostasis, including fatty acid oxidation and the inhibition of lipogenesis in the liver, the inhibition of AMPK may be a key factor in the development of fatty liver and liver injury arising from alcohol consumption Journal of ood and Nutrition esearch 511 [6,7,8,9]. Therefore, alcohol-induced CYP2E1 activation plays a key role in the development of liver damage. According to the 2014 survey The State of World isheries and Aquaculture, there were approximately 16 million tons of aquatic plants, of which 14.9 million tons were produced by aquaculture [10]. ecent studies have established several components in aquatic plants that are biologically active or show effects on health [11]. In the present study, we assessed the potential hepatoprotective effects of Capsosiphon fulvescens, which is a green algae belonging to the Ulotrichaceae family, traditionally eaten in the southwestern regions of Korea. Although some studies have reported the antioxidant [12], anticancer [13], and immunomodulatory effects [14] of C. fulvescens, the hepatoprotective effects have not been studied. Therefore, we investigated the hepatoprotective effects of 10% ethanol extract from C. fulvescens against ethanol-induced oxidative stress in HepG2 cells overexpressing CYP2E1 (HepG2/2E1). 2. Methods 2.1. Extraction C. fulvescens was harvested from Wando (Jeonnam, Korea). Dried, whole C. fulvescens was ground into powder. The powder of C. fulvescens (25 g) was refluxed with 1.5 L of 10% ethanol at 250 C for 3 h. The extract was filtered with Whatman paper No. 6 and concentrated in rotary evaporator under reduced pressure. The concentrate was lyophilized and stored at -20 C until used. The yield of 10% ethanol extract of C. fulvescens (CE10) was 25.2 ± 0.6 % Cell Culture and Transfection The HepG2 cell line was obtained from the American Type Culture Collection (ockville, MD, USA). The vector containing human CYP2E1 cdna was transfected into the cells by using Lipofectamine 2000 TM transfection reagent (Invitrogen, Carlsbad, CA, USA) to produce CYP2E1-overexpressing HepG2 cells (HepG2/2E1). After 24 h, the cells were trypsinized and seeded at a low cell density into 10-cm culture dishes in minimum essential medium (MEM) containing 10% fetal bovine serum (BS) with 24 μg plasmid DNA. The transfected cells were grown in MEM supplemented with 10% BS and 1% antibiotics (100 U/mL penicillin A and 100 U/mL streptomycin) and were maintained at 37 C in a humidified atmosphere of 5% CO Cytotoxicity The cells were seeded at cells/well in a 24-well culture plate and grown as described above. After incubation for 16 h, the medium was removed and the cultured cells were washed twice with 1 PBS. Then, 1 ml MEM containing 3% BS with CE10 was transferred into each well and 250 mm ethanol was added. After incubation for 5 days, a colorimetric cell viability assay was performed. reshly prepared XTT-PMS solution (0.25 ml; composed of 1 mg XTT and 10 mg PMS/mL phosphate-buffered saline [PBS]) was added to each well and incubated for an additional 2 h. After incubation, the absorbance was measured at a wavelength of 450 nm by using a spectrophotometer. The cytotoxicity was expressed as a percentage relative to the control wells, which contained no sample Hepatoprotective Effects The cells were seeded at cells/well in a 24-well culture plate and grown as described above. After incubation for 16 h, the medium was removed and the cultured cells were washed twice with 1 PBS. Then, 1 ml MEM containing 3% BS with CE10 was transferred into each well and 250 mm ethanol was added. After incubation for 5 days, the cytotoxicity was measured according to the XTT assay described above Measurement of Intracellular OS Intracellular OS levels were detected using the fluorescence probe 2,7 -dichlorofluorescein diacetate (DC-DA). The cells were seeded at cells/well in a 24-well culture plate and grown as described above. After incubation for 16 h, the medium was removed and the cultured cells were washed twice with 1 PBS. Then, 1 ml MEM containing 3% BS with CE10 was transferred into each well and 250 mm ethanol was added. After incubation for 5 days, the cells were incubated with 30 μm DC-DA for an additional 30 min at 37 C. The fluorescence intensity in the cells was measured on a plate fluorescence reader with an excitation wavelength of 485 nm and an emission wavelength of 530 nm Measurement of Antioxidant Enzyme Activity The cells were grown in a 100-mm culture dish. At 16 h after seeding, the growth medium was removed and the cells were washed twice with PBS. Then, 1 ml MEM containing 3% BS with CE10 was transferred into each well and 250 mm ethanol was added. After incubation for 5 days, the cells were harvested and lysed using CelLytic MT cell lysis reagent. SOD activity was assayed by the method of McCord and ridovich (1969) [15], CAT activity was determined as described by Aebi (1984) [16], hepatic glutathione-s-transferase (GST) activity was assayed according to the method of Habig and Jakoby (1981) [17], GPx activity was estimated by the method of Pagila and Valentine (1967) [18], and G activity was measured using an adaptation of the method of Calberg and Mannervik (1975) [19]. The level of glutathione (GSH), a key intracellular antioxidant, was measured by method of Akerboom and Sies (1981) [20]. The concentration of malondialdehyde (MDA), the end product of lipid peroxidation, was assayed by monitoring thiobarbituric acid reactive substance formation as described by Draper and Hadley (1990) [21]. The amount of protein was estimated using the Bradford assay. 512 Journal of ood and Nutrition esearch 2.7. Measurement of Intracellular Lipid Droplets The cells were seeded at cells/well in a 24-well plate. At 16 h after seeding, the growth medium was removed and washed twice with PBS. Then, 1 ml MEM containing 3% BS with CE10 was transferred into each well and 250 mm ethanol was added. After incubation for 5 days, the cells were harvested and the level of intracellular lipid droplets was measured using Adipoed Assay eagent kits (Lonza, Walkersville, MD, USA) Isolation of Total NA and eal-time PC Total NA was extracted using the easy-blue TM total NA extraction kit (Intron Biotechnology, Gyeonggi-do, Korea) and complementary DNA was synthesized from purified total NA in the reaction buffer by using the iscript cdna Synthesis Kit (Bio-ad Laboratories, Hercules, CA, USA). eal-time PC was performed using a SYB green T-PC kit obtained from Qiagen (Venlo, Netherlands) and custom-designed primers (Table 1). The cdna was amplified for 40 cycles of denaturation (95 C for 30 s), annealing (58 C for 30 s), and extension (72 C for 45 s). The results of the real-time T-PC were processed with the 7500 System SDS software version (Applied Biosystems, oster City, CA, USA), which was also used to process the quantitative data Statistical Analysis Data are presented as the mean ± SD. The data were statistically evaluated with one-way ANOVA using SPSS statistical procedures for Windows (SPSS PASW Statistics 22.0, SPSS Inc. Chicago, IL, USA) and Duncan s multiple range test was used to compare significant differences between the groups at p esults 3.1. Cytotoxicity and Hepatoprotective Effects of C. fulvescens Extract in HepG2/2E1 Cells Treated with Ethanol CE10 showed no signs of cytotoxicity at μg/ml in HepG2/2E1 cells (igure 1). We investigated the effect of 200 μg/ml and 500 μg/ml CE10 in ethanol-treated HepG2/2E1 cells. Gene β-actin CYP2E1 AMPK SEBP-1c ACC CPT-1 PPA-α Table 1. Primer sets used for real-time PC Sequence 5'- ACGGCCAGGTCATCACTATTG-3' 5'- CAAGAAGGAAGGCTGGAAAAGA-3' 5'- CGTGGAAATGGAGAAGGAAA-3' 5'- GGTGATGAACCGCTGAATCT-3' 5'- GGCACCCTCCCATTTGATG-3' 5'- ACACCCCCTCGGATCTTCTT-3' 5'- CGGAACCATCTTGGCAACA-3' 5'- GCCGGTTGATAGGCAGCTT-3' 5'- TGCAGATCTTAGCGGACCAA-3' 5'- GCCTGCGTTGTACAGAGCAA-3' 5'- TGTTGGGTATGCTGTTCATGACA-3' 5'- GCGGCCTGGGTAGGAAGA-3' 5'- AACATCCAAGAGATTTCGCAATC-3' 5'- CCGTAAAGCCAAAGCTTCCA-3' igure 1. Viability of the HepG2/2E1 cells following treatment for 5 days with different concentrations of 10% ethanol extract of Capsosiphon fulvescens (CE10). The data are expressed as the mean ± SD (n=3) and significant differences were analyzed using Duncan s multiple-range test. NS=not significant. Journal of ood and Nutrition esearch 513 igure 2. Hepatoprotective effects of 10% ethanol extract of Capsosiphon fulvescens (CE10) in ethanol-treated HepG2/2E1 cells. The data are expressed as the mean ± SD (n=3), and different letters indicate (a b c) a significant difference at p 0.05, as determined by a Duncan's multiple range test igure 3. Effects of 10% ethanol extract of Capsosiphon fulvescens (CE10) on the production of intracellular OS in ethanol-treated HepG2/2E1 cells. The data are expressed as the mean ± SD (n=3), and different letters indicate (a b c) a significant difference at p 0.05, as determined by a Duncan's multiple range test Table 2. Effects of Capsosiphon fulvescens extracted by 10% ethanol (CE10) on antioxidant enzyme activity in ethanol-treated HepG2/2E1 cells CAT SOD GST G GPx GSH Control ± a ± 9.61 a ± 2.04 a ± 8.18 a ± 1.78 a ± 1.31 a Ethanol ± c ± 5.09 b 8.38 ± 1.95 b ± 8.47 b ± 1.20 b ± 1.16 b CE ± b ± 9.93 a ± 0.95 a ± 4.40 a ± 3.45 a ± 1.53 a CE ± b ± 4.24 a ± 1.97 a ± 2.85 a ± 1.89 a ± 1.69 a The data are expressed as the mean ± SD (n=3), and different letters indicate (a b c) a significant difference at p 0.05, as determined by a Duncan's multiple range test. The protective effects of 200 μg/ml and 500 μg/ml CE10 against ethanol-induced toxicity are illustrated in igure 2. The viability of ethanol-treated HepG2/2E1 cells significantly decreased (63.38 ± 6.71%) compared with that of the control cells. When the ethanol-treated HepG2/2E1 cells were treated with 200 μg/ml and 500 μg/ml CE10, viability significantly increased to ± 5.91% and ± 7.14%, respectively, compared with that of ethanol-induced HepG2/2E1 cells (p 0.05) Effects of C. fulvescens Extract on the Production of Intracellular OS In ethanol-treated HepG2/2E1 cells, the level of intracellular OS ( ± ) significantly increased when compared with that in the normal control group ( ± ). A significant decrease in the level of intracellular OS was observed in the cells treated with 200 μg/ml CE10 ( ± ) and 500 514 Journal of ood and Nutrition esearch μg/ml CE10 ( ± ) compared with that in ethanol-induced HepG2/2E1 cells (p 0.05) (igure 3) Effects of C. fulvescens Extract on Antioxidant Enzyme Activity Compared with the normal control group, the ethanolinduced HepG2/2E1 cells showed significant decreases in the activities of CAT, SOD, GST, G, GPx, and GSH. When the ethanol-induced HepG2/2E1 cells were treated with 200 μg/ml and 500 μg/ml CE10, the activities of CAT, SOD, GST, G, GPx, and GSH significantly increased compared with that in ethanol-treated HepG2/2E1 cells (p 0.05) (Table 2) Effects of C. fulvescens Extract on Lipid Peroxidation We measured the levels of malondialdehyde (MDA), which is the end product of lipid peroxidation. The level of MDA was significantly increased in the ethanol-induced HepG2/2E1 cells compared with the normal control group. However, treatment with 200 μg/ml and 500 μg/ml CE10 resulted in a significant decrease in the level of MDA compared with the ethanoltreated HepG2/2E1 cells (p 0.05) (igure 4) Effects of C. fulvescens Extract on the Production of Intracellular Lipid Droplets We found that the production of intracellular lipid droplets markedly increased in ethanol-treated HepG2/2E1 cells compared with that in the normal control group. The groups that were also treated with CE10 showed significant decreases in the production of intracellular lipid droplets compared with that in the ethanol-treated HepG2/2E1 cells (p 0.05) (igure 5) Effects of C. fulvescens Extract on mna Expression The ethanol-treated HepG2/2E1 cells showed a significant increase in the mna expression of CYP2E1 compared with that in the normal control group. Treatment with 200 μg/ml and 500 μg/ml CE10 showed a significant decrease in the mna expression of CYP2E1 compared with that in the ethanol-induced HepG2/2E1 cells (p 0.05) (igure 6). igure 4. Effects of 10% ethanol extract of Capsosiphon fulvescens (CE10) on lipid peroxidation (production of malondialdehyde) in ethanol-treated HepG2/2E1 cells. The data are expressed as the mean ± SD (n=3), and different letters indicate (a b) a significant difference at p 0.05, as determined by a Duncan's multiple range test igure 5. Effects of 10% ethanol extract of Capsosiphon fulvescens (CE10) on the production of intracellular lipid droplets in the ethanol-treated HepG2/2E1 cells. The data are expressed as the mean ± SD (n=3), and different letters indicate (a b c) a significant difference at p 0.05, as determined by a Duncan's multiple range test Journal of ood and Nutrition esearch 515 igure 6. Effects of 10% ethanol extract of Capsosiphon fulvescens (CE10) on the mna expression of CYP2E1 in ethanol-treated HepG2/2E1 cells. The data are expressed as the mean ± SD (n=3), and different letters indicate (a b c) a significant difference at p 0.05, as determined by a Duncan's multiple range test igure 7. Effects of 10% ethanol extract of Capsosiphon fulvescens (CE10) on the mna expression of AMPK, SEBP-1c, ACC, CPT-1, and PPAα in ethanol-treated HepG2/2E1 cells. The data are expressed as the mean ± SD (n=3), and different letters indicate (a b c) a significant difference at p 0.05, as determined by a Duncan's multiple range test In addition, significant increases in the mna expression of SEBP-1c and ACC and decreases in the mna expression of AMPK, CPT1, and PPA-α were observed in the ethanol-treated HepG2/2E1 cells compared with that in the normal control group. However, the ethanol-treated HepG2/2E1 cells that were also treated with 200 μg/ml and 500 μg/ml CE10 showed a significant decrease in the mna expression of SEBP-1c and ACC and increases in the mna expression of AMPK, CPT1, and PPA-α, compared with the levels of the ethanol-treated HepG2/2E1 cells (p 0.05) (igure 7). 4. Discussion The liver participates in approximately 500 different functions; one of the most important of these is energy metabolism. In the liver, fatty acids, triacylglycerols, and cholesterol are exported as fatty packages, such as very low-density lipoprotein (VLDL), and delivered to the body via the blood. However, lipids cannot be stored in liver cells [22]. The accumulation of excess lipid droplets in liver cells is called fatty liver (steatosis). atty liver is vulnerable to further inflammation, termed steatohepatitis, which may cause liver damage. As liver cells do not normally export or break down fats, the consumption of excess calories causes fatty liver [23]. In addition, the most common cause of fatty liver is excessive alcohol consumption, which induces alcoholic liver diseases such as alcoholic hepatitis and cirrhosis [3,23]. In the liver, the primary enzymes involved in alcohol metabolism are ADH, CYP2E1, and catalase. Several reports have shown that chronic or excessive alcohol consumption increased the activation of CYP2E1 in the oxidative pathways of alcohol metabolism, which may contribute to the pathogenesis of alcoholic liver damage through OS production during the catalytic circle [2,24]. In this study, we used HepG2 cells tr
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