Fish Physiology and Biochemistry

, Volume 43, Issue 6, pp 1487–1500 | Cite as

Silymarin inhibits adipogenesis in the adipocytes in grass carp Ctenopharyngodon idellus in vitro and in vivo

  • Peizhen Xiao
  • Zhou Yang
  • Jian Sun
  • Jingjing Tian
  • Zhiguang Chang
  • Xuexian Li
  • Baotong Zhang
  • Yuantu Ye
  • Hong Ji
  • Ermeng Yu
  • Jun Xie


In this study, two experiments were performed to explore the function of silymarin in adipogenesis in grass carp (Ctenopharyngodon idellus) using in vitro and in vivo models. In experiment 1, differentiated grass carp pre-adipocytes were treated with silymarin for 6 days. Treatment with 100 μg mL−1silymarin (SM100 group) significantly reduced triglyceride accumulation at day 6. The adipogenic gene expression levels of PPARγ, C/EBPα, SREBP1c, FAS, SCD1, and LPL, and the protein expression level of PPARγ were significantly down-regulated in the SM100 group. Additionally, the SM100 group had significantly lower reactive oxygen species production and reduced glutathione contents compared with the control in vitro. In experiment 2, the juvenile grass carp (mean body weight= 27.4 ± 0.17 g) were fed six isonitrogenous and isocaloric diets in a factorial design containing 0, 100, or 200 mg kg−1 silymarin (SM0, SM100, SM200) associated with either 4 or 8% lipid levels (low lipid, LL, and high lipid, HL, respectively) for 82 days. The results demonstrated that dietary silymarin supplementation significantly reduced the elevated intraperitoneal fat index in grass carp fed with high-lipid diets, and the gene expression of adipogenesis (PPARγ, FAS) when supplemented with dietary silymarin was notably lower than when no silymarin was supplemented under the high-lipid diets. Thus, our data suggest that silymarin suppressed lipid accumulation in grass carp both in vitro and in vivo, and the effect might be due to an influence on the expression of adipogenesis factors and ROS production partly associated with effects on antioxidant capability.


Silymarin Adipogenesis Grass carp Adipocytes 



Bovine serum albumin


CCAAT enhancer-binding protein-α


2,7-dichlorofluorescein diacetate


Dulbecco’s modified Eagle’s medium


Dimethyl sulfoxide


Fatty acid synthase


Final average body weight


Reduced glutathione


High lipid


Intraperitoneal fat


Intraperitoneal fat index


Low lipid


Lipoprotein lipase


Phosphate-buffered saline


Peroxisome proliferators-activated receptor-γ


Reactive oxygen species


Stearoyl-CoA desaturases 1




Superoxide dismutase


Sterol regulatory element-binding protein-1c





Financial support was provided by the Modern Agro-industry Technology Research System (No. CARS-46-17) and the fund of Beijing Sunpu Biochem. Tech. Co., Ltd., China. Thanks are also due to H.J. Chen, X.C. Shi, C. X. Lei, and A. Jin for their assistance in the study.


  1. Asghar Z, Masood Z (2008) Evaluation of antioxidant properties of silymarin and its potential to inhibit peroxyl radicals in vitro. Pak J Pharm Sci 21:249–254PubMedGoogle Scholar
  2. Boadi WY, Amartey PK, Lo A (2016) Effect of quercetin, genistein and kaempferol on glutathione and glutathione—redox cycle enzymes in 3T3 -L1 preadipocytes. Drug Chem Toxicol 39:239–247. doi: 10.3109/01480545.2015.1082135 CrossRefPubMedGoogle Scholar
  3. Bou M, Todorčević M, Fontanillas R, Capilla E, Gutiérrez J, Navarro I (2014) Adipose tissue and liver metabolic responses to different levels of dietary carbohydrates in gilthead sea bream (Sparus aurata). Comp Biochem Phys A 175:72–81CrossRefGoogle Scholar
  4. Bouraoui L, Gutierrez J, Navarro I (2008) Regulation of proliferation and differentiation of adipocyte precursor cells in rainbow trout (Oncorhynchus mykiss). J Endocrinol 198:459–469CrossRefPubMedGoogle Scholar
  5. Bouraoui L, Cruz-Garcia L, Gutiérrez J, Capilla E, Navarro I (2012) Regulation of lipoprotein lipase gene expression by insulin and troglitazone in rainbow trout (Oncorhynchus mykiss) adipocyte cells in culture. Comp Biochem Phys A 161:83–88CrossRefGoogle Scholar
  6. Castro JP, Grune T, Speckmann B (2016) The two faces of ROS in adipocyte function and dysfunction. Biol Chem 397:709–724CrossRefPubMedGoogle Scholar
  7. China Fisheries Yearbook (2016) Fisheries department of agriculture ministry of China. China Agriculture Press, Beijing, pp 30–31Google Scholar
  8. Chou C, Chen Y, Hsu M, Tsai W, Chang C, Chiu C (2012) Effect of silymarin on lipid and alcohol metabolism in mice following long-term alcohol consumption. J Food Biochem 36:369–377CrossRefGoogle Scholar
  9. Crocenzi FA, Roma MG (2006) Silymarin as a new hepatoprotective agent in experimental cholestasis: new possibilities for an ancient medication. Curr Med Chem 13:1055–1074CrossRefPubMedGoogle Scholar
  10. Du Z, Liu Y, Tian L, Wang J, Wang Y, Liang G (2005) Effect of dietary lipid level on growth, feed utilization and body composition by juvenile grass carp (Ctenopharyngodon idella). Aquac Nutr 11:139–146CrossRefGoogle Scholar
  11. Du Z, Liu Y, Tian L, He J, Cao J, Liang G (2006) The influence of feeding rate on growth, feed efficiency and body composition of juvenile grass carp (Ctenopharyngodon idella). Aquacult Int 14:247–257CrossRefGoogle Scholar
  12. Du Z, Clouet P, Degrace P, Zheng W, Frøyland L, Tian L, Liu Y (2008) Hypolipidaemic effects of fenofibrate and fasting in the herbivorous grass carp (Ctenopharyngodon idella) fed a high-fat diet. Brit J Nutr 100:1200–1212CrossRefPubMedGoogle Scholar
  13. Eberlé D, Hegarty B, Bossard P, Ferré P, Foufelle F (2004) SREBP transcription factors: master regulators of lipid homeostasis. Biochimie 86:839–848. doi: 10.1016/j.biochi.2004.09.018 CrossRefPubMedGoogle Scholar
  14. Fridovich I (1995) Superoxide radical and superoxide dismutases. Annu Rev Biochem 64:97–112. doi: 10.1146/ CrossRefPubMedGoogle Scholar
  15. Furukawa S, Fujita T, Shimabukuro M, Iwaki M, Yamada Y, Nakajima Y, Nakayama O, Makishima M, Matsuda M, Shimomura I (2004) Increased oxidative stress in obesity and its impact on metabolic syndrome. J Clin Invest 114:1752–1761CrossRefPubMedPubMedCentralGoogle Scholar
  16. Gomez-Zorita S, Tréguer K, Mercader J, Carpéné C (2013) Resveratrol directly affects in vitro lipolysis and glucose transport in human fat cells. J Physiol Biochem 69:585–593CrossRefPubMedGoogle Scholar
  17. Guo L, Li X, Tang Q (2015a) Transcriptional regulation of adipocyte differentiation: a central role for CCAAT/enhancer-binding protein (C/EBP) β. J Biol Chem 290:755–761CrossRefPubMedGoogle Scholar
  18. Guo X, Liang XF, Fang L, Yuan X, Zhou Y, Zhang J, Li B (2015b) Effects of dietary non-protein energy source levels on growth performance, body composition and lipid metabolism in herbivorous grass carp (Ctenopharyngodon idella Val.) Aquac Res 46:1197–1208CrossRefGoogle Scholar
  19. He A, Ning L, Chen L, Chen Y, Xing Q, Li J, Qiao F, Li D, Zhang M, Du Z (2015) Systemic adaptation of lipid metabolism in response to low- and high-fat diet in nile tilapia (Oreochromis niloticus). Physiological Reports 3:e12485CrossRefPubMedPubMedCentralGoogle Scholar
  20. Jensen-Urstad AP, Semenkovich CF (2012) Fatty acid synthase and liver triglyceride metabolism: housekeeper or messenger? BBA-Mol Cell Biol L 1821:747–753CrossRefGoogle Scholar
  21. Ji H, Cao Y, Lin Y, Liu P, Lu R, Su S, Yang G, Oku H (2010) Primary culture of grass carp preadipocyte in vitro. Acta Hydrobiologica Sinica 33:1226–1230CrossRefGoogle Scholar
  22. Ji H, Li J, Liu P (2011) Regulation of growth performance and lipid metabolism by dietary n-3 highly unsaturated fatty acids in juvenile grass carp, Ctenopharyngodon idellus. Comp Biochem Phys B 159:49–56CrossRefGoogle Scholar
  23. Jin Y, Tian L, Zeng S, Xie S, Yang H, Liang G, Liu Y (2013) Dietary lipid requirement on non-specific immune responses in juvenile grass carp (Ctenopharyngodon idella). Fish Shellfish Immun 34:1202–1208CrossRefGoogle Scholar
  24. Joseph SB, Laffitte BA, Patel PH, Watson MA, Matsukuma KE, Walczak R, Collins JL, Osborne TF, Tontonoz P (2002) Direct and indirect mechanisms for regulation of fatty acid synthase gene expression by liver X receptors. J Biol Chem 277:11019–11025CrossRefPubMedGoogle Scholar
  25. Ka SO, Kim KA, Kwon KB, Park JW, Park BH (2009) Silibinin attenuates adipogenesis in 3T3-L1 preadipocytes through a potential upregulation of the insig pathway. Int J Mol Med 23:633–637PubMedGoogle Scholar
  26. Kageyama H, Hirano T, Okada K, Ebara T, Kageyama A, Murakami T, Shioda S, Adachi M (2003) Lipoprotein lipase mRNA in white adipose tissue but not in skeletal muscle is increased by pioglitazone through PPAR-γ. Biochem Bioph Res Co 305:22–27CrossRefGoogle Scholar
  27. Kanda Y, Hinata T, Kang SW, Watanabe Y (2011) Reactive oxygen species mediate adipocyte differentiation in mesenchymal stem cells. Life Sci 89:250–258CrossRefPubMedGoogle Scholar
  28. Krautbauer S, Eisinger K, Neumeier M, Hader Y, Buettner R, Schmid PM, Aslanidis C, Buechler C (2014) Free fatty acids, lipopolysaccharide and IL-1α induce adipocyte manganese superoxide dismutase which is increased in visceral adipose tissues of obese rodents. PLoS One 9:e86866CrossRefPubMedPubMedCentralGoogle Scholar
  29. Le Lay S, Lefrère I, Trautwein C, Dugail I, Krief S (2002) Insulin and sterol-regulatory element-binding protein-1C (SREBP-1C) regulation of gene expression in 3T3-L1 adipocytes identification of CCAAT/enhancer-binding protein β as an SREBP-1C target. J Biol Chem 277:35625–35634CrossRefPubMedGoogle Scholar
  30. Lee DY, Liu Y (2003) Molecular structure and stereochemistry of silybin A, silybin B, isosilybin a, and isosilybin B, isolated from Silybum marianum (milk thistle). J Nat Prod 66:1171–1174CrossRefPubMedGoogle Scholar
  31. Lee SM, Jeon IG, Lee JY (2002) Effects of digestible protein and lipid levels in practical diets on growth, protein utilization and body composition of juvenile rockfish (Sebastes schlegeli). Aquaculture 211:227–239CrossRefGoogle Scholar
  32. Lee H, Lee YJ, Choi H, Ko EH, Kim JW (2009) Reactive oxygen species facilitate adipocyte differentiation by accelerating mitotic clonal expansion. J Biol Chem 284:10601–10609CrossRefPubMedPubMedCentralGoogle Scholar
  33. Lee OH, Seo DH, Park CS, Kim YC (2010) Puerarin enhances adipocyte differentiation, adiponectin expression, and antioxidant response in 3T3-L1 cells. Biofactors 36:459–467CrossRefPubMedGoogle Scholar
  34. Lee OH, Seo MJ, Choi HS, Lee BY (2012) Pycnogenol® inhibits lipid accumulation in 3T3-L1 adipocytes with the modulation of reactive oxygen species (ROS) production associated with antioxidant enzyme responses. Phytother Res 26:403–411PubMedGoogle Scholar
  35. Lee B, Lee M, Lefevre M, Kim HR (2014) Anthocyanins inhibit lipogenesis during adipocyte differentiation of 3T3-L1 preadipocytes. Plant Food Hum Nutr 69:137–141CrossRefGoogle Scholar
  36. Lewis-McCrea LM, Lall SP (2007) Effects of moderately oxidized dietary lipid and the role of vitamin E on the development of skeletal abnormalities in juvenile Atlantic halibut (Hippoglossus hippoglossus). Aquaculture 262:142–155CrossRefGoogle Scholar
  37. Li X, Liu W, Lu K, Xu W, Wang Y (2012) Dietary carbohydrate/lipid ratios affect stress, oxidative status and non-specific immune responses of fingerling blunt snout bream, Megalobrama amblycephala. Fish Shellfish Immun 33:316–323CrossRefGoogle Scholar
  38. Liu P, Ji H, Li C, Chen L, Du Z (2015) Morphology, mitochondrial development and adipogenic-related genes expression during adipocytes differentiation in grass carp (Ctenopharyngodon idellus). Sci Bull 60:1241–1251CrossRefGoogle Scholar
  39. Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using realtimequantitative PCR and the 2 - ΔΔCT method. Methods 25:402–408CrossRefPubMedGoogle Scholar
  40. Lu K, Xu W, Li X, Liu W, Wang L, Zhang C (2013) Hepatic triacylglycerol secretion, lipid transport and tissue lipid uptake in blunt snout bream (Megalobrama amblycephala) fed high-fat diet. Aquaculture 408:160–168CrossRefGoogle Scholar
  41. Ludovico A, Gabriella A, Raffaele C, Natasa M, Francesco C (2011) Milk thistle for treatment of nonalcoholic fatty liver disease. Hepat Mon:173–177Google Scholar
  42. MacDougald OA, Lane MD (1995) Transcriptional regulation of gene expression during adipocyte differentiation. Annu Rev Biochem 64:345–373CrossRefPubMedGoogle Scholar
  43. Masella R, Benedetto RD, Varì R, Filesi C, Giovannini C (2005) Novel mechanisms of natural antioxidant compounds in biological systems: involvement of glutathione and glutathione-related enzymes. J Nutr Biochem 16:577–586CrossRefPubMedGoogle Scholar
  44. Nambiar DK, Deep G, Singh RP, Agarwal C, Agarwal R (2014) Silibinin inhibits aberrant lipid metabolism, proliferation and emergence of androgen-independence in prostate cancer cells via primarily targeting the sterol response element binding protein 1. Oncotarget 5:10017–10033CrossRefPubMedPubMedCentralGoogle Scholar
  45. Nencini C, Giorgi G, Micheli L (2007) Protective effect of silymarin on oxidative stress in rat brain. Phytomedicine 14:129–135CrossRefPubMedGoogle Scholar
  46. Ntambi JM, Kim YC (2000) Adipocyte differentiation and gene expression. J Nutr 130:3122S–3126SPubMedGoogle Scholar
  47. Ntambi JM, Miyazaki M (2004) Regulation of stearoyl-CoA desaturases and role in metabolism. Prog Lipid Res 43:91–104CrossRefPubMedGoogle Scholar
  48. Oku H, Umino T (2008) Molecular characterization of peroxisome proliferator-activated receptors (PPARs) and their gene expression in the differentiating adipocytes of red sea bream, Pagrus major. Comp Biochem Phys B 151:268–277CrossRefGoogle Scholar
  49. Oku H, Tokuda M, Okumura T, Umino T (2006) Effects of insulin, triiodothyronine and fat soluble vitamins on adipocyte differentiation and LPL gene expression in the stromal-vascular cells of red sea bream, Pagrus major. Comp Biochem Phys B 144:326–333CrossRefGoogle Scholar
  50. de Oliveira DR, Schaffer LF, Busanello A, Barbosa CP, Peroza LR, de Freitas CM, Krum BN, Bressan GN, Boligon AA, Athayde ML, de Menezes IRA, Fachinetto R (2015) Silymarin has antioxidant potential and changes the activity of Na+/K+-ATPase and monoamine oxidase in vitro. Ind Crop Prod 70:347–355CrossRefGoogle Scholar
  51. Park SJ, Choe YG, Kim JH, Chang KT, Lee HS, Lee DS (2016) Isoliquiritigenin impairs insulin signaling and adipocyte differentiation through the inhibition of protein-tyrosine phosphatase 1B oxidation in 3T3-L1 preadipocytes. Food Chem Toxicol 93:5–12CrossRefPubMedGoogle Scholar
  52. Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45CrossRefPubMedPubMedCentralGoogle Scholar
  53. Podder B, Kim YS, Zerin T, Song HY (2012) Antioxidant effect of silymarin on paraquat-induced human lung adenocarcinoma A549 cell line. Food Chem Toxicol 50:3206–3214CrossRefPubMedGoogle Scholar
  54. Ricchi M, Odoardi MR, Carulli L, Anzivino C, Ballestri S, Pinetti A, Fantoni LI, Marra F, Bertolotti M, Banni S, Lonardo A, Carulli N, Loria P (2009) Differential effect of oleic and palmitic acid on lipid accumulation and apoptosis in cultured hepatocytes. J Gastroen Hepat 24:830–840CrossRefGoogle Scholar
  55. Rosen ED, Spiegelman BM (2001) PPARgamma: a nuclear regulator of metabolism, differentiation, and cell growth. J Biol Chem 276:37731–37734CrossRefPubMedGoogle Scholar
  56. Rosen ED, Walkey CJ, Puigserver P, Spiegelman BM (2000) Transcriptional regulation of adipogenesis. Genes Dev 14:1293–1307PubMedGoogle Scholar
  57. Rosen ED, Hsu CH, Wang X, Sakai S, Freeman MW, Gonzalez FJ, Spiegelman BM (2002) C/EBPα induces adipogenesis through PPARγ: a unified pathway. Genes Dev 16:22–26CrossRefPubMedPubMedCentralGoogle Scholar
  58. Seo MJ, Lee OH, Choi HS, Lee BY (2012) Extract from edible red seaweed (Gelidium amansii) inhibits lipid accumulation and ROS production during differentiation in 3T3-L1 cells. Prev Nutr Food Sci 17:129–135CrossRefPubMedPubMedCentralGoogle Scholar
  59. Seo MJ, Seo YJ, Pan CH, Lee OH, Kim KJ, Lee BY (2016) Fucoxanthin suppresses lipid accumulation and ROS production during differentiation in 3T3-L1 adipocytes. Phytother Res 30:1802–1808CrossRefPubMedGoogle Scholar
  60. Shaker E, Mahmoud H, Mna S (2010) Silymarin, the antioxidant component and Silybum marianum extracts prevent liver damage. Food Chem Toxicol 48:803–806CrossRefPubMedGoogle Scholar
  61. Sheridan MA, Kao YH (1998) Regulation of metamorphosis-associated changes in the lipid metabolism of selected vertebrates. Integr Comp Biol 38:350–368Google Scholar
  62. Škottová N, Kazdová L, Oliyarnyk O, Večeřa R, Sobolová L, Ulrichová J (2004) Phenolics-rich extracts from Silybum marianum and Prunella vulgaris reduce a high-sucrose diet induced oxidative stress in hereditary hypertriglyceridemic rats. Pharmacol Res 50:123–130CrossRefPubMedGoogle Scholar
  63. Stéphan G, Guillaume J, Lamour F (1995) Lipid peroxidation in turbot (Scophthalmus maximus) tissue: effect of dietary vitamin E and dietary n-6 or n-3 polyunsaturated fatty acids. Aquaculture 130:251–268CrossRefGoogle Scholar
  64. Suh HJ, Cho SY, Kim EY, Choi HS (2015) Blockade of lipid accumulation by silibinin in adipocytes and zebrafish. Chem Biol Interact 227:53–62CrossRefPubMedGoogle Scholar
  65. Surai PF (2015) Silymarin as a natural antioxidant: an overview of the current evidence and perspectives. Antioxidants 4:204–247CrossRefPubMedPubMedCentralGoogle Scholar
  66. Vegusdal A, Sundvold H, Gjøen T, Ruyter B (2003) An in vitro method for studying the proliferation and differentiation of Atlantic salmon preadipocytes. Lipids 38:289–296CrossRefPubMedGoogle Scholar
  67. Wang JF, Zhang X, Groopman JE (2004) Activation of vascular endothelial growth factor receptor-3 and its downstream signaling promote cell survival under oxidative stress. J Biol Chem 279:27088–27097CrossRefPubMedGoogle Scholar
  68. Wang J, Liu Y, Tian L, Mai K, Du Z, Wang Y, Yang H (2005) Effect of dietary lipid level on growth performance, lipid deposition, hepatic lipogenesis in juvenile cobia (Rachycentron canadum). Aquaculture 249:439–447CrossRefGoogle Scholar
  69. Wang W, Zhang Y, Lu W, Liu K (2015) Mitochondrial reactive oxygen species regulate adipocyte differentiation of mesenchymal stem cells in hematopoietic stress induced by arabinosylcytosine. PLoS One 10:e0120629CrossRefPubMedPubMedCentralGoogle Scholar
  70. Xiao P, Ji H, Ye Y, Zhang B, Chen Y, Tian JJ, Liu P, Chen L, Du Z (2017) Dietary silymarin supplementation promotes growth performance and improves lipid metabolism and health status in grass carp (Ctenopharyngodon idellus) fed diets with elevated lipid levels. Fish Physiol Biochem 43:1–19CrossRefGoogle Scholar
  71. Yang Z, Sun J, Ji H, Shi XC, Li Y, Du ZY, Chen LQ (2017) Pigment epithelium-derived factor improves TNFα-induced hepatic steatosis in grass carp (Ctenopharyngodon idella). Dev Comp Immunol 71:8–17CrossRefPubMedGoogle Scholar
  72. Yao J, Zhi M, Gao X, Hu P, Li C, Yang X (2013) Effect and the probable mechanisms of silibinin in regulating insulin resistance in the liver of rats with non-alcoholic fatty liver. Braz J Med Biol Res 46:270–277CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2017

Authors and Affiliations

  • Peizhen Xiao
    • 1
  • Zhou Yang
    • 1
  • Jian Sun
    • 1
  • Jingjing Tian
    • 1
  • Zhiguang Chang
    • 1
  • Xuexian Li
    • 1
  • Baotong Zhang
    • 2
  • Yuantu Ye
    • 3
  • Hong Ji
    • 1
  • Ermeng Yu
    • 4
  • Jun Xie
    • 4
  1. 1.College of Animal Science and TechnologyNorthwest A & F UniversityYanglingPeople’s Republic of China
  2. 2.Open Lab for Aquatic Animal NutritionBeijing Research Institute for Nutritional ResourcesBeijingPeople’s Republic of China
  3. 3.Key Laboratory of Aquatic Nutrition of Jiangsu Province, School of Biology and Basic Medical SciencesSoochow UniversitySoochowPeople’s Republic of China
  4. 4.Key Laboratory of Tropical & Subtropical Fishery Resource Application & Cultivation, Ministry of AgriculturePearl River Fisheries Research Institute, Chinese Academy of Fishery SciencesGuangzhouPeople’s Republic of China

Personalised recommendations