Molecular and Cellular Biochemistry

, Volume 450, Issue 1–2, pp 1–23 | Cite as

Silibinin-induced autophagy mediated by PPARα-sirt1-AMPK pathway participated in the regulation of type I collagen-enhanced migration in murine 3T3-L1 preadipocytes

  • Xiaoling Liu
  • Qian Xu
  • Xinyu Long
  • Weiwei Liu
  • Yeli Zhao
  • Toshihiko Hayashi
  • Shunji Hattori
  • Hitomi Fujisaki
  • Takaaki Ogura
  • Shin-ichi Tashiro
  • Satoshi Onodera
  • Masayuki Yamato
  • Takashi IkejimaEmail author


Preadipocyte migration is a fundamental and important process for the development of tissue organization, especially in the development of primitive adipose tissue and adipocyte tissue wound healing. However, excessive migration may result in abnormal development and fibrosis-related diseases such as hypertrophic scar. We previously reported that type I collagen (collagen I) enhanced migration of 3T3-L1 preadipocytes via phosphorylation and/or acetylation of NF-κB p65, and the enhanced cell migration is repressed by silibinin treatment through sirt1. It is known that sirt1 has an ability to deacetylate acetylated NF-κB p65, but little is known about the effect of sirt1 on phosphorylated NF-κB p65. This study aims to examine the potential effect of sirt1 on the regulation of phosphorylated NF-κB p65 and the underlying mechanism. Autophagy is involved in many physiological and pathological processes, including regulation of cell migration as well as in cellular homeostasis. The present study demonstrates that silibinin induces autophagy in a dose-dependent manner in 3T3-L1 cells. Autophagy is under the regulation of sirt1/AMPK pathway, and inhibits collagen I-enhanced migration of 3T3-L1 cells through negative regulation of NF-κB p65 phosphorylation but not acetylation. The expression of peroxisome proliferator-activated receptor α (PPARα) is up-regulated with silibinin accompanying up-regulation of autophagy through activating sirt1 in 3T3-L1 cells. Taken together, these findings indicate that silibinin-induced autophagy is mediated by up-regulation of PPARα-sirt1-AMPK, contributing to repression of type I collagen-enhanced migration in murine 3T3-L1 preadipocytes through down-regulation of phosphorylated NF-κB p65.


Autophagy Collagen Migration PPARα/sirt1/AMPK NF-κB p65 



This study was supported by Innovation and Entrepreneurship Training Program of Shenyang Pharmaceutical University (201610163008).

Compliance with ethical standards

Conflict and interest



  1. 1.
    Bear JE, Haugh JM (2014) Directed migration of mesenchymal cells: where signaling and the cytoskeleton meet. Curr Opin Cell Biol 30:74–82. CrossRefGoogle Scholar
  2. 2.
    Trepat X, Chen Z, Jacobson K (2012) Cell migration. Compr Physiol 2:2369–2392. Google Scholar
  3. 3.
    Michaelis UR (2014) Mechanisms of endothelial cell migration. Cell Mol Life Sci 71:4131–4148. CrossRefGoogle Scholar
  4. 4.
    Torii T, Miyamoto Y, Sanbe A, Nishimura K, Yamauchi J, Tanoue A (2010) Cytohesin-2/ARNO, through its interaction with focal adhesion adaptor protein paxillin, regulates preadipocyte migration via the downstream activation of Arf6. J Biol Chem 285:24270–24281. CrossRefGoogle Scholar
  5. 5.
    Pachon-Pena G, Serena C, Ejarque M, Petriz J, Duran X, Oliva-Olivera W, Simo R, Tinahones FJ, Fernandez-Veledo S, Vendrell J (2016) Obesity determines the immunophenotypic profile and functional characteristics of human mesenchymal stem cells from adipose tissue. Stem Cells Transl Med 5:464–475. CrossRefGoogle Scholar
  6. 6.
    Guo L, Huang JX, Liu Y, Li X, Zhou SR, Qian SW, Zhu H, Huang HY, Dang YJ, Tang QQ (2013) Transactivation of Atg4b by C/EBPbeta promotes autophagy to facilitate adipogenesis. Mol Cell Biol 33:3180–3190. CrossRefGoogle Scholar
  7. 7.
    Coly PM, Gandolfo P, Castel H, Morin F (2017) The autophagy machinery: a new player in chemotactic cell migration. Front Neurosci 11:78. CrossRefGoogle Scholar
  8. 8.
    Li WD, Hu N, Lei FR, Wei S, Rong JJ, Zhuang H, Li XQ (2015) Autophagy inhibits endothelial progenitor cells migration via the regulation of MMP2, MMP9 and uPA under normoxia condition. Biochem Biophys Res Commun 466:376–380. CrossRefGoogle Scholar
  9. 9.
    Zhan Z, Xie X, Cao H, Zhou X, Zhang XD, Fan H, Liu Z (2014) Autophagy facilitates TLR4- and TLR3-triggered migration and invasion of lung cancer cells through the promotion of TRAF6 ubiquitination. Autophagy 10:257–268. CrossRefGoogle Scholar
  10. 10.
    Huang R, Liu W (2015) Identifying an essential role of nuclear LC3 for autophagy. Autophagy 11:852–853. CrossRefGoogle Scholar
  11. 11.
    Cao L, Liu C, Wang F, Wang H (2013) SIRT1 negatively regulates amyloid-beta-induced inflammation via the NF-kappaB pathway. Braz J Med Biol Res 46:659–669. CrossRefGoogle Scholar
  12. 12.
    Lynch CJ, Shah ZH, Allison SJ, Ahmed SU, Ford J, Warnock LJ, Li H, Serrano M, Milner J (2010) SIRT1 undergoes alternative splicing in a novel auto-regulatory loop with p53. PLoS ONE 5:e13502. CrossRefGoogle Scholar
  13. 13.
    Liu X, Xu Q, Liu W, Yao G, Zhao Y, Xu F, Hayashi T, Fujisaki H, Hattori S, Tashiro SI, Onodera S, Yamato M, Ikejima T (2017) Enhanced migration of murine fibroblast-like 3T3-L1 preadipocytes on type I collagen-coated dish is reversed by silibinin treatment. Mol Cell Biochem. Google Scholar
  14. 14.
    Mihaylova MM, Shaw RJ (2011) The AMPK signalling pathway coordinates cell growth, autophagy and metabolism. Nat Cell Biol 13:1016–1023. CrossRefGoogle Scholar
  15. 15.
    Lan F, Cacicedo JM, Ruderman N, Ido Y (2008) SIRT1 modulation of the acetylation status, cytosolic localization, and activity of LKB1. Possible role in AMP-activated protein kinase activation. J Biol Chem 283:27628–27635. CrossRefGoogle Scholar
  16. 16.
    Talero E, Alcaide A, Avila-Roman J, Garcia-Maurino S, Vendramini-Costa D, Motilva V (2016) Expression patterns of sirtuin 1-AMPK-autophagy pathway in chronic colitis and inflammation-associated colon neoplasia in IL-10-deficient mice. Int Immunopharmacol 35:248–256. CrossRefGoogle Scholar
  17. 17.
    Jiao M, Ren F, Zhou L, Zhang X, Zhang L, Wen T, Wei L, Wang X, Shi H, Bai L, Zheng S, Zhang J, Chen Y, Han Y, Zhao C, Duan Z (2014) Peroxisome proliferator-activated receptor alpha activation attenuates the inflammatory response to protect the liver from acute failure by promoting the autophagy pathway. Cell Death Dis 5:e1397. CrossRefGoogle Scholar
  18. 18.
    Wang W, Lin Q, Lin R, Zhang J, Ren F, Ji M, Li Y (2013) PPARalpha agonist fenofibrate attenuates TNF-alpha-induced CD40 expression in 3T3-L1 adipocytes via the SIRT1-dependent signaling pathway. Exp Cell Res 319:1523–1533. CrossRefGoogle Scholar
  19. 19.
    Ko JW, Shin NR, Park SH, Lee IC, Ryu JM, Kim HJ, Cho YK, Kim JC, Shin IS (2017) Silibinin inhibits the fibrotic responses induced by cigarette smoke via suppression of TGF-beta1/Smad 2/3 signaling. Food Chem Toxicol 106:424–429. CrossRefGoogle Scholar
  20. 20.
    Rigby CM, Roy S, Deep G, Guillermo-Lagae R, Jain AK, Dhar D, Orlicky DJ, Agarwal C, Agarwal R (2017) Role of p53 in silibinin-mediated inhibition of ultraviolet B radiation-induced DNA damage, inflammation and skin carcinogenesis. Carcinogenesis 38:40–50. CrossRefGoogle Scholar
  21. 21.
    Jiang C, Jin S, Jiang Z, Wang J (2016) Inhibitory effects of silibinin on proliferation and lung metastasis of human high metastasis cell line of salivary gland adenoid cystic carcinoma via autophagy induction. OncoTargets Ther 9:6609–6618. CrossRefGoogle Scholar
  22. 22.
    Xu Q, Liu W, Liu X, Wang H, Yao G, Zang L, Hayashi T, Tashiro S, Onodera S, Ikejima T (2016) Silibinin negatively contributes to primary cilia length via autophagy regulated by histone deacetylase 6 in confluent mouse embryo fibroblast 3T3-L1 cells. Mol Cell Biochem 420:53–63. CrossRefGoogle Scholar
  23. 23.
    Li F, Ma Z, Guan Z, Chen Y, Wu K, Guo P, Wang X, He D, Zeng J (2015) Autophagy induction by silibinin positively contributes to its anti-metastatic capacity via AMPK/mTOR pathway in renal cell carcinoma. Int J Mol Sci 16:8415–8429. CrossRefGoogle Scholar
  24. 24.
    Sato K, Ebihara T, Adachi E, Kawashima S, Hattori S, Irie S (2000) Possible involvement of aminotelopeptide in self-assembly and thermal stability of collagen I as revealed by its removal with proteases. J Biol Chem 275:25870–25875. CrossRefGoogle Scholar
  25. 25.
    Xu Q, Liu W, Liu X, Otkur W, Hayashi T, Yamato M, Fujisaki H, Hattori S, Tashiro SI, Ikejima T (2017) Type I collagen promotes primary cilia growth through down-regulating HDAC6-mediated autophagy in confluent mouse embryo fibroblast 3T3-L1 cells. J Biosci Bioeng. Google Scholar
  26. 26.
    Fujisaki H, Hattori S (2002) Keratinocyte apoptosis on type I collagen gel caused by lack of laminin 5/10/11 deposition and Akt signaling. Exp Cell Res 280:255–269CrossRefGoogle Scholar
  27. 27.
    Fujisaki H, Adachi E, Hattori S (2008) Keratinocyte differentiation and proliferation are regulated by adhesion to the three-dimensional meshwork structure of type IV collagen. Connect Tissue Res 49:426–436. CrossRefGoogle Scholar
  28. 28.
    Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(− Delta Delta C(T)) method. Methods 25:402–408. CrossRefGoogle Scholar
  29. 29.
    Liu W, Otkur W, Zhang Y, Li Q, Ye Y, Zang L, He H, Hayashi T, Tashiro S, Onodera S, Ikejima T (2013) Silibinin protects murine fibroblast L929 cells from UVB-induced apoptosis through the simultaneous inhibition of ATM-p53 pathway and autophagy. FEBS J 280:4572–4584. CrossRefGoogle Scholar
  30. 30.
    Liu W, Otkur W, Li L, Wang Q, He H, Ye Y, Zhang Y, Hayashi T, Tashiro S, Onodera S, Ikejima T (2013) Autophagy induced by silibinin protects human epidermoid carcinoma A431 cells from UVB-induced apoptosis. J Photochem Photobiol B 123:23–31. CrossRefGoogle Scholar
  31. 31.
    Zheng N, Zhang P, Huang H, Liu W, Hayashi T, Zang L, Zhang Y, Liu L, Xia M, Tashiro S, Onodera S, Ikejima T (2015) ERalpha down-regulation plays a key role in silibinin-induced autophagy and apoptosis in human breast cancer MCF-7 cells. J Pharmacol Sci 128:97–107. CrossRefGoogle Scholar
  32. 32.
    Jacquin E, Leclerc-Mercier S, Judon C, Blanchard E, Fraitag S, Florey O (2017) Pharmacological modulators of autophagy activate a parallel noncanonical pathway driving unconventional LC3 lipidation. Autophagy 13:854–867. CrossRefGoogle Scholar
  33. 33.
    Garcia-Maurino S, Alcaide A, Dominguez C (2012) Pharmacological control of autophagy: therapeutic perspectives in inflammatory bowel disease and colorectal cancer. Curr Pharm Des 18:3853–3873CrossRefGoogle Scholar
  34. 34.
    Vequaud E, Seveno C, Loussouarn D, Engelhart L, Campone M, Juin P, Barille-Nion S (2015) YM155 potently triggers cell death in breast cancer cells through an autophagy-NF-kB network. OncoTarget 6:13476–13486. CrossRefGoogle Scholar
  35. 35.
    Zhang H, Chen Z, Miranda RN, Medeiros LJ, McCarty N (2016) TG2 and NF-kappaB signaling coordinates the survival of mantle cell lymphoma cells via IL6-mediated autophagy. Cancer Res 76:6410–6423. CrossRefGoogle Scholar
  36. 36.
    Fujishima Y, Nishiumi S, Masuda A, Inoue J, Nguyen NM, Irino Y, Komatsu M, Tanaka K, Kutsumi H, Azuma T, Yoshida M (2011) Autophagy in the intestinal epithelium reduces endotoxin-induced inflammatory responses by inhibiting NF-kappaB activation. Arch Biochem Biophys 506:223–235. CrossRefGoogle Scholar
  37. 37.
    Xi C, Zhou J, Du S, Peng S (2016) Autophagy upregulation promotes macrophages to escape mesoporous silica nanoparticle (MSN)-induced NF-kappaB-dependent inflammation. Inflamm Res 65:325–341. CrossRefGoogle Scholar
  38. 38.
    Li X, Wang Y, Xiong Y, Wu J, Ding H, Chen X, Lan L, Zhang H (2016) Galangin induces autophagy via deacetylation of LC3 by SIRT1 in HepG2 cells. Sci Rep 6:30496. CrossRefGoogle Scholar
  39. 39.
    Huang R, Xu Y, Wan W, Shou X, Qian J, You Z, Liu B, Chang C, Zhou T, Lippincott-Schwartz J, Liu W (2015) Deacetylation of nuclear LC3 drives autophagy initiation under starvation. Mol Cell 57:456–466. CrossRefGoogle Scholar
  40. 40.
    Lee IH, Cao L, Mostoslavsky R, Lombard DB, Liu J, Bruns NE, Tsokos M, Alt FW, Finkel T (2008) A role for the NAD-dependent deacetylase Sirt1 in the regulation of autophagy. Proc Natl Acad Sci USA 105:3374–3379. CrossRefGoogle Scholar
  41. 41.
    Wang WR, Liu EQ, Zhang JY, Li YX, Yang XF, He YH, Zhang W, Jing T, Lin R (2015) Activation of PPAR alpha by fenofibrate inhibits apoptosis in vascular adventitial fibroblasts partly through SIRT1-mediated deacetylation of FoxO1. Exp Cell Res 338:54–63. CrossRefGoogle Scholar
  42. 42.
    Bonzo JA, Brocker C, Jiang C, Wang RH, Deng CX, Gonzalez FJ (2014) Hepatic sirtuin 1 is dispensable for fibrate-induced peroxisome proliferator-activated receptor-alpha function in vivo. Am J Physiol Endocrinol Metab 306:E824–E837. CrossRefGoogle Scholar
  43. 43.
    Lee JM, Wagner M, Xiao R, Kim KH, Feng D, Lazar MA, Moore DD (2014) Nutrient-sensing nuclear receptors coordinate autophagy. Nature 516:112–115. Google Scholar
  44. 44.
    Jiao M, Ren F, Zhou L, Zhang X, Zhang L, Wen T, Wei L, Wang X, Shi H, Bai L, Zhang X, Zheng S, Zhang J, Chen Y, Han Y, Zhao C, Duan Z (2014) Peroxisome proliferator-activated receptor alpha activation attenuates the inflammatory response to protect the liver from acute failure by promoting the autophagy pathway. Cell Death Dis 5:e1397. CrossRefGoogle Scholar
  45. 45.
    Tuloup-Minguez V, Hamai A, Greffard A, Nicolas V, Codogno P, Botti J (2013) Autophagy modulates cell migration and beta1 integrin membrane recycling. Cell Cycle 12:3317–3328. CrossRefGoogle Scholar
  46. 46.
    Neal MD, Sodhi CP, Dyer M, Craig BT, Good M, Jia H, Yazji I, Afrazi A, Richardson WM, Beer-Stolz D, Ma C, Prindle T, Grant Z, Branca MF, Ozolek J, Hackam DJ (2013) A critical role for TLR4 induction of autophagy in the regulation of enterocyte migration and the pathogenesis of necrotizing enterocolitis. J Immunol 190:3541–3551. CrossRefGoogle Scholar
  47. 47.
    Muller PA, Caswell PT, Doyle B, Iwanicki MP, Tan EH, Karim S, Lukashchuk N, Gillespie DA, Ludwig RL, Gosselin P, Cromer A, Brugge JS, Sansom OJ, Norman JC, Vousden KH (2009) Mutant p53 drives invasion by promoting integrin recycling. Cell 139:1327–1341. CrossRefGoogle Scholar
  48. 48.
    Indelicato M, Pucci B, Schito L, Reali V, Aventaggiato M, Mazzarino MC, Stivala F, Fini M, Russo MA, Tafani M (2010) Role of hypoxia and autophagy in MDA-MB-231 invasiveness. J Cell Physiol 223:359–368. Google Scholar
  49. 49.
    Sanjuan MA, Dillon CP, Tait SW, Moshiach S, Dorsey F, Connell S, Komatsu M, Tanaka K, Cleveland JL, Withoff S, Green DR (2007) Toll-like receptor signalling in macrophages links the autophagy pathway to phagocytosis. Nature 450:1253–1257. CrossRefGoogle Scholar
  50. 50.
    Han X, Tai H, Wang X, Wang Z, Zhou J, Wei X, Ding Y, Gong H, Mo C, Zhang J, Qin J, Ma Y, Huang N, Xiang R, Xiao H (2016) AMPK activation protects cells from oxidative stress-induced senescence via autophagic flux restoration and intracellular NAD(+) elevation. Aging Cell 15:416–427. CrossRefGoogle Scholar
  51. 51.
    Chang C, Su H, Zhang D, Wang Y, Shen Q, Liu B, Huang R, Zhou T, Peng C, Wong CC, Shen HM, Lippincott-Schwartz J, Liu W (2015) AMPK-dependent phosphorylation of GAPDH triggers Sirt1 activation and is necessary for autophagy upon glucose starvation. Mol Cell 60:930–940. CrossRefGoogle Scholar
  52. 52.
    Wu Y, Li X, Zhu JX, Xie W, Le W, Fan Z, Jankovic J, Pan T (2011) Resveratrol-activated AMPK/SIRT1/autophagy in cellular models of Parkinson’s disease. Neurosignals 19:163–174. CrossRefGoogle Scholar
  53. 53.
    Lee YH, Chen HY, Su LJ, Chueh PJ (2015) Sirtuin 1 (SIRT1) deacetylase activity and NAD(+)/NADH ratio are imperative for capsaicin-mediated programmed cell death. J Agric Food Chem 63:7361–7370. CrossRefGoogle Scholar
  54. 54.
    Lin QQ, Geng YW, Jiang ZW, Tian ZJ (2017) SIRT1 regulates lipopolysaccharide-induced CD40 expression in renal medullary collecting duct cells by suppressing the TLR4-NF-kappaB signaling pathway. Life Sci 170:100–107. CrossRefGoogle Scholar
  55. 55.
    Pei F, Wang HS, Chen Z, Zhang L (2016) Autophagy regulates odontoblast differentiation by suppressing NF-kappaB activation in an inflammatory environment. Cell Death Dis 7:e2122. CrossRefGoogle Scholar
  56. 56.
    Thu YM, Richmond A (2010) NF-kappaB inducing kinase: a key regulator in the immune system and in cancer. Cytokine Growth Factor Rev 21:213–226. CrossRefGoogle Scholar
  57. 57.
    Djavaheri-Mergny M, Codogno P (2007) Autophagy joins the game to regulate NF-kappaB signaling pathways. Cell Res 17:576–577. CrossRefGoogle Scholar
  58. 58.
    Trocoli A, Djavaheri-Mergny M (2011) The complex interplay between autophagy and NF-kappaB signaling pathways in cancer cells. Am J Cancer Res 1:629–649Google Scholar
  59. 59.
    Hsu MH, Savas U, Griffin KJ, Johnson EF (2001) Identification of peroxisome proliferator-responsive human genes by elevated expression of the peroxisome proliferator-activated receptor alpha in HepG2 cells. J Biol Chem 276:27950–27958. CrossRefGoogle Scholar
  60. 60.
    Hayashida S, Arimoto A, Kuramoto Y, Kozako T, Honda S, Shimeno H, Soeda S (2010) Fasting promotes the expression of SIRT1, an NAD+-dependent protein deacetylase, via activation of PPARalpha in mice. Mol Cell Biochem 339:285–292. CrossRefGoogle Scholar
  61. 61.
    Purushotham A, Schug TT, Xu Q, Surapureddi S, Guo X, Li X (2009) Hepatocyte-specific deletion of SIRT1 alters fatty acid metabolism and results in hepatic steatosis and inflammation. Cell Metab 9:327–338. CrossRefGoogle Scholar
  62. 62.
    Xu Q, Liu W, Liu X, Liu W, Wang H, Yao G, Zang L, Hayashi T, Tashiro S, Onodera S, Ikejima T (2016) Silibinin negatively contributes to primary cilia length via autophagy regulated by histone deacetylase 6 in confluent mouse embryo fibroblast 3T3-L1 cells. Mol Cell Biochem 420:53–63. CrossRefGoogle Scholar
  63. 63.
    Wang HJ, He WQ, Chen L, Liu WW, Xu Q, Xia MY, Hayashi T, Fujisaki H, Hattori S, Tashiro S, Onodera S, Ikejima T (2015) Type I collagen gel protects murine fibrosarcoma L929 cells from TNFalpha-induced cell death. Biochem Biophys Res Commun 457:693–699. CrossRefGoogle Scholar
  64. 64.
    Jones TJ, Adapala RK, Geldenhuys WJ, Bursley C, AbouAlaiwi WA, Nauli SM, Thodeti CK (2012) Primary cilia regulates the directional migration and barrier integrity of endothelial cells through the modulation of hsp27 dependent actin cytoskeletal organization. J Cell Physiol 227:70–76. CrossRefGoogle Scholar
  65. 65.
    Clement DL, Mally S, Stock C, Lethan M, Satir P, Schwab A, Pedersen SF, Christensen ST (2013) PDGFRalpha signaling in the primary cilium regulates NHE1-dependent fibroblast migration via coordinated differential activity of MEK1/2-ERK1/2-p90RSK and AKT signaling pathways. J Cell Sci 126:953–965. CrossRefGoogle Scholar

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© Springer Science+Business Media, LLC, part of Springer Nature 2018

Authors and Affiliations

  • Xiaoling Liu
    • 1
  • Qian Xu
    • 1
  • Xinyu Long
    • 1
  • Weiwei Liu
    • 1
  • Yeli Zhao
    • 1
  • Toshihiko Hayashi
    • 1
  • Shunji Hattori
    • 2
  • Hitomi Fujisaki
    • 2
  • Takaaki Ogura
    • 2
  • Shin-ichi Tashiro
    • 3
  • Satoshi Onodera
    • 4
  • Masayuki Yamato
    • 5
  • Takashi Ikejima
    • 1
    Email author
  1. 1.China-Japan Research Institute of Medical and Pharmaceutical SciencesShenyang Pharmaceutical UniversityShenyangChina
  2. 2.Nippi Research Institute of BiomatrixNippi, IncorporatedTorideJapan
  3. 3.Department of Medical Education and Primary CareKyoto Prefectural University of MedicineKyotoJapan
  4. 4.Department of Clinical and Pharmaceutical SciencesShowa Pharmaceutical UniversityTokyoJapan
  5. 5.Institute of Advanced Biomedical Engineering and ScienceTokyo Women’s Medical UniversityTokyoJapan

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