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Natural flavonoid silibinin promotes the migration and myogenic differentiation of murine C2C12 myoblasts via modulation of ROS generation and down-regulation of estrogen receptor α expression

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Abstract

Skeletal muscle regeneration is a complex process, involving the proliferation, migration, and differentiation of myoblasts. Recent studies suggest that some natural flavanones stimulate myogenesis. However, the effect of plant estrogen, silibinin, on the regulation of myoblast behaviors is unclarified. In this study, we investigated the effects of silibinin on immortalized murine myoblast C2C12 in the aspects of proliferation, migration, differentiation along with underlying mechanisms. The results show that silibinin at concentrations below 50 μM enhanced the migration and differentiation of C2C12 cells, but had no effect on cell proliferation. Silibinin significantly promoted the production of ROS, which appeared to play important roles in the migration and differentiation of the myoblasts. Interestingly, among ROS, the superoxide anion and hydroxyl radical were associated with the migration, whereas hydrogen peroxide contributed to the myogenic differentiation. We used ER agonist and antagonist to explore whether estrogen receptors (ERs), which are affected by silibinin treatment in the silibinin-enhanced C2C12 migration and differentiation. Migration was independent of ERs, whereas the differentiation was associated with decreased ERα activity. In summary, silibinin treatment increases ROS levels, leading to the promotion of migration and myogenic differentiation. Negative regulation ERα of differentiation but not of migration may suggest that ERα represses hydrogen peroxide generation. The effect of silibinin on myoblast migration and differentiation suggests that silibinin may have therapeutic benefits for muscle regeneration.

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References

  1. Prior B, Modlesky C, Evans E, Sloniger M, Saunder M, Lewis R, Cureton K (2001) Muscularity and the density of the fat-free mass in athletes. J Appl Physiol 90:1523–1531. https://doi.org/10.1152/jappl.2001.90.4.1523

    Article  CAS  PubMed  Google Scholar 

  2. Baghdadi MB, Tajbakhsh S (2018) Regulation and phylogeny of skeletal muscle regeneration. Dev Biol 433:200–209. https://doi.org/10.1016/j.ydbio.2017.07.026

    Article  CAS  PubMed  Google Scholar 

  3. Braun T, Gautel M (2011) Transcriptional mechanisms regulating skeletal muscle differentiation, growth and homeostasis. Nat Rev Mol Cell Biol 12:349–361. https://doi.org/10.1038/nrm3118

    Article  CAS  PubMed  Google Scholar 

  4. Galluzzo P, Rastelli C, Bulzomi P, Acconcia F, Pallottini V, Marino M (2009) 17Beta-estradiol regulates the first steps of skeletal muscle cell differentiation via ER-alpha-mediated signals. Am J Physiol Cell Physiol 297:C1249–C1262. https://doi.org/10.1152/ajpcell.00188.2009

    Article  CAS  PubMed  Google Scholar 

  5. Ogawa M, Yamaji R, Higashimura Y, Harada N, Ashida H, Nakano Y, Inui H (2011) 17Beta-estradiol represses myogenic differentiation by increasing ubiquitin-specific peptidase 19 through estrogen receptor alpha. J Biol Chem 286:41455–41465. https://doi.org/10.1074/jbc.M111.276824

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Zhou DR, Eid R, Miller KA, Boucher E, Mandato CA, Greenwood MT (2019) Intracellular second messengers mediate stress inducible hormesis and Programmed Cell Death: a review. Biochim Biophys Acta Mol Cell Res 1866:773–792. https://doi.org/10.1016/j.bbamcr.2019.01.016

    Article  CAS  PubMed  Google Scholar 

  7. Urao N, Ushio-Fukai M (2013) Redox regulation of stem/progenitor cells and bone marrow niche. Free Radic Biol Med 54:26–39. https://doi.org/10.1016/j.freeradbiomed.2012.10.532

    Article  CAS  PubMed  Google Scholar 

  8. Sun QA, Hess DT, Nogueira L, Yong S, Bowles DE, Eu J, Laurita KR, Meissner G, Stamler JS (2011) Oxygen-coupled redox regulation of the skeletal muscle ryanodine receptor-Ca2+ release channel by NADPH oxidase 4. Proc Natl Acad Sci USA 108:16098–16103. https://doi.org/10.1073/pnas.1109546108

    Article  PubMed  Google Scholar 

  9. Youm TH, Woo SH, Kwon ES, Park SS (2019) NADPH oxidase 4 contributes to myoblast fusion and skeletal muscle regeneration. Oxid Med Cell Longev 2019:3585390. https://doi.org/10.1155/2019/3585390

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Le Moal E, Pialoux V, Juban G, Groussard C, Zouhal H, Chazaud B, Mounier R (2017) Redox control of skeletal muscle regeneration. Antioxid Redox Signal 27:276–310. https://doi.org/10.1089/ars.2016.6782

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Lee S, Tak E, Lee J, Rashid MA, Murphy MP, Ha J, Kim SS (2011) Mitochondrial H2O2 generated from electron transport chain complex I stimulates muscle differentiation. Cell Res 21:817–834. https://doi.org/10.1038/cr.2011.55

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Handayaningsih AE, Iguchi G, Fukuoka H, Nishizawa H, Takahashi M, Yamamoto M, Herningtyas EH, Okimura Y, Kaji H, Chihara K, Seino S, Takahashi Y (2011) Reactive oxygen species play an essential role in IGF-I signaling and IGF-I-induced myocyte hypertrophy in C2C12 myocytes. Endocrinology 152:912–921. https://doi.org/10.1210/en.2010-0981

    Article  CAS  PubMed  Google Scholar 

  13. Bosutti A, Degens H (2015) The impact of resveratrol and hydrogen peroxide on muscle cell plasticity shows a dose-dependent interaction. Sci Rep 5:80–93. https://doi.org/10.1038/srep08093

    Article  CAS  Google Scholar 

  14. Powers SK, Talbert EE, Adhihetty PJ (2011) Reactive oxygen and nitrogen species as intracellular signals in skeletal muscle. J Physiol 589:2129–2138. https://doi.org/10.1113/jphysiol.2010.201327

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Lim S, Shin JY, Jo A, Jyothi KR, Nguyen MN, Choi TG, Kim J, Park JH, Eun YG, Yoon KS, Ha J, Kim SS (2013) Carbonyl reductase 1 is an essential regulator of skeletal muscle differentiation and regeneration. Int J Biochem Cell Biol 45:1784–1793. https://doi.org/10.1016/j.biocel.2013.05.025

    Article  CAS  PubMed  Google Scholar 

  16. Hansen JM, Klass M, Harris C, Csete M (2007) A reducing redox environment promotes C2C12 myogenesis: implications for regeneration in aged muscle. Cell Biol Int 31:546–553. https://doi.org/10.1016/j.cellbi.2006.11.027

    Article  CAS  PubMed  Google Scholar 

  17. Ferreira LF, Reid MB (1985) (2008) Muscle-derived ROS and thiol regulation in muscle fatigue. J Appl Physiol 104:853–860. https://doi.org/10.1152/japplphysiol.00953.2007

    Article  CAS  Google Scholar 

  18. Sun Y, Yang J, Liu W, Yao G, Xu F, Hayashi T, Onodera S, Ikejima T (2019) Attenuating effect of silibinin on palmitic acid-induced apoptosis and mitochondrial dysfunction in pancreatic beta-cells is mediated by estrogen receptor alpha. Mol Cell Biochem 460:81–92. https://doi.org/10.1007/s11010-019-03572-1

    Article  CAS  PubMed  Google Scholar 

  19. Zheng N, Liu L, Liu WW, Li F, Hayashi T, Tashiro SI, Onodera S, Ikejima T (2017) Crosstalk of ROS/RNS and autophagy in silibinin-induced apoptosis of MCF-7 human breast cancer cells in vitro. Acta Pharmacol Sin 38:277–289. https://doi.org/10.1038/aps.2016.117

    Article  CAS  PubMed  Google Scholar 

  20. Acharya S, Stark TD, Oh ST, Jeon S, Pak SC, Kim M, Hur J, Matsutomo T, Hofmann T, Hill RA, Balemba OB (2017) (2R,3S,2″'R,3″R)-manniflavanone protects proliferating skeletal muscle cells against oxidative stress and stimulates myotube formation. J Agric Food Chem 65:3636–3646. https://doi.org/10.1021/acs.jafc.6b05161

    Article  CAS  PubMed  Google Scholar 

  21. Nie Y, Chen H, Guo C, Yuan Z, Zhou X, Zhang Y, Zhang X, Mo D, Chen Y (2017) Palmdelphin promotes myoblast differentiation and muscle regeneration. Sci Rep 7:41608. https://doi.org/10.1038/srep41608

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Neuhaus P, Oustanina S, Loch T, Kruger M, Bober E, Dono R, Zeller R, Braun T (2003) Reduced mobility of fibroblast growth factor (FGF)-deficient myoblasts might contribute to dystrophic changes in the musculature of FGF2/FGF6/mdx triple-mutant mice. Mol Cell Biol 23:6037–6048. https://doi.org/10.1128/mcb.23.17.6037-6048.2003

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Dedieu S, Poussard S, Mazeres G, Grise F, Dargelos E, Cottin P, Brustis JJ (2004) Myoblast migration is regulated by calpain through its involvement in cell attachment and cytoskeletal organization. Exp Cell Res 292:187–200. https://doi.org/10.1016/j.yexcr.2003.08.014

    Article  CAS  PubMed  Google Scholar 

  24. Gao Z, Liu R, Ye N, Liu C, Li X, Guo X, Zhang Z, Li X, Yao Y, Jiang X (2018) FOXO1 inhibits tumor cell migration via regulating cell surface morphology in non-small cell lung cancer cells. Cell Physiol Biochem 48:138–148. https://doi.org/10.1159/000491670

    Article  CAS  PubMed  Google Scholar 

  25. Malewicz B, Wang Z, Jiang C, Guo J, Cleary MP, Grande JP, Lu J (2006) Enhancement of mammary carcinogenesis in two rodent models by silymarin dietary supplements. Carcinogenesis 27:1739–1747. https://doi.org/10.1093/carcin/bgl032

    Article  CAS  PubMed  Google Scholar 

  26. Dupuis ML, Conti F, Maselli A, Pagano MT, Ruggieri A, Anticoli S, Fragale A, Gabriele L, Gagliardi MC, Sanchez M, Ceccarelli F, Alessandri C, Valesini G, Ortona E, Pierdominici M (2018) The natural agonist of estrogen receptor beta silibinin plays an immunosuppressive role representing a potential therapeutic tool in rheumatoid arthritis. Front Immunol 9:1903. https://doi.org/10.3389/fimmu.2018.01903

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Bosutti A, Degens H (2015) The impact of resveratrol and hydrogen peroxide on muscle cell plasticity shows a dose-dependent interaction. Sci Rep 5:8093. https://doi.org/10.1038/srep08093

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. He L, He T, Farrar S, Ji L, Liu T, Ma X (2017) Antioxidants maintain cellular redox homeostasis by elimination of reactive oxygen species. Cell Physiol Biochem 44:532–553. https://doi.org/10.1159/000485089

    Article  PubMed  Google Scholar 

  29. Liu C, McFarland D, Velleman S (2005) Effect of genetic selection on MyoD and myogenin expression in turkeys with different growth rates. Poult Sci 84:376–384. https://doi.org/10.1093/ps/84.3.376

    Article  CAS  PubMed  Google Scholar 

  30. Lee NK, Choi YG, Baik JY, Han SY, Jeong DW, Bae YS, Kim N, Lee SY (2005) A crucial role for reactive oxygen species in RANKL-induced osteoclast differentiation. Blood 106:852–859. https://doi.org/10.1182/blood-2004-09-3662

    Article  CAS  PubMed  Google Scholar 

  31. Malinska D, Kudin AP, Bejtka M, Kunz WS (2012) Changes in mitochondrial reactive oxygen species synthesis during differentiation of skeletal muscle cells. Mitochondrion 12:144–148. https://doi.org/10.1016/j.mito.2011.06.015

    Article  CAS  PubMed  Google Scholar 

  32. Li J, Stouffs M, Serrander L, Banfi B, Bettiol E, Charnay Y, Steger K, Krause KH, Jaconi ME (2006) The NADPH oxidase NOX4 drives cardiac differentiation: role in regulating cardiac transcription factors and MAP kinase activation. Mol Biol Cell 17:3978–3988. https://doi.org/10.1091/mbc.e05-06-0532

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Ogawa M, Kitano T, Kawata N, Sugihira T, Kitakaze T, Harada N, Yamaji R (2017) Daidzein down-regulates ubiquitin-specific protease 19 expression through estrogen receptor beta and increases skeletal muscle mass in young female mice. J Nutr Biochem 49:63–70. https://doi.org/10.1016/j.jnutbio.2017.07.017

    Article  CAS  PubMed  Google Scholar 

  34. Fan S, Qi M, Yu Y, Li L, Yao G, Tashiro S, Onodera S, Ikejima T (2012) P53 activation plays a crucial role in silibinin induced ROS generation via PUMA and JNK. Free Radic Res 46:310–319. https://doi.org/10.3109/10715762.2012.655244

    Article  CAS  PubMed  Google Scholar 

  35. Goncalves RLS, Watson MA, Wong HS, Orr AL, Brand MD (2020) The use of site-specific suppressors to measure the relative contributions of different mitochondrial sites to skeletal muscle superoxide and hydrogen peroxide production. Redox Biol 28:101341. https://doi.org/10.1016/j.redox.2019.101341

    Article  CAS  PubMed  Google Scholar 

  36. Forman H, Fukuto J, Torres M (2004) Redox signaling: thiol chemistry defines which reactive oxygen and nitrogen species can act as second messengers. Am J Physiol Cell Physiol 287:246–256. https://doi.org/10.1152/ajpcell.00516.2003

    Article  Google Scholar 

  37. Kosmidou I, Vassilakopoulos T, Xagorari A, Zakynthinos S, Papapetropoulos A, Roussos C (2002) Production of interleukin-6 by skeletal myotubes role of reactive oxygen species. Am J Respir Cell Mol Biol 26:587–593. https://doi.org/10.1165/ajrcmb.26.5.4598

    Article  CAS  PubMed  Google Scholar 

  38. Sosa V, Moline T, Somoza R, Paciucci R, Kondoh H, LLeonart ME (2013) Oxidative stress and cancer: an overview. Ageing Res Rev 12:376–390. https://doi.org/10.1016/j.arr.2012.10.004

    Article  CAS  PubMed  Google Scholar 

  39. Touyz R (2005) Reactive oxygen species as mediators of calcium signaling by angiotensin II: implications in vascular physiology and pathophysiology. Antioxid Redox Signal 7:1302–1313. https://doi.org/10.1089/ars.2005.7.1302

    Article  CAS  PubMed  Google Scholar 

  40. Vara D, Pula G (2014) Reactive oxygen species: physiological roles in the regulation of vascular cells. Curr Mol Med 14:1103–1125. https://doi.org/10.2174/1566524014666140603114010

    Article  CAS  PubMed  Google Scholar 

  41. Tamborindeguy MT, Matte BF, Ramos GO, Alves AM, Bernardi L, Lamers ML (2018) NADPH-oxidase-derived ROS alters cell migration by modulating adhesions dynamics. Biol Cell 110:225–236. https://doi.org/10.1111/boc.201800011

    Article  CAS  PubMed  Google Scholar 

  42. L’Honore A, Drouin J, Buckingham M, Montarras D (2014) Pitx2 and Pitx3 transcription factors: two key regulators of the redox state in adult skeletal muscle stem cells and muscle regeneration. Free Radic Biol Med 75(Suppl 1):S21–S53. https://doi.org/10.1016/j.freeradbiomed.2014.10.781

    Article  Google Scholar 

  43. L’Honore A, Commere PH, Negroni E, Pallafacchina G, Friguet B, Drouin J, Buckingham M, Montarras D (2018) The role of Pitx2 and Pitx3 in muscle stem cells gives new insights into P38alpha MAP kinase and redox regulation of muscle regeneration. eLife 7. https://doi.org/10.7554/eLife.32991

    Article  PubMed  PubMed Central  Google Scholar 

  44. Aoyama S, Jia H, Nakazawa K, Yamamura J, Saito K, Kato H (2016) Dietary genistein prevents denervation-induced muscle atrophy in male rodents via effects on estrogen receptor-alpha. J Nutr 146:1147–1154. https://doi.org/10.3945/jn.115.226316

    Article  CAS  PubMed  Google Scholar 

  45. Vasconsuelo A, Milanesi L, Boland R (2010) Participation of HSP27 in the antiapoptotic action of 17beta-estradiol in skeletal muscle cells. Cell Stress Chaperones 15:183–192. https://doi.org/10.1007/s12192-009-0132-y

    Article  CAS  PubMed  Google Scholar 

  46. Li QY, Chen L, Zhu YH, Zhang M, Wang YP, Wang MW (2011) Involvement of estrogen receptor-beta in farrerol inhibition of rat thoracic aorta vascular smooth muscle cell proliferation. Acta Pharmacol Sin 32:433–440. https://doi.org/10.1038/aps.2011.1

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Hsieh DJ, Kuo WW, Lai YP, Shibu MA, Shen CY, Pai P, Yeh YL, Lin JY, Viswanadha VP, Huang CY (2015) 17Beta-estradiol and/or estrogen receptor beta attenuate the autophagic and apoptotic effects induced by prolonged hypoxia through HIF-1alpha-mediated BNIP3 and IGFBP-3 signaling blockage. Cell Physiol Biochem 36:274–284. https://doi.org/10.1159/000374070

    Article  CAS  PubMed  Google Scholar 

  48. Ambhore NS, Katragadda R, Raju Kalidhindi RS, Thompson MA, Pabelick CM, Prakash YS, Sathish V (2018) Estrogen receptor beta signaling inhibits PDGF induced human airway smooth muscle proliferation. Mol Cell Endocrinol 476:37–47. https://doi.org/10.1016/j.mce.2018.04.007

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Fan D, Liu SY, van Hasselt CA, Vlantis AC, Ng EK, Zhang H, Dong Y, Ng SK, Chu R, Chan AB, Du J, Wei W, Liu X, Liu Z, Xing M, Chen GG (2015) Estrogen receptor alpha induces prosurvival autophagy in papillary thyroid cancer via stimulating reactive oxygen species and extracellular signal regulated kinases. J Clin Endocrinol Metab 100:E561–E571. https://doi.org/10.1210/jc.2014-3257

    Article  CAS  PubMed  Google Scholar 

  50. Nadal-Serrano M, Sastre-Serra J, Pons DG, Miro AM, Oliver J, Roca P (2012) The ERalpha/ERbeta ratio determines oxidative stress in breast cancer cell lines in response to 17beta-estradiol. J Cell Biochem 113:3178–3185. https://doi.org/10.1002/jcb.24192

    Article  CAS  PubMed  Google Scholar 

  51. Cook KL, Clarke PA, Parmar J, Hu R, Schwartz-Roberts JL, Abu-Asab M, Warri A, Baumann WT, Clarke R (2014) Knockdown of estrogen receptor-alpha induces autophagy and inhibits antiestrogen-mediated unfolded protein response activation, promoting ROS-induced breast cancer cell death. FASEB J 28:3891–3905. https://doi.org/10.1096/fj.13-247353

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Authors and Affiliations

  1. Wuya College of Innovation, Shenyang Pharmaceutical University, Shenyang, 110016, Liaoning, People’s Republic of China

    Xinyu Long, Yanfang Gao, Weiwei Liu, Xiaoling Liu, Toshihiko Hayashi & Takashi Ikejima

  2. Department of Chemistry and Life Science, School of Advanced Engineering, Kogakuin University, 2665-1, Nakanomachi, Hachioji, Tokyo, 192-0015, Japan

    Toshihiko Hayashi

  3. Nippi Research Institute of Biomatrix, Ibaraki, 649-1211, Japan

    Kazunori Mizuno & Shunji Hattori

  4. Key Laboratory of Computational Chemistry-Based Natural Antitumor Drug Research & Development Liaoning Province, 103 Wenhua Road, Shenyang, 110016, Liaoning, China

    Takashi Ikejima

  5. China-Japan Research Institute of Medical and Pharmaceutical Sciences, Shenyang Pharmaceutical University, 103 Wenhua Road, Shenyang, 110016, Liaoning, China

    Takashi Ikejima

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  1. Xinyu Long
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  2. Yanfang Gao
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Long, X., Gao, Y., Liu, W. et al. Natural flavonoid silibinin promotes the migration and myogenic differentiation of murine C2C12 myoblasts via modulation of ROS generation and down-regulation of estrogen receptor α expression. Mol Cell Biochem 474, 243–261 (2020). https://doi.org/10.1007/s11010-020-03849-w

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