DOI QR코드

DOI QR Code

산화스트레스에 의해 유도된 C2C12 근세포 손상과, 신경절제에 의해 근감소가 유도된 C57BL/6 마우스에서 열처리 사균체 엔테로코커스 패칼리스 EF-2001의 효과

Effect of Heat-Killed Enterococcus faecalis, EF-2001 on C2C12 Myoblast Damage Induced by Oxidative Stress and Muscle Volume Decreased by Sciatic Denervation in C57BL/6 Mice

  • 장상진 (연세대학교 생명과학기술학부) ;
  • 이명헌 (연세대학교 생명과학기술학부) ;
  • 김완중 (연세대학교 생명과학기술학부) ;
  • 채유리 (연세대학교 생명과학기술학부) ;
  • 이와사 마사히로 (연세대학교 생명과학기술학부) ;
  • 한권일 (연세대학교 생명과학기술학부) ;
  • 김완재 (주식회사 한국베름 연구개발센터) ;
  • 김택중 (연세대학교 생명과학기술학부)
  • Chang, Sang-Jin (Division of Biological Science and Technology, Yonsei University) ;
  • Lee, Myung-Hun (Division of Biological Science and Technology, Yonsei University) ;
  • Kim, Wan-Joong (Division of Biological Science and Technology, Yonsei University) ;
  • Chae, Yuri (Division of Biological Science and Technology, Yonsei University) ;
  • Iwasa, Masahiro (Division of Biological Science and Technology, Yonsei University) ;
  • Han, Kwon-Il (Division of Biological Science and Technology, Yonsei University) ;
  • Kim, Wan-Jae (Research & Development Center, Korea BeRM Co. Ltd.) ;
  • Kim, Tack-Joong (Division of Biological Science and Technology, Yonsei University)
  • 투고 : 2018.12.06
  • 심사 : 2018.12.28
  • 발행 : 2019.02.28

초록

노화, 상해, 유전병 및 산화 스트레스와 같은 다양한 원인으로 인해 근육 위축을 유발한다. 그 동안의 연구에 의하면 열처리 사균체 엔테로코커스 패칼리스(EF-2001)는 항알레르기, 항염증 및 항종양 효과를 보였다. 그러나 항산화 및 항근위축에 대한 효과는 잘 알려져 있지 않고 있다. 본 연구에서는 EF-2001이 근육 위축에 미치는 영향을 연구 하였다. 산화 스트레스에 의한 EF-2001의 세포손상 보호 효과를 확인하기 위해 C2C12 근섬유 아세포는 H2O2로 처리되어, 산화 스트레스를 유도하여 세포 손상을 유발하였다. 그러나 EF-2001 처리로 인해 근세포 손상이 감소됨을 확인 하였다. 우리는 산화스트레스에 의한 EF-2001의 근세포손상의 감소 효과에 대한 메커니즘을 확인하였다. EF-2001는 산화스트레스로 유도된 근세포내의 HSP70 및 SOD1 단백질의 발현을 감소시켰다. 또한, 근세포내에 Atrogin-1/MAFbx 및 MuRF1의 mRNA 수준은 산화 스트레스 조건 하에서 증가하였으나, EF-2001에 의해 감소하였다. 더나아가 근감소를 유도한 좌골신경 절제 모델동물을 통한 근육량을 확인하기 위해 마이크로 CT를 활용해 2차원과 3차원으로 분석하였다. 근육량은 좌골 신경 절제 후에 감소하였고, EF-2001의 경구 투여에 의해 근육량이 회복되었다. 본 결과는 열처리 사균체 엔테로코커스 패칼리스인 EF-2001이 노인들에게 자주 발생하는 근감소증의 예방 및 개선할 수 있는 기능성 식품으로 다양한 분야에서 유용하게 활용 될 수 있음을 시사한다.

Muscle dysfunction may arise from skeletal muscle atrophy caused by aging, injury, oxidative stress, and hereditary disease. Powdered heat-killed Enterococcus faecalis (EF-2001) has anti-allergy, anti-inflammatory, and anti-tumor effects. However, its antioxidant and anti-atrophy effects are poorly characterized. In this study, we examined the effects of EF-2001 on muscle atrophy. To determine the protective effect of EF-2001 on oxidative stress, C2C12 myoblasts were treated with $H_2O_2$ to induce oxidative stress. This induced cell damage, which was reduced by treatment with EF-2001. The mechanism of EF-2001's effect was examined in response to oxidative stress. Treatment with EF-2001 reversed the expression of HSP70 and SOD1 proteins. Also, mRNA levels of Atrogin-1/MAFbx and MuRF1 increased under oxidative stress conditions but decreased following EF-2001 treatment. To evaluate muscle volume, two and three dimensional models of the muscles were analyzed using micro-CT. As expected, muscle volume decreased after sciatic denervation and recovered after oral administration of EF-2001. Therefore, EF-2001 is a candidate for the treatment of muscular atrophy, and future discovery of the additional effects of EF-2001 may yield further applications as a functional food with useful activities in various fields.

키워드

SMGHBM_2019_v29n2_215_f0002.png 이미지

Fig. 2 Effect of EF-2001 on apoptosis in C2C12 myoblasts induced by oxidative stress.

SMGHBM_2019_v29n2_215_f0003.png 이미지

Fig. 3. Effect of EF-2001 on HSP70 and SOD1 protein expression in C2C12 myoblasts induced by oxidative stress.

SMGHBM_2019_v29n2_215_f0004.png 이미지

Fig. 4. Effect of EF-2001 on Atrogin-1/MAFbx and MuRF1 mRNA expression in C2C12 myoblasts induced by oxidative stress.

SMGHBM_2019_v29n2_215_f0005.png 이미지

Fig. 5. Effect of EF-2001 on muscle atrophy induced by sciatic denervation in C57BL/6 mice.

SMGHBM_2019_v29n2_215_f0006.png 이미지

Fig. 6. EF-2001 can restore muscle atrophy caused by oxidative stress in vitro and in vivo.

SMGHBM_2019_v29n2_215_f0007.png 이미지

Fig. 1. Effects of Effect of EF-2001 on the viability of C2C12 myoblasts induced by oxidative stress.

참고문헌

  1. Berridge, M. V., Herst, P. M. and Tan, A. S. 2005. Tetrazolium dyes as tools in cell biology: new insights into their cellular reduction. Biotechnol. Annu. Rev. 11, 127-152. https://doi.org/10.1016/S1387-2656(05)11004-7
  2. Booth, F. W. 1982. Effect of limb immobilization on skeletal muscle. J. Appl. Physiol. Respir. Environ. Exerc. Physiol. 52, 1113-1118.
  3. Choi, E. J., Iwasa, M., Han, K. I., Kim, W. J., Tang, Y., Han, W. C., Kim, E. K. and Park, Z. Y. 2016. Effect of Enterococcus faecalis EF-2001 on experimentally induced atopic eczema in mice. Food Sci. Biotechnol. 25, 1087-1093. https://doi.org/10.1007/s10068-016-0175-7
  4. Choi, M. S., Chang, S. J., Chae, Y., Lee, M. H., Kim, W. J., Iwasa, M., Han, K. I., Kim, W. J. and Kim, T. J. 2018. Anti-inflammatory Effect of Heat-Killed Enterococcus faecalis EF-2001. J. Life Sci. 28, 1361-136. https://doi.org/10.5352/JLS.2018.28.11.1361
  5. Frost, R. A., Nystrom, G. J., Jefferson, L. S. and Lang, C. H. 2007. Hormone, cytokine, and nutritional regulation of sepsis-induced increases in atrogin-1 and MuRF1 in skeletal muscle. Am. J. Physiol. Endocrinol. Metab. 292, E501-512. https://doi.org/10.1152/ajpendo.00359.2006
  6. Gu, Y. H., Choi, H., Yamashita, T., Kang, K. M., Iwasa, M., Lee, M. J., Lee, K. H. and Kim, C. H. 2017. Pharmaceutical Production of Anti-tumor and Immune-potentiating Enterococcus faecalis-2001 ${\beta}$-glucans: Enhanced Activity of Macrophage and Lymphocytes in Tumor-implanted Mice. Curr. Pharm. Biotechnol. 18, 653-661.
  7. Haycock, J. W., MacNeil, S., Jones, P., Harris, J. B. and Mantle, D. 1996. Oxidative damage to muscle protein in Duchenne muscular dystrophy. Neuroreport 8, 357-361. https://doi.org/10.1097/00001756-199612200-00070
  8. Jaattela, M., Wissing, D., Bauer, P. A. and Li, G. C. 1992. Major heat shock protein hsp70 protects tumor cells from tumor necrosis factor cytotoxicity. EMBO J. 11, 3507-3512. https://doi.org/10.1002/j.1460-2075.1992.tb05433.x
  9. Jackson, M. J. and Farrell, S. O. 1993. Free radicals and muscle damage. Br. Med. Bull. 49, 630-641. https://doi.org/10.1093/oxfordjournals.bmb.a072636
  10. Lee, Y. H., Kim, W. J., Lee, M. H., Kim. S. Y., Seo, D. H., Kim. H. S., Gelinsky, M. and Kim, T. J. 2016. Anti-skeletal muscle atrophy effect of Oenothera odorata root extract via reactive oxygen species-dependent signaling pathways in cellular and mouse model. Biosci. Biotechnol. Biochem. 80, 80-88. https://doi.org/10.1080/09168451.2015.1075861
  11. Lee, Y. H., Seo, D. H., Park, J. H., Kabayama, K., Opitz, J., Lee, K. H., Kim, H. S. and Kim, T. J. 2015. Effect of Oenothera odorata root extract on microgravity and disuse-induced muscle atrophy. Evid. Based Complement. Alternat. Med. 2015, 130513.
  12. Li, C. Y., Lee, J. S., Ko, Y. G., Kim, J. I. and Seo, J. S. 2000. Heat shock protein 70 inhibits apoptosis down stream of cytochrome c release and upstream of caspase-3 activation. J. Biol. Chem. 275, 25665-25671. https://doi.org/10.1074/jbc.M906383199
  13. Mrosek, M., Meier, S., Ucurum-Fotiadis, Z., von Castelmur, E., Hedbom, E., Lustig, A., Grzesiek, S., Labeit, D., Labeit, S. and Mayans, O. 2008. Structural analysis of B-Box 2 from MuRF1: identification of a novel self-association pattern in a RING-like fold. Biochemistry 47, 10722-10730. https://doi.org/10.1021/bi800733z
  14. Natanek, S. A., Riddoch-Contreras, J., Marsh, G. S., Hopkinson, N. S., Moxham, J., Man, W. D., Kemp, P. R. and Polkey, M. I. 2013. MuRF-1 and atrogin-1 protein expression and quadriceps fiber size and muscle mass in stable patients with COPD. COPD 10, 618-624. https://doi.org/10.3109/15412555.2013.781577
  15. Ogata, T., Oishi, Y., Higashida, K., Higuchi, M. and Muraoka, I. 2009. Prolonged exercise training induces long-term enhancement of HSP70 expression in rat plantaris muscle. Am. J. Physiol. Regul. Integr. Comp. Physiol. 296, R1557-1563. https://doi.org/10.1152/ajpregu.90911.2008
  16. Oksala, N. K., Ekmekci, F. G., Ozsoy, E., Kirankaya, S., Kokkola, T., Emecen, G., Lappalainen, J., Kaarniranta, K. and Atalay, M. 2014. Natural thermal adaptation increases heat shock protein levels and decreases oxidative stress. Redox Biol. 3, 25-28. https://doi.org/10.1016/j.redox.2014.10.003
  17. Powers, S. K. 2014. Can antioxidants protect against disuse muscle atrophy? Sports Med. 44, S155-165. https://doi.org/10.1007/s40279-014-0255-x
  18. Rodriguez, M. C. and Tarnopolsky, M. A. 2003. Patients with dystrophinopathy show evidence of increased oxidative stress. Free Radic. Biol. Med. 34, 1217-1220. https://doi.org/10.1016/S0891-5849(03)00141-2
  19. Sugawara, T., Lewen, A., Gasche, Y., Yu, F. and Chan, P. H. 2002. Overexpression of SOD1 protects vulnerable motor neurons after spinal cord injury by attenuating mitochondrial cytochrome c release. FASEB J. 16, 1997-1999. https://doi.org/10.1096/fj.02-0251fje
  20. Tsang, C. K., Liu, Y., Thomas, J., Zhang, Y. and Zheng, X. S. 2014. Superoxide dismutase 1 acts as a nuclear transcription factor to regulate oxidative stress resistance. Nat. Commun. 5, 3446. https://doi.org/10.1038/ncomms4446
  21. Zylicz, M. and Wawrzynow, A. 2001. Insights into the function of Hsp70 chaperones. IUBMB Life 51, 283-287. https://doi.org/10.1080/152165401317190770