Protective Effect of Membrane-Free Stem Cell Extract against Oxidative Stress in LLC-PK1 Cells

무막줄기세포추출물의 LLC-PK1 세포에서의 산화적 스트레스 개선 효과

  • Kim, Min Jeong (Department of Food Science and Nutrition & Kimchi Research Institute, Pusan National University) ;
  • Kim, Ji Hyun (Department of Food Science and Nutrition & Kimchi Research Institute, Pusan National University) ;
  • Park, Hye Sook (T-STEM Co., Ltd.) ;
  • Kim, Young Sil (T-STEM Co., Ltd.) ;
  • Cho, Eun Ju (Department of Food Science and Nutrition & Kimchi Research Institute, Pusan National University)
  • 김민정 (부산대학교 식품영양학과 및 김치연구소) ;
  • 김지현 (부산대학교 식품영양학과 및 김치연구소) ;
  • 박혜숙 ((주)티스템) ;
  • 김영실 ((주)티스템) ;
  • 조은주 (부산대학교 식품영양학과 및 김치연구소)
  • Received : 2019.05.07
  • Accepted : 2019.08.02
  • Published : 2019.08.31


Oxidative stress in kidneys can precede the development of chronic renal injury. We investigated the antioxidative effect of membrane-free stem cell extract (MFSCE) from adipose tissue in LLC-$PK_1$ renal proximal tubule cells. Treatment of LLC-$PK_1$ cells with MFSCE showed the up-regulation of heme-oxygenase-1, thioredoxin reductase 1, and NADPH quinine oxidoreductase-1 protein expressions, which are proteins related with antioxidative activities. When oxidative stress was induced by 3-morpholinosydnonimine (SIN-1), cell viability was decreased, indicating that LLC-$PK_1$ cells were damaged by SIN-1. However, MFSCE significantly elevated cell viability from 58.84% to 64.43% at the concentration of $2.5{\mu}g/mL$ in oxidative stress-induced LLC-$PK_1$ cells. Furthermore, MFSCE ameliorated inflammation and apoptosis in SIN-1-treated LLC-$PK_1$ cells by modulating protein expressions. Inducible nitric oxide synthase and cyclooxygenase-2 protein expressions were down-regulated when LLC-$PK_1$ cells were treated with MFSCE. Apoptosis-related proteins, including B-cell lymphoma-2-associated X protein/B-cell lymphoma-2 ratio, cleaved caspase-3, and cleaved-poly (ADP-ribose) polymerase, were also down-regulated. It indicated that MFSCE protected apoptosis against oxidative stress in LLC-$PK_1$ cells. Taken together, these results suggested that MFSCE had a protective effect against SIN-1-induced oxidative stress in LLC-$PK_1$ cells. Therefore, MFSCE could be a promising therapeutic agent for oxidative stress-induced renal injury.

신장에서 발생한 산화적 스트레스는 조직을 손상시키고 이는 만성신장질환으로 이어질 수 있다. 본 연구에서는 LLC-$PK_1$ 신장세포를 이용하여 산화적 스트레스 개선 효과를 살펴보았다. LLC-$PK_1$ 세포에 무막줄기세포추출물을 처리했을 때 체내 항산화 단백질인 heme-oxygenase-1, thioredoxin reductase 1, 및 NADPH quinine oxidoreductase-1의 발현이 증가함을 확인하였다. LLC-$PK_1$에 산화적 스트레스를 유도하기 위하여 3-morpholinosydnonimine (SIN-1)을 처리한 결과 세포생존율이 감소하여 산화적 스트레스로 인해 세포가 손상됨을 확인하였다. 그러나 무막줄기세포추출물을 처리하였을 때 세포생존율이 증가하였으며, $2.5{\mu}g/mL$에서 세포생존율이 58.84%에서 64.43%까지 증가하였다. 또한 무막줄기세포추출물은 LLC-$PK_1$ 세포에서 SIN-1으로 유도된 염증 및 세포사멸을 조절하였다. 염증 관련 단백질인 inducible nitric oxide synthase와 cyclooxygenase-2는 무막줄기세포 추출물을 처리했을 때 단백질 발현이 감소하였고, 세포사멸과 관련된 B-cell lymphoma-2-associated X protein/B-cell lymphoma-2 비율과 cleaved caspase-3, cleaved-poly (ADP-ribose) polymeras의 단백질 발현이 감소함을 확인하였다. 결과적으로 무막줄기세포출물은 SIN-1을 처리한 LLC-$PK_1$ 세포에서 산화적 스트레스에 대한 보호 효과가 있음을 알 수 있었으며, 이들 결과를 바탕으로 무막줄기세포추출물의 항산화 기능성 소재로서의 활용 가능성을 확인하였다.


  1. S. I. Liochev, "Reactive oxygen species and the free radical theory of aging", Free Radical Biology and Medicine, Vol.60, pp.1-4, 2013. DOI:
  2. R. P. Patel, J. McAndrew, H. Sellak, C. R. White, H. Jo, B. A. Freeman, V. M. Darley-Usmar, "Biological aspects of reactive nitrogen species", Biochimica et Biophysica Acta (BBA) - Bioenergetics, Vol.1411, No.2-3, pp.385-400, 1999. DOI:
  3. H. S. Kim, Y. H. Kang, "Antioxidant activity of ethanol extracts of non-edible parts (stalk, stem. leaf, seed) from oriental melon", Korean Journal of Plant Resources, Vol.23, No.5, pp.451-457, 2010.
  4. B. Halliwell, "Antioxidant defence mechanisms: from the beginning to the end (of the beginning)", Free radical research, Vol.31, No.4, pp.261-272, 1999. DOI:
  5. K. Hensley, K. A. Robinson, S. P. Gabbita, S. Salsman, R. A. Floyd, "Reactive oxygen species, cell signaling, and cell injury", Free Radical Biology and Medicine, Vol.28, No.10, pp.1456-1462, 2000. DOI:
  6. D. M. Small, J. S. Coombes, N. Bennett, D. W. Johnson, G. C. Gobe, "Oxidative stress, anti-oxidant therapies and chronic kidney disease" Nephrology, Vol.17, No.4, pp.311-321, 2012. DOI:
  7. E. Ozbek, "Induction of oxidative stress in kidney", International journal of nephrology, Vol.2012, No., pp.1-9, 2012. DOI:
  8. N. D. Vaziri, "Roles of oxidative stress and antioxidant therapy in chronic kidney disease and hypertension", Current opinion in nephrology and hypertension, Vol.13, No.1, pp.93-99, 2004.
  9. J. S. Lee, J. L. Song, J. H. Kil, B. J. Jeong, J. S. Jeong, T. G. Huh, K. Y. Park, "Protective effects of phellinus linteus and curry-added cooked mixed grain rice extracts on oxidative stress-induced LLC-$PK_{1}$ cell damage", Journal of the Korean Society of Food Science and Nutrition, Vol.43, No.11, pp.1674-1680, 2014. DOI:
  10. S. H. Park, "Antioxidative effect of tamoxifen in proximal tubule cells", Korean Journal of Laboratory Animal Science, Vol.19, No., pp.110-116, 2013.
  11. J. Gimble, F. Guilak, "Adipose-derived adult stem cells: isolation, characterization, and differentiation potential", Cytotherapy, Vol.5, No.5, pp.362-369, 2003. DOI:
  12. M. Raff, "Adult stem cell plasticity: fact or artifact?", Annual review of cell and developmental biology, Vol.19, No.11, pp.1-22, 2003. DOI:
  13. J. M. Gimble, A. J. Katz, B. A. Bunnell, "Adipose-derived stem cells for regenerative medicine", Circulation research, Vol.100, No.9, pp.1249-1260, 2007. DOI:
  14. T. Liu, M. Lee, J. J. Ban, W. Im, I. Mook-Jung, M. Kim, "Cytosolic extract of human adipose stem cells reverses the amyloid beta-induced mitochondrial apoptosis via P53/Foxo3a pathway", PloS one, Vol.12, No.1, pp.e0168859, 2017. DOI:
  15. T. Mosmann, "Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays", Journal of immunological methods, Vol.65, No.1-2, pp.55-63, 1983.
  16. H. Hagar, N. Ueda, S. V. Shah, "Role of reactive oxygen metabolites in DNA damage and cell death in chemical hypoxic injury to LLC-$PK_{1}$ cells", American Journal of Physiology-Renal Physiology, Vol.271, No.1, pp.F209-F215, 1996. DOI:
  17. A. K. Salahudeen, "Role of lipid peroxidation in $H_{2}O_{2}$-induced renal epithelial (LLC-$PK_{1}$) cell injury", American Journal of Physiology-Renal Physiology, Vol.268, No.1, pp.F30-F38, 1995. DOI:
  18. S. P. Andreoli, J. A. McAteer, S. A. Seifert, S. A. Kempson, "Oxidant-induced alterations in glucose and phosphate transport in LLC-$PK_{1}$ cells: mechanisms of injury", American Journal of Physiology-Renal Physiology, Vol.265, No.3, pp.F377-F384, 1993. DOI:
  19. H. Hagar, N. Ueda, S. V. Shah, "Endonuclease induced DNA damage and cell death in chemical hypoxic injury to LLC-$PK_{1}$ cells", Kidney international, Vol.49, No.2, pp.355-361, 1996. DOI:
  20. M. Tanito, M. P. Agbaga, R. E. Anderson, "Upregulation of thioredoxin system via Nrf2-antioxidant responsive element pathway in adaptive-retinal neuroprotection in vivo and in vitro ", Free Radical Biology and Medicine, Vol.42, No.12, pp.1838-1850, 2007. DOI:
  21. A. Loboda, M. Damulewicz, E. Pyza, A. Jozkowicz, J. Dulak, "Role of Nrf2/HO-1 system in development, oxidative stress response and diseases: an evolutionarily conserved mechanism", Cellular and molecular life sciences, Vol.73, No.17, pp.3221-3247, 2016. DOI:
  22. M. Cebula, E. E. Schmidt, E. S. Arner, "TrxR1 as a potent regulator of the Nrf2-Keap1 response system", Antioxidants and redox signaling, Vol.23, No.10, pp.823-853, 2015. DOI:
  23. D. Ross, J. K. Kepa, S. L. Winski, H. D. Beall, A. Anwar, D. Siegel, "NAD (P) H: quinone oxidoreductase 1 (NQO1): chemoprotection, bioactivation, gene regulation and genetic polymorphisms", Chemico-biological interactions, Vol.129, No.1-2, pp.77-97, 2000. DOI:
  24. Y. Koriyama, Y. Nakayama, S. Matsugo, S. Kato, "Protective effect of lipoic acid against oxidative stress is mediated by Keap1/Nrf2-dependent heme oxygenase-1 induction in the RGC-5 cellline", Brain research, Vol.1499, pp.145-157, 2013. DOI:
  25. A. Sakurai, M. Nishimoto, S. Himeno, N. Imura, M. Tsujimoto, M. Kunimoto, S. Hara, "Transcriptional regulation of thioredoxin reductase 1 expression by cadmium in vascular endothelial cells: role of NF-E2- related factor-2", Journal of cellular physiology, Vol.203, No.3, pp.529-537, 2005. DOI:
  26. D. Mustacich, G. Powis, "Thioredoxin reductase", Biochemical Journal, Vol.346, No.1, pp.1-8, 2000. DOI:
  27. J. I. Abe, B. C. Berk, "Reactive oxygen species as mediators of signal transduction in cardiovascular disease", Trends in Cardiovascular Medicine, Vol.8, No.2, pp.59-64, 1998. DOI:
  28. G. H. Choi, H. C. Shin, "The effects of Lycium Chinense Milie on the $H_{2}O_{2}$-treated LLC-$PK_{1}$ cell's redox status and $NF-{\kappa}B$ Signaling", The Journal of Internal Korean Medicine, Vol.30, No.1, pp.36-50, 2009.
  29. P. Tripathi, A. Aggarwal, "NF-${\kappa}B$ transcription factor: a key player in the generation of immune response", Current Science, Vol.90, No.4, pp.519-531, 2006.
  30. S. Ruiz, P. E. Pergola, R. A. Zager, N. D. Vaziri, "Targeting the transcription factor Nrf2 to ameliorate oxidative stress and inflammation in chronic kidney disease", Kidney international, Vol.83, No.6, pp.1029-1041, 2013. DOI:
  31. R. Rajakariar, M. M. Yaqoob, D. W. Gilroy, "COX-2 in inflammation and resolution", Molecular interventions, Vol.6, No.4, pp.199-207, 2006. DOI:
  32. J. R. Vane, J. A. Mitchell, I. Appleton, A. Tomlinson, D. Bishop-Bailey, J. Croxtall, D. A. Willoughby, "Inducible isoforms of cyclooxygenase and nitric-oxide synthase in inflammation", Proceedings of the National Academy of Sciences, Vol.91, No.6, pp.2046-2050, 1994. DOI:
  33. J. H. Yoon, S. G. Park, M. J. Lee, J. Y. Park, K. S. Seo, K. C. Woo, C. E. Lee, "Antioxidant and anti-inflammatory effects of Bletilla striata Reichenbach fil. fractions as cosmetic", Journal of Life Science, Vol.23, No.9, pp.1073-1078, 2013. DOI:
  34. M. S. Park, B. S. Kim, P. Devarajan, "Hypoxia/re-oxygenation injury induces apoptosis of LLC-$PK_{1}$ cells by activation of caspase-2", Pediatric Nephrology, Vol.22, No.2, pp.202-208, 2007. DOI:
  35. A. Havasi, S. C. Borkan, "Apoptosis and acute kidney injury", Kidney international, Vol.80, No.1, pp.29-40, 2011. DOI:
  36. A. Gross, J. M. McDonnell, S. J. Korsmeyer, "BCL-2 family members and the mitochondria in apoptosis", Genes and development, Vol.13, No.15, pp.1899-1911, 1999.
  37. J. Yang, X. Liu, K. Bhalla, C. N. Kim, A. M. Ibrado, J. Cai, T. I. Peng, D. P. Jones, X. Wang, "Prevention of apoptosis by Bcl-2: release of cytochrome c from mitochondria blocked", Science, Vol.275, No.5303, pp.1129-1132, 1997. DOI:
  38. T. J. Fan, L. H. Han, R. S. Cong, J. Liang, "Caspase family proteases and apoptosis", Acta biochimica et biophysica Sinica, Vol.37, No.11, pp.719-727, 2005. DOI:
  39. A. H. Boulares, A. G. Yakovlev, V. Ivanova, B. A. Stoica, G. Wang, S. Iyer, M. Smulson, "Role of poly (ADP-ribose) polymerase (PARP) cleavage in apoptosis Caspase 3-resistant PARP mutant increases rates of apoptosis in transfected cells", Journal of Biological Chemistry, Vol.274, No.33, pp.22932-22940, 1999. DOI:
  40. Y. K. Na, J. J. Ban, M. Lee, W. Im, M. Kim, "Wound healing potential of adipose tissue stem cell extract", Biochemical and biophysical research communications, Vol.485, No.1, pp.30-34, 2017. DOI:
  41. W. S. Kim, B. S. Park, S. H. Park, H. K. Kim, J. H. Sung, "Antiwrinkle effect of adipose-derived stem cell: activation of dermal fibroblast by secretory factors", Journal of dermatological science, Vol.53, No.2, pp.96-102, 2009. DOI:
  42. C. H. Won, G. H. Park, X. Wu, T. N. Tran, K. Y. Park, B. S. Park, D. Y. Kim, O. Kwon, K. H. Kim, "The Basic mechanism of hair growth stimulation by adipose-derived stem cells and their secretory factors", Current stem cell research and therapy, Vol.12, No.7, pp.535-543, 2017. DOI:
  43. M. Lee, L. Tian, W. Im, M. Kim, "Exosomes from adipose-derived stem cells ameliorate Huntington's disease phenotypes in an in vitro model", European Journal of Neuroscience, Vol.44, No.4, pp.2114-2119, 2016. DOI:
  44. Z. Wu, Y. Yu, L. Niu, A. Fei, S. Pan, "IGF-1 protects tubular epithelial cells during injury via activation of ERK/MAPK signaling pathway", Scientific reports, Vol.6, pp.28066, 2016. DOI: