DOI QR코드

DOI QR Code

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

Abstract

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.

Keywords

References

  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: https://doi.org/10.1016/j.freeradbiomed.2013.02.011
  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: https://doi.org/10.1016/S0005-2728(99)00028-6
  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: https://doi.org/10.1080/10715769900300841
  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: https://doi.org/10.1016/S0891-5849(00)00252-5
  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: https://doi.org/10.1111/j.1440-1797.2012.01572.x
  7. E. Ozbek, "Induction of oxidative stress in kidney", International journal of nephrology, Vol.2012, No., pp.1-9, 2012. DOI: http://dx.doi.org/10.1155/2012/465897
  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. https://doi.org/10.1097/00041552-200401000-00013
  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: https://doi.org/10.3746/jkfn.2014.43.11.1674
  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: https://doi.org/10.1080/14653240310003026
  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: https://doi.org/10.1146/annurev.cellbio.19.111301.143037
  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: https://doi.org/10.1161/01.RES.0000265074.83288.09
  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: https://doi.org/10.1371/journal.pone.0168859
  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. https://doi.org/10.1016/0022-1759(83)90303-4
  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: https://doi.org/10.1152/ajprenal.1996.271.1.F209
  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: https://doi.org/10.1152/ajprenal.1995.268.1.F30
  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: https://doi.org/10.1152/ajprenal.1993.265.3.F377
  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: https://doi.org/10.1038/ki.1996.52
  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: https://doi.org/10.1016/j.freeradbiomed.2007.03.018
  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: https://doi.org/10.1007/s00018-016-2223-0
  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: https://doi.org/10.1089/ars.2015.6378
  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: https://doi.org/10.1016/S0009-2797(00)00199-X
  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: https://doi.org/10.1016/j.brainres.2012.12.041
  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: https://doi.org/10.1002/jcp.20246
  26. D. Mustacich, G. Powis, "Thioredoxin reductase", Biochemical Journal, Vol.346, No.1, pp.1-8, 2000. DOI: https://doi.org/10.1042/bj3460001
  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: https://doi.org/10.1016/S1050-1738(97)00133-3
  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: https://doi.org/10.1038/ki.2012.439
  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: https://doi.org/10.1124/mi.6.4.6
  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: https://doi.org/10.1073/pnas.91.6.2046
  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: https://doi.org/10.5352/JLS.2013.23.9.1073
  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: https://doi.org/10.1007/s00467-006-0256-6
  35. A. Havasi, S. C. Borkan, "Apoptosis and acute kidney injury", Kidney international, Vol.80, No.1, pp.29-40, 2011. DOI: https://doi.org/10.1038/ki.2011.120
  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. https://doi.org/10.1101/gad.13.15.1899
  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: https://doi.org/10.1126/science.275.5303.1129
  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: https://doi.org/10.1111/j.1745-7270.2005.00108.x
  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: https://doi.org/10.1074/jbc.274.33.22932
  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: https://doi.org/10.1016/j.bbrc.2017.01.103
  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: https://doi.org/10.1016/j.jdermsci.2008.08.007
  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: https://doi.org/10.2174/1574888X12666170829161058
  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: https://doi.org/10.1111/ejn.13275
  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: https://doi.org/10.1038/srep28066