생명과학회지 (Journal of Life Science)

  • 제19권5호
  • /
  • Pages.598-606
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  • 2009
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  • 1225-9918(pISSN)
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  • 2287-3406(eISSN)
한국생명과학회 (Korean Society of Life Science)
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배양대뇌신경세포 저산소증모델에서 유해산소생성억제 및 사립체막전위 소실방지에 의한 반하(半夏)의 신경세포사 억제 효능

Neuroprotective Effects of Pinelliae Rhizoma Water-Extract by Suppression of Reactive Oxygen Species and Mitochondrial Membrane Potential Loss in a Hypoxic Model of Cultured Rat Cortical Cells.

  • 권건록 (동국대학교 한의과대학 내과학교실) ;
  • 문일수 (동국대학교 의과대학 해부학교실) ;
  • 이원철 (부산대학교 한의학전문대학원)
  • 발행 : 2009.05.30
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초록

본 연구는 저산소증에서 반하가 대뇌신경세포에 미치는 영향을 알아보기 위하여 $E_18$의 배양 흰쥐 대뇌신경세포를 반하로 전처리한 후, LDH assay와 tryphan blue 염색으로 세포 생존율을 측정하였고, $H_2DCF-DA$, JC-1 염색으로 MMP, ROS 및 RNS 변화를 조사하였다. 이에 반하는 저산소증으로 유발된 대뇌신경세포를 2.5 ${\mu}g/ml$까지 농도의존적으로 세포 생존률을 증가시켰으며, 시간에 따른 생존율을 살펴보면 저산소증 유발 후 1 시간에는 별 차이를 보이지 않았지만 3 일, 5 일 후에는 각각 10.2%, 17.8%로 매우 유의한 증가를 보였다. 저산소증에서 반하가 MMP에 미치는 영향을 보기 위해 저산소증 유발직전과 유발 후 1 일, 3 일, 5 일에 JC-1으로 염색하고 미토콘드리아의 염색강도를 측정한 결과 적색형광은 실험군에서 전반적으로 대조군에 비하여 강하게 염색되는 미토콘드리아의 비율을 증가시킨 반면 녹색형광은 대조군과 뚜렷한 차이를 보이지 않았다. 즉 반하가 저산소증으로 유발된 MMP의 소실을 감소시킴을 알 수 있다. 또한 반하는 전반적으로 $H_2DCF-DA$에 염색되는 세포 비율을 현저하게 낮추는 것으로 나타나 저산소증으로 유발된 ROS 및 RNS의 생성을 유의성 있게 감소시켰다. 따라서 반하는 저산소증에서 ROS의 생성을 억제하고 MMP의 소실을 막아 세포의 에너지고갈을 방지함으로서 신경세포를 보호하는 것으로 이해된다.

Oxidative stress by free radicals is a major cause of neuronal cell death. Excitotoxicity in hypoxia/ischemia causes an increase in reactive oxygen species (ROS) and a loss of mitochondrial membrane potential (MMP), resulting in dysfunction of the mitochondria and cell death. Pinelliae Rhizoma (PR) is a traditional medicine for incipient stroke. We investigated the effects of PR Water-Extract on the modulation of ROS and MMP in a hypoxic model using cultured rat cortical cells. PR Water-Extract was added to the culture medium at various concentrations (0.25${\sim}$5, 5.0 ${\mu}g/ml$) on day in vitro 12(DIV12), given a hypoxic shock (2% $O_2$/5% $CO_2$, $37^{\circ}C$, 3 hr), and cell viability was assessed on DIV15 by Lactate Dehydrogenase Assay (LDH assays). PR Water-Extract showed a statistically significant effect on neuroprotection (10${\sim}$15% increase in viability; p<0.01) at 1.0 and 2.5 ${\mu}g/ml$ in normoxia and hypoxia. Measurement of ROS production by $H_2DCF-DA$ stainings showed that PR Water-Extract efficiently reduced the number and intensity of ROS-producing neurons, especially at 1 hr post shock and DIV15. When MMP was measured by JC-1 stainings, PR Water-Extract efficiently maintained high-energy charged mitochondria. These results indicate that PR Water-Extract protects neurons in hypoxia by preventing ROS production and preserving the cellular energy level.

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참고문헌

  1. Andreadis, A. A., S. L. Hazen, S. A. A. Comhair, and S. C. Erzurum. 2003. Oxidative and nitrosative events in asthma. Free Radic. Biol. Med. 35, 213-225 https://doi.org/10.1016/S0891-5849(03)00278-8
  2. Arechabala, B., C. Coiffard, P. Rivalland, L. J. Coiffard, and Y. de Roeck-Holtzhauer. 1999. Comparison of cytotoxicity of various surfactants tested on normal human fibroblast cultures using the neutral red test, MTT assay and LDH release. J. Appl. Toxicol. 19, 163-165 https://doi.org/10.1002/(SICI)1099-1263(199905/06)19:3<163::AID-JAT561>3.0.CO;2-H
  3. Beltran, B., A. Mathur, M. R. Duchen, J. D. Erusalimsky, and S. Moncadan. 2000. The effect of nitric oxide on cell respiration: A key to understanding its role in cell survival or death. Proc. Natl. Acad. Sci. USA 97, 14602-14607 https://doi.org/10.1073/pnas.97.26.14602
  4. Brewer, G. J., J. R. Torricelli, E. K. Evege, and P. J. Price. 1993. Optimized survival of hippocampal neurons in B27-supplemented Neurobasal, a new serum-free medium combination. J. Neurosci. Res. 35, 567-676 https://doi.org/10.1002/jnr.490350513
  5. Evenson, D. P., Z. Darzynkiewicz, and M. R. Melamed. 1982. Simultaneous measurement by flow cytometry of sperm cell viability and mitochondrial membrane potential related to cell motility. J. Histochem. Cytochem. 30, 279-280 https://doi.org/10.1177/30.3.6174566
  6. Fleury, C., B. Mignotte, and J. L. Vayssiere. 2002. Mitochondrial reactive oxygen species in cell death signaling. Biochimie. 84, 131-141 https://doi.org/10.1016/S0300-9084(02)01369-X
  7. Friedman, J. E. and G. G. Haddad. 1993. Major differences in Ca2+, response to anoxia between neonatal and adult rat CA1 neurons: role of Ca2+ and Na+. J. Neurosci. 13, 63-72
  8. Fubini, B. and A. Hubbard. 2003. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) generation by silica in inflammation and fibrosis. Free Radic. Biol. Med. 34, 1507-1516 https://doi.org/10.1016/S0891-5849(03)00149-7
  9. Fuh, Jr., S. J. Wang, E. B. Larson, and H. C. Liu. 1996. Prevalence of stroke in kinmen. Stroke. 27, 1338-1341 https://doi.org/10.1161/01.STR.27.8.1338
  10. Halestrap, A. P., P. M. Kerr, S. Javadov, and K. Y. Woodfield. 1998. Elucidating the molecular mechanism of the permeability transition pore and its role in reperfusion injury of the heart. Biochim. Biophys. Acta. 1366, 79-94 https://doi.org/10.1016/S0005-2728(98)00122-4
  11. Halliwell, B. and J. M. Gutteridge. 1985. The importance of free radicals and catalytic metal ions in human diseases. Mol. Aspects Med. 8, 89-193 https://doi.org/10.1016/0098-2997(85)90001-9
  12. Hansen, A. 1977. Extracellular potassium concentration in juvenile and adult brain cortex during anoxia. Acta Physiol. Scand. 99, 412-420 https://doi.org/10.1111/j.1748-1716.1977.tb10394.x
  13. Heales, S. J., J. P. Bolanos, V. C. Stewart, P. S. Brookes, J. M. Land, and J. B. Clark. 1999. Nitric oxide, mitochondria and neurological disease. Biochim. Biophys. Acta. 1410, 215-228 https://doi.org/10.1016/S0005-2728(98)00168-6
  14. Hoe, J. 1999. Dong-Eui-Bo-Gam. pp. 965, 961, 1955, Byuinmoonhwasa, Seoul
  15. Ji, H. J. and S. I. Lee. 2007. Hanyack-Gyugyuk-Juhea. pp. 329, Korean index Co., Seoul
  16. Kerr, J. F. R., A. H. Wyllie, and A. R. Currie. 1972. Apoptosis: a basic biological phenomenon with wideranging implications in tissue kinetics. Br. J. Cancer 26, 239-245 https://doi.org/10.1038/bjc.1972.33
  17. Kim, H. C. 2001. Pharmacology of Korea. pp. 346-347, Jipmoondang, Seoul.
  18. Kirino, T. 1982. Delayed neuronal death in the gerbil hippocampus following ischemia. Brain Res. 239, 57-69 https://doi.org/10.1016/0006-8993(82)90833-2
  19. Kirino, T., A. Tamura, and K. Sano. 1985. Selective vulnerability of the hippocampus to ischemia-reversible and irreversible types of ischemic cell damage. Prog. Brain Res. 63, 39-58 https://doi.org/10.1016/S0079-6123(08)61974-3
  20. Kirkeby, O. J., S. Kutzsche, C. Risoe, and I. R. Rise. 2000. Cerebral nitric oxide concentration and microcirculation during hypercapnia, hypoxia, and high intracranial pressure in pigs. J. Clin. Neurosci. 7, 531-538 https://doi.org/10.1054/jocn.2000.0788
  21. Kroemer, G., B. Dallaporta and M. Resche-Rigon. 1998. The mitochondrial death/life regulator in apoptosis and necrosis. Annu. Rev. Physiol. 60, 619-642 https://doi.org/10.1146/annurev.physiol.60.1.619
  22. Lemasters, J. J., T. Qian, C. A. Bradham, D. A. Brenner, W. E. Cascio, L. C. Trost, Y. Nishimura, A. L. Nieminen, and B. Herman. 1999. Mitochondrial dysfunction in the pathogenesis of necrotic and apoptotic cell death. Bioenerg. Biomembr. 31, 305-319 https://doi.org/10.1023/A:1005419617371
  23. Lobner, D. 2000. Comparison of the LDH and MTT assays for quantifying cell death: validity for neuronal apoptosis. J. Neurosci. Methods 96, 147-152 https://doi.org/10.1016/S0165-0270(99)00193-4
  24. Nieminen, A. L. 2003. Apoptosis and necrosis in health and disease: role of mitochondria. Int. Rev. Cytol. 224, 29-55 https://doi.org/10.1016/S0074-7696(05)24002-0
  25. Oshio, H., M. Tsukui, and T. Matsuoka. 1978. Isolation of l-ephedrine from 'pinelliae tuber'. Chem. Pharm. Bull. (Tokyo) 26, 2096-2097 https://doi.org/10.1248/cpb.26.2096
  26. Rego, A. C. and C. R. Oliveira. 2003. Mitochondrial dysfunction and reactive oxygen species in excitotoxicity and apoptosis: implications for the pathogenesis of neurodegenerative diseases. Neurochem. Res. 28, 1563-1574 https://doi.org/10.1023/A:1025682611389
  27. Schild, L., J. Huppelsberg, S. Kahlert, G. Keilhoff, and G. Reiser. 2003. Brain mitochondria are primed by moderate Ca2+ rise upon hypoxia；reoxygenation for functional breakdown and morphological disintegration. J. Biol. Chem. 278, 25454-25460 https://doi.org/10.1074/jbc.M302743200
  28. Zamzami, N., T. Hirsch, B. Dallaporta, P. X. Petit, and G. Kroemer. 1997. Mitochondrial implication in accidental and programmed cell death: apoptosis and necrosis. J. Bioenerg. Biomembr. 29, 185-193 https://doi.org/10.1023/A:1022694131572
  29. Zimmermann, K. C. and D. R. Green. 2001. How cells die: Apoptosis pathways. J. Allergy Clin. Immunol. 108, 99-103 https://doi.org/10.1067/mai.2001.117819

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