생명과학회지 (Journal of Life Science)

  • 제19권1호
  • /
  • Pages.21-33
  • /
  • 2009
  • /
  • 1225-9918(pISSN)
  • /
  • 2287-3406(eISSN)
한국생명과학회 (Korean Society of Life Science)
권호 표지
DOI QR코드

DOI QR Code

배양신경세포의 저산소증모델에서 대황 물추출물에 의한 유전자 표현 변화의 microarray 분석

Microarray Analysis of Gene Expression by Rhei Rhizoma Water Extracts in a Hypoxia Model of Cultured Neurons

  • 이현숙 (동국대학교 의과대학 해부학교실) ;
  • 송진영 (동국대학교 의과대학 해부학교실) ;
  • 문일수 (동국대학교 의과대학 해부학교실)
  • Lee, Hyun-Sook (Department of Anatomy, College of Medicine, Dongguk University) ;
  • Song, Jin-Young (Department of Anatomy, College of Medicine, Dongguk University) ;
  • Moon, Il-Soo (Department of Anatomy, College of Medicine, Dongguk University)
  • 발행 : 2009.01.30
PDF 다운로드
⟨ 이전 논문 다음 논문 ⟩

초록

대황(Rhei Rhizoma; RR, 대황(大黃))은 Rheum officinale Baill.와 Rheum palmatum L.(polygonaceae)의 땅속부분으로 남아시아의 민속의학에서 간 및 신장의 손상을 치료하는데 널리 이용되고 있다. 본 연구에서는 배양한 흰쥐 해마신경세포의 저산소증모델을 이용하여 대황의 물추출물이 유전자 표현에 미치는 영향을 microarray 방법을 이용하여 조사하였다. 배양 후 10일 (DIV10)에 추출물을 배지에 $2.5{\mu}g/ml$ 농도로 첨가하고, DIV13에 저산소증(2% $O_2$/5% $CO_2$, $37^{\circ}C$, 3 h)을 유발한 후 24 시간 후에 total RNA를 분리하여 microarray에 사용하였다. MA-plot에 의하면 표현이 연화된 대부분의 유전자는 ${\pm}2$배 이내로 증감되었다. 이 가운데 Global M 값이 0.2(즉, 15%)보다 더 증가한 유전자는 472종, Global M 값이 -0.2(즉, -15%)보다 더 감소한 유전자는 725종이였다. 세포의 생존과 관련된 유전자 가운데 세포자연사 억제유전자인 Tegt(2.4배), Nfkb1 (2.4배), Veg (1.8배), Ngfr (1.6배) 등이 크게 증가하였으며, 반면에 자연사 촉진유전자인 Bad (-64%), Cstb (-66%)는 감소하였다. 스트레스를 극복하는데 필요한 유전자인 Defb3 (2.7배), Cygb (2.2배), Ahsg (2.18배), Alox5 (2배) 등도 크게 증가하였다. 그리고 세포 성장을 촉진하는 유전자인 Erbb2 (1.84배), Mapk12 (1.8배)도 크게 증가하였다. 따라서 대황의 물추출물은 세포생존에 필요한 유전자를 증가시키고, 세포사를 유도하는 유전자는 감소시킴으로서 저산소층 스트레스에서 신경세포의 사망을 억제하는 것으로 해석된다.

In this study, we investigated the effect of Rhei Rhizoma (RR; 大黃) water extract on gene expression in a hypoxia model of cultured rat hippocampal neurons. RR water extract $(2.5{\mu}g/ml)$ was added to the culture media on day 10 in vitro (DIV10), and a hypoxic shock (2% $O_2$/5% $CO_2$, $37^{\circ}C$, 3 h) was given on DIV13. After maintaining the cultures in normoxia for 24 hr, total RNA was isolated and used for microarray analysis. The MA-plot indicated that most genes were up- or downregulated within 2-fold. There were more downregulated genes (725 ea) than upregulated ones (472 ea) when larger than Global M value 0.2 (i.e., >15% increase) or smaller than Global M value -0.2 (i.e., >15% decrease) were considered. Antiapoptosis genes such as Tegt (2.4-fold), Nfkb1 (2.4-fold) Veg (1.8-fold), Ngfr (1.6-fold) were upregulated, while pro-apoptosis genes such as Bad (-64%), Cstb (-66%) were downregulated. Genes for combating environmental stress (stress response genes) such as Defb3 (2.7-fold), Cygb (2.2-fold), Ahsg (2.18-fold), Alox5 (2-fold) were upregulated. Genes for cell proliferation (cell cycle-related genes) such as Erbb2 (1.84-fold), Mapk12 gene (1.8-fold) was upregulated. Therefore, RR water extracts upregulate many pro-survival genes while downregulating many pro-death genes. It is interpreted that these genes, in combination with other regulated genes, can promote neuronal survival in a stress such as hypoxia.

키워드

참고문헌

  1. Bonizzi, G. and M. Karin. 2004. The two NF-B activation pathways and their role in innate and adaptive immunity. Trends Immunol. 25, 280-288 https://doi.org/10.1016/j.it.2004.03.008
  2. 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. 35, 567-576 https://doi.org/10.1002/jnr.490350513
  3. Burmester, T., B. Ebner, B. Weich and T. Hankeln 2002. Cytoglobin: a novel globin type ubiquitously expressed in vertebrate tissues. Mol. Biol. Evol. 19, 416-421 https://doi.org/10.1093/oxfordjournals.molbev.a004096
  4. Chae, H. J., H. R. Kim, C. Xu, B. Bailly-Maitre, M. Krajewska, S. Krajewski, S. Banares and et. al. 2004. BI-1 regulates an apoptosis pathway linked to endoplasmic reticulum stress. Mol. Cell 15, 355-366 https://doi.org/10.1016/j.molcel.2004.06.038
  5. Chae, H. J., N. Ke, H. R. Kim, S. Chen, A. Godzik, M. Dickman and J. C. Reed. 2003. Evolutionarily conserved cytoprotection provided by Bax Inhibitor-1 homologs from animals, plants, and yeast. Gene 323, 101-113 https://doi.org/10.1016/j.gene.2003.09.011
  6. Citri, A., K. B. Skaria and Y. Yarden. 2003. The deaf and the dumb: the biology of ErbB-2 and ErbB-3. Exp. Cell Res. 284, 54-65 https://doi.org/10.1016/S0014-4827(02)00101-5
  7. Crack, P. J., J. M. Taylor, N. J. Flentjar, J. Haan, P. Hertzog, R. C. Iannello and I. Kola. 2001. Increased infarct size and exacerbated apoptosis in the Gpx-1 knockout mouse brain in response to ischemia/reperfusion injury. J. Neurochem. 78, 1389-1399 https://doi.org/10.1046/j.1471-4159.2001.00535.x
  8. Dohm, C. P., S. Siedenberg, J. Liman, A. Esposito, F. S. Wouters, J. C. Reed, M. Bahr and P. Kermer. 2006, Bax inhibitor-1 protects neurons from oxygen-glucose deprivation. J. Mol. Neurosci. 29, 1-8 https://doi.org/10.1385/JMN:29:1:1
  9. Dudoit, S., Y. H. Yang, M. J. Callow and T. P. Speed. 2002. Statistical methods for identifying differentially expressed genes in replicated cDNA microarray experiments. Statistica Sinica 12, 111-140
  10. Fordel, E., E. Geuens, S. Dewilde, P. Rottiers, P. Carmeliet, J. Grooten and L. Moens, 2004. Cytoglobin expression is upregulated in all tissues upon hypoxia: an in vitro and in vivo study by quantitative real-time PCR. Biochem. Biophys. Res. Commun. 319, 342-348 https://doi.org/10.1016/j.bbrc.2004.05.010
  11. Fujimura, M., Y. Morita-Fujimura, N. Noshita, T. Sugawara, M. Kawase and P. H. Chan. 2000. The cytosolic antioxidant copper/zinc-superoxide dismutase prevents the early release of mitochondrial cytochrome c in ischemic brain after transient focal cerebral ischemia in mice. J. Neurosci. 20, 2817-2824
  12. Fulton, C., G. M. Anderson, M. Zasloff, R. Bull and A. Quinn. 1997. Expression of natural peptide antibiotics in human skin. Lancet 350, 1750-1751 https://doi.org/10.1016/S0140-6736(05)63574-X
  13. Garcia, J. R, F. Jaumann, S. Schulz and A. Krause, J. Rodriguez-Jimenez, U. Forssmann, K. Adermann, E. Kluver, C. Vogelmeier, D. Becker, R. Hedrich, W. G. Forssmann and R. Bals. 2001. Identification of a novel, multifunctional $\beta$-defensin (human $\beta$-defensin 3) with specific antimicrobial activity. Its interaction with plasma membranes of Xenopusoocytes and the induction of macrophage chemoattraction.Cell Tissue Res. 306, 257-264 https://doi.org/10.1007/s004410100433
  14. Gennaro, R. and M. Zanetti. 2001. Structural features and biological activities of the cathelicidin-derived antimicrobial peptides. Biopolymers 55, 31-49 https://doi.org/10.1002/1097-0282(2000)55:1<31::AID-BIP40>3.0.CO;2-9
  15. Gerson, S. L. 2004. MGMT: its role in cancer aetiology and cancer therapeutics. Nat. Rev. Cancer 4, 296-307 https://doi.org/10.1038/nrc1319
  16. Ghosh, S. and D. Baltimore. 1990. Activation in vitro of NF-kappa B by phosphorylation of its inhibitor I kappa B. Nature 344, 678-682 https://doi.org/10.1038/344678a0
  17. Goslin, K., H. Asmussen and G. Banker. 1998. Rat hippocampal neurons in low-density culture, pp. 339-370, In Banker, G. and K. Goslin (eds.), 2nd eds. Culturing nerve cells. Cambridge, MA: MIT Press
  18. Green, D. R. and J. C. Reed. 1998. Mitochondria and apoptosis. Science 281, 1309-1312 https://doi.org/10.1126/science.281.5381.1309
  19. Grosjean, J., S. Kiriakidis, K. Reilly, M. Feldmann and E. Paleolog. 2006. Vascular endothelial growth factor signalling in endothelial cell survival: a role for NF-$\kappa$B. Biochem. Biophys. Res. Commun. 340, 984-994 https://doi.org/10.1016/j.bbrc.2005.12.095
  20. Hankeln, T., S. Wystub, T. Laufs, M. Schmidt, F. Gerlach, S. Saaler-Reinhardt, S. Reuss and T. Burmester. 2004. The cellular and subcellular localization of neuroglobin and cytoglobin- A clue to their function?. IUBMB Life 56, 671-679 https://doi.org/10.1080/15216540500037794
  21. Harder, J., J. Bartels, E. Christophers and J. M. Schroder. 2001. Isolation and characterization of human $\beta$-defensin-3, a novel human inducible peptide antibiotic. J. Biol. Chem. 276, 5707-5713 https://doi.org/10.1074/jbc.M008557200
  22. Harder, J., J. Bartels, E. Christophers and J. M. Schroder. 1997. A peptide antibiotic from human skin. Nature 387, 861 https://doi.org/10.1038/43088
  23. Hardison, R. C. 1996. A brief history of hemoglobins: plant, animal, protist, and bacteria. Proc. Natl. Acad. Sci. USA 93, 5675-5679 https://doi.org/10.1073/pnas.93.12.5675
  24. Hayden, M. S. and S. Ghosh. 2005. Signaling to NF-kappaB. Genes Dev. 18, 2195-2224 https://doi.org/10.1101/gad.1228704
  25. Huang, D. C. and A. Strasser. 2000. BH3-Only proteins—Essential initiators of apoptotic cell death. Cell 103, 839-842 https://doi.org/10.1016/S0092-8674(00)00187-2
  26. Jin, K. L., X. O. Mao and D. A. Greenberg. 2000. Vascular endothelial growth factor: direct neuroprotective effect in in vitro ischemia. Proc. Natl. Acad. Sci. USA 97, 10242-10247 https://doi.org/10.1073/pnas.97.18.10242
  27. Kaiser, V. and G. Diamond. 2001. Expression of mammalian defensin genes. J. Leukoc. Biol. 68, 779-784
  28. Kaplan, D. R. and R. M. Stephens. 1994. Neurotrophin signal transduction by the Trk receptor. J. Neurobiol. 25, 1404-1417 https://doi.org/10.1002/neu.480251108
  29. Karin, M. and Y. N. Ben. 2000. Phosphorylation meets ubiquitination: the control of NF-kappaB activity. Annu. Rev. Immunol. 18, 621-663 https://doi.org/10.1146/annurev.immunol.18.1.621
  30. Kinouchi, H., C. J. Epstein, T. Mizui, E. Carlson, S. F. Chen and P. H. Chan. 1991. Attenuation of focal cerebral ischemic injury in transgenic mice overexpressing CuZn superoxide dismutase. Proc. Natl. Acad. Sci. USA 88, 11158-11162 https://doi.org/10.1073/pnas.88.24.11158
  31. Kondo, T., A. G. Reaume, T. T. Huang, E. Carlson, K. Murakami, S. F. Chen, E. K. Hoffman, R. W. Scott, C. J. Epstein and P. H. Chan. 1997. Reduction of CuZn-superoxide dismutase activity exacerbates neuronal cell injury and edema formation after transient focal cerebral ischemia. J. Neurosci. 17, 4180-4189
  32. Krappmann, D. and C. Scheidereit. 2005. A pervasive role of ubiquitin conjugation in activation and termination of IkappaB kinase pathways. EMBO Rep. 6, 321-326 https://doi.org/10.1038/sj.embor.7400380
  33. Lee, G. H., H. K. Kim, S. W. Chae, D. S. Kim, K. C. Ha, M. Cuddy, C. Kress, J. C. Reed, H. R. Kim and H. J. Chae. 2007. Bax inhibitor-1 regulates endoplasmic reticulum stress-associated reactive oxygen species and heme oxygenase-1 expression. J. Biol. Chem. 282, 21618-21628 https://doi.org/10.1074/jbc.M700053200
  34. Lee, H. S., J. Y. Song and I. S. Moon. 2008 Water extracts of Rhei Rhizoma prevent production of reactive oxygen species and loss of mitochondrial membrane potential in a hypoxia model of cultured neurons. J. Life Sci. (In press)
  35. Li, D., X. Q. Chen, W. J. Li, Y. H. Yang, J. Z. Wang and A. C. Yu. 2007. Cytoglobin up-regulated by hydrogen peroxide plays a protective role in oxidative stress. Neurochem. Res. 32, 1375-1380 https://doi.org/10.1007/s11064-007-9317-x
  36. Liu, A. Y., D. Destoumieux, A. V. Wong, D, H, Park, E. V. Valore, L. Liu and T. Ganz. 2002. Human $\beta$-defensin-2 product[63]ion in keratinocytes is regulated by interleukin-1, bacteria, and the state of differentiation. J. Invest. Dermatol. 118, 275-281 https://doi.org/10.1046/j.0022-202x.2001.01651.x
  37. Margison, G. P. and M. F. Santibanez-Koref. 2002. O6-alkylguanine-DNA alkyltransferase: role in carcinogenesis and chemotherapy. Bioessays 24, 255-266 https://doi.org/10.1002/bies.10063
  38. Marinissen, M. J., M. Chiariello and J. S. Gutkind. 2001. Regulation of gene expression by the small GTPase Rho through the ERK6 (p38 gamma) MAP kinase pathway. Genes Dev. 15, 535-553 https://doi.org/10.1101/gad.855801
  39. Merx, M. W., C. Schafer, R. Westenfeld, V. Brandenburg, S. Hidajat, C. Weber, M. Ketteler and W. Jahnen-Dechent. 2005. Myocardial stiffness, cardiac remodeling, and diastolic dysfunction in calcification-prone fetuin-A-deficient mice. J. Am. Soc. Nephrol. 16, 3357-3364 https://doi.org/10.1681/ASN.2005040365
  40. Nakagawa, T., T. Yokozawa, Y. A. Kim, K. S. Kang and T. Tanaka. 2005. Activity of wen-pi-tang, and purified constituents of rhei rhizoma and glycyrrhizae radix against glucose-mediated protein damage. Am. J. Chin. Med. 33, 817-829 https://doi.org/10.1142/S0192415X05003375
  41. Nishijima, K., Y. S. Ng, L. Zhong, J. Bradley, W. Schubert, N. Jo, J. Akita, S. J. Samuelsson, G. S. Robinson, A. P. Adamis and D. T. Shima. 2007. Vascular endothelial growth factor-A is a survival factor for retinal neurons and a critical neuroprotectant during the adaptive response to ischemic injury. Am. J. Pathol. 171, 53-67 https://doi.org/10.2353/ajpath.2007.061237
  42. Niyonsaba, F., I. Nagaoka and H. Ogawa. 2006. Human defensins and cathelicidins in the skin: beyond direct antimicrobial properties. Crit. Rev. Immunol. 26, 545-576 https://doi.org/10.1615/CritRevImmunol.v26.i6.60
  43. Pennacchio, L. A., D. M. Bouley, K. M. Higgins, M. P. Scott, J. L. Noebels and R. M. Myers. 1998. Progressive ataxia, myoclonic epilepsy and cerebellar apoptosis in cystatin B-deficient mice. Nat. Genet. 20, 251-258 https://doi.org/10.1038/3059
  44. Radmark, O. 2002. Arachidonate 5-lipoxygenase. Prostaglandins Other Lipid Mediat. 68, 211-234 https://doi.org/10.1016/S0090-6980(02)00032-1
  45. Ren, J. and A. J. Davidoff. 2002. alpha2-Heremans Schmid glycoprotein, a putative inhibitor of tyrosine kinase, prevents glucose toxicity associated with cardiomyocyte dysfunction. Diabetes Metab. Res. Rev. 18, 305-310 https://doi.org/10.1002/dmrr.299
  46. Rhyu, D. Y., K. S. Kang, M. Sekiya and T. Yokozawa. 2007. Antioxidant effect of Wen-Pi-Tang and its component crude drugs on oxidative stress. Am. J. Chin. Med. 35, 127-137 https://doi.org/10.1142/S0192415X07004680
  47. Rinne, R., P. Saukko, M. Jarvinen and A. E. Lehesjoki. 2002. Reduced cystatin B activity correlates with enhanced cathepsin activity in progressive myoclonus epilepsy. Ann. Med. 34, 380-385
  48. Sagara, K., T. Oshima and T. Yoshida. 1987. Rapid and simple determination of sennosides A and B in Rhei Rhizoma by ion-pair high-performance liquid chromatography. J. Chromatogr. 403, 253-261 https://doi.org/10.1016/S0021-9673(00)96359-3
  49. Schmidt, M., F. Gerlach, A. Avivi, T. Laufs, S. Wystub, J. C. Simpson, E. Nevo, S. Saaler-Reinhardt, S. Reuss, T. Hankeln and T. Burmester. 2004. Cytoglobin is a respiratory protein in connective tissue and neurons, which is up-regulated by hypoxia. J. Biol. Chem. 279, 8063-8069 https://doi.org/10.1074/jbc.M310540200
  50. Shannon, P., L. A. Pennacchio, M. K. Houseweart, B. A. Minassian, R. M. Myers. 2002. Neuropathological changes in a mouse model of progressive myoclonus epilepsy: cystatin B deficiency and Unverricht-Lundborg disease. J. Neuropathol. Exp. Neurol. 61, 1085-1091
  51. Sondell, M., F. Sundler and M. Kanje. 2000. Vascular endothelial growth factor is a neurotrophic factor which stimulates axonal outgrowth through the flk-1 receptor. Eur. J. Neurosci. 12, 4243-4254 https://doi.org/10.1046/j.0953-816X.2000.01326.x
  52. Sondell, M., G. Lundborg and M. Kanje. 1999. Vascular endothelial growth factor has neurotrophic activity and stimulates axonal outgrowth, enhancing cell survival and Schwann cell proliferation in the peripheral nervous system. J. Neurosci. 19, 5731-5740
  53. Sun, Y., K. Jin, A. Peel, X. O. Mao, L. Xie and D. A. Greenberg. 2003. Neuroglobin protects the brain from experimental stroke in vivo. Proc. Natl. Acad. Sci. USA 100, 3497-3500 https://doi.org/10.1073/pnas.0637726100
  54. Sun, Y., K. Jin, X. O. Mao, Y. Zhu and D. A. Greenber., 2001. Neuroglobin is up-regulated by and protects neurons from hypoxic-ischemic injury. Proc. Natl. Acad. Sci. USA 98, 15306-15311 https://doi.org/10.1073/pnas.251466698
  55. Tian, J., H. Du, H. Yang, X. Liu and Z. Li. 1997. A clinical study on compound da huang (radix et Rhizoma rhei) preparations for improvement of senile persons' memory ability. J. Tradit. Chin. Med. 17, 168-173
  56. Tong, W. G., X. Z. Ding, R. C. Witt and et al. 2002. Lipoxygenase inhibitors attenuate growth of human pancreatic cancer xenografts and induce apoptosis through the mitochondrial pathway. Mol. Cancer Ther. 1, 929-935 https://doi.org/10.1096/fj.00-0866fje
  57. Tuynder, M., L. Susini, S. Prieur, S. Besse, G. Fiucci, R. Amson and A. Telerman. 2002. Biological models and genes of tumor reversion: Cellular reprogramming though tpt1/TCTP and SIAH-1. Proc. Natl. Acad. Sci. USA 23, 14976-14981
  58. Walter, L., P. Marynen, J. Szpirer, G. Levan and E. G$\ddot{u}$nther. 1995. Identification of a novel conserved human gene, TEGT. Genomics 28, 301-304 https://doi.org/10.1006/geno.1995.1145
  59. Wittenberg, J. B. and B. A. Wittenberg. 1990. Mechanisms of cytoplasmic hemoglobin and myoglobin function. Annu. Rev. Biophys. Chem. 19, 217-241 https://doi.org/10.1146/annurev.bb.19.060190.001245
  60. Wittenberg, J. B. and B. A. Wittenberg. 2003. Myoglobin function reassessed. J. Exp. Biol. 206, 2011-2020 https://doi.org/10.1242/jeb.00243
  61. Xu, Q. and J. C. Reed. 1998. Bax inhibitor-1, a mammalian apoptosis suppressor identified by functional screening in yeast. Mol. Cell 1, 337-346 https://doi.org/10.1016/S1097-2765(00)80034-9
  62. Yamaoka, T., F. Yan, H. Cao, S. S. Hobbs, R. S. Dise, W. Tong and D. B. Polk. 2008. Transactivation of EGF receptor and ErbB2 protects intestinal epithelial cells from TNF-induced apoptosis. Proc. Natl. Acad. Sci. USA 105, 11772-11777 https://doi.org/10.1073/pnas.0801463105
  63. Yang, Z., X. Mo, Q. Gong, Q. Pan, X. Yang, W. Cai, C. Li, J. X. Ma, Y. He and G. Gao. 2008. Critical effect of VEGF in the process of endothelial cell apoptosis induced by high glucose. Apoptosis 13, 1331-1343 https://doi.org/10.1007/s10495-008-0257-y
  64. Yuan, X. M., W. Li, H. Dalen, J. Lotem, R. Kama, L. Sachs and U. T. Brunk. 2002. Lysosomal destabilization in p53-induced apoptosis. Proc. Natl. Acad. Sci. USA 99, 6286-6291 https://doi.org/10.1073/pnas.092135599
  65. Zha, J., H. Harada, E. Yang, J. Jockel and S. J. Korsmeyer. 1996. Serine phosphorylation of death agonist BAD in response to survival factor results in binding to 14-3-3 not BCL-X(L). Cell 87, 619-628 https://doi.org/10.1016/S0092-8674(00)81382-3
  66. Zhao, C., I. Wang and R. I. Lehrer. 1996. Widespread expression of beta-defensin hBD-1 in human secretory glands and epithelial cells. FEBS Lett. 396, 319-322 https://doi.org/10.1016/0014-5793(96)01123-4

피인용 문헌

  1. Classification of the Efficacy of Herbal Medicine Alterations in Neuronal Hypoxia Models through Analysis of Gene Expression vol.35, pp.4, 2014, https://doi.org/10.13048/jkm.14042