Effect of Korea Red Ginseng Extract on PC12 Cell Death Induced by Serum Deprivation

홍삼 수용성 추출물이 PC12 세포사멸에 미치는 영향

  • Lee, Sang-Hyun (Hong-Ik Oriental Clinic) ;
  • Yun, Young-Gab (Dept. of Oriental Medical Prescription, College of Oriental Medicine, Won-Kwang University)
  • 이상현 (홍익한의원) ;
  • 윤용갑 (원광대학교 한의과대학 방제학교실)
  • Received : 2009.03.09
  • Accepted : 2009.04.08
  • Published : 2009.04.30
  • 39 0


Abstract

Objectives : This study was to evaluate the pharmacological effect of Korea Red Ginseng aqueous extract (KRGE) on serum-deprived apoptosis of neuronal-like pheochromocytoma PC12 cells and to investigate its underlying action mechanism. Methods : KRGE was prepared by extracting Korea Red Ginseng with hot water and concentrating using a vacuum evaporator. Cell viability was determined after incubation of cells with KRGE or chemical inhibitor in serum-deprived medium for 60 h by counting intact nuclei following lysing of the cell membrane. Caspase activities were measured using chromogenic substrates and signal-associated protein phosphorylation and cytochrome c release were determined by Western blot analyses using their specific antibodies. Results : Serum deprivation induced PC12 cell death, which was accompanied by typical morphological features of apoptotic cell, such as nuclear fragmentation, caspase-3 activation, and cytochrome c release. This apoptotic cell death was significantly inhibited by KRGE and caspase-3 inhibitor, but not by the addition of NMA, ODQ, and PD98059. KRGE promoted phosphorylation of Akt and Bad, and this phosphorylation was inhibited by the PI3K inhibitor LY92004. In addition, this inhibitor also reversed KRGE-mediated protection of PC 12 cells from serum deprivation. These results suggested that KRGE protects PC12 cells from serum deprivation-induced apoptosis through the activation of PI3K/Akt-dependent Bad phosphorylation and cytochrome c release, resulting in caspase-3 activation. Conclusions : KRGE should be considered as a potential therapeutic drug for brain diseases including stroke induced by apoptosis of neuronal cells.

References

  1. Zhou W, Chai H, Lin PH, Lumsden AB, Yao Q, Chen CJ. Molecular mechanisms and clinical applications of ginseng root for cardiovascular disease. Med Sci Monit. 2004;10(8):RA187-92.
  2. Kaneko H, Nakanishi K. Proof of the mysterious efficacy of ginseng: basic and clinical trials: clinical effects of medical ginseng, Korean red ginseng: specifically, its anti-stress action for prevention of disease. J Pharmacol Sci. 2004;95(2):158-62. https://doi.org/10.1254/jphs.FMJ04001X5
  3. Miles AN, Knuckey NW. Apoptotic neuronal death following cerebral ischaemia. J Clin Neurosci. 1998;5(2):125-45. https://doi.org/10.1016/S0967-5868(98)90027-3
  4. Marone R, Cmiljanovic V, Giese B, Wymann MP. Targeting phosphoinositide 3-kinase: moving towards therapy. Biochim Biophys Acta. 2008;1784(1):159-85.
  5. Zimmermann KC, Green DR. How cells die: apoptosis pathways. J Allergy Clin Immunol. 2001;108:S99-103. https://doi.org/10.1067/mai.2001.117819
  6. Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME. Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science. 1995;270(5240):1326-31. https://doi.org/10.1126/science.270.5240.1326
  7. Yun TK, Lee YS, Lee YH, Kim SI, Yun HY. Anticarcinogenic effect of Panax ginseng C.A.Meyer and identification of active compounds. J Korean Med Sci. 2001;16(Suppl) :S6-18. https://doi.org/10.3346/jkms.2001.16.S.S6
  8. Lee SJ, Kim KM, Namkoong S, Kim CK, Kang YC, Lee H, Ha KS, Han JA, Chung HT, Kwon YG, Kim YM. Nitric oxide inhibition of homocysteine-induced human endothelial cell apoptosis by down-regulation of p53-dependent Nox aexpression through the formation of S-nitrosohomocysteine. J Biol Chem. 2005;280(7):5781-8. https://doi.org/10.1074/jbc.M411224200
  9. Yap E, Tan WL, Ng I, Ng YK. Combinatorial-approached neuroprotection using pan-caspase inhibitor and poly (ADP-ribose) polymerase (PARP) inhibitor following experimental stroke in rats; is there additional benefit? Brain Res. 2008;1195:130-8. https://doi.org/10.1016/j.brainres.2007.12.024
  10. Del Peso L, Gonzalez-Garcia M, Page C, Herrera R, Nunez G.Interleukin-3-induced phosphorylation of BAD through the protein kinase Akt. Science. 1997;278(5883):687-9. https://doi.org/10.1126/science.278.5338.687
  11. Schonhoff CM, Bulseco DA, Brancho DM, Parada LF, Ross AH. The Ras-ERK pathway is required for the induction of neuronal nitric oxide synthase in differentiating PC12 cells. J Neurochem. 2001;78(3):631-9. https://doi.org/10.1046/j.1471-4159.2001.00432.x
  12. Ha KS, Kim KM, Kwon YG, Bai SK, Nam WD, Yoo YM, Kim PK, Chung HT, Billiar TR, Kim YM. Nitric oxide prevents 6-hydroxydopamine-induced apoptosis in PC12 cells through cGMP-dependent PI3 kinase/Akt activation. FASEBJ. 2003;17(9):1036-47. https://doi.org/10.1096/fj.02-0738com
  13. 신천호. 問答式本草學. 서울:영신문화사. 1992:343-4.
  14. Attele AS, Wu JA, Yuan CS. Ginseng pharmacology: multiple constituents and multiple actions. Biochem Pharmacol. 1999;58(11):1685-93. https://doi.org/10.1016/S0006-2952(99)00212-9
  15. Renolleau S, Fau S, Goyenvalle C, Joly LM, Chauvier D, Jacotot E, Mariani J, Charriaut-Marlangue C. Specific caspase inhibitor Q-VD-OPh prevents neonatal strokein P7 rat: a role for gender. J Neurochem. 2007;100(4):1062-71. https://doi.org/10.1111/j.1471-4159.2006.04269.x
  16. Casciola-Rosen L, Nicholson DW, Chong T, Rowan KR, Thornberry NA, Miller DK, Rosen A. Apopain/CPP32 cleaves proteins that are essential for cellular repair: a fundamental principle of apoptotic death. J Exp Med. 1996;183(5):1957-64. https://doi.org/10.1084/jem.183.5.1957
  17. Thorburn A. Death receptor-induced cell killing. Cell Signal. 2004;16(2):139-44. https://doi.org/10.1016/j.cellsig.2003.08.007
  18. Tachikawa E, Kudo K, Harada K, Kashimoto T, Miyate Y, Kakizaki A, Takahashi E. Effects of ginseng saponins on responses induced by various receptor stimuli. Eur J Pharmacol. 1999;369(1):23-32. https://doi.org/10.1016/S0014-2999(99)00043-6
  19. Cheng Y, Shen LH, Zhang JT. Anti-amnestic and anti-aging effects of ginsenoside Rg1 and Rb1 and its mechanism of action. Acta Pharmacol Sin. 2005;26(2):143-9. https://doi.org/10.1111/j.1745-7254.2005.00034.x
  20. 조대연. 동의보감에 수록된 인삼이 주약으로 배오된 방제의 활용범위, 병증, 주치, 병리 및 구성내용 조사. 원광대학교 대학원. 석사학위논문. 2002.
  21. Zinkel S, Gross A, Yang E. BCL2 family in DNA damage and cell cycle control. Cell Death Differ. 2006;13(8):1351-9. https://doi.org/10.1038/sj.cdd.4401987
  22. 신길구. 申氏本草學. 서울:수문사. 1982:2-8.
  23. Chen LW, Wang YQ, Wei LC, Shi M, Chan YS. Chinese herbs and herbal extracts for neuroprotection of dopaminergic neurons and potential therapeutic treatment of Parkinson's disease. CNS Neurol Disord Drug Targets. 2007;6(4):273-81. https://doi.org/10.2174/187152707781387288
  24. Kim YM, Kim TH, Seol DW, Talanian RV, Billiar TR. Nitric oxide suppression of apoptosis occurs in association with an inhibition of Bcl-2 cleavage and cytochrome crelease. J Biol Chem. 1998;273(47):31437-41. https://doi.org/10.1074/jbc.273.47.31437
  25. Shibata S. Chemistry cancer preventing activities of ginseng saponins and some related triterpenoid compounds. J Korean Med Sci. 2001;16(Suppl):S28-37. https://doi.org/10.3346/jkms.2001.16.S.S28
  26. Bhatia M. Apoptosis versus necrosis in acute pancreatitis. Am J Physiol Gastrointest Liver Physiol. 2004;286(2):G189-96. https://doi.org/10.1152/ajpgi.00304.2003
  27. Rausch WD, Liu S, Gille G, Radad K. Neuroprotective effects of ginsenosides. Acta Neurobiol Exp (Wars). 2006;66(4):369-75.
  28. Yang L, TaoL Y, Chen XP. Roles of NF-kB in central nervous system damage and repair. Neurosci Bull. 2007;23(5):307-13. https://doi.org/10.1007/s12264-007-0046-6
  29. Kim YM, Chung HT, Kim SS, Han JA, Yoo YM, Kim KM, Lee GH, Yun HY, Green A, Li J, Simmons RL, Billiar TR. Nitric oxide protects PC12 cells from serum deprivation-induced apoptosis by cGMP-dependent inhibition of caspase signaling. J Neurosci. 1999;19(16):6740-7.