Effect of Moutan Cortex Radicis on gene expression profile of differentiated PC12 rat cells oxidative-stressed with hydrogen peroxide

모단피의 PC12 cell 산화억제 효과 및 neuronal 유전자 발현 profile 분석에 대한 연구

  • Kim Hyun Hee (Department of Oriental Medicine, Graduate School of Kyunghee University) ;
  • Rho Sam Woong (Department of Oriental Medicine, Graduate School of Kyunghee University) ;
  • Na Youn Gin (Purimed R&D Institute) ;
  • Bae Hyun Su (Department of Oriental Medicine, Graduate School of Kyunghee University) ;
  • Shin Min Kyu (Department of Oriental Medicine, Graduate School of Kyunghee University) ;
  • Kim Chung Suk (Korea Institute of Oriental Medicine) ;
  • Hong Moo Chang (Department of Oriental Medicine, Graduate School of Kyunghee University)
  • 김현희 (경희대학교 한의과대학 생리학교실) ;
  • 노삼웅 (경희대학교 한의과대학 생리학교실) ;
  • 나영인 (퓨리메드(주) 기업부설연구소) ;
  • 배현수 (경희대학교 한의과대학 생리학교실) ;
  • 신민규 (경희대학교 한의과대학 생리학교실) ;
  • 김정숙 (국립한의학연구소) ;
  • 홍무창 (경희대학교 한의과대학 생리학교실)
  • Published : 2003.04.01

Abstract

Yukmijihwang-tang has been widely used as an and-aging herbal medicine for hundred years in Asian countries. Numerous studies show that Yukmijihwangtang has anti-oxidative effect both in vivo and in vitro. It has been reported that Moutan Cortex Radicis extract (MCR) was the most effective herb in Yukmijihwang-tang on undifferentiated PC12 cells upon oxidative-stressed with hydrogen peroxide. The purpose of this study is to; 1) evaluate the recovery of neuronal damage by assessing the anti-oxidant effect of MCR on PC12 cells differentiated with nerve growth factor (NGF), 2) identify candidate genes responsible for anti-oxidative effect on differentiated PC12 cells by oligonucleotide chip microarray. PC12 cells, which were differentiated by treating with NGF, were treated without or with hydrogen peroxide in the presence or absence of various concentration of MCR. Cell survival was determined by using MTS assay. Measurement of intracellular reactive oxygen species (ROS) generation was determined using the H2DCFDA assay The viability of cells treated with MCR was significantly recovered from stressed PC12 cell. In addition, wide rage of concentrations of MCR shows dose-dependent inhibitory effect on ROS production in oxidative-stressed cells. Total RNAs of cells without treatment(Control group), only treated with H₂O₂ (stressed group) and treated with both H₂O₂ and of MCR (MCR group) were isolated, and cDNAs was synthesized using oligoT7(dT) primer. The fragmented cRNAs, synthesized from cDNAs, were applied to Affymetrix GeneChip Rat Neurobiology U34 Array. mRNA of Calcium/calmodulin-dependent protein kinase II delta subunit(CaMKII), neuron glucose transporter (GLUT3) and myelin/oligodendrocyte glycoprotein(MOG) were downregulated in Stressed group comparing to Control group. P2X2-5 receptor (P2X2R-5), P2X2-4 receptor (P2X2R-4), c-fos, 25 kDa synaptosomal attachment protein(SNAP-25a) and GLUT3 were downregulated, whereas A2 adenosine receptor (A2AR), cathechol-O-methyltransferase(COMT), glucose transporter 1 (GLUT1), EST223333, heme oxygenase (HO), VGF, UI-R-CO-ja-a-07-0-Ul.s1 and macrophage migration inhibitory factor (MIF) were upregulated in MCA group comparing to Control group. Expression of Putative potassium channel subunit protein (ACK4), P2X2A-5, P2X2A-4, Interferon-gamma inducing factor isoform alpha precursor (IL-18α), EST199031, P2XR, P2X2 purinoceptor isoform e (P2X2R-e), Precursor interleukin 18 (IL-18) were downregulated, whereas MOO, EST223333, GLUT-1, MIF, Neuronatin alpha, UI-R-C0-ja-a-07-0-Ul.s1, A2. adenosine receptor, COMT, neuron-specific enolase (NSE), HO, VGF, A rat novel protein which is expressed with nerve injury (E12625) were upregulated in MCR group comparing to Stressed group. The results suggest that decreased viability and AOS production of PC12 cell by H₂O₂ may be, at lease, mediated by impaired glucose transporter expression. It is implicated that the MCR treatment protect PC12 cell from oxidative stress via following mechanisms; improving glucose transport into the cell, enhancing expression of anti-oxidative genes and protecting from dopamine cytotoxicity by increment of COMT and MIF expression. The list of differentially expressed genes may implicate further insight on the action and mechanism behind the anti-oxidative effects of herbal extract Moutan Cortex Radicis.

Keywords

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