Title page
Contents
INTRODUCTION 5
MATERIALS and METHODS 8
Materials 8
Cell Culture 9
Cell Viability Assay 9
Morphological Observation of Nuclear Change 10
Measurement of Apoptosis in Cells 10
Flow Cytometric Measurement of Mitochondrial Transmembrane Potential 11
Measurement of Cytochrome c Release 11
Measurement of Caspase-3 Activity 13
Measurement of Intracellular Reactive Oxygen Species (ROS) Formation 13
Measurement of Total Glutathione 14
Statistical Analysis 15
RESULTS 16
Effect of Lamotrigine on Rotenone-or MPP+-Induced Nuclear Damage and Cell Death 16
Effect of Lamotrigine on Rotenone-or MPP+-Induced Changes in the Mitochondrial Membrane Permeability 22
Effects of Lamotrigine on Rotenone-or MPP+-Induced Formation of ROS and Depletion of GSH 26
DISCUSSION 30
SUMMARY 34
REFERENCES 35
국문초록 40
Abstract 42
Fig. 1. Effect of lamotrigine on the rotenone-or MPP+-induced cell death. PC12 cells were pre-treated with lamotrigine (1-50 μM) for 15 min, exposed to 2.5 μM rotenone or 500 μM MPP+ for 24 h, and cell viability was determined. Data are expressed as the percentage of cell viability and represent means ± S.E.M. (n = 6). +p [ 0.05, compared to control (percentage of control); and *p [ 0.05, compared to neurotoxin alone. 19
Fig. 2. Effect of oxidant scavengers on neurotoxin-induced cell death. PC12 cells were pre-treated with various scavengers (1 mM N-acetylcysteine [NAC], 20 μM trolox, 25 μM carboxy-PTIO [PTIO] and 30 μM MnTBAP [TBAP]) for 15 min, exposed to 2.5 μM rotenone or 500 μM MPP+ for 24 h, and cell viability was determined. Data represent means ± S.E.M. (n = 6). +p [ 0.05 compared to control; and *p [ 0.05 compared to neurotoxin alone. 20
Fig. 3. Effect of lamotrigine on nuclear damage due to rotenone. PC12 cells were treated with 2.5 μM rotenone in the presence of 5 μM lamotrigine for 24 h. In experiment A, nuclear morphological changes were observed by fluorescence microscopy after nuclei staining with Hoechst 33258. (a) control cells; (b) cells treated with rotenone alone; (c) cells treated with rotenone and lamotrigine; (d) cells treated with lamotrigine alone. a-d are representative of four different experiments. In experiment B, the 21
Fig. 4. Effect of lamotrigine on the loss of the mitochondrial transmembrane potential. PC12 cells were treated with neurotoxins in the presence of 5 μM lamotrigine for 24 h. Data are expressed as the percentage of cells with depolarized mitochondria and represent mean in four separate experiments. +p [ 0.05, compared to control; and *p [ 0.05, compared to neurotoxins. Lamotrigine was expressed as Lamo. 24
Fig. 5. Effect of lamotrigine on cytochrome c release and activation of caspase-3. PC12 cells were treated with neurotoxins in the presence of lamotrigine (1-10μM). Data are expressed as nanograms/ml for cytochrome c release (A) and units for caspase-3 activity (B), and represent means ± S.E.M. (n = 6). +p [ 0.05, compared to control; and *p [ 0.05, compared to neurotoxins. The levels of cytochrome c in the cytosolic fractions were also analyzed by Western blotting with anti-cytochrome c antibody (A). Dat 25
Fig. 6. Effect of lamotrigine on the rotenone-or MPP+-induced ROS formation. PC12 cells were treated with neurotoxins in the presence of lamotrigine (1-50μM). The values are expressed as arbitrary units (a.u.) of fluorescence. Data represent means ± S.E.M. (n = 6). +p [ 0.05, compared to control; and *p [ 0.05, compared to neurotoxins. Lamotrigine was expressed as Lamo. 28
Fig. 7. Effect of lamotrigine on the rotenone-or MPP+-induced GSH depletion. PC12 cells were treated with neurotoxins in the presence of lamotrigine (1-50μM) for 24 h. The values are expressed as nmol/mg protein for GSH contents. Data represent means ± S.E.M. (n = 6). +p [ 0.05, compared to control; and *p [ 0.05, compared to neurotoxins. 29