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NEUROPHARMACOLOGY

Protective Effect of Glycyrrhizin on 1-Methyl-4-phenylpyridinium-Induced Mitochondrial Damage and Cell Death in Differentiated PC12 Cells

Department of Neurology, Gangneung Asan Hospital, College of Medicine, University of Ulsan, Gangwon-do, South Korea (S.B.Y.); and Department of Pharmacology, College of Medicine, Chung-Ang University, Seoul, South Korea (S.B.Y., S.E.P., C.S.L.)

Received January 7, 2007; accepted February 16, 2007.

	Abstract

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Defects in mitochondrial function have been shown to participate in the induction of neuronal cell injury. The aim of the present study was to assess the preventive effect of licorice compounds glycyrrhizin and 18-glycyrrhetinic acid against the toxicity of parkinsonian neurotoxin 1-methyl-4-phenylpyridinium (MPP⁺) in relation to the mitochondria-mediated cell death process and role of oxidative stress. MPP⁺ induced the nuclear damage, the changes in the mitochondrial membrane permeability, leading to the cytochrome c release and caspase-3 activation, the formation of reactive oxygen species, and the depletion of glutathione (GSH) in differentiated PC12 cells. Glycyrrhizin up to 100 µM significantly attenuated cell death and depletion of GSH due to MPP⁺ concentration-dependently. Meanwhile, 18-glycyrrhetinic acid showed a maximal inhibitory effect at 10 µM; beyond this concentration, the inhibitory effect declined. The protective effect of licorice compounds was also detected in the rotenone-treated PC12 cells. Glycyrrhizin and 18-glycyrrhetinic acid prevented the MPP⁺-induced formation of the mitochondrial permeability transition. The results show that both glycyrrhizin and a metabolite, 18-glycyrrhetinic acid, exhibit a depressant effect against the MPP⁺ toxicity. Glycyrrhizin and 18-glycyrrhetinic acid may prevent the cytotoxicity of MPP⁺ by suppressing the mitochondrial permeability transition formation. The preventive effect seems to be ascribed to the inhibitory effect on the formation of reactive oxygen species and depletion of GSH.

MPTP produces an irreversible and severe parkinsonian-like syndrome in human and nonhuman primates (Przedborski and Jackson-Lewis, 1998; Przedborski et al., 2000). The inhibition of complex I in the mitochondrial electron transport chain induced by MPP⁺, the active metabolite of MPTP, results in impaired ATP production, loss of mitochondrial membrane potential, and formation of ROS (Cassarino et al., 1997; Schulz et al., 1997). The membrane permeability transition of mitochondria is known as a central event in the course of toxic and oxidative forms of cell injury, as well as apoptosis (Mignotte and Vayssière, 1998). Along with respiratory chain inhibition, MPP⁺-induced neuronal cell death is suggested to be mediated by formation of the mitochondrial permeability transition, which leads to the release of cytochrome c and activation of caspases, and by disturbance of intracellular Ca²⁺ homeostasis (Cassarino et al., 1999; Lee et al., 2002, 2005).

Licorice root is a traditional herbal remedy that has been used for the treatment of various pathologic conditions, including chronic hepatitis and gastric ulcer (Shibata, 2000). Increasing evidence indicates that glycyrrhizin (GL), a triterpenoid saponin found in Glycyrrhiza glabrata, and its hydrolyzed metabolite, 18-glycyrrhetinic acid (GA), reveal anti-inflammatory, anticancer, and antihepatotoxic effects (Jeong et al., 2002; Matsui et al., 2004; Agarwal et al., 2005). GL and GA have antioxidant ability and reduce oxidative damage due to carbon tetrachloride, tert-butyl hydroperoxide, or ischemia-reperfusion injury (Nagai et al., 1991; Jeong et al., 2002; Kinjo et al., 2003). GA attenuates tumor necrosis factor-, lipopolysaccharide-induced cell death in human hepatoblastoma cell line, and cultured liver cells (Yoshikawa et al., 1999; Zheng and Lou, 2003).

Mitochondrial dysfunction, increased oxidative stress, and inflammation are involved in neurodegenerative process in Parkinson's disease (Olanow and Tatton, 1999; Jenner, 2003). Therefore, the compounds that have properties, such as mitochondrial function enhancement, anti-inflammatory activity, and antioxidant ability may provide the neuroprotective effect on the degeneration of striatal dopaminergic neurons (Bonuccelli and Del Dotto, 2006). GA and GL are known to exhibit an antioxidant and anti-inflammatory effect. However, the effect of licorice compounds against neuronal cell injury due to exposure of parkinsonian toxin MPP⁺ remains uncertain. Therefore, the aim of the present study was to assess the preventive effect of GL and GA against the neurotoxicity of MPP⁺ in relation to the mitochondria-mediated cell death process and role of oxidative stress.

	Materials and Methods

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Materials. TiterTACS colorimetric apoptosis detection kit was purchased from Trevigen, Inc. (Gaithersburg, MD), Quantikine M rat/mouse cytochrome c assay kit were from R&D systems (Minneapolis, MN), anti-cytochrome c (A-8) was from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA), horseradish peroxidase-conjugated anti-mouse IgG was from EMD-Calbiochem (La Jolla, CA), SuperSignal West Pico chemiluminescence substrate for cytochrome c detection in Western blot was from Pierce Biotechnology Inc. (Rockford, IL), ApoAlert CPP32/caspase-3 assay kit was from Clontech Laboratories Inc. (Palo Alto, CA), and Mn-TBAP was from OXIS International Inc. (Portland, OR). MPP⁺, glycyrrhizin (glycyrrhizic acid ammonium salt), 18-glycyrrhetinic acid, catalase (from bovine liver; 10,000–25,000 U/mg protein), carboxy-PTIO, MTT, Hoechst 33258, DiOC₆(3), DCFH₂-DA, 5,5'-dithio-bis-(2-nitrobenzoic acid), glutathione reductase, phenylmethylsulfonyl fluoride, and RPMI 1640 medium were obtained from Sigma-Aldrich Inc. (St. Louis, MO).

Cell Culture. Rat PC12 cells (adrenal gland; pheochromocytoma) were obtained from Korean Cell Line Bank (Seoul, South Korea). PC12 cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated horse serum, 5% heat-inactivated fetal bovine serum (FBS), 100 U/ml penicillin, and 100 µg/ml streptomycin according to the manual of the cell line bank. Cells were differentiated by treating with 100 ng/ml 7S nerve growth factor for 9 days (Tatton et al., 2002). Cells were washed with RPMI 1640 medium containing 1% FBS 24 h before experiments and replated onto the 96- and 24-well plates.

Cell Viability Assay. Cell viability was measured by using the MTT assay, which is based on the conversion of MTT to formazan crystals by mitochondrial dehydrogenases (Mosmann, 1983). PC12 cells (4 x 10⁴) were treated with MPP⁺ in the presence of licorice compounds for 24 h at 37°C. The medium (200 µl) was incubated with 10 µl of 10 mg/ml MTT solution for 2 h at 37°C. After centrifugation at 412g for 10 min, culture medium was removed, and 100 µl of dimethyl sulfoxide was added to each well to dissolve the formazan. Absorbance was measured at 570 nm using a microplate reader (Spectra MAX 340; Molecular Devices Co., Sunnyvale, CA). Cell viability was expressed as a percentage of the value in control cultures.

Morphological Observation of Nuclear Change. PC12 cells (1 x 10⁶ cells/ml) were treated with MPP⁺ for 24 h at 37°C, and the nuclear morphological change was assessed using the Hoechst dye 33258 (Oberhammer et al., 1992). Cells were incubated with 1 µg/ml Hoechst 33258 for 3 min at room temperature, and nuclei were visualized using an Olympus Microscope with a WU excitation filter (Olympus, Tokyo, Japan).

Measurement of Apoptosis in Cells. Apoptosis was assessed by measuring the DNA fragmentation, which occurs following the activation of endonucleases. PC12 cells (1 x 10⁵) were treated with MPP⁺ for 24 h at 37°C, washed with phosphate-buffered saline (PBS), and fixed with formaldehyde solution. Nucleotide (dNTP) was incorporated at the 3' ends of DNA fragments using terminal deoxynucleotidyl transferase, and the nucleotide was detected using a streptavidin-horseradish peroxidase and TACS-Sapphire according to the TiterTACS protocol. Data were expressed as absorbance at 450 nm.

Flow-Cytometric Measurement of Mitochondrial Transmembrane Potential. Changes in the mitochondrial transmembrane potential during the MPP⁺-induced apoptosis in PC12 cells were quantified by flow cytometry with the cationic lipophilic dye DiOC₆(3) (Berthier et al., 2004). Cells (1 x 10⁶/ml) were treated with neurotoxin for 24 h at 37°C, DiOC₆(3) (40 nM) was added to the medium, and cells were incubated for 15 min at 37°C. After centrifugation at 412g for 10 min, the supernatants were removed, and the pellets were suspended in PBS containing 0.5 mM EDTA. For analysis, a FACScan cytofluorometer (BD Biosciences, San Jose, CA) with argon laser excitation at 501 nm was used to assess 10,000 cells from each sample.

Measurement of Cytochrome c Release. The release of cytochrome c from mitochondria into the cytosol was assessed by performing Western blot analysis and a solid phase, enzyme-linked immunosorbent assay. PC12 cells (5 x 10⁶ cells/ml for Western blotting and 5 x 10⁵ cells/ml for ELISA) were harvested by centrifugation at 412g for 10 min, washed twice with PBS, suspended in buffer (250 mM sucrose, 10 mM KCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 0.5 mM dithiothreitol, 0.1 mM phenylmethylsulfonyl fluoride, and 20 mM HEPES-KOH at pH 7.5) and homogenized further by successive passages through a 26-gauge hypodermic needle. The homogenates were centrifuged at 100,000g for 30 min, and the supernatant was used for analysis of cytochrome c. Protein concentration was determined by the method of Bradford according to the manufacturer's instructions (Bio-Rad Laboratories, Hercules, CA).

For Western blotting, supernatants were mixed with SDS-polyacrylamide gel electrophoresis sample buffer and boiled for 5 min. Samples (30 µg protein/well) were loaded onto each lane of 12% SDS-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes (GE Healthcare Chalfont St. Giles, Buckinghamshire, UK). Membranes were blocked for 2 h in TBS (50 mM Tris-HCl, pH 7.5 and 150 mM NaCl) containing 0.1% Tween 20 and 5% nonfat dried milk. The membranes were labeled with anti-cytochrome c (diluted 1:1000 in TBS containing 0.1% Tween 20) overnight at 4°C with gentle agitation. After four washes in TBS containing 0.1% Tween 20, the membranes were incubated with horseradish peroxidase-conjugated anti-mouse IgG (1:2000) for 2 h at room temperature. Protein bands were treated with SuperSignal West Pico chemiluminescence substrate and identified by detecting the enhanced chemiluminescence in Luminescent image analyzer (Lite for Las-1000 plus version 1.1; Fuji Photo Film Co., Tokyo, Japan).

For ELISA for cytochrome c, the supernatants and cytochrome c conjugate were added into the 96-well microplates coated with monoclonal antibody specific for rat/mouse cytochrome c. The procedure was performed according to the manufacturer's instructions. Absorbance of samples was measured at 450 nm in a microplate reader. A standard curve was constructed by adding diluted solutions of cytochrome c standard, handled like samples, to the microplates coated with monoclonal antibody. The amount was expressed as nanograms/milliliter by reference to the standard curve.

Measurement of Caspase-3 Activity. The activation of caspase-3 that occurred during the apoptotic process in cells was assessed (Mignotte and Vayssière, 1998). PC12 cells (2 x 10⁶ cells/ml) were treated with neurotoxin for 24 h at 37°C, and caspase-3 activity was determined according to the user's manual for the ApoAlert CPP32/Caspase-3 assay kit. The supernatant obtained by a centrifugation of lysed cells was added to the reaction mixture containing dithiothreitol and caspase-3 substrate (N-acetyl-Asp-Glu-Val-Asp-p-nitroanilide) and incubated for 1 h at 37°C. Absorbance of the chromophore p-nitroanilide produced was measured at 405 nm. The standard curves were obtained from the absorbance of p-nitroanilide standard reagent diluted with cell lysis buffer (up to 20 nM). One unit of the enzyme was defined as the activity producing 1 nmol of p-nitroanilide.

Measurement of Intracellular ROS Formation. The dye DCFH₂-DA, which is oxidized to fluorescent 2',7'-dichlorofluorescein (DCF) by hydroperoxides, was used to measure relative levels of cellular peroxides (Fu et al., 1998). PC12 cells (4 x 10⁴) were treated with MPP⁺ for 24 h at 37°C, washed, suspended in FBS-free RPMI 1640 medium, incubated with 50 µM dye for 30 min, and washed with PBS. The cell suspensions were centrifuged at 412g for 10 min, the medium were removed, and cells were dissolved with 1% Triton X-100. Fluorescence changes were measured at an excitation wavelength of 485 nm and an emission wavelength of 530 nm using a fluorescence microplate reader (Spectrafluor; TECAN, Salzburg, Austria).

Measurement of Total Glutathione. The total glutathione (reduced form GSH + oxidized form glutathione disulfide) was determined using glutathione reductase (van Klaveren et al., 1997). PC12 cells (4 x 10⁴) were treated with MPP⁺ for 24 h at 37°C and centrifuged at 412g for 10 min in a microplate centrifuge, the media were removed, and cells were washed twice with PBS. Cells were dissolved with 2% 5-sulfosalicylic acid (100 µl) and incubated in 100 µl of the reaction mixture containing 22 mM sodium EDTA, 600 µM NADPH, 12 mM 5,5'-dithio-bis-(2-nitrobenzoic acid), and 105 mM NaH₂PO₄, pH 7.5, at 37°C. Glutathione reductase (20 µl of 100 U/ml) was added, and the mixture was incubated for an additional 10 min. Absorbance was measured at 412 nm using a microplate reader. The standard curve was obtained from absorbance of the diluted commercial GSH that was incubated in the mixture as in samples.

Statistical Analysis. Data are expressed as mean ± S.E.M. Statistical analysis was performed by one-way analysis of variance. When significance was detected, post hoc comparisons between the different groups were made using the Duncan's test for multiple comparisons. A probability less than 0.05 was considered to be statistically significant.

	Results

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Prevention of MPP⁺-Induced Cell Death and Nuclear Damage by Licorice Compounds. The preventive effect of licorice compounds GL and GA on the cytotoxicity of MPP⁺ was assessed in PC12 cells that are differentiated by nerve growth factor. The incidence of cell death after exposure to 500 µM MPP⁺ for 24 h was approximately 46%. GL (1–100 µM) significantly reduced the 500 µM MPP⁺-induced cell death with a maximal inhibitory at 50 µM; beyond this concentration, the inhibitory effect declined (Fig. 1). We also examined the effect of GA, a metabolite of GL, against the MPP⁺-induced cell viability loss in PC12 cells. The current data revealed that GA showed a maximal inhibitory effect at 10 µM; beyond this concentration, the inhibitory effect declined (Fig. 1). Although GA at 100 µM and GA at 25 µM caused approximately 3 and 11% cell death, respectively, they prevented the cell death due to MPP⁺. The preventive effect of licorice compounds against dell death was also observed in PC12 cells treated with rotenone, a mitochondrial complex I inhibitor. Treatment with 2.5 µM rotenone for 24 h caused approximately 44% cell death. As expected, GL and GA significantly reduced the rotenone-induced cell death and exhibited a maximal inhibitory effect at 10 and 5 µM, respectively (Fig. 2).

View larger version (20K): [in this window] [in a new window]   Fig. 1. Prevention of MPP+-induced cell death by licorice compounds. PC12 cells were pretreated with licorice compounds (1–100 µM GL in A or 1–25 µM GA in B) for 15 min and exposed to 500 µM MPP+ for 24 h, and cell viability was determined. Data represent mean ± S.E.M. (n = 6). +, P < 0.05 compared to control (percentage of control); *, P < 0.05 compared to MPP+ alone.

View larger version (18K): [in this window] [in a new window]   Fig. 2. Inhibition of rotenone-induced cell death by licorice compounds. PC12 cells were pretreated with licorice compounds (1–100 µM GL in A or 1–25 µM GA in B) for 15 min and exposed to 2.5 µM rotenone for 24 h, and cell viability was determined. Data represent mean ± S.E.M. (n = 6). +, P < 0.05 compared to control; *, P < 0.05 compared to rotenone alone.

To examine whether the toxic effect of MPP⁺ against PC12 cells is mediated by oxidative stress, we assessed the effect of various scavengers. Treatment with 1 mM thiol compound N-acetylcysteine, 10 µg/ml catalase (a scavenger of hydrogen peroxide), 20 µM Trolox (a scavenger of hydroxyl radicals and peroxynitrite), 25 µM carboxy-PTIO (a scavenger of nitric oxide), and 30 µM Mn-TBAP (a scavenger of peroxynitrite and cell-permeable metalloporphyrin that mimics superoxide dismutase) prevented cell death due to 500 µM MPP⁺ exposure (Fig. 3).

View larger version (18K): [in this window] [in a new window]   Fig. 3. Effect of scavengers on MPP+-induced cell death. PC12 cells were pretreated with the scavengers [1 mM N-acetylcysteine (NAC), 10 µg/ml catalase (CAT), 20 µM Trolox, 25 µM carboxy-PTIO (PTIO), and 30 µM Mn-TBAP (TBAP)] for 15 min and exposed to 500 µM MPP+ for 24 h, and cell viability was determined. Data represent mean ± S.E.M. (n = 6). +, P < 0.05 compared to control; *, P < 0.05 compared to MPP+.

View larger version (35K): [in this window] [in a new window]   Fig. 4. Preventive effect of licorice compounds on MPP+-induced nuclear damage. PC12 cells were pretreated with 50 µM GL or 10 µM GA for 15 min and exposed to 500 µM MPP+ for 24 h. In experiment A, cells were observed by fluorescence microscopy after nuclei staining with Hoechst 33258. Figure represents microscopic morphology of the control cells (a), cells treated with MPP+ (b), cells treated with MPP+ plus 50 µM GL (c), and cells treated with MPP+ plus 10 µM GA (d). a to d are representative of four different experiments. In experiment B, the 3' ends of DNA fragments were detected as described under Materials and Methods. Data are expressed as absorbance and represent mean ± S.E.M. (n = 6). +, P < 0.05 compared to control; *, P < 0.05 compared to MPP+ alone.

During the process of apoptosis, DNA fragmentation is caused by activation of endonucleases. To clarify the preventive effect of licorice compounds on the MPP⁺-induced nuclear damage, we further assessed the protective effect by performing the quantitative analysis for DNA fragmentation. The amount of fragmented DNA was measured by monitoring the binding of dNTP to the 3' ends of DNA fragments and detected by a quantitative colorimetric assay. PC12 cells were treated with 500 µM MPP⁺ in the presence or absence of licorice compounds. Control cells showed absorbance of 0.219 ± 0.006 (mean ± S.E.M. of six experiments), whereas exposure to 500 µM MPP⁺ for 24 h increased the absorbance approximately 2.5-fold (Fig. 4B). The addition of GL and GA (50 and 10 µM each) significantly prevented the fragmentation of DNA due to MPP⁺ exposure.

Preventive Effect of Licorice Compounds on MPP⁺-Induced Changes in Mitochondrial Membrane Permeability. We assessed the preventive effect of licorice compounds against the MPP⁺-induced cell viability loss by investigating the effect on changes in the mitochondrial membrane permeability. Changes in the mitochondrial transmembrane potential in PC12 cells exposed to MPP⁺ were quantified by flow cytometry with the dye DiOC₆(3). When PC12 cells were treated with 500 µM MPP⁺ for 24 h, the percentage of cells with depolarized mitochondria (characterized by low values of the transmembrane potential) increased. As seen in the present data, GL and GA (50 and 10 µM each) significantly attenuated the MPP⁺-induced increase in cells with depolarized mitochondria (Fig. 5A).

View larger version (36K): [in this window] [in a new window]   Fig. 5. Preventive effect of licorice compounds on loss of the mitochondrial transmembrane potential, release of cytochrome c, and activation of caspase-3 due to MPP+. PC12 cells were treated with 500 µM MPP+ in the presence of licorice compounds (1–50 µM; 50 µM GL and 10 µMGA for the membrane potential and cytochrome c in Western blot) or 1 mM N-acetylcysteine (NAC) for 24 h. Data are expressed as the percentage of cells that have depolarized mitochondria for the mitochondrial membrane potential (A), nanograms/milliliter for cytochrome c release (B), and units for caspase-3 activity (C) and represent mean ± S.E.M. (n = 3–6). +, P < 0.05 compared to control; *, P < 0.05 compared to MPP+ alone.

Prevention of MPP⁺-Induced Formation of ROS and Depletion of GSH by Licorice Compounds. To determine whether the preventive effect of licorice compounds against the cytotoxicity of MPP⁺ was ascribed to the depressant effect on oxidative stress, we investigated the effect on the formation of ROS and the depletion of GSH in PC12 cells. The formation of ROS within cells was determined by monitoring a conversion of DCFH₂-DA to DCF. PC12 cells treated with 500 µM MPP⁺ showed a significant increase in DCF fluorescence. As seen in cell death findings, GL and GA (1–100 µM) prevented the MPP⁺-induced increase in DCF fluorescence and showed a maximal inhibitory effect at 50 and 10 µM, respectively; beyond these concentrations, the inhibitory effect declined (Fig. 6).

View larger version (20K): [in this window] [in a new window]   Fig. 6. Inhibition of MPP+-induced ROS formation by licorice compounds. PC12 cells were pretreated with 1 to 100 µM GL or 1 to 25 µM GA for 15 min and then exposed to 500 µM MPP+. Data are expressed as arbitrary units of fluorescence and represent mean ± S.E.M. (n = 6). +, P < 0.05 compared to control; *, P < 0.05 compared to MPP+ alone.

View larger version (18K): [in this window] [in a new window]   Fig. 7. Preventive effect of licorice compounds on MPP+-induced decrease in the GSH contents. PC12 cells were pretreated with GL (1–100 µM) in A or GA (1–25 µM) in B for 15 min and then exposed to 500 µM MPP+. Data are expressed as nanomole of GSH/milligram of protein and represent mean ± S.E.M. (n = 6). +, P < 0.05 compared to control; *, P < 0.05 compared to MPP+ alone.

	Discussion

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The toxic effect of MPP⁺ was assessed in differentiated PC12 cells by measuring the nuclear damage, mitochondrial dysfunction, and cell death. PC12 cells upon the nerve growth factor stimulation not only display abundant neuritic growth but also adopt a neurochemical dopaminergic phenotype (Kadota et al., 1996). Formation of the mitochondrial permeability transition causes a release of cytochrome c from mitochondria and subsequent activation of caspase-3 that is involved in apoptotic cell death (Mignotte and Vayssière, 1998). In PC12 cells treated with MPP⁺, the condensation and fragmentation of nuclei and significant increase in caspase-3 activity indicate apoptotic cell death. MPP⁺ induces apoptosis in neuronal cells by causing loss of the mitochondrial transmembrane potential, leading to the release of mitochondrial cytochrome c and subsequent activation of caspase-3 (Cassarino et al., 1999; Lee et al., 2005). Consistent with this finding, in this study, the MPP⁺-induced apoptotic cell death seemed to be mediated by the loss of the mitochondrial membrane potential and cytochrome c release that resulted in activation of caspase-3.

Licorice compound GA attenuates the tumor necrosis factor-induced apoptotic cell death without cell surface Fas activation (Yoshikawa et al., 1999). However, it has been shown that GA induces cell death in various cancer cell lines (Hibasami et al., 2006). Furthermore, GL enhances Fas-mediated apoptosis without alteration of caspase-3-like activity (Ishiwata et al., 1999). In addition, it is uncertain whether the preventive effect of licorice compounds comes from their inhibitory action on formation of the mitochondrial permeability transition. Therefore, the present study was conducted to assess the effect of GL and GA against the MPP⁺-induced cell damage in relation to the mitochondria-mediated cell death process. As seen in the present data, GL up to 100 µM and its metabolite GA up to 25 µM significantly prevented the MPP⁺-induced cell viability loss in PC12 cells. Although there is a difference of inhibitory potency on the basis of concentration, a metabolite GA as well as GL exhibits a cytoprotective effect. The protective effect of GL and GA against cell damage was also detected in the rotenone-treated PC12 cells. Intravenous administration of GL achieved maximal plasma concentration of 36 µM in healthy men and 120 µM in hepatitis patients (Yamamura et al., 1992; van Rossum et al., 1999). In patients with chronic hepatitis, the administration with GL suppository (a mixture of 300 mg glycyrrhizinic ammonium salt and 60 µg of sodium capric acid) yielded approximately 6 µM serum GL levels (Fujioka et al., 2003). Orally administered licorice achieved 0.4 to 10 µM plasma GA levels in humans (de Groot et al., 1988; Yamamura et al., 1992). The current work was performed under acute exposure to high concentration of MPP⁺. Therefore, at the concentrations achievable in humans, both compounds seemed to exert an effective depressant effect against the neurotoxicity of parkinsonian toxins, such as MPP⁺ and rotenone. The present data suggest that GL less than 100 µM and GA less than 25 µM may prevent the toxicity of MPP⁺ against PC12 cells by suppressing loss of the mitochondrial transmembrane potential, release of mitochondrial cytochrome c, and activation of caspase-3. Meanwhile, the toxicity at the high concentrations seems to nullify the protective of licorice compounds.

The MPP⁺ treatment causes the respiratory chain inhibition, leading to the formation of ROS and nitrogen species (Jenner, 2003). ROS act upon mitochondria, causing a disruption of mitochondrial membrane potential and the release of cytochrome c (Fleury et al., 2002). The formation of ROS in PC12 cells exposed to MPP⁺ and the inhibitory effect of antioxidants, including Trolox and carboxy-PTIO, suggest that MPP⁺ induces the formation of ROS and nitrogen species that is involved in mitochondrial dysfunction and cell death. Meanwhile, the inhibitory effect of catalase indicates that the cytotoxicity of MPP⁺ seems to be mediated by extracellularly released hydrogen peroxide, a cell-permeable oxidant. GL and GA attenuate either renal injury due to ischemia-reperfusion or FeCl₂ plus ascorbate-induced lipid peroxidation in liver homogenates through scavenging action on free radicals (Yokozawa et al., 2000; Jeong et al., 2002). However, GA induces apoptosis of HL60 cells by increasing production of ROS (Makino et al., 2006). These reports represent uncertainty whether licorice compounds have antioxidant ability and reveal a cytoprotective effect. We examined whether the preventive effect of licorice compounds against the mitochondrial damage was ascribed to their inhibitory action on oxidative stress. An increased ROS production causes changes in the levels of intracellular antioxidants, such as GSH, NADH, or NADPH, that result in impairment of mitochondrial function (Fleury et al., 2002). The oxidation and depletion of cellular GSH can modulate opening of the mitochondrial permeability transition pore (Constantini et al., 1996; Hall, 1999). The mitochondrial GSH depletion is suggested to trigger the apoptotic pathway (Hall, 1999). As seen in the present study, GL and GA attenuated the formation of ROS and depletion of GSH due to exposure of MPP⁺. The inhibitory effect of licorice compounds on the MPP⁺-induced cell death approximately correlated with the effect on GSH depletion. Therefore, GL and GA seem to prevent the MPP⁺-induced changes in the mitochondrial membrane permeability by suppressing ROS formation and interfering with cellular GSH depletion.

Overall, the results show that licorice compounds GL and GA seem to reduce the MPP⁺-induced viability loss in PC12 cells by suppressing the mitochondrial permeability transition, leading to the activation of caspase-3. The preventive effect may be accomplished by the inhibitory action on the formation of ROS and depletion of GSH. The findings suggest that licorice compounds seem to exhibit a protective effect against neuronal cell injury, which is mediated by oxidative mitochondrial damage. GL and GA may provide a beneficial protective effect against the neurotoxicity due to exposure of parkinsonian toxins.

	Footnotes

 
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.

doi:10.1124/jpet.107.119602.

ABBREVIATIONS: ROS, reactive oxygen species; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; MPP⁺, 1-methyl-4-phenylpyridinium iodide; GL, glycyrrhizin; GA, 18-glycyrrhetinic acid; carboxy-PTIO, 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide; Mn-TBAP, Mn(III) tetrakis(4-benzoic acid)porphyrin chloride; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; DiOC₆(3), 3,3'-dihexyloxacarbocyanine iodide; DCFH₂-DA, 2',7'-dichlorofluorescein diacetate; PBS, phosphate-buffered saline; FBS, fetal bovine serum; GSH, glutathione; ELISA, enzyme-linked immunosorbent assay; DCF, fluorescent 2',7'-dichlorofluorescein.

Address correspondence to: Dr. Chung Soo Lee, Department of Pharmacology, College of Medicine, Chung-Ang University, Seoul 156-756, South Korea. E-mail: leecs{at}cau.ac.kr

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