Rosmarinic acid protects human dopaminergic neuronal cells against hydrogen peroxide-induced apoptosis

Toxicology 250 (2008) 109–115

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Toxicology journal homepage: www.elsevier.com/locate/toxicol

Rosmarinic acid protects human dopaminergic neuronal cells against hydrogen peroxide-induced apoptosis Hyo Jung Lee a,d,1 , Hong-Suk Cho a,b,1 , Euteum Park a,b , Seung Kim a , Sook-Young Lee b , Chun-Sung Kim c , Do Kyung Kim c , Sung-Jun Kim a , Hong Sung Chun a,b,∗ a

Department of Biotechnology (BK21 Program), Chosun University, Gwangju 501-759, Republic of Korea Research Center for Proteineous Materials, Chosun University, Gwangju 501-759, Republic of Korea Department of Oral Physiology, College of Dentistry (BK21 Program), Chosun University, Gwangju 501-759, Republic of Korea d Creation and Love Women’s Hospital, Gwangju 502-800, Republic of Korea b c

a r t i c l e

i n f o

Article history: Received 12 April 2008 Received in revised form 18 June 2008 Accepted 20 June 2008 Available online 4 July 2008 Keywords: Rosmarinic acid Dopaminergic cell Heme oxygenase-1 Apoptosis

a b s t r a c t In this study, we investigated the protective effects of rosmarinic acid (RA) on H2 O2 -induced neurotoxicity in human dopaminergic cell line, SH-SY5Y. Results showed that RA significantly attenuated H2 O2 -induced reactive oxygen species (ROS) generation and apoptotic cell death. Rosmarinic acid effectively suppressed the up-regulation of Bax and down-regulation of Bcl-2. Furthermore, RA stimulated the antioxidant enzyme heme oxygenase-1 (HO-1). We also demonstrated that the HO-1 induction by RA was associated with the protein kinase A (PKA) and phosphatidylinositiol-3-kinase (PI3K) signaling pathways. These results suggest that RA can protect SH-SY5Y cells under oxidative stress conditions by regulating apoptotic process. Thus, RA should be clinically evaluated for the prevention of neurodegenerative diseases. © 2008 Elsevier Ireland Ltd. All rights reserved.

1. Introduction Parkinson’s disease (PD) is a common neurodegenerative disease characterized by the selective loss of substantia nigra (SN) dopaminergic neurons. Even though there are several pathogenic mechanisms of PD, the most widely accepted mechanism of dopaminergic cell death in PD is a vicious cycle of oxidative stress (Zhang et al., 2000). Several lines of evidence strongly suggested that oxidative stress induced by reactive oxygen species (ROS) is common central player in affected human PD brains and experimental animal models of PD (Nagatsu and Sawada, 2007; Singh

Abbreviations: DAPI, 4 ,6-diamidino-2-phenylindole; DCF, 2 ,7 dichlorofluorescein; H2 DCFDA, 2 ,7 -dichlorodihydrofluorescein diacetate; H2 O2 , hydrogen peroxide; HO-1, heme oxygenase-1; MAP kinase, mitogen-activated protein kinase; PD, Parkinson’s disease; PI3K, phosphatidylinositol-3-kinase; PKA, protein kinase A; PKC, protein kinase C; RA, rosmarinic acid; ROS, reactive oxygen species; SN, substantia nigra; SOD, superoxide dismutase; XTT, 2,3-bis(2methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide; ZnPP, zinc (II) protoporphyrin IX. ∗ Corresponding author at: Department of Biotechnology and Research Center for Proteineous Materials, Chosun University, 375 Seosuk-dong, Dong-gu, Gwangju 501-759, Republic of Korea. Tel.: +82 62 230 6609; fax: +82 62 230 6609. E-mail address: [email protected] (H.S. Chun). 1 These authors contributed equally to this study. 0300-483X/$ – see front matter © 2008 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.tox.2008.06.010

et al., 2007). Hydrogen peroxide (H2 O2 ) is a major ROS and can induce apoptosis in many different cell types (Jenner, 2003; Wang et al., 2008). Although several antioxidant molecules in the brain such as superoxide dismutase (SOD), glutathione peroxidase and ascorbate can remove the ROS, oxidative stress is caused by the imbalance between the rate of oxidants production and the level of antioxidants (Nagatsu and Sawada, 2007; Jenner, 2003). For these reasons, much interest has focused on the antioxidant defenses including supplement with exogenous antioxidant. Recently, naturally occurring antioxidants have received great attention because they are perceived as safe and functional compounds to treat the neurodegenerative diseases. Rosmarinic acid (RA) is a naturally occurring hydroxylated polyphenolic compound in various plant families such as Lamiaceae herbs, Boraginaceae, sea grass family Zosteraceae, and fern family Blechnaceae (Petersen and Simmonds, 2003). It has been known that RA has multiple biological activities such as antioxidative, anti-inflammatory and antiviral activities. RA displayed a strong antiviral and anti-inflammatory effect against Japanese encephalitis (Swarup et al., 2007). Additional studies have revealed that RA effectively inhibited ROS-mediated damage in macrophages and astrocytes (Qiao et al., 2005; Gao et al., 2005). Furthermore, other studies reported that RA could prevent apoptosis in cardiac muscle cells, astrocytes and PC12 cells (Kim et al., 2005; Iuvone et al., 2006). On the other hand, the effect of RA against the dopaminergic cell

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death and pathogenesis of PD has not been studied. Therefore, we examined the neuroprotective effects of RA against H2 O2 -induced dopaminergic neuronal damage. In this study, we found for the first time that RA-mediated neuroprotection in SH-SY5Y cells was involved in the attenuation of apoptotic cell death and modulation of antioxidative molecule heme oxygenase-1 (HO-1).

of RA (14–56 ␮M) for another 30 min. Then cells were washed twice with phenol red-free DMEM/F12 and were incubated with H2 O2 (100 ␮M) for 1 h. Cellular fluorescence was measured in a fluorescence microplate reader (SpecraMax Gemini EM, Molecular Devices, Sunnyvale, CA, USA) at excitation wavelength 485 nm and emission wavelength 520 nm.

2. Materials and methods

SH-SY5Y cells grown under various experimental conditions were washed twice with PBS, and then lysed with RIPA buffer (PBS, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 0.1 mg/ml phenylmethylsulfonyl fluoride, 30 mg/ml aprotinin and 1 mM Na3 VO4 ). The cells were scraped off the plate, transferred to a microfuge tube, and passed through a 21 gauge needle to shear DNA and reduce the viscosity. After 30 min incubation on ice, cell lysates were microfuged at 14,000 × g for 10 min at 4 ◦ C. The supernatants were used as the total cell lysates. Protein concentration was determined by the BCA protein assay kit (BioRad, Hercules, CA, USA) using bovine serum albumin as a standard. Protein samples (20 ␮g) were separated on 10–15% sodium dodecyl sulfate (SDS)–polyacrylamide gel and then electrotransferred to polyvinylidine difluoride (PVDF) membrane. The membrane was incubated with primary antibody for Bcl-2 (1:4000 dilution), Bax (1:1000 dilution), HO-1 (1:4000 dilution), or actin (1:4000 dilution), and then reacted with horse radish peroxidase (HRP)-conjugated secondary antibody. Immunoreactive bands were detected by ECL chemiluminescence kit (GE Healthcare, USA).

2.1. Reagents Rosmarinic acid was obtained from InB:Hauser (Denver, CO, USA). H2 O2 and other chemicals were purchased from Sigma (St Louis, MO, USA). 2 ,7 Dichlorodihydrofluorescein diacetate (H2 DCFDA) was obtained from Molecular Probes (Invitrogen, Carlsbad, CA, USA). Antibodies against Bcl-2, Bax, HO-1 and actin were obtained from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). The PKC inhibitor Gö6976, the specific PKA inhibitor PKI, PI3 kinase inhibitor LY294002 and p38 MAP kinase inhibitor SB203580 were purchased from Calbiochem (San Diego, CA, USA). 2.2. Cell culture and treatments The human dopaminergic neuronal cell line, SH-SY5Y, was cultured in DMEM/F12 medium (Life Technologies, Rockville, MD, USA) supplemented with 10% FBS and penicillin (100 U/ml)–streptomycin (100 ␮g/ml) at 37 ◦ C in 5% CO2 . Usually, 1 day before any treatment, the culture medium was changed to DMEM/F12 medium with 0.5% FBS to reduce the serum effect. When indicated, RA was added 30 min prior to the treatment of H2 O2 . For prevent the direct interaction between RA and H2 O2 in the culture medium, at the end of the RA pretreatment, the medium was changed to fresh low-serum DMEM/F12 medium. For RA-mediated HO-1 induction signaling pathway studies, Gö6976, PKI, LY294002 and SB203580 were applied individually 1 h before RA treatment. RA was dissolved in dimethyl sulfoxide (DMSO) as a stock solution (20 mg/ml; approximately 56 mM) and diluted to the desired final concentrations (5–20 ␮g/ml; approximately 14–56 ␮M). The DMSO content in the final concentration of RA should never exceed 0.1%. Gö6976, LY294002 and SB203580 were dissolved in DMSO as a 1000× stock solution. PKI was dissolved in sterile deionized distilled water. In a single experiment each treatment was performed in triplicate. To estimate cell viability, XTT (2,3-bis-(2-methoxy-4nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide) reduction assay was used in combination with the total cell counting, using trypan blue dye exclusion as previously described (Chun et al., 2001a). 2.3. Nuclear staining for assessment of apoptosis Nuclear staining with DAPI (4 ,6 -diamidino-2-phenylindole) was performed to evaluate apoptosis. SH-SY5Y cells were cultured in 24-well plates at a seeding density of 1 × 105 cells per well for 24 h, and then treated with H2 O2 for 18 h with or without pretreatment with RA for 30 min. The treated SH-SY5Y cells were fixed with 1% paraformaldehyde (in phosphate buffered saline; PBS) for 30 min at room temperature and washed twice with PBS. Permeate the cells with ice-cold ethanol for 5 min at room temperature and washed twice with PBS. The fixed cells were stained with DAPI (300 nM) for 5 min at room temperature in dark, washed twice with PBS and examined under fluorescent microscopy (IX71, Olympus, Japan). Percentage of apoptotic cells, which coincided with morphological criteria of apoptosis such as DNA fragmentation, nuclear condensation and segmentation, was counted as a ratio of apoptotic nuclei over the total number of nuclei. At least 10 fields with a minimum of 50 cells were counted.

2.6. Immunoblot analysis

2.7. Statistical analysis The data were expressed as the mean ± S.E.M. Data were first analyzed using one-way factorial analysis of variance (ANOVA). Student’s t-test or Turkey’s test was then performed to compare treated samples, and p < 0.05 was considered significant.

3. Results 3.1. RA protects SH-SY5Y cells against H2 O2 -induced cytotoxicity SH-SY5Y cells are widely used to study dopaminergic pathogenesis, because this cell line expresses some representative dopaminergic markers, such as tyrosine hydroxylase and dopamine transporter (Wang et al., 2008; Hasegawa et al., 2003). Therefore, SH-SY5Y cells can be a suitable model system to study the role of RA against H2 O2 -mediated dopaminergic cell death. In this study, the effect of RA on H2 O2 -induced SH-SY5Y cell viability loss was assessed by XTT assay and with trypan blue exclusion test. H2 O2 (100 ␮M) induced approximately 50% cell loss after 18 h treatment (Fig. 1). Initial studies were performed to examine the potential cytotoxic response of SH-SY5Y cells to RA. Cells were treated with various concentrations of RA (14–56 ␮M) for 18 h. Those concentrations of RA did not show any cytotoxicity in SH-SY5Y cells (data not shown). To investigate whether RA could protect against H2 O2 -induced dopaminergic cell death, SH-SY5Y

2.4. Caspase-3 activity assay Caspase-3 activity was measured by previously described method (Chun et al., 2001b). Cells were scraped from the plate with cold lysis buffer (50 mM HEPES, pH 7.4, 0.1% CHAPS, 1 mM DTT, 0.1 mM EDTA) and the subsequent lysates were incubated for 5 min on ice and clarified by centrifugation (10,000 × g) at 4 ◦ C for 10 min. 10 ␮g of protein samples were incubated at 25 ◦ C with 200 ␮M of caspase-3 substrates (AcDEVD-pNA; Biomol, Plymouth Meeting, PA, USA). Formation of p-nitroaniline (pNA) from the reaction was measured using a microplate reader with 405 nm wavelength. After recording data for 30–60 min, the activity was calculated as pmol substrate hydrolyzed/min. 2.5. Measurement of intracellular reactive oxygen species 2 ,7 -Dichlorodihydrofluorescein diacetate (H2 DCFDA) was used to detect intracellular ROS. The nonfluorescent H2 DCFDA becomes fluorescent product, 2 ,7 dichlorofluorescein (DCF), in the presence of wide variety of ROS including nitric oxide, secondary and tertiary peroxides. SH-SY5Y cells were cultured 70–80% confluence in 96-well plates, and then loaded with 10 ␮M H2 DCFDA for 30 min at 37 ◦ C. After removal of excess H2 DCFDA, cells were exposed to the indicated concentrations

Fig. 1. Cell death induced by H2 O2 and neuroprotective effect of RA in SH-SY5Y cells. Indicated concentrations of RA were added to the SH-SY5Y cell cultures for 30 min before the addition of H2 O2 (100 ␮M). Cell viability was assessed by XTT reduction assay and trypan blue exclusion test at 18 h after the H2 O2 addition. Data are the mean ± S.E.M. from three independent experiments in triplicate. The asterisk represents statistical significance in comparison with control value (*p < 0.05). Abbreviations: Con, untreated control; RA, rosmarinic acid.

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cells were pretreated with 14–56 ␮M RA for 30 min, followed by 100 ␮M H2 O2 treatment for 18 h. As shown in Fig. 1, H2 O2 -induced loss of cell viability was significantly attenuated by RA treatment dose dependently. Pretreatment with high concentration of RA (56 ␮M) produced almost completely blocking of H2 O2 -induced cell death. 3.2. Effect of RA on apoptosis rate and intracellular ROS level The nuclear morphological changes were assessed using DAPI staining. A significant proportion of H2 O2 -mediated cell death was apoptotic, based on DAPI-stained chromatin condensation as well as the occurrence of DNA fragmentation (Fig. 2b). Those hallmarks of apoptosis were not revealed in untreated control cells and RA treated cells (Fig. 2a and d). Of interest, RA pretreatment significantly blocked the H2 O2 -induced nuclear damage and retained nuclear morphology as normal regular and oval shape (Fig. 2c). As quantified in Fig. 2e, the basal level of apoptotic nuclei in the untreated cells was 5.0 ± 0.9%. When cells were incubated with H2 O2 for 18 h, apoptotic rate was reached to 22.3 ± 2.1% of total cells. However, a significantly reduced percentage of cells (7.1 ± 1.9%) showed apoptotic morphology when cells were pretreated with RA prior to exposure to H2 O2 . Next, we determined caspase-3 activity as another marker of apoptosis. As shown in Fig. 2f, the exposure of SH-SY5Y cells to 100 ␮M H2 O2 for various time points (6, 12, 18 and 24 h) increased caspase-3 activity by 2.4-, 2.2-, 3.4- and 2.9-fold, respectively. RA (56 ␮M) pretreatment strongly attenuated the effects of H2 O2 on caspase-3 activity. Pretreatment of RA reduced the caspase-3 activity by approximately 50% in each time points when compared with H2 O2 treated cells. Incubation of cells with RA alone did not change the basal caspase-3 activity. These results positively verify the antiapoptotic effect of RA in human dopaminergic neurons. To determine the changes of intracellular ROS in human dopaminergic cells during the H2 O2 -induced cell death and RAmediated protection, we measured ROS production in SH-SY5Y cells using fluorescent dye H2 DCFDA. As shown in Fig. 3, the levels of intracellular ROS markedly increased after 30 min and 1 h treatment with H2 O2 . However, RA significantly suppressed H2 O2 induced ROS production dose dependently. The amount of ROS in cultures pretreated with RA (14–56 ␮M) before the H2 O2 challenge for 30 min and 1 h decreased by 16–20% (14 ␮M RA), 36–37% (28 ␮M RA), and 50–50.3% (56 ␮M RA), respectively. 3.3. RA modulates Bcl-2 and Bax protein expression in H2 O2 -treated SH-SY5Y cells Several studies have demonstrated that Bcl-2 is down-regulated but Bax is up-regulated during apoptotic cell death (Adams and Cory, 2007; O’Malley et al., 2003). Therefore, we examined whether the expressions of anti-apoptotic Bcl-2 and pro-apoptotic Bax are modulated by RA. Consistent with previous studies, after treatment with 100 ␮M H2 O2 for 12 h, the expression of Bcl-2 was markedly decreased but the expression of Bax was increased (Fig. 4). However, pretreatment with RA (56 ␮M) almost completely inhibited the upregulation of Bax and effectively retained the level of Bcl-2 in SHSY5Y cells (Fig. 4). RA treatment alone maintained Bcl-2 and Bax expression when compared with untreated control. 3.4. RA up-regulates HO-1 expression HO-1 is an inducible enzyme with strong antioxidant properties. HO-1 induction is the most sensitive cellular marker for oxidative stress representing adaptive and protective response to multiple oxidative insults (Ahmad et al., 2006; Baranano and Snyder,

Fig. 2. Effect of RA on the H2 O2 -induced changes in apoptotic nuclear morphology and activation of caspase-3 in SH-SY5Y cells. (a–d) Representative fluorescence photomicrographs show the nuclei morphology of SH-SY5Y cells. (a) Untreated control cells. (b) 100 ␮M H2 O2 treated cells. (c) Treated with 56 ␮M RA for 30 min before the addition of 100 ␮M H2 O2 . (d) Treated with 56 ␮M RA alone. (e) The apoptosis rate was assessed by the ratio of apoptotic nuclei/total nuclei after 18 h treatment. (f) The temporal activation of caspase-3 was measured at various time points. 10 ␮g of each protein sample was used for the measurement of caspase-3 activity using caspase-3 specific substrate, Ac-DEVD-pNA. Activity was calculated as pmol of substrate hydrolyzed/(min (␮g protein)). Results are expressed as mean ± S.E.M. of three independent experiments. *p < 0.05, compared with H2 O2 treated cells. **p < 0.05, compared with untreated control cells. Arrows indicate chromatin condensation, reduced nuclear size, and nuclear fragmentation typically observed in apoptotic cells. Scale bar indicates 10 ␮m.

2001). As shown in Fig. 5, treatment with SH-SY5Y cells with H2 O2 (100 ␮M) led to significant increase in HO-1 expression. Of great interest, RA alone increased by almost 1.4-fold the expression of HO-1, compared with untreated control cells. Moreover, preincubation of RA (56 ␮M) for 30 min, followed by H2 O2 (100 ␮M) treatment for 12 h, induced a marked boosted expression of HO-

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Fig. 3. RA dose dependently decreases the H2 O2 -induced ROS generation in SHSY5Y cells. Cells were incubated with H2 DCFDA (10 ␮M) for 30 min, followed by incubation with various concentrations of RA (14–56 ␮M) for another 30 min. The intracellular ROS production was determined by measuring the DCF-derived fluorescence after incubation of the cells with H2 O2 (100 ␮M) for 30 min and 1 h, respectively. The fluorescence intensity monitored after 30 min incubation with H2 O2 was normalized to the arbitrary unit 1. All values represent mean ± S.E.M. of three independent experiments in triplicate. *p < 0.05, compared with H2 O2 treated cells.

1 as compared with H2 O2 treatment alone. However, RA failed to stimulate HO-2 expression (data not shown). These suggest that RA specifically up-regulates the HO-1 isoform and could suppress, at least in part, the oxidative stress via induction of HO-1 in SH-SY5Y cells. To determine the meaning of HO-1 expression on the cell viability, we compared the cytotoxicity induced by H2 O2 in SH-SY5Y cells pretreated or not with HO-1 inhibitor, ZnPP. In addition SHSY5Y cells were pretreated with ZnPP, followed by RA plus H2 O2 treatment to examine whether the protection effects of RA depend

Fig. 5. Immunoblot analysis of HO-1 expression in SH-SY5Y cells. RA (56 ␮M) was added to the medium for 30 min before the addition of H2 O2 (100 ␮M), and then the cells were incubated for 12 h. The expression of HO-1 was determined by Western blot analysis and estimated by densitometric analysis of each protein band. The results are expressed as mean ± S.E.M. from four-independent experiments. *p < 0.05, compared with untreated control (Con).

on HO-1 expression. As shown in Fig. 6a, pretreatment with ZnPP increased the sensitivity of SH-SY5Y cells to H2 O2 -induced cytotoxicity and abolished RA’s protective effect. In addition, 10 ␮M ZnPP pretreatment induced a significant increase of caspase-3 activation. As shown in Fig. 6b, the inhibition of H2 O2 - or RA plus H2 O2 induced HO-1 expression with a ZnPP pretreatment increased caspase-3 activity compared to H2 O2 treatment alone. These results suggest that the stimulated HO-1 can protect against H2 O2 -induced dopaminergic cell death and the induction of HO-1 is essential for RA to attenuate H2 O2 -induced cytotoxicity. 3.5. Induction of HO-1 by RA requires PKA, PI3K and p38 MAPK pathways

Fig. 4. Effects of RA on Bcl-2 and Bax expressions. SH-SY5Y cells were treated with H2 O2 (100 ␮M) for 12 h. RA (56 ␮M) was added to the medium for 30 min before the addition of H2 O2 . Expression of Bcl-2 and Bax were examined by immunoblots using specific antibodies and intensity of each band was estimated by densitometric analysis. Anti-actin antibody was used for normalization. All values represent mean ± S.E.M. of three independent experiments. *p < 0.05 and **p < 0.01.

Several studies suggested that various signaling pathways promote HO-1 up-regulation (Ryter et al., 2006; Kang et al., 2002). To identify the signaling pathways induced by RA to elevate HO-1 expression, SH-SY5Y cells were preincubated for 1 h with 200 nM Gö6976 (PKC inhibitor), 0.5 ␮M PKI (PKA inhibitor), 1 ␮M LY294002 (PI3 kinase inhibitor) or 1 ␮M SB203580 (p38 MAP kinase inhibitor), and then stimulated with 56 ␮M RA for 30 min and treated with 100 ␮M H2 O2 for 12 h. As shown in Fig. 7a and b, pretreatment of SH-SY5Y cells with inhibitors of the PKA and PI3K, respectively, resulted in a significant reduction of the stimulated HO-1 expression by RA plus H2 O2 . However, HO-1 expression was not affected by PKC inhibitor or p38 MAPK inhibitor treatment. This result suggests that the PKA and PI3K pathways are required for the induction of HO-1 by RA plus H2 O2 in SH-SY5Y cells. To further test if those pathways act as a potential signal for neuroprotection, we examined the effect of PKA or PI3K pathways inhibition on the extent of apoptosis in SH-SY5Y cells (Fig. 7c). As quantified by the data in Fig. 7c, both PKA inhibition and PI3K inhibition significantly increased the number of apoptotic cells compared to the cells without inhibitors. However, p38 inhibition produced only a small increase in apoptotic rate correlating with less decrease in HO-1 expression in Fig. 7a. Thus, these results support that RA regulates the expression of HO-1, at least in part, through the PKA and PI3K pathways and those pathways mainly exert a neuroprotective effect in this experimental model.

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Fig. 6. Effect of heme oxygenase-1 inhibitor on cell viability and caspase-3 activity. (a) SH-SY5Y cells were pretreated with heme oxygenase-1 inhibitor, ZnPP (10 ␮M) for 10 min and RA (56 ␮M) for 30 min. After this pretreatment, the medium was replaced and then cells were exposed to H2 O2 (100 ␮M) for 18 h. Cell viability was assessed by XTT reduction assay. (b) Caspase-3 activity was determined under the conditions of (a). Data are the mean ± S.E.M. from three independent experiments in triplicate. *p < 0.05, compared with untreated control cells. # p < 0.05, compared with ZnPP treated cells. Abbreviations: ZnPP, zinc (II) protoporphyrin IX; RA, rosmarinic acid.

4. Discussion

Fig. 7. Induction of HO-1 blocked by PKA inhibition and PI3K inhibition. SHSY5Y cells were preincubated for 1 h with either 200 nM Gö6976 (protein kinase C inhibitor), 0.5 ␮M PKI (protein kinase A inhibitor), 1 ␮M LY294002 (PI3 kinase inhibitor) or 1 ␮M SB203580 (p38 MAPK inhibitor) as indicated and then stimulated with RA (56 ␮M) for 30 min before being exposed to 100 ␮M H2 O2 . (a) Cells were left untreated (Con) or treated with rosmarinic acid (56 ␮M) plus H2 O2 (100 ␮M) and harvested after 12 h. Protein extracts were prepared and analyzed by Western blotting with specific antibody for HO-1. (b) The fold changes of HO-1 expression were plotted as a graph after densitometric analysis. Values represent the mean ± S.E.M. from three independent experiments. *p < 0.05, compared to the rosmarinic acid plus H2 O2 treated condition without inhibitors. (c) Effect of blocking the PKA or PI3K pathways on the extent of apoptosis in SH-SY5Y cells. The percentage of apoptotic cells was determined by counting the number of nuclei showing apoptotic morphology over the total nuclei number. Cells were stained with DAPI after 18 h treatment. Values represent ±S.E.M. from three independent experiments. *p < 0.05, compared to the rosmarinic acid plus H2 O2 treated condition without inhibitors.

The involvement of oxidative stress in the etiology and progression of neurodegenerative diseases including PD has led to interest in the use of naturally occurring antioxidants. Recent study revealed that RA is the most potent antioxidant among the hydroxycinnamic group of polyphenols (Soobrattee et al., 2005). In this study, we found that RA could modulate H2 O2 -induced cell death in human dopaminergic neurons. In addition, our results showed that RA exerted its protective effect through ROS scavenging (Fig. 3). These results propose that the anti-oxidative effect is a possible mechanism for RA-mediated protection. However, RA pretreatment did not completely attenuate the ROS production induced by H2 O2 (Fig. 3). This means that other mechanisms could also be exist in the protective processes regulated by RA. Our previous studies using murine dopaminergic cells showed the activation of apoptotic molecules during dopaminergic cell death (Chun et al., 2001a,b). In accordance with those previous studies, our results showed the essential morphologic form of apoptosis, activation of caspase-3, and pro-apoptotic Bax elevation and anti-apoptotic Bcl-2 down-regulation when treated with H2 O2 in SH-SY5Y cells (Figs. 2 and 4). Of interest RA pretreatment dramatically attenuated the Bax expression and apoptotic cell death. From these results it is clear that RA provides some protection against the apoptotic cell death by oxidative stress. However, when analyzing our results (Figs. 1 and 2) in detail, H2 O2 treatment induced a decrease of around 50% of cell viability but the same treatment generated only an increase of 22% of the apoptotic nuclei. It means that there are other cell death mechanisms (i.e. necrosis) in our experimental conditions. Several studies have shown that H2 O2 induces neuronal cell death via the process of apoptosis and/or necrosis depending on its concentration (Cole and Perez-Polo, 2002; Fordel et al., 2006). High concentrations of H2 O2 -induced rapid cell death, mainly necrotic cell death with no evidence of apoptosis. However, moderate concentrations (less than 100 ␮M) of H2 O2 -induced both apoptosis and necrosis. Our results demonstrated complete protective effect of RA (56 ␮M) against the H2 O2 -induced cell death in SH-SY5Y (Fig. 1). Therefore, it is possible that RA inhibits both apoptotic and necrotic processes. However, apoptosis is the most important way of neuronal cell death and is associated with pathological conditions of neurodegenerative diseases including Parkinson’s disease (Honig and Rosenberg, 2000; Mattson, 2000). Hence, the present study investigated the mechanism of apoptotic cell death after exposure to H2 O2 and protective effect of RA in SH-SY5Y cells. The major finding of this study is the critical role for HO-1 expression-related signals contributing to the dopaminergic neu-

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ronal survival by RA. As far as we are aware, this study is the first to investigate the regulating effects of RA on the HO-1 expression. Several evidences suggest that HO-1 has neuroprotective effects against oxidative stress-induced neuronal damage (Ahmad et al., 2006; Baranano and Snyder, 2001; Ryter et al., 2006). The heme oxygenase family is composed of three well-characterized isoenzymes: inducible HO-1 and constitutively expressed HO-2 and HO-3. Heme oxygenase isoforms are different gene products and dissimilar in their tissue distribution and mode of regulation (Maines, 2004). Nevertheless, they catalyze the oxidative degradation of heme to biliverdin, carbon monoxide and iron. Especially, the biliverdin is continuously converted to bilirubin, which is a potent endogenous anti-oxidant. While HO-2 may accounts for most central nervous system HO activity under normal conditions, HO-1 appears to be more involved in protection against oxidative stress (Takahashi et al., 2004). Although HO-1 protein levels are normally low in neurons, HO-1 can be highly up-regulated in cerebral ischemia (Nimura et al., 1996) and during the formation of neurofibrillary tangles in Alzheimer’s brain (Takeda et al., 2004) or markedly accumulated in neuronal Lewy bodies of Parkinson’s patients (Schipper et al., 1998). Heme and other stress stimuli that produce oxidative stress (UV light, heavy metals, glutathione depletion and H2 O2 ) enhance the HO-1 expression in practically all tissues and cells including neurons (Schipper, 2004). It has been suggested that an elevation of HO-1 by various stimuli may protective cellular response to delay the cell death. In accordance with that postulation, Fig. 5 demonstrated that the pretreatment with RA boosted HO-1 expression in SH-SY5Y cells. HO-1 decomposes the heme into equimolar amounts of carbon monoxide (CO), iron and biliverdin. Biliverdin is further converted to bilirubin, which is a potent endogenous anti-oxidant (Ryter et al., 2006). Moderate concentrations of bilirubin and CO derived from HO-1 catalysis exert antioxidant actions and cytoprotective effect (Salinas et al., 2003). On the other hand, an extremely high level of HO-1 is toxic, because it generates excessive free iron, which can damage the mitochondrial membrane and induce more oxidative stress (Ryter et al., 2006). Our results showed that RA was able to up-regulate HO-1 moderately (Fig. 5). Therefore, we were interested in determining the potential role of HO-1 in the H2 O2 -induced human dopaminergic cell damage and in the RA-mediated neuroprotection. Reduction in HO-1 expression by HO-1 inhibitor, ZnPP significantly increased the sensitivity of SH-SY5Y cells to the H2 O2 induced cytotoxicity. Furthermore, protective effects induced by RA were greatly reduced by ZnPP (Fig. 6). These results suggest that stimulated HO-1 may increase cell resistance to oxidative injury and delay the dopaminergic cell death. Accumulating evidence indicated that many inducers of HO1 activate protein phosphorylation-dependent signaling cascades that ultimately converge to the transcription factors regulation the HO-1 gene. The MAPK cascades have been principally implicated in HO-1 activation (Kietzmann et al., 2003; Krönke et al., 2003). Furthermore, signaling pathways mediated by the PI3K, PKA, or PKC has also been known to engage in HO-1 induction (Ryter et al., 2006; Krönke et al., 2003; Martin et al., 2004). As shown in Fig. 7, the results of this study confirmed that the PKA and PI3K pathways are positively relate to the regulation of HO-1 expression in SH-SY5Y cells. However, HO-1 expression was not affected by PKCand p38-pathways (Fig. 7). Based on the strong correlation between HO-1 stimulation and resistance to apoptosis, we tested whether those pathway are directly related to the neuroprotection in SHSY5Y cells. PKA- and PI3K-mediated pathways were demonstrated as anti-apoptotic pathways responded to RA in human dopaminergic neurons. From these results, we conclude that up-regulation of HO-1 expression by RA pretreatment is a neuroprotective response mediated by the PKA and PI3K pathways.

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