Neuroprotective Effect of Rosmarinus officinalis Extract on Human Dopaminergic Cell line, SH-SY5Y

Abstract

Hydrogen peroxide (H₂O₂) is a major Reactive Oxygen Species (ROS), which has been implicated in many neurodegenerative conditions including Parkinson’s disease (PD). Rosmarinus officinalis (R. officinalis) has been reported to have various pharmacological properties including anti-oxidant activity. In this study, we investigated the neuroprotective effects of R. officinalis extract on H₂O₂-induced apoptosis in human dopaminergic cells, SH-SY5Y. Our results showed that H₂O₂-induced cytotoxicity in SH-SY5Y cells was suppressed by treatment with R. officinalis. Moreover, R. officinalis was very effective in attenuating the disruption of mitochondrial membrane potential and apoptotic cell death induced by H₂O₂. R. officinalis extract effectively suppressed the up-regulation of Bax, Bak, Caspase-3 and -9, and down-regulation of Bcl-2. Pretreatment with R. officinalis significantly attenuated the down-regulation of tyrosine hydroxylase (TH), and aromatic amino acid decarboxylase (AADC) gene in SH-SY5Y cells. These findings indicate that R. officinalis is able to protect the neuronal cells against H₂O₂-induced injury and suggest that R. officinalis might potentially serve as an agent for prevention of several human neurodegenerative diseases caused by oxidative stress and apoptosis.

Introduction

Parkinson’s disease (PD) is the most common neurodegenerative movement disorder, affecting over six million people worldwide (Licker et al. 2009) and is expected to impose an ever increasing impact on our society emotionally, socially, and financially. PD is characterized by selective degeneration of dopaminergic (DArgic) neurons of the substantia nigra and formation of fibrillar cytoplasmic inclusions known as Lewy bodys (LBs). Although the mechanism underlying selective degeneration of DArgic neurons and formation of LBs are not known completely, oxidative stress (Beal 2003; Zhang et al. 2000), mitochondrial dysfunction (Greenamyre et al. 2001; Orth and Schapira 2002), programmed cell death (Ziv et al. 1998), protein misfolding (Dawson and Dawson 2003; McNaught and Olanow 2003), and other unknown factors, which might be endogenous or exogenous (Calne and Langston 1983) have been reported to play important roles. Accumulating evidences show that oxidative stress as one of the important pathways leading to neuronal cell death in PD. Selective degeneration of DArgic neurons by chronic oxidative stress is thought to be the result of apoptosis. Although the source of increased oxidative stress is not completely known, environmental factor, excitotoxin, dopamine homeostasis, and others have gained more attention (Sayre et al. 2008). Oxidative stress may induce mitochondrial dysfunction, genetic mutation, and protein aggregation, and ultimately cause cell death (Mattson et al. 2002).

Nowadays, there is an increasing interest in focusing on natural products with antioxidant capacities that may be promising therapeutics for PD. Rosmarinus officinalis (R. officinalis) is one of the most common traditional medicinal herb. The fresh and dried leaves are used frequently in traditional Mediterranean cuisine as a flavoring agent and as a food preservative. Since R. officinalis is widely consumed, no toxicity has been reported for this herb (Aruoma et al. 2003). Historically, R. officinalis has been used as a medicinal agent to treat renal colic and dysmenorrhea. Extracts of R. officinalis are used in aromatherapy to treat anxiety-related conditions and to increase alertness. It has been reported to have various properties including anti-oxidant activity (Cheung and Tai 2007). Antioxidant activity of R. officinalis had been reported to be more potent than synthetic phenolic anti-oxidant (McCarthy et al. 2001). Antioxidant efficiency of R. officinalis is due to high content of phenolic compounds, such as monoterpenes (eteric olis), diterpene phenols (carnosic acid, carnosol, rosmanol, epirosmanol, isorosmanol, methyl carnosate), phenolic acids (rosmarinic acid), flavonols, and triterpene acids (ursolic acid, oleanolic acid, butilinic acid) (Leung and Foster 1996). Most of these compounds are available in the market and are expensive. The phenolic compounds such as carnosol, carnosic acid, and rosmarinic acid inhibit production of nitric oxide (NO) (Lo et al. 2002) and protect dopaminergic neuronal cells (Kim et al. 2006; Lee et al. 2008; Park et al. 2008). Recently, Posadas et al. (2009) have shown that R. officinalis decrease cerebral catalase activity, lipid peroxidation and ROS level in rat brain.

Nevertheless, no information is available regarding the effect of R. officinalis extract against the pathogenesis of PD. Therefore, this study was designed to evaluate the neuroprotective effects of R. officinalis extract against H₂O₂-induced DArgic neuronal damage. We observed that R. officinalis-mediated neuronal cell protection in SH-SY5Y cell was involved in the attenuation of pro-apoptotic factor induced by H₂O₂. Furthermore, we revealed that the increase of TH and AADC by R. officinalis was responsible for the dopamine production in catecholamine biosynthesis.

Materials and Methods

Materials

Dulbecco’s Modified Eagle Medium:Nutrients Mix F-12 (1:1, DMEM/F-12), Fetal Bovine Serum (FBS), penicillin, and streptomycin were obtained from Gibco BRL (Gaithersburg MD, USA). Hydrogen peroxide (H₂O₂), DMSO, Hoechst 33324, Propidium iodide (PI), and Rhodamine 123 were obtained from Sigma–Aldrich (St. Louis, MO, USA). Anti-TH antibody was purchased from Affinity BioReagents, Inc. (Golden, CO, USA). Anti-AADC antibody was purchased from Abcam (Cambridge, MA, USA). Caspase-9, Caspase-3, Bcl-2, Bak, and Bax antibodies were obtained from SANTA CRUZ (Santa cruz, CA, USA). Anti-actin antibody was purchased from Biomeda crop (Foster City, CA, USA). WEST-ZOL plus was obtained from INTRON biotech (Seongnam, Korea). Bicinchoninic acid (BCA) protein assay kit was obtained from Pierce (Rockford, IL, USA). Protein inhibitor cocktail was obtained from Calbiochem (Darmstadt, Germany). Cytotoxicity Detection Kit (LDH assay) was purchased from Roche Applied Science (Rotkreuz, Switzerland). DeadEnd^TM Fluorometric TUNEL System was purchased from Promega coporation (Madison, WI, USA).

Preparation of R. officinalis Extract

Rosmarinus officinalis was obtained from the Agricultural Research and Development Promotion Center (Jang-Heung, Korea), and was authenticated by Professor Myung-Kon Kim, Department of Bio-food technology, Chonbuk National University, South Korea. A voucher specimen was deposited at Chonbuk National University. One hundred grams of fresh R. officinalis leaves was extracted with 1L of methanol (70%) in a ratio of 1:10 W/V for 1 week at room temperature. The extract was filtered through Whatman No.1 filter paper, and concentrated using a rotary vacuum evaporator. The concentrate was freeze dried and its yield was 7.8%. The final extract was a dark green powder. This powder was then dissolved in phosphate buffered saline (PBS) and filtered through 0.2-μm membrane filter (Millipore, Bedford, MA, USA) and stored at 4°C.

Cell Culture and Treatments

The human DA neuronal cell line, SH-SY5Y was obtained from ATCC (Rockville MD). Cells were cultured in DMEM/F12 medium (GIBCO, Gaithersburg) supplemented with 10% FBS and penicillin (100 units/ml)-streptomycin (100 μg/ml) at 37°C in 5% CO₂. Media were changed every 2 days. Typically, 1 day before any treatment, the culture medium was changed to DMEM/F12 medium with 0.5% FBS to reduce the serum effect. In order to examine possible toxic effects, SH-SY5Y cells were treated with the R. officinalis extract in a concentration ranging from 1 to 100 μg/ml for 12 h. Similarly, cells were treated with H₂O₂ at concentrations ranging from 1 to 300 μM for 12 h. A total of 10 μg/ml of R. officinalis extract, which was non toxic and 150 μM H₂O₂ was chosen to evaluate the neuroprotective effects by examining cell viability. R. officinalis extract was added 1 h prior to treatment with H₂O₂. In a single experiment, each treatment was performed in triplicate.

Analysis of Cell Viability

Cell viability was determined by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. SH-SY5Y cells were seeded in 96-well plates at a density of 1 × 10⁴ cell/well and incubated for 24 h prior to experimental treatments. The cells were then subjected to the treatments of interest. After 12-h incubation, MTT (0.5 mg/ml) was added to each well. Following an additional 3-h incubation at 37°C, 100 μl of DMSO was added to dissolve the formazan crystals. The absorbance was then measured at 540 nm using a VERSAmax micro plate reader (Molecular Devices, CA, USA). Wells without cells were used as blanks and were subtracted as background from each sample. Results were expressed as a percentage of control.

Lactate Dehydrogenase (LDH) Release Assay

Cells dying by apoptosis or necrosis released LDH into the supernatant. The amount of LDH in the supernatant was measured with a cytotoxicity detection kit (Roche). In brief, the cells (1 × 10⁴ cell/well) were seeded in 96-well plates and then treated with H₂O₂ for indicated periods after being pretreated with or without R. officinalis extract for 1 h. For analysis, 100 μl supernatant was extracted from each well and was placed in separate wells of a new 96-well plate, and 100 μl catalyst solutions was added to each well and incubated at 37°C for 30 min. Absorbance was measured at 490 nm using a VERSAmax microplate reader (Molecular Devices, Sunnyvale, CA, USA). Total cellular LDH was determined by lysing the cells with 2% Triton X-100 (high control); the assay medium served as a low control and was subtracted from all absorbance measurements; Cytotoxicity (%) = (exp.value − low control)/(high control − low control) × 100.

Nuclear Staining with Hoechst 33342

Nuclear morphology was assessed by staining with the supravital DNA dye Hoechst 33342. The cells (1 x 10³ cells/well) were seeded in 8-well chamber slide for 24 h and then treated with H₂O₂ for indicated periods after pretreated with or without R. officinalis extract for 1 h. The cells were washed twice with PBS and then fixed in 4% paraformaldehyde for 15 min. After two rinses with PBS, the cells were stained with 10 μg/ml DNA dye Hoechst 33342 in PBS for 30 min at 37°C. Slides were washed twice with PBS and examined under fluorescent microscope (Nikon, Eclipse TE 2000-U, Japan) and photographed at 100× magnification.

TUNEL assay

For in situ detection of fragmented DNA, TUNEL assay was performed using DeadEnd^TM Fluorometric TUNEL System (Promega coporation, USA). Cells were cultured on 8-well chamber slide at a density of 1x10³ cells/chamber. After treatment with H₂O₂ and R. officinalis extract as indicated, cells were washed with PBS and fixed by incubation in 4% paraformaldehyde for 20 min at 4°C. The fixed cells were then washed and permeabilized with 0.2% Triton X-100 in PBS for 5 min. After rinses with PBS, the cells were incubated with terminal deoxynucleotidyl transferase recombinant (rTdT)-catalyzed reaction and nucleotide mixture for 60 min at 37°C in dark, and then immersed in stop/wash buffer for 15 min at room temperature. The cells were then washed with PBS to remove unincorporated fluorescein-12-dUTP. After washing, cells were incubated in 1 μg/ml propidium iodide (PI) solution for 15 min in dark. The cells were observed with fluorescent microscope (Nikon, Eclipse TE 2000-U, Japan) and photographed at 100× magnification.

Measurement of Mitochondrial Membrane Potential

Mitochondrial membrane potential was determined using the fluorescent dye Rhodamine 123. In brief, the cells were treated with H₂O₂ for indicated periods after being pretreated with or without R. officinalis extract for 1 h. Cells were washed with PBS and fixed by incubation in 4% paraformaldehyde for 15 min at room temperature. After rinses with PBS, the fixed cells were incubated with 10 μg/ml Rhodamine 123 for 60 min at 37°C. The cells were washed and monitored by fluorescent microscope (Nikon, Eclipse TE 2000-U, Japan) and photographed. The fluorescence intensity was determined using a Spectra Max Gemini EM fluorometer (Molecular Devices, Sunnyvale, CA, USA) at 490-nm excitation and 515-nm emission.

Immunoblotting

After treatment, cells were washed once with PBS and then lysed using ice-cold RIPA buffer with protease inhibitor cocktail. Cell lysates were centrifuged at 12,000 rpm for 25 min, and the protein concentrations were determined by the bicinchoninic acid (BCA) method using bovine serum albumin (BSA) as standard. The proteins were separated by 10% SDS-PAGE and transferred to polyvinylidine difluoride (PVDF) membrane. The Membrane was blocked with 5% (v/v) nonfat dry milk in Tris-buffered saline with Tween 20 (TBS-T) (10 mM Tris–HCl, 150 mM NaCl, and 0.1% Tween 20, pH 7.5) and incubated with primary antibody for Bcl-2, Bax, and Bak (1:1000 dilution), Caspase-3 and Caspase-9 (1:2000 dilution), TH and AADC (1:1000 dilution), or Actin (1:4000 dilution) overnight at 4°C. The membrane was washed in TBS-T and incubated for 2 h at room temperature with horseradish peroxidase (HRP)-conjugated secondary antibody. In order to reveal the reaction bands, the membrane was reacted with WEST-ZOL (plus) western blot detection system (Intron Biotechnology, Inc., Korea) and exposed on X-ray film (BioMax MS-1, Eastman Kodak, USA).

Statistical Analysis

The data were expressed as the means ± S.D. Statistical evaluation of the data was performed with the Student’s t-test when two value sets were compared. P < 0.05 was considered statistically significant.

Result

R. officinalis Protects against H₂O₂-Induced Cytotoxicity

SH-SY5Y cells were treated with different concentrations of R. officinalis extract (0, 1, 10, 50 and 100 μg/ml) for 12 h and the cell viability was determined by MTT assay. As shown in Fig. 1a, when exposed to R. officinalis concentrations of 50 μg/ml or lower, the viability of SH-SY5Y cells was the same as untreated control cells. However, a decrease of cell viability was observed with 100 μg/ml R. officinalis extract.

Fig. 1.

Rosmarinus officinalis attenuate H₂O₂-induced cell death. a SH-SY5Y cells were treated with various concentrations of R. officinalis for 12 h, and cell viability was determined by MTT assay. b SH-SY5Y cells were treated with different concentrations of H₂O₂ for 12 h, and cell viability was determined by MTT assay. c SH-SY5Y cells were pretreated with 10 μg/ml R. officinalis extract for 1 h, then treated with H₂O₂ (150 μM) for 12 h, and Cell viability was measured using the MTT assay. d SH-SY5Y cells were pretreated with 10 μg/ml R. officinalis extract for 1 h, then treated with H₂O₂ (150 μM) for 12 h, and Cell toxicity was measured by LDH assay. Data are the mean ± SD from three independent experiments in triplicate. C, control. ^* P < 0.05 versus control group. ^# P < 0.05, compared to H₂O₂ or R. officinalis-treated group

In order to evaluate whether H₂O₂ influences neuronal cytotoxicity, SH-SY5Y cells were treated with various concentrations of H₂O₂ (0, 1, 10, 50, 150, and 300 μM) for 12 h. As shown in Fig. 1b, H₂O₂ induced a dose-dependent cytotoxicity in SH-SY5Y cells.

In order to determine the protective effects of R. officinalis against H₂O₂-induced loss of cell viability, SH-SY5Y cells were pretreated with 10 μg/ml R. officinalis extract for 1 h, followed by treatment with 150 μM H₂O₂ for 12 h. As shown in Fig. 1c, H₂O₂-induced loss of cell viability was significantly attenuated by R. officinalis treatment (P < 0.05).

In order to further investigate the protective effect of R. officinalis, the release of LDH was measured (Fig. 1d). LDH release is increased as the number of dead cells increases. As shown in Fig. 1d, release of LDH was increased significantly after exposure to 150 μM H₂O₂ (P < 0.05), indicating that H₂O₂ caused cytotoxicity in SH-SY5Y cells. In contrast, R. officinalis-treated cells showed decreased release of LDH compared with H₂O₂-exposed cell group. The protective effect of R. officinalis on H₂O₂-induced cytotoxicity determined by LDH assay was similar to that determined by MTT assay. R. officinalis rescued the viability of cells against the neurotoxicity induced by H₂O₂, suggesting the protective effect of R. officinalis.

R. officinalis Protects against H₂O₂-Induced Apoptosis

Cell body shrinkage, nuclear condensation, and DNA fragmentation are hallmarks of apoptosis. We investigated whether R. officinalis extract prevents apoptosis induced by H₂O₂ in SH-SY5Y cells. Treatment with H₂O₂ (150 μM) elicited cell death as evidenced by Hoechst 33342 staining, PI staining, and TUNEL assay (Fig. 2b, f, j). The apoptotic features. such as nuclear condensation and DNA fragmentation, were not revealed in untreated control cells and R. officinalis extract-treated cells (Fig. 2a, e, i, and c, g, k). Pretreatment of R. officinalis significantly inhibited the H₂O₂-induced apoptosis (Fig. 2d, h, and l). This result demonstrates that R. officinalis decreased the level of cell death, nuclear condensation, and DNA fragmentation, and indicates that R. officinalis has an anti-apoptotic effect in SH-SY5Y cells.

Fig. 2.

Effect of R. officinalis on the H₂O₂ induced changes in apoptotic nuclear morphology and DNA fragmentation in SH-SY5Y cells. Fluorescence photomicrographs of Hoechast 33324 stained cells (a–d); (a) untreated control, (b) 150 μM H₂O₂ treated cells, (c) 10 μg/ml R. officinalis treated cells, (d) cells were pretreated with 10 μg/ml R. officinalis extract for 1 h then treated with H₂O₂ (150 μM) for 12 h. PI stained cells (e–h); (e) untreated control, (f) 150 μM H₂O₂ treated cells, (g) 10 μg/ml R. officinalis treated cells, (h) cells were pretreated with 10 μg/ml R. officinalis extract for 1 h then treated with H₂O₂ (150 μM) for 12 h. TUNEL-assayed cells (i–l); (i) untreated control, (j) 150 μM H₂O₂ treated cells, (k) 10 μg/ml R. officinalis treated cells, (l) cells were pretreated with 10 μg/ml R. officinalis extract for 1 h, and then treated with H₂O₂ (150 μM) for 12 h. Arrows indicate chromatin condensation, reduced nuclear size, and nuclear fragmentation typically observed in apoptotic cells. Each image is representative of three experiments. Pictures were taken using a fluorescent microscope (Nikon, Eclipse TE 2000-U, Japan) and photographed at 100× magnification

R. officinalis Prevents H₂O₂-Induced Reduction of the Mitochondrial Membrane Potential

A collapse of the mitochondrial trans-membrane potential has been linked to several models for apoptosis (Prehn et al. 1996). The examination of whether H₂O₂ cells induced apoptosis, and its rescue by R. officinalis involve on mitochondrial membrane potential was carried out using Rodamine 123. As shown in Fig. 3, when SH-SY5Y cells were exposed to 150 μM H₂O₂ for 12 h, the mitochondrial membrane potential was significantly decreased to 71% (P < 0.05) (Fig. 3A, B (b)). However, the cells pre-incubated with R. officinalis prior to the addition of H₂O₂, showed a markedly increased in the mitochondrial membrane potential by 83% (P < 0.05) as compared with H₂O₂-treated cells (Fig. 3A, B (d)). These results showed that R. officinalis extract suppressed the H₂O₂-induced decrease of mitochondrial membrane potential.

Fig. 3.

Effect of R. officinalis on H₂O₂-induced decrease of mitochondrial membrane potential. SH-SY5Y cells were pretreated with R. officinalis (10 μg/ml) for 1 h followed by 150 μM H₂O₂for 12 h. Cells were incubated with Rhodamine 123 and the fluorescence intensity was determined using a Spectra Max Gemini EM fluorometer (Molecular Devices, Sunnyvale, CA, USA) at 490-nm excitation and 515 -nm emission (A), membrane potential was monitored by fluorescent microscope (Nikon, Eclipse TE 2000-U, Japan) (B). The reduced fluorescence of Rhodamine 123 was determined as the reduced mitochondrial membrane potential. Results are expressed as mean ± SD of three independent experiments. ^*  P < 0.05 versus control group. ^#  P < 0.05, compared to H₂O₂ or R. officinalis-treated group

R. officinalis Modulates Bcl-2 and Bax Protein Expression in H₂O₂ Treated SH-SY5Y Cells

The Bax to Bcl-2 expression ratio can be used to determine whether a cell has undergone apoptosis. In H₂O₂-treated cells, the expression of Bcl-2 protein was down-regulated whereas the expression of Bax protein was up-regulated (Fig. 4a), which resulted in a high Bax to Bcl-2 ratio (Fig. 4b). However, pretreatment with R. officinalis attenuated the change in Bax and Bcl-2, which was induced by H₂O₂, resulting in a decrease in the Bax to Bcl-2 ratio (Fig. 4). R. officinalis extract treatment alone maintained the Bax to Bcl-2 ratio compared with control.

Fig. 4.

Effects of R. officinalis on mitochondrial apoptotic proteins, Bcl-2, and Bax expression. SH-SY5Y cells were pretreated with R. officinalis (10 μg/ml) for 1 h followed by treatment with 150 μM H₂O₂for 12 h. Expressions of Bcl-2 and Bax were assessed by immunoblots, and intensity of each band was estimated by densitometric analysis. Actin was used as an internal loading control. Results are expressed as mean ± SD of three independent experiments. ^*  P < 0.05 versus control group. ^#  P < 0.05, compared to H₂O₂ or R. officinalis-treated group

R. officinalis Attenuates the Levels of Apoptotic Proteins Induced by H₂O₂

We then investigated whether pro-apoptotic factor gene expressions were affected by R. officinalis. Pro-apoptotic factor Bak protein levels were measured by western blot. As shown in Fig. 5, H₂O₂ increased level of Bak compared with control group. However, pretreatment with R. officinalis decreased the Bak protein level. Since Caspase-3 and Caspase-9 play an important role in apoptosis, their expression levels were also examined. As shown in Fig. 5, expression of Caspase-3 and Caspase-9 were markedly increased with the treatment of H₂O₂. In contrast, R. officinalis pretreatment significantly attenuated the Caspase-3 and Caspase-9 expressions in cells treated with H₂O₂ (Fig. 5). These results suggest that R. officinalis inhibit downstream apoptotic signaling including the Caspase-3, Caspase-9, and Bak.

Fig. 5.

Effect of R. officinalis against H₂O₂-induced apoptosis in SH-SY5Y cells. Cells were pretreated with R. officinalis (10 μg/ml) for 1 h followed by 150 μM H₂O₂ for 12 h. Expressions of the pro-apoptotic factors Bak, Caspase-3, and Caspase-9 were examined by immunoblot assay. Actin was used as an internal loading control. All data were representative of three independent experiments

R. officinalis Modulates the Catecholamine Expression in H₂O₂-Treated SH-SY5Y Cells

TH is the first and rate-limiting enzyme, and AADC is the second enzyme in the catecholamine biosynthesis pathway. As these enzymes play a key role, we investigated the effect of R. officinalis on protein level expression of TH and AADC. As shown in Fig. 6, H₂O₂ treatment significantly reduced both TH and AADC expression. However, R. officinalis treatment increased TH and AADC protein level compared with H₂O₂-treated group and maintained the level of TH and AADC even after the H₂O₂ treatment.

Fig. 6.

Effect of R. officinalis on the expressions of the TH and AADC protein in SH-SY5Y cells. Cells were pretreated with R. officinalis (10 μg/ml) for 1 h followed by 150 μM H₂O₂ for 12 h. Expressions of the TH and AADC were detected by immunoblot assay. Actin was used as an internal loading control. All data were representative of three independent experiments

Discussion

Many studies have shown that the oxidative stress as a major cause of loss of DArgic neurons in the SNc. Chronic Oxidative stress induces Reactive oxygen species (ROS) such as H₂O₂ and superoxide anion which results in mitochondrial dysfunction, protein misfolding, genetic mutation, and finally cell death (Shibata and Kobayashi 2008). Suppression of ROS by antioxidants might be an effective strategy in inhibiting oxidative stress-induced cell death. Therefore, the use of anti-oxidant agents as a way of neuroprotection could be a potential therapy to slow or ameliorate the progression of neurodegenerative diseases (Yuan et al. 2007).

In this study, we showed a protective effect of R. officinalis against H₂O₂-induced cell death in human DArgic cells, SH-SY5Y. The extract of R. officinalis decreased H₂O₂-induced cell death in SH-SY5Y cells. Apoptosis is the process of cell death characterized by cell shrinkage, nuclear condensation, DNA fragmentation and membrane blabbing. These apoptotic features in situ were detected by Hoechst 33342, TUNEL and PI staining (Fig. 2). Interestingly, R. officinalis significantly attenuate these features, indicates that R. officinalis may possess an inhibitory effect on H₂O₂-induced apoptosis. Moreover, in this study, we observed that H₂O₂ significantly reduced the mitochondrial membrane potential. The changes in membrane potential were detected by Rodamine123. However, the treatment with R. officinalis prevented depolarization of mitochondrial membrane potential induced by H₂O₂ (Fig. 3).

Apoptosis is mediated through extrinsic pathway by death receptor and intrinsic pathway by mitochondria. Ultimately, these pathways activate caspases, and activated caspases induce cell death. Bcl-2 family consists of two groups; anti-apoptotic group (Bcl-2 and Bcl-xL), and pro-apoptotic group (Bak, Bax, Bid), and they play an important role in mitochondrial related apoptosis pathway. Bcl-2, one of anti-apoptotic factors, residing in the outer mitochondrial membrane inhibits Cytochrome c release (Borner 2003). The pro-apoptotic factors, Bak and Bax, reside in the cytosol. Translocation of Bax to the mitochondrial membrane might lead to loss of mitochondrial membrane potential and an increase in mitochondrial permeability. Increased mitochondrial permeability results in the release of Cytochrome c from the mitochondria (Chinnaiyan et al. 1996). Released Cytochrome c triggers activation of Caspase-9 which in turn activates Caspase-3, and activated Caspase-3 induces cell death. In this study, we investigated whether R. officinalis has any effect on the expression of Bax, and Bcl-2 in H₂O₂-treated cells using western blot. After treatment of R. officinalis, Bax was decreased, but Bcl-2 was increased. Also, we investigated Caspase-3, Caspase-9, and Bak expressions using western blot. This result is consistent with the result from Bax. The treatment of R. officinalis decreased the expressions of Caspase-3 and Caspase-9, more than those seen with H₂O₂-treated cells. Therefore, the effect of R. officinalis on H₂O₂-treated cells may be mediated by regulation of Bcl-2, Bak, Bax, Caspase-3 and Caspase-9 expressions, and regulation of antioxidant enzyme.

The expressions of TH and AADC play a critical role in the survival and differentiation of DArgic neurons. The degeneration of DArgic neurons in PD, resulting in depletion of catecholamine such as TH and AADC and ultimately decreased dopamine in SNc (Schapira 1999). In this study, we found that R. officinalis is a potent inducer of TH and AADC and can modulate the expression of these enzymes in human DArgic cells, SH-SY5Y, against oxidative insult.

The mechanism involved in the protective effects of R. officinalis extract in H₂O₂-induced damage in human dopaminergic cells,SH-SY5Y, is not fully understood. A probable underlying mechanism of this protection may be associated with the presence of flavonoids in the extract, which are a source of antioxidants, since oxidative process are important in the pathogenesis of several disorders including PD. The components with potential antioxidant activity are promising candidates for use as new therapeutic agents against PD. Although the protective mechanisms have to be determined, our results indicate that R. officinalis may be a highly valuable candidate for the treatment of neurodegenerative disorders.

In summary, these results show that R. officinalis decreased H₂O₂-induced cell death, morphological change of nuclei, apoptosis-related gene expressions (Bak, Bax, Caspase-3, and Caspase-9), and increased mitochondrial membrane potential, Bcl-2 expression, and catecholamines-TH and AADC expressions in human dopaminergic cells, SH-SY5Y. Based on these results, we concluded that R. officinalis exhibits its neuroprotective effect thorough the inhibition of apoptosis-related gene expression. This study may offer a new therapeutic strategy in treating Parkinson’s disease, although further research into the neuroprotective mechanisms of R. officinalis will be necessary.