Neuroprotective Effect of Sesame Seed Oil in 6-Hydroxydopamine Induced Neurotoxicity in Mice Model: Cellular, Biochemical and Neurochemical Evidence

ORIGINAL PAPER

Neuroprotective Effect of Sesame Seed Oil in 6-HydroxydopamineInduced Neurotoxicity in Mice Model: Cellular, Biochemicaland Neurochemical Evidence

Saif Ahmad • M. Badruzzaman Khan • M. Nasrul Hoda •

Kanchan Bhatia • Rizwanul Haque • Inayat Saleem Fazili •

Arshad Jamal • Jafar Salamt Khan • Deepshikha Pande Katare

Received: 3 September 2011 / Revised: 12 October 2011 / Accepted: 27 October 2011 / Published online: 17 November 2011

� Springer Science+Business Media, LLC 2011

Abstract Natural antioxidants have shown a remarkable

reduction in oxidative stress due to excess formation of

reactive oxygen species by enhancing antioxidant mecha-

nism in the neurodegenerative disorders. Sesame seed oil

(SO) is one of the most important edible oil in India as well

as in Asian countries and has potent antioxidant properties

thus the present study evaluated the neuroprotective effect

of SO by using 6-Hydroxydopamine (6-OHDA)-induced

Parkinson’s disease model in mice. The mice were fed an

SO mix diet for 15 days and then 6-OHDA was injected

into the right striatum of mice brain. Three weeks after

6-OHDA infusion, mice were sacrificed and the striatum

was removed. The neuroprotective role of SO on the

activities of antioxidant and non-antioxidant enzymes such

S. Ahmad (&) � I. S. Fazili � A. Jamal � D. P. Katare (&)

Department of Biotechnology, Jamia Hamdard (Hamdard

University), New Delhi 110 062, India

e-mail: [email protected]

D. P. Katare

e-mail: [email protected]

Present Address:S. Ahmad

Department of Ophthalmology, School of Medicine, Georgia

Health Sciences University (GHSU), Augusta, GA 30912, USA

M. B. Khan � M. N. Hoda � K. Bhatia � R. Haque

Department of Medical Elementology and Toxicology, Faculty

of Science, Jamia Hamdard (Hamdard University), New Delhi

110062, India

Present Address:M. B. Khan

Center for Molecular Chaperone/Radiobiology and Cancer

Virology, School of Medicine, Georgia Health Sciences

University (GHSU), Augusta, GA 30912, USA

Present Address:M. N. Hoda

Department of Neurology, School of Medicine, Georgia Health

Sciences University (GHSU), Augusta, GA 30912, USA

Present Address:K. Bhatia

Experimental Medicine, School of Medicine, Georgia Health

Sciences University (GHSU), Augusta, GA 30912, USA

Present Address:R. Haque

Department of Microbiology and Immunology, C6749 Penn

State College of Medicine, 500 University Drive, Hershey, PA

17033, USA

Present Address:I. S. Fazili

Center of Animal Biotechnology, Sher-i-Kashmir University of

Agricultural Sciences and Technology, Shuhama (Alusteng),

Srinagar 190 006, India

Present Address:A. Jamal

Biological Science Department, College of Science and Arts

Rabigh, King Abdulaziz University, Jeddah, Saudi Arabia

J. S. Khan

Council of Scientific and Industrial Research(CSIR),

Anusandhan Bhawan, 2 Rafi Marg, New Delhi 110001, India

Present Address:D. P. Katare

Amity Institute of Biotechnology, Amity University, Sector-125,

Noida, Uttar Pradesh 201303, India

123

Neurochem Res (2012) 37:516–526

DOI 10.1007/s11064-011-0638-4

as glutathione reductase (GR), glutathione-S-transferase

(GST), glutathione peroxidase (GPx), catalase (CAT) and

content of glutathione (GSH) and thiobarbituric acid reactive

substance (TBARS) were studied in the striatum. The

activities of all the above-mentioned enzymes decreased

significantly in 6-OHDA group (Lesioned) when compared

with Sham. The pretreatment of SO on antioxidant mecha-

nism and dopamine level in the brain had shown some sig-

nificant improvement in Lesion?SO (L?SO) group when

compared with Lesioned group. However, NADPH oxidase

subunit, Nox2 and inflammatory stimulator Cox2 expression

was increased as well as antioxidant MnSOD level was

decreased in Lesioned group while SO showed the inhibitory

effect on the activation of Nox2 and Cox2 and restored

MnSOD expression in L?SO group. Increased tyrosine

hydroxylase (TH) expression in substantia nigra as well as

dopamine and its metabolite DOPAC level in L?SO group

also support our findings that SO may inhibit activation of

NADPH oxidase dependent inflammatory mechanism due to

6-OHDA induced neurotoxicity in mice.

Keywords Sesame seed oil (SO) � Parkinson disease

(PD) � Neurotoxicity � NADPH oxidase � Antioxidants �Tyrosine hydroxylase

Introduction

Free radical mediated oxidative stress plays an important

role in the functional impairments of brain and linked to

neuronal cell death in certain neurodegenerative disorders

[1]. However, brain cells possess many defense mechanisms

against reactive oxygen species (ROS). ROS (superoxide

radical, O2-; hydrogen peroxide, H2O2; hydroxyl radical,

OH-), are mainly produced in mitochondria. Sometimes it

can be inadequate and may lead to oxidative stress in which

the production of ROS befuddles the antioxidant defenses of

the organism [2]. Reports indicate that oxygen derived rad-

icals are implicated in lipid peroxidation (LPO) events, and

are critical in neuronal injury as brain may be particularly

vulnerable to oxidative stress because of PUFAs, which are

particularly susceptible to free radicals mediated damage [2].

Parkinson’s disease (PD) is the second most common neu-

rodegenerative disorder and it is characterized by the pref-

erential degeneration of dopaminergic neurons in the

substantia nigra pars compacta and a depletion of neuro-

transmitter dopamine in striatum [3]. Although the patho-

genesis of PD still remains unclear. Evidences suggest that

oxidative stress caused by an over production of ROS, is

involved in the etiology of Parkinson’s disease [2].

6-hydoxydopamine (6-OHDA, a hydroxylated analogue

of dopamine) is used to induce PD in animal as well as in vitro

studies [4, 5]. The neurotoxic effect of 6-OHDA is mainly due

to the oxidation of 6-OHDA, resulting in the generation of

cytotoxic free radicals via two major mechanisms, being a

substrate for monomine oxidase (MAO) and its auto-oxida-

tion activity, inhibits the mitrochondrial respiratory chain

which are believed to play a pivotal role in the degeneration of

nigrostriatal dopaminirgic system [6]. Furthermore, it has

been reported that 6-OHDA leads to a reduction in endoge-

nous cellular antioxidants such as superoxide dismutase

(SOD), glutathione (GSH) content, Catalase (CAT) and

increased the LPO, and protein damage [7].

Natural antioxidants have been reported as free radical

scavengers, which can ameliorate the risk of oxidative dam-

age and protect from apoptosis and cell death in several

human diseases including neurodegneration, hypertension

and cancer [8, 9]. Sesame seed oil (SO) has been considered a

potent antioxidant source which is derived from the plant

Sesame indicum L., and a famous oil seed crop and a com-

ponent of the traditional health food in India as well as in other

Asian countries [10]. SO contains a higher proportion of

monounsaturated fatty acids (MUFA) than saturated fatty

acids (SFA), and the main fatty acid compositions are oleic,

linoleic, palmetic, steric and archidic. [11]. SO also contains

good amounts of phenol, sesamin, sesamol and sesamolin and

relatively small amounts of tocopherol, which contribute to its

superior oxidative stability and plays an important role in

plant defense [12]. Recent finding has shown that SO

increases cell resistance to LPO and potently attenuates oxi-

dative stress triggered by the endotoxin lipopolysaccharide

(LPS) in rats [13]. SO in comparison to other dietary oils such

as groundnut, canola and sunflower oil depicted better pro-

tection against increased blood pressure, hyperlipidemea and

LPO by increasing enzymatic and non-enzymatic antioxi-

dants [14]. Sesamin, sesamolin and tocopherols are powerful

antioxidants in SO, and have been reported to possess a broad

spectrum of pharmacological effects including neuroprotec-

tive, anti-inflammatory, antihypertensive and antimutagenic

[15–17]. Sesamin and sesamolin have been shown inhibitory

effect on LPS-activated p38 mitogen activated protein kinase

(MAPK) and nuclear factor kB (NF-kB) activation in mi-

croglial cells [18]. It has been shown that sesamin down

regulates cyclin D1 protein expression in human tumor cells,

induces neural differentiation in PC12 cells through activation

of ERK1/2 signalling, and improves endothelial dysfunction

in renovascular hypertensive rats [19, 20]. Reports demon-

strate that sesamin and sesamolin potently inhibited phos-

phorylation of JNK and caspase-3 under hypoxia in BV-2

cells [21]. Furthermore, sesamol has been found to inhibit

LPO, DNA cleavage and involved in activation of endoge-

nous antioxidant enzymes such as GSH, GST, Catalase, and

also protects DNA damage in mice [22].

To the best of our knowledge, no report has shown on

the preventive role of SO in 6-OHDA induced oxidative

stress in mice model of PD. In this study, we hypothesized

Neurochem Res (2012) 37:516–526 517

123

that SO may regulate NADPH oxidase mediated anti-

inflammatory signaling and prevent neuronal cell death in

neurodegeneration by enhancing antioxidant defense

mechanism. Therefore, SO may be a good source of anti-

oxidant dietary supplement and thus, we examined the

neuroprotective role of SO in mice model of PD.

Materials and Methods

Chemicals and Drugs

Glutathione (GSH), NADPH, ATP, qubain, 1-chloro-2,

4-dinitrobenzene (CDNB), 5-50-dithiobis-2-nitrobenzoic

acid (DTNB), oxidized glutathione (GSSG), glutathione

reductase (GR), thiobarbituric acid (TBA), 6-hydroxydop-

amine (6-OHDA), apomorphine hydrochloride, EDTA,

nitroblue tetrazolium (NBT), bovine serum albumin (BSA),

3, 4-dihydroxyphenyl acetic acid (DOPAC), 3, 4-dihydr-

oxybenzylamine (DHBA),Triton X-100, EDTA, EGTA,

NaF, Na4P2O7, Na3VO4, glycerol, SDS, deoxycholate,

phenylmethanesulfonyl fluoride (PMSF) and protease

inhibitor cocktail, were purchased from Sigma–Aldrich,

USA, Antibodies (Tyrosine Hydroxilase, gp91phox [sc-

207820], MnSOD [sc-133254] and Cox-2 [sc-166475])

were purchased from Santa Cruz Biotechnology, CA, USA,

b-actin [ab6276] was purchased from abcam, Cambridge,

MA, USA and other chemicals were AR grade.

Dietary Sesame Seed Oil

Authentic Sesame seed was provided by CSIR, New Delhi,

India. Oil was extracted from the sesame seeds according

to method of Fazli et al. [23]. In brief, the sesame seeds

were ground in a disintegrator. The ground seeds were

extracted with n-hexane for 48 h, and then filtered. This

process was repeated three times using fresh solvent each

time to extract most of the oils from the ground seeds. Free

fatty acids were determined by Gas Liquid Chromatogra-

phy (GLC) as per the protocol of Fazli et al. [23] and

sesamin and sesamol content were analyzed according to

method of Budowski et al. [24] (Data has already been

published in our earlier study Ahmad et al. [11]). Control

(fat free) diet was provided by Animal House Facility,

Hamdard University, New Delhi, India. SO was mixed in

the control diet in appropriate amount (200 ml SO (20%)

was mixed in 1 kg diet). Small pellets (*20 gm wt) were

prepared manually and dried at room temperature.

Animals and Experimental Protocol

Male swiss albino mice weighing 25–35 g (3–4 months-

old) were obtained from the Animal House Facility, Jamia

Hamdard (Hamdard University), New Delhi, India. Mice

were housed in the group of four animals per cage in

polypropylene cages at room temperature (25�C) in stan-

dard environment and were allowed free access to experi-

mental (20% SO mixed in fat free diet) and palette diet as

well as water ad libitum. Animals were divided in four

groups, eight animals each. First group (Sham) and second

group Lesion, i.e. 6-OHDA induced, were fed pellet diet,

third group (Lesioned?SO) was fed experimental diet and

the fourth group (Sham?SO) was also fed experimental

diet alone for 15 days. They were maintained on a 12/12 h

dark-light cycle. The animals were used in accordance with

the procedure approved by the Hamdard University Animal

Ethics Committee (Jamia Hamdard, New Delhi, India).

6-Hydroxydopamine (6-OHDA) Lesions and Tissue

Preparation

In brief, after 15 days pretreatment with SO, 6-OHDA was

injected into the striatum; a sham lesion with the same vol-

ume (2 ll) of solvent was performed into control mice stri-

atum. Mice were anaesthetized using chloralhydrate

(400 mg/kg, i.p.) and placed into a stereotactic frame adap-

ted for mice (David Kopf Instruments, USA). The skull was

exposed by making a small incision in the skin and lesions

were made by the unilateral injection of 6-hydroxydop-

amine. 6-OHDA was dissolved at a concentration of 2 lg/ll

saline in 0.2% ascorbic acid and 2 ll was injected at a rate of

0.5 ll/min. The needle was left in place for 5 min after the

injection before retraction. The injection was performed

using a Hamilton syringe at the following co-ordinates: AP:

?0.04 cm, ml: ± 0.18 cm, DV: -0.35 cm [43].

After 3 weeks of 6-OHDA injection, animals were kil-

led and brains were taken out quickly and kept on ice.

Striatum was dissected out. For enzymes and GSH assays,

striatum was homogenized in phosphate buffer (10% w/v,

pH 7.0) and centrifuge at 10,000 rpm for 10 min at 4�C to

get post mitochondrial supernatant (PMS).

Biochemical Assays

Assay of Lipid Peroxidation (LPO)

The procedure of Utley et al. [26] as modified by Islam et al.

[27] was used for estimating the rate of LPO. 1 ml of

homogenate (2.5% in chilled KCl) was pipetted in a glass

radioimmunoassay vial of 20 ml capacity and incubated at

37 ± 1�C in a metabolic shaker (120 rpm/min) for 60 min.

Similarly, 1.0 ml of the same homogenate was pipetted in a

centrifuge tube and incubated at 0�C. After 1 h of incubation,

1.0 ml 5% chilled TCA was added followed by 1.0 ml of

0.67% TBA in each vial and proper mixing was performed

after each addition. The aliquot from each vial was

518 Neurochem Res (2012) 37:516–526

123

transferred to a centrifuge tube and centrifuged at 3,500 rpm

for 15 min. Thereafter, the supernatant was placed in a

boiling water bath. After 10 min, the test tubes were cooled

and the absorbance of the color was read at 535 nm. The rate

of LPO was expressed as n mol TBARS formed/h/mg protein.

Estimation of Reduced Glutathione (GSH) Activity

Reduced glutathione was assayed by the method of Jollow

et al. [25]. One ml PMS (10%) was precipitated with 1.0 ml

sulfosalicylic acid (4%). The samples were kept at 4�C for

1 h and then subjected to centrifugation at 1,200g for 15 min

at 4�C. The assay mixture was contained 0.1 ml filtered

aliquot, 2.7 ml phosphate buffer (0.1 M, pH 7.4) and 0.2 ml

DTNB (0.4% in phosphate buffer 0.4 M, pH 7.4) in a total

volume of 3.0 ml. The activity was read immediately at

412 nm. The results were expressed as n mol GSH/g tissue.

Estimation of Glutathione-S-Transferase activity (GST)

Glutathione-S-transferase (GST) activity was measured by

the method of Habig et al. [28]. The reaction mixture

consisted of 1.425 ml phosphate buffer (0.1 M, pH 7.4),

0.2 ml reduced glutathione (1 mM), 0.025 ml CDNB

(1 mM) and 0.3 ml PMS (10%) in a total volume of 2.0 ml.

The changes in absorbance were recorded at 340 nm and

enzyme activity was calculated as nmole CDNB conjugate

formed/min/mg protein using a molar extinction coefficient

of 9.6 9 103 M-1 cm-1.

Glutathione Peroxide Activity (GPx)

GPx was estimated according to the procedure described by

Mohandas et al. [29]. The reaction mixture consisted of

1.44 ml phosphate buffer (0.05 M, pH 7.0), 0.1 ml EDTA

(1 mM), 0.1 ml of sodium azide (1 mM), 0.05 ml of gluta-

thione reductase (1 EU/ml), 0.10 ml of glutathione (1 mM),

0.10 ml of NADPH (0.2 mM), 0.01 ml of hydrogen perox-

ide (0.25 mM) and 0.1 ml of PMS in the final volume of

2 ml. The disappearance of NADPH at 340 nm was recorded

at room temperature. The enzyme activity was calculated as

nmol NADPH oxidized/min/mg/protein by using the molar

extinction coefficient 6.22 9 103 M-1 cm-1.

Glutathione Reductase Activity (GR)

Glutathione reductase activity was assayed by the method of

Carlberg and Mannervik [30], as modified by Mohandas

et al. [29]. The assay mixture consisted of 1.65 ml phosphate

buffer (0.1 M, pH 7.6), 0.1 ml NADPH (0.1 mM), 0.1 ml

EDTA (0.5 mM) and 0.05 ml oxidized glutathione (1 mM)

and 0.1 ml of PMS in total volume of 2 ml. The enzyme

activity was estimated at room temperature by measuring the

disappearance of NADPH at 340 nm and was calculated as

nmol NADPH oxidized/min/mg protein using molar

extinction coefficient of 6.22 9 10-3 M-1 cm-1.

Catalase Activity (CAT)

Catalase activity was assayed by the method of Claiborne

[31]. Briefly, the assay mixture consisted of 1.95 ml

phosphate buffer (0.05 M, pH 7.0), 1.0 ml hydrogen per-

oxide (0.019 M) and 0.05 ml PMS in total volume of

3.0 ml. The change in absorbance was recorded at 240 nm.

Catalase activity was calculated as nmol of H2O2 con-

sumed/min/mg/protein.

Superoxide Dismutase Activity (SOD)

SOD activity was measured by the method of Beauchamp

et al. [32]. The reaction mixture consisted of 0.5 M phos-

phate buffer pH 7.4, 0.1 ml PMS, 1.0 mM xanthine, 57 lM

NBT. It was incubated for 15 min at room temperature and

the reaction was initiated by the addition of 50 mU xan-

thine oxidase. The rate of reaction was measured by

recording change in the absorbance at 550 nm due to for-

mation of formazan, a reduction product of NBT.

Quantification of Dopamine and its Metabolite

The striatal tissue levels of DA and its metabolite DOPAC

were measured by high-performance liquid chromatography

(Waters, USA), using Photodiode Array Detector (PDA

detector) by the method of Zafar et al. (2003). The striatum

(20% wt./vol.) was sonicated in 0.4 N perchloric acid con-

taining 100 ng/ml of the internal standard DHBA (2,

3-dihydroxybenzoic acid), followed by centrifugation at

15,0009g for 10 min at 4�C and the filtered through a 0.20-

lm membrane that was injected manually through a 20-ll

loop over the C18 column for separation and quantification.

The mobile phase consisted of 0.1 M potassium phosphate

(pH 4.0), 10% methanol, and 1.0 mM heptane sulfonic acid.

Samples were separated on C18 column using a flow rate of

1.0 ml/min. The concentrations of DA and its metabolite

DOPAC were calculated using a standard curve generated by

determining ratio between three known amounts of the amine

or its metabolites and a constant amount of internal standard

DHBA and represented as nanograms per milligram of tissue.

Western-Blot Analysis

Frozen brain tissues from -80�C were taken out and

washed with cold PBS (pH 7.5) and lysed in lysis buffer

containing Tris–HCl (50 mM, pH 8.0), NaCl (150 mM),

Triton X-100 (1%), EDTA (1 mM), EGTA (1 mM), NaF

(1 mM), Na4P2O7 (20 mM), Na3VO4 (2 mM), glycerol

Neurochem Res (2012) 37:516–526 519

123

(10%), SDS (0.1%), deoxycholate (0.5%), PMSF (100ug/

ml) and protease inhibitor cocktail. The lysate was centri-

fuged at 14,0009g for 10 min at 4�C and the supernatant

was collected. Equal amount of protein were separated on

10% SDS–polyacrylamide gels. Proteins were transferred

onto polyvinylidene difluoride (PVDF) membranes and

blocked with 5% non-fat dry milk in Tris-buffer saline with

0.1% Tween 20 (TBS-T) for 1 h at room temperature. The

membranes were incubated overnight with different pri-

mary antibodies (anti-gp91phox [1:500], anti-COX-2

[1:500], anti-MnSOD [1:1,000] and anti-b actin [1:5,000]).

After three washings with TBS-T, membranes were incu-

bated with secondary horseradish peroxidase-conjugated

antibody at room temperature for 1 h, followed by three

washings with TBS-T. The bands were visualized on a

Biomax X-ray film (Kodak) by using enhanced chemilu-

minescence and quantified by densitometric analysis. All

data from the three independent experiments were

expressed as a ratio to optical density (OD).

Immunohistochemical Analysis

Immunohistochemical analysis was done for TH?ve neu-

rons in substantia nigra of mice brain by using MOM kit

(Vector Laboratories, CA, USA). In brief, brain paraffin

slides for each group were de-paraffinized following by

antigen retrieval in citrate buffer. The endogenous perox-

idase activity was blocked by 3% hydrogen peroxide. Brain

slides were incubated into blocking reagent for 1 h and

after washing with PBS, slides were incubated into anti-TH

antibody (1:500) for 30 min at room temperature. After

incubation, slides were washed three times and further

incubated into biotinylated secondary antibody for 10 min.

After subsequent washing and following by ABC reagent

incubation, the colour for peroxidase-linked antibody was

developed with DAB as chromogen. Reaction was stopped

into water and hematoxylin was used for the nuclei stain-

ing, then sections were dehydrated and mounted with cover

slip by using aqueous mounting medium (Vector Labora-

tories, CA, USA) and pictures were taken by using Carl

Zeiss microscope (USA). The immunohistochemistry pro-

tocol comes with the MOM kit.

Estimation of Protein

Protein was estimated by the method of Bradford [33]

using bovine serum albumin (BSA) as a standard.

Statistical Analysis

Results are expressed as Mean ± SE (n = 6). Differences

between the means of experimental and control groups

were analyzed statistically by using student’s t-test and

one-way analysis of variance (ANOVA) followed by Tu-

key–Kramer post hoc test for multiple comparisons. A

value P \ 0.05 was considered significant.

Results

Effect of SO on LPO and GSH

Figure 1a, b shows the effect of dietary SO on the TBARS

and GSH content in 6-OHDA induced neurotoxicity.

TBARS is the marker for LPO in oxidative stress and

excess generation of ROS is implicated in this event and

GSH, antioxidant which is known to quench increased

ROS generation. The content of TBARS in mice brain was

significantly elevated (P B 0.001) in Lesioned (L) group as

compared to Sham group and its content was significantly

decreased in L?SO group (P B 0.01). The level of GSH

content was significantly depleted (P B 0.001) in Lesioned

group as compared to Sham group and significantly

attenuated in L?SO group (P B 0.05) when compared

with L group. No significant changes were observed

between SO and Sham group.

Effect of SO on SOD and Catalase

Figure 2a, b shows the activity of superoxide dismutase

(SOD) and Catalase (CAT). SOD, endogenous antioxidant

enzyme which dismutase superoxide radicals into hydrogen

peroxide and later CAT converts H2O2 into H2O. SOD and

CAT were significantly decreased in L group (P B 0.001)

as compared to Sham group. Enzymatic activities were

observed to improve significantly in Lesioned?SO group

as compared to Lesioned group (P B 0.05). No significant

changes were observed between SO and Sham group.

Effect of SO on GPx, GR and GST

GPx, GR and GST, endogenous antioxidant enzymes have

been reported significant role in reducing oxidative stress

by inhibiting excess ROS generation in the cells. Table 1

shows the significant decrease (P B 0.01) in the activity of

endogenous antioxidants GPx, GR and GST in L group

when compared with Sham. Enzymatic activities were

observed to improve significantly in Lesioned?SO group

(P B 0.01, P B 0.05) as compared to Lesioned group.

There was no significant difference found between SO

alone and Sham group.

Estimation of Dopamine and DOPAC levels

Table 2 shows the significant decreased (P \ 0.001) in the

level of DA and DOPAC in striatal region of 6-OHDA-

520 Neurochem Res (2012) 37:516–526

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lesioned mice as compared to Sham group which indicates

a significant loss of dopaminergic neurons in Lesioned

group. SO?Lesioned group exhibited significant increase

in DA and DOPAC level comparison to Lesioned group

mice that shows functional viability of dopaminergic neu-

rons. No significant change was observed in the SO alone

pretreated sham group (SO?S) as compared to Sham

group.

Effect of SO on Nox2, Cox-2 and SOD Protein

Expression in Mice Brain

To confirm 6-OHDA induced neurotoxicity on signal

transduction, we performed Western blot analysis to

investigate the expression of the NADPH oxidase subunit

Nox-2, a marker of oxidative stress and has been impli-

cated in neurodegenerative disorders. Results show an

Fig. 1 a Neuroprotective effect of SO on the content of

TBARS (TBARS formed/h/mg protein) in mice striatum.

Values are expressed as mean ± SE (n = 6). ***P \ 0.001

sham versus Lesion; ##P \ 0.01 Lesion versus Lesion?SO.

b Neuroprotective effect of SO on the content of GSH (GSH/g

tissue) in mice striatum.Values are expressed as mean ± SE

(n = 6). ***P \ 0.001 sham versus Lesion; #P \ 0.05 Lesion

versus Lesion?SO

Fig. 2 a Neuroprotective effect of SO on the content of SOD (nmole

of epinephrine protected from oxidation/min/mg protein) in mice

striatum. Values are expressed as mean ± SE (n = 6). ***P \ 0.001

sham versus Lesion; #P \ 0.05 Lesion versus Lesion?SO.

b Neuroprotective effect of SO on the content of CAT (nmol of

H2O2 consumed/min/mg/protein) in mice striatum. Values are

expressed as mean ± SE (n = 6). *P \ 0.05 sham versus Lesion;#P \ 0.05 Lesion versus Lesion?SO

Table 1 Neuroprotective effect of SO on activities of antioxidant enzymes in mice striatum in different treatment groups

Antioxidant Enzymes

Groups GST GR GPx

I (Sham) 119.46 ± 5.11 235.87 ± 15.86 316.51 ± 16.14

II (SO) 120.30 ± 6.82 226.75 ± 16.94 309.69 ± 20.98

III (L) 106.80 ± 7.34** 172.34 ± 13.64*** 249.84 ± 17.72***

IV (L?SO) 114.33 ± 5.14# 197.65 ± 17.21# 283.62 ± 15.51##

Values are express as means ± SE (n = 6). GST expressed as nmol CDNB conjugates/min/mg protein, GR as nmol NADPH oxidized/min/mg

protein, and GPx as nmol NADPH oxidized/min/mg protein. The values observed were significant at ***P \ 0.001 and **P \ 0.01 when

compared to Sham versus Lesion, #P \ 0.05 and ##P \ 0.01 when compared to Lesion versus Lesion?SO

Neurochem Res (2012) 37:516–526 521

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enhanced level of expression of Nox-2 in Lesioned group

(P B 0.01) when compared with Sham group and the

L?SO group shows the least expression (P B 0.05)

(Fig. 3). The expression of Cox-2, an inflammatory marker

which is known to stimulate inflammatory mechanism, was

enhanced in Lesioned group (P B 0.01) when compared

with Sham group but the Lesioned?SO group (P B 0.05)

showed less expression as compared with lesion alone

(Fig. 4). The antioxidant enzyme MnSOD expression was

significantly depleted (P B 0.01) in Lesioned group as

compared with Sham group but it shows an increased

expression level in Lesioned?SO group (P B 0.05)

(Fig. 5).

Table 2 Neuroprotective effect of SO on dopamine and DOPAC

levels in 6-OHDA induced mice brain striatum

Group DA (ng/mg tissue) DOPAC (ng/mg tissue)

Sham 5.76 ± 0.29 1.23 ± 0.093

SO 5.97 ± 0.38 1.28 ± 0.086

Lesion 1.85 ± 0.18*** 0.55 ± 0.073***

L?SO 3.92 ± 0.37# 0.91 ± 0.090#

The values are expressed as mean SEM (n = 6). 6-OHDA signifi-

cantly decreases dopamine and DOPAC level in Lesion group as

compared to Sham group. Treatment of SO shows significant atten-

uation in the level of dopamine and DOPAC in L?SO group as

compared to L group. Values in parentheses show the increase or

decrease with respect to their control. #P \ 0.05, L versus L?SO and

***P \ 0.001, L versus Sham

Sham SO Lesion L+SO

Nox-2

β-actin

a

b

Fig. 3 Effect of SO on the protein expression of Nox2 in mice brain

by Western blotting. a represents Nox2 and b-actin expression as well

as b is the histogram represents densitometry analysis normalized to

the corresponding b-actin. Values are expressed as mean ± SE

(n = 6). ***P \ 0.01 sham versus Lesion; #P \ 0.05 Lesion versus

Lesion?SO

Sham SO Lesion L+SO

COX-2

β-actin

a

b

Fig. 4 Effect of SO on the protein expression of Cox2 in mice brain by

Western blotting. a represents Nox2 and b-actin expression as well as

b is the histogram represents densitometry analysis normalized to the

corresponding b-actin. Values are expressed as mean ± SE (n = 6).

***P \ 0.01 sham versus Lesion; #P \ 0.05 Lesion versus Lesion?SO

Sham SO Lesion L+SO

MnSOD

β-actin

a

b

Fig. 5 Effect of SO on the protein expression of MnSOD in mice

brain by Western blotting. a represents Nox2 and b-actin expression

as well as b is the histogram represents densitometry analysis

normalized to the corresponding b-actin. Values are expressed as

mean ± SE (n = 6). ***P \ 0.01 sham versus Lesion; #P \ 0.05

Lesion versus Lesion?SO

522 Neurochem Res (2012) 37:516–526

123

Tyrosine Hydroxylase (TH) Immunohistochemistry

Figure 6 shows the neuroprotective role of SO and func-

tional viability of dopaminergic neurons in the SNpc. In the

Lesioned group, we found that the Tyrosine hydroxylase

positive neurons were degenerated and died as compared to

Sham group. Whereas TH?ve neurons in Lesioned?SO

group were more pronounced as compared to Lesioned

group and exhibited a better survival. SO pretreatment did

not show any remarkable effects in the SO compared with

the Sham group (data not shown).

Discussion

In the present study, we evaluated the neuroprotective role

of SO and its antioxidant properties in 6-OHDA infused PD

mice model. Recent findings suggest that the pathogenesis

of several neurodegenerative diseases, including PD, Alz-

heimer’s disease, multiple sclerosis and amyotrophic lat-

eral sclerosis, may involve the generation of ROS, which

causes oxidative stress [1, 8]. Oxidative stress has been

associated with oxidant-antioxidant imbalance, and thought

to underlie defects in energy metabolism and induce cel-

lular degeneration [2]. The excess production of ROS can

lead to oxidative damage in the brain of PD, which

increases the LPO and DNA damage in the substantia nigra

[34, 35]. Here our observations showed that SO decreases

the 6-OHDA induced neurotoxicity and restores of striatal

dopamine and its metabolites in mice brain by enhancing

and activating antioxidant defense mechanisms which

indicates that SO may act as an intracellular ROS scav-

enger. The earlier reports suggest that the antioxidant

compounds present in SO such as sesamin, sesamol and

sesamolin inhibit excess production of ROS in various

human diseases like hypertension, endothelial dysfunction

and hepatic disorder and also inhibit 20-Hydroxyeicosa-

tetraenoic acid synthesis which is involved in renal dys-

function [17, 36].

In our investigation, we demonstrated that the infusion

of 6-OHDA in brain increased LPO and diminished GSH

activity in lesion group as compared to sham. Brain cells

are vulnerable to oxidative stress and then excess genera-

tion of ROS may alter the antioxidant defense mechanism

in the cells and increase the LPO. However, SO treatment

with lesion group decreases the TBARS level and increases

GSH activity in the brain striatum when compared with the

lesion group. It may be concluded that SO significantly

diminishes the generation of hydroxyl radicals, which

resulted in the suppression of LPO.

Cellular antioxidant enzymes such as superoxide dis-

mutases (SODs), catalase (CAT), glutathione peroxidase

(GPx), glutathione reductase (GR) and reduced glutathione

(GST) and non-enzymatic glutathione (GSH) in the brain

play important role in cellular defense against oxidative

damage by ROS [2]. SOD coverts superoxide radicals to

form H2O2 and CAT and GPx are involved in the detoxi-

fication of H2O2 both at high and low concentrations [37,

38]. In the earlier studies, researchers have demonstrated

that the brain contains low CAT levels and hence GPx has

a major role in quenching H2O2 and other peroxides which

otherwise lead to production of hydroxyl and peroxyl

radicals [7]. GR is another important enzyme for the

maintenance of intracellular concentration of GSH [39].

However, GSH serves as primary antioxidant defense

against oxidative stress and plays an important role in the

behavioral activity [40] and GST involves in the detoxifi-

cation of oxidized metabolites of catecholamines (o-qui-

none), and may serve as antioxidant, preventing

degenerative processes [41]. We found that antioxidant

enzymatic activities were reduced in lesion group as

compared to sham and treatment of SO shows significant

Sham Lesion Lesion + SO

TH+neurons

TH+neurons

50µm50µm 50µm

Fig. 6 Immunohistochemical analysis of pre-treatment effect of SO

on TH expression in right SNpc in mice infused with 6-OHDA. The

expression of tyrosine hydroxylase was significantly reduced in

Lesion group (20X) compared to Sham group (20X), while the

Lesioned group pretreated with SO has shown a moderate staining of

tyrosine hydroxylase and better survival of dopaminergic neurons

(20X). However, the SO group has not shown any significant change

in tyrosine hydroxylase staining when compared with Sham (picture

not shown)

Neurochem Res (2012) 37:516–526 523

123

improvement in enzymatic activities in lesion?SO group

when compared with lesion. Additionally, the brain dopa-

mine has long been associated with the neurotoxicity,

which undergoes spontaneous oxidation to toxic quinone

and other electrophilic species, causing PD [42] and the

administration of natural antioxidants reverse this process

and stop the reduction of brain dopamine levels [43], and it

may be possible due to reduction in auto-oxidation of

dopamine. Thus, our results are consistent with earlier

studies where natural antioxidants play potential role in

balancing antioxidant enzymatic activity [44, 45]. Our

results are also in agreement with Cheng et al. [15], who

has reported the neuroprotective role of sesamin and ses-

amolin on gerbil brain in cerebral ischemia.

Furthermore, we investigated the expression of signaling

molecules NADPH oxidase subunit Nox2, and the

inflammatory agent Cox-2, as well as MnSOD in mice

brain. NADPH oxidase, a multiunit enzyme first discovered

in neutrophils, has emerged as a major molecule of ROS

generator in neurons, glial cells and cerebral blood vessels

and produces a large amount of O2- in leukocytes, and has

a role in various neurodegenerative diseases as gp91phox

increases in the brain after intracerebral hemorrhage,

resulting in enhanced LPO [46]. In this study, 6-OHDA

infusion in mice brain significantly enhanced the Nox2

expression in lesion group and SO treatment effectively

down regulated its expression in SO?Lesion group. This

demonstrates that the SO in presence of powerful antioxi-

dants, inhibits activation of NADPH oxidase signaling,

lowering the ROS generation in the brain, and preventing

neuronal damage. Nakano et al. [47] reported that sesamin,

a component of SO decreases superoxide production and

suppresses mRNA expression of NADPH oxidase in aorta

of hypertensive rat. Our results are in agreement with other

study that the microglial cell, gp91phox appeared to be

involved in the induction of neuronal damage in neurode-

generative disorders and study suggests that inhibition of

NADPH oxidase is an important therapeutic target for

neuroprotection [48].

Cox-2 plays an important role in neurodegeneration and

inflammatory mechanism in the brain and it was reported

that selective Cox-2 inhibition prevents progressive dopa-

mine neuron degeneration in PD [49]. SO treatment sig-

nificantly decreased the elevated level of Cox-2 protein

expression in 6-OHDA induced mice brain, which shows

that SO may inhibit the inflammatory cytokines activation

via Cox-2 signaling. Several reports also indicate that ses-

amin and sesamol have anti-inflammatory effects as they

inhibit N-formyl-methionyl-phenylalanine (fMLF)-induced

inflammatory response through a NF-kB–related signaling

pathway, down regulate IL-6 and cyclin D1 expression in

human cells, suppresses LPS induced cytokines production

and also have modulatory effect on TH, and iNOS protein

expression in dopaminergic cells [50–52]. Another natural

antioxidant resveratrol, was reported to decrease Cox-2 and

TNF-a mRNA expression in 6-OHDA infused rat’s brain

[44], which shows that natural compounds can modulate the

pro-inflammatory mechanism.

Antioxidant enzyme SOD, which converts the super-

oxide radicals into H2O2, is further metabolized to water by

catalase has shown important role in the neurodegeneration

and the impaired mitochondrial complex I activity and

reduced SOD activity have already been reported in PD

brain [53]. Our results indicate that SO restored the

MnSOD protein expression in lesion?SO group and the

elevated MnSOD level as compared with lesion demon-

strates that SO enhances the ability of SOD to eliminate

superoxide radicals formed during 6-OHDA induced oxi-

dative stress. Lahaie-Collins et al. [16] reported similar

finding that sesamin modulates superoxide dismutase and

catalase in dopaminergic cells under 1-methyl-4-phen-

ylpyridinium (MPP?)-induced oxidative stress.

Tyrosine hydroxylase, a rate-limiting enzyme in the

formation of DA and marker for the DA neuron survival

showed increased localization in ipsilateral Spec in

lesion?SO group as compared to lesion and HPLC analysis

of dopamine and its metabolite DOPAC level in the stria-

tum was also significantly high in the treatment group,

which suggests that SO may block the activation of cell

death receptor signaling. Previously, it has been reported

that the sesamin and sesamolin inhibit c-jun N-terminal

kinase (JNK), p38 MAPKs and caspase-3 activation under

hypoxia in PC12 cells [19, 21]. Our results are also an

agreement with other studies, which show that natural

antioxidants such as Gingo biloba, Curcuma oil, curcumin,

resveratrol, and baicalein can reduce oxidative damage in

neurodegeneration and act as neuroprotective agents [6, 7,

44, 54, 55]. Thus, SO may act as a potent ROS scavenger

and anti-inflammatory in neurodegenerative disorders.

Nevertheless, further study is needed to evaluate molecular

mechanisms.

In conclusion, the present study demonstrated a pro-

tective role of SO due to 6-OHDA induced oxidative stress

in mice brain by enhancing the activities of antioxidant

enzymes, decreasing the TBARS content, TH positive

expression and increased dopamine and its metabolite

DOPAC level. SO also shows the inhibitory effect on Nox2

activation that is implicated in excess generation of ROS in

neurodegenerative disorders including PD. Inflammatory

marker, Cox-2 expression also suppressed in lesion group

when compared with lesioned?SO group, and apart from

this antioxidant enzyme, MnSOD expression was also

restored in lesioned?SO group. These neuroprotective

effects may be due to the presence of potent antioxidants

such as sesamin, sesamolin, and sesamol in SO. Thus, our

finding suggests that SO may be a good neuroprotective

524 Neurochem Res (2012) 37:516–526

123

agent. However, further evaluation of SO on molecular

level is essential.

Acknowledgments Authors are thankful to CSIR, Ministry of Sci-

ence and Technology, New Delhi, Govt. of India for providing

authentic sesame seed as a gift sample to carry out this research work.

This work was supported by women young scientist award (Depart-

ment of Science and Technology (DST), New Delhi, Govt. of India)

to DPK. We greatly acknowledge Ms Sylvia Megyardi, Oral Biology

and Anatomy, GHSU, Augusta, GA, USA for reviewing and editing

this manuscript.

Conflict of interest There is no conflict of interest with any person

or other organization.

References

1. Andersen JK (2004) Oxidative stress in neurodegeneration: cause

or consequence? Nat Med 10:S18–S25

2. Cui K, Luo X, Xu K, Ven Murthy MR (2004) Role of oxidative

stress in neurodegeneration: recent developments in assay

methods for oxidative stress and nutraceutical antioxidants. Prog

Neuropsychopharmacol Biol Psychiatry 28:771–799

3. Hara H, Hiramatsu H, Adachi T (2007) Pyrroloquinoline quinone

is a potent neuroprotective nutrient against 6-hydroxydopamine-

induced neurotoxicity. Neurochem Res 32:489–495

4. Chao J, Yu MS, Ho YS et al (2008) Dietary oxyresveratrol pre-

vents parkinsonian mimetic 6-hydroxydopamine neurotoxicity.

Free Radic Biol Med 45:1019–1026

5. Gao QG, Chen WF, Xie JX et al (2009) Ginsenoside Rg1 protects

against 6- OHDA-induced neurotoxicity in neuroblastoma SK-N-

SH cells via IGF-I receptor and estrogen receptor pathways.

J Neurochem 109:1338–1347

6. Im HI, Nam E, Lee ES et al (2006) Baicalein Protects 6-OHDA -

induced neuronal damage by suppressing oxidative stress. Korean

J Physiol Pharmacol 10:309–315

7. Ahmad M, Saleem S, Ahmad AS, Yousuf S et al (2005) Ginkgo

biloba affords dose-dependent protection against 6-hydroxydop-

amine-induced parkinsonism in rats:neurobehavioural, neuro-

chemical and immunohistochemical evidences. J Neurochem 93:

94–104

8. Sun AY, Wang Q, Simonyi A et al (2008) Botanical phenolics

and brain health. Neuromol Med 10:259–274

9. Zhao B (2009) Natural antioxidants protect neurons in Alzhei-

mer’s disease and Parkinson’s disease. Neurochem Res 34:

630–638

10. Shad KF, Al-Salam S, Hamza AA (2007) Sesame oil as a pro-

tective agent against doxorubicin induced cardio toxicity in rat.

Am J Pharmacol Toxicol 2:159–163

11. Ahmad S, Yousuf S, Ishrat T et al (2005) Effect of dietary sesame

oil as antioxidant on brain hippocampus of rat in focal cerebral

ischemia. Life Sci 79:1921–1928

12. Jeng KC, Hou RC, Wang JC et al (2005) Sesamin inhibits lipo-

polysaccharide- induced cytokine production by suppression of

p38 mitogen-activated protein kinase and nuclear factor-kappaB.

Immunol Lett 97:101–106

13. Hsu DZ, Liu MY (2002) Sesame oil attenuates multiple organ

failure and increases survival rate during endotoxemia in rats.

Crit Care Med 30:1859–1862

14. Sankar D, Sambandam G, Ramakrishna Rao M et al (2005)

Modulation of blood pressure, lipid profiles and redox status in

hypertensive patients taking different edible oils. Clin Chim Acta

355:97–104

15. Cheng FC, Jinn TR, Hou RCW (2006) Neuroprotective effects of

sesamin and sesamolin on gerbil brain in cerebral ischemia. Int J

Biomed Sci 2:284–288

16. Lahaie-Collins V, Bournival J, Plouffe M et al (2008) Sesamin

modulates tyrosine hydroxylase, superoxide dismutase, catalase,

inducible NO synthase and interleukin-6 expression in dopami-

nergic cells under MPP?-induced oxidative stress. Oxid Med

Cell Longev 1:54–62

17. Kong X, Yang JR, Guo LQ et al (2009) Sesamin improves

endothelial dysfunction in renovascular hypertensive rats fed with

a high-fat, high-sucrose diet. Eur J Pharmacol 620:84–89

18. Jeng KCG, Hou RCW (2005) Sesamin and sesamolin: nature’s

therapeutic lignans. Curr Enzym Inhib 1:11–20

19. Hamada N, Fujita Y, Tanaka A et al (2009) Metabolites of ses-

amin, a major lignan in sesame seeds, induce neuronal differen-

tiation in PC12 cells through activation of ERK1/2 signaling

pathway. J Neural Transm 116:841–852

20. Yokota T, Matsuzaki Y, Koyama M et al (2007) Sesamin, a

lignan of sesame, down-regulates cyclin D1 protein expression in

human tumor cells. Cancer Sci 98:1447–1453

21. Hou RC, Wu CC, Yang CH et al (2004) Protective effects of

sesamin and sesamolin on murine BV-2 microglia cell line under

hypoxia. Neurosci Lett 367:10–13

22. Joshi R, Kumar MS, Satyamoorthy K et al (2005) Free radical

reactions and antioxidant activities of sesamol: pulse radiolytic

and biochemical studies. J Agric Food Chem 53:2696–2703

23. Fazli IS, Abdin MZ, Jamal A et al (2005) Interactive effect of

sulphur and nitrogen on lipid accumulation, acetyl- CoA con-

centration and acetyl-CoA carboxylase activity in developing

seeds of oilseed crops (Brassica campestris L. and Eruca sativa

Mill.). Plant Sci 168:29–36

24. Budowski P, Connor RT, Field ET (1950) Sesameoil: IV.

Determination of free and bound sesamol. J Am Oil Chem Soc

27:307–310

25. Jollow DJ, Mitchell JR, Zampaghone N et al (1974) Bromoben-

zene induced oxide as the hepatotoxic intermediate. Pharmacol-

ogy 11:161–169

26. Utley HC, Bernhein F, Hochslein P (1967) Effects of sulfhydryl

reagent on peroxidation in microsomes. Arch Biochem Biophys

260:521–531

27. Islam F, Zia S, Sayeed I et al (2002) Selenium induced alteration

on lipids, lipid peroxidation, and thiol group in circadian rhythm

centers of rat. Biol Trace Elem Res 90:1–12

28. Habig WH, Pabst MJ, Jokoby WB (1974) Glutathione-S-trans-

ferase:the first enzymatic step in mercapturic acid formation.

J Biol Chem 249:7130–7139

29. Mohandas J, Marshall JJ, Duggin GG et al (1984) Differential

distribution of glutathione and glutathione related enzymes in

rabbit kidneys: possible implication in analgesic neuropathy.

Cancer Res 44:5086–5091

30. Carlberg I, Mannervik B (1975) Glutathione reductase levels in

rat brain. J Biol Chem 250:5475–5480

31. Caliborne A (1985) Catalase activity. In: Wakd G (ed) CRC hand

book of methods for oxygen radical research. CRC Press, Boca

Raton, pp 283–284

32. Beauchamp C, Fridovich I (1971) Superoxide dismutase:

improved assays and an assay applicable to acrylamide gels. Ann

Biochem 44:276–287

33. Bradford MM (1976) Rapid and sensitive method for the quan-

titation of microgram quantities of protein utilizing the principle

of protein-dye binding. Anal Biochem 72:248–254

34. Halliwell B (2006) Oxidative stress and neurodegeneration:

where are we now? J Neurochem 97:1634–1658

Neurochem Res (2012) 37:516–526 525

123

35. Cheung YT, Lau WK, Yu MS et al (2009) Effects of all-trans-

retinoic acid on human SH-SY5Y neuroblastoma as in vitro

model in neurotoxicity research. Neurotox 30:127–135

36. Wu JH, Hodgson JM, Clarke MW et al (2009) Inhibition of 20-

hydroxyeicosatetraenoic acid synthesis using specific plant lign-

ans: in vitro and human studies. Hyperten 54:1151–1158

37. Fridovich I (1995) Superoxide radical and superoxide dismutases.

Annu Rev Biochem 64:97–112

38. Boillee S, Cleveland DW (2008) Revisiting oxidative damage in

ALS: microglia, Nox, and mutant SOD1. J Clin Invest 118:474–478

39. Huang J, Philbert MA (1996) Cellular responses of cultured

cerebellar astrocytes to ethacrynic acid-induced perturbation of

subcellular glutathione homeostasis. Brain Res 711:184–192

40. Cruz-Auguado R, Almaguer-Melian W, Diaz MC (2001)

Behavioural and biochemical effects of glutathione depletion in

the rat brain. Brain Res Bull 55:327–333

41. Baez S, Segura-Aguilar J, Widersten M (1997) Glutathione

transferases catalyse the detoxication of oxidized metabolites (o-

quinones) of catecholamines and may serve as an antioxidant

system preventing degenerative cellular processes. Biochem J

324:25–28

42. Halliwell B, Gutteridge JM, Cross C (1992) Free radicals, anti-

oxidants, and human disease: where are we now? J Lab Clin Med

119:598–620

43. Zafar KS, Siddiqui A, Sayeed I et al (2003) Dose-dependent

protective effect of selenium in rat model of Parkinson’s disease:

neurobehavioral and neurochemical evidences. J Neurochem

84:438–446

44. Khan MM, Ahmad A, Ishrat T et al (2010) Resveratrol attenuates

6- hydroxydopamine-induced oxidative damage and dopamine

depletion in rat model of Parkinson’s disease. Brain Res 1328:

139–151

45. Liu T, Jin H, Sun QR et al (2010) The neuroprotective effects of

tanshinone IIA on b-amyloid-induced toxicity in rat cortical

neurons. Neuropharmacol 59:595–604

46. Brown DI, Griendling KK (2009) Nox proteins in signal trans-

duction. Free Radic Biol Med 47:1239–1253

47. Nakano D, Kurumazuka D, Nagai Y et al (2008) Dietary sesamin

suppresses aortic NADPH oxidase in DOCA salt hypertensive

rats. Clin Exp Pharmacol Physiol 35:324–326

48. Anantharam V, Kaul S, Song C et al (2007) Pharmacological

inhibition of neuronal NADPH oxidase protects against 1-methyl-

4-phenylpyridinium (MPP?)-induced oxidative stress and apop-

tosis in mesencephalic dopaminergic neuronal cells. Neurotoxi-

cology 28:988–997

49. Teismann P, Vila M, Choi DK (2003) COX-2 and neurodegen-

eration in Parkinson’s disease. Ann N Y Acad Sci 991:272–277

50. Cui Y, Hou X, Chen J et al (2010) Sesamin inhibits bacterial

formylpeptide- induced inflammatory responses in a murine air-

pouch model and inTHP-1 human monocytes. Nutr J 140:

377–381

51. Utsunomiya T, Chavali SR, Zhong WW et al (2000) Effects of

sesamin- supplemented dietary fat emulsions on the ex vivo

production of lipopolysaccharide-induced prostanoids and tumor

necrosis factor alpha in rats. Am J Clin Nutr 72:804–808

52. Jin F, Wu Q, Lu YF et al (2008) Neuroprotective effect of res-

veratrol on 6-OHDA-induced Parkinson’s disease in rats. Eur J

Pharmacol 600:78–82

53. Callio J, Oury TD, Chu CT (2005) Manganese superoxide dis-

mutase protects against 6-hydroxydopamine injury in mouse

brains. J Biol Chem 280:18536–18542

54. Wang J, Du XX, Jiang H et al (2009) Curcumin attenuates

6-hydroxydopamine- induced cytotoxicity by anti-oxidation and

nuclear factor-kappa B modulation in MES23.5 cells. Biochem

Pharmacol 78:178–183

55. Rathore P, Dohare P, Varma S et al (2008) Curcuma oil: reduces

early accumulation of oxidative product and is anti-apoptogenic

in transient focal ischemia in rat brain. Neurochem Res 33:

1672–1682

526 Neurochem Res (2012) 37:516–526

123