TREATMENT OF NIGELLA SATIVA IN EXPERIMENTAL SEPSIS MODEL IN RATS

TREATMENT OF NIGELLA SATIVA


IN EXPERIMENTAL SEPSIS MODEL IN RATS

OZLEM ALICI1, HAVVA SAHIN KAVAKLI2*, CEMILE KOCA3

AND NERIMAN DEFNE ALTINTAS4

1Department of Infectious Diseases and Clinical Bacteriology, Medical Park Hospital, Istanbul, Turkey

2Department of Emergency Medicine, Ankara Ataturk Training and Research Hospital, Ankara, Turkey

3Department of Biochemistry, Faculty of Medicine, Fatih University Ankara, Turkey

4Department of Medical Intensive Care, Ankara Ataturk Training and Research Hospital, Ankara, Turkey

ABSTRACT

This experimental study was designed to determine effects of Nigella sativa oil (NSO) on endothelin-1 (ET-1)

level and oxidative stress parameters, superoxide dismutase (SOD) and malondialdehyde (MDA) in a rat sepsis

model. Twenty four adult Wistar albino rats were divided randomly into three groups: sham group (group 1),

sepsis group (group 2), sepsis group pretreated with NSO (group 3). Serum ET-1, tissue SOD and tissue MDA

levels were measured in all groups. Compared to group 1, ET-1 and MDA levels were higher in group 2. ET-1

and MDA levels in NSO pretreated group 3 were lower with respect to group 2 (p<0.03, and p<0.02,

respectively). Additionally, SOD levels in group 3 were found to be higher than group 2 (p<0.02). Based on our

results, it can be concluded that NSO may have a positive impact on ET-1 levels and oxidative stress induced by

sepsis in experimental rat models.

Keywords: Sepsis, Nigella sativa oil, endothelin, superoxide dismutase, malondialdehyde.

INTRODUCTION

Sepsis, is an inflammatory process in response to an

infectious stimulus, and it is the number one cause of

mortality in the intensive care units other than coronary

units (Dare et al., 2009, Ribeiro et al., 2009). It is

characterized by systemic inflammation and multi-organ

dysfunction which may eventually lead to death (Dare et

al., 2009). Despite advances in the understanding of its

pathophysiology, there are limited pharmacotherapeutic

options for sepsis, septic shock, and related

complications (Gupta et al., 2005). Several researchers

have linked reactive oxygen species (ROS) with the onset,

progression and outcome of sepsis, both in experimental

and in clinical studies (Guo and Ward, 2007; Zapelini et

al., 2008). ROS are important signaling molecules but

their overproduction results in detrimental oxidative stress

and must be avoided by the organism (Andrades et al.,

2009b).

Endothelins (ET) are a family of powerful vasoconstrictor

peptides that have numerous roles. ET-1 is the principal

isoform produced by the endothelium and it acts locally to

maintain the basal vascular tone. Its levels increase in a

variety of clinical conditions involving the endothelium,

including sepsis which has marked endothelial

dysfunction (Piechota et al., 2007; Shah, 2007).

Nigella sativa oil (NSO) is an agent that has been shown

to have antioxidant and antiinflammatory activities in

different clinical conditions (Coban et al., 2010; Hamdy

and Taha, 2009; Yildiz et al., 2010). In this study, we

aimed to evaluate the effects of NSO on ET-1 levels and

oxidative stress in a rat sepsis model.

MATERIALS AND METHODS

Study design

The study was approved by the Animal Research Ethics

Committee of Fatih University, School of Medicine

before 24 adult male Wistar rats (weight range, 250 to

270 g) were used for the experiment. All animals had

access to food and water ad libitum throughout the study.

The rats were divided randomly into three groups of eight

animals each: control group (group 1), sepsis group

(group 2), and sepsis induced group pretreated with NSO

(group 3). Control group (group 1) received only 1 ml

intraperitoneal (i.p.) injection of saline.

Sepsis in group 2 and group 3 were induced by

intraperitoneal (i.p.) injection of 2 x 106 CFU of

Escherichia coli ATCC 25922 which was grown in brainheart

infusion broth. In the logarithmic phase of the

growth, the suspension was centrifuged at 1000 g for 15

minutes, the supernatant was discarded, and the bacteria

were resuspended and diluted in sterile saline. The rats

received an i.p. inoculum of 1 mL of saline containing 2x

1010 CFU of E. coli ATCC 25922 (Ghiselli et al., 2004).

Rats in group 3 received NSO 50 g/kg orally 30 minutes

before the bacterial challenge, whereas rats in group 2

received only saline.

*Correspondence author: e-mail: havvasahin1973@yahoo.com

Treatment of Nıgella satıva

228 Pak. J. Pharm. Sci., Vol.24, No.2, April 2011, pp.227-231

Sample collection

24 hour after bacterial challenge, all animals were

sacrified using ketamine and cardiac puncture. Blood

samples were drawn from vena cava inferior for the

determination of endothelin levels, centrifuged at 3000g

for 10 minutes and stored at –80oC. Liver tissue samples

were subsequently removed and stored at -800C for the

determination of tissue associated malondialdehyde

(MDA) activity and superoxide dismutase (SOD) levels,

as the parameters of oxidative stress.

Quantitative determination of serum endothelin levels

Rat big ET-1 levels were measured using commercially

available enzyme linked immunosorbent assay (ELISA)

kits (Assay Designs, MI, USA) following the

manufacturer’s instructions. The results are presented as

pg/ml.

Quantitative determination of tissue malondialdehyde

levels

The tissue MDA level was determined based on pink

color formation with thiobarbituric acid (TBA) at 532

nm(Okhawa et al., 1979). After mixing with 10% (w/v)

trichloroacetic acid to precipitate the protein, sample was

reacted with 0.67% (w/v) TBA. At the end of incubation

in a boiling water-bath for 10 min, sample was

centrifuged and supernatant’s absorbance was read at 532

nm. Results were expressed as nmol per gram wet tissue,

according to the standard graphic prepared from

measurements with a standard solution.

Quantitative determination of tissue superoxide dismutase

levels

SOD activity was determined according to the method by

Sun et al. (1988). One unit of SOD was defined as the

amount causing 50% inhibition in the nitroblue

tetrazolium (NBT) reduction rate. The SOD activity is

expressed as U mg−1 protein.

STATISTICAL ANALYSIS

For statistical evaluation, we used the statistical software

package SPSS 15.0 and a probability value of less than

0.05 was accepted as statistically significant. As the data

were independent and showed normal distribution,

statistical analysis was performed using analysis of

variance (ANOVA) followed by Tukey test when

comparing groups. The results are given as the mean ±

standard deviation (SD).

RESULTS

ET-1, MDA and SOD levels for all groups are presented

in table 1 as mean ± SD.

Serum endothelin-1 levels

ET-1 levels were determined as it is one of the major

peptides in the pathogenesis of sepsis (fig. 1). ET-1 levels

in group 2 were significantly higher compared to group 1

(14.05±4.26 vs. 7.26±3.61, p=0.016). When sepsis was

induced in rats pretreated with NSO (group 3), ET-1

levels were found to be similar to group 1 (p>0.05) and

significantly lower compared to group 2 (8.52±2.70 vs

14.05±4.26, p<0.033).

Fig. 1: Endothelin-1 levels in group 2 were significantly

higher compared to group 1 (p=0.016). In group 3,

endothelin-1 levels were significantly lower compared to

group 2 (p<0.033).

Tissue malondialdehyde levels

Tissue MDA levels were determined as a product of lipid

peroxidation, a result of oxidative stress (fig. 2). MDA

Table 1. Results of laboratory studies in group 1, group 2 and group 3, mean±SD (P results while comparing the

groups have been presented beneath the table).

ET(pg/mL) MDA(nmol g−1 protein) SOD(U mg−1protein)

Group 1 7.26±3.61 6.89±1.31 6,92±2.11

Group 2 14.05±4.26 10.93±1.89 1.76±0.46

Group 3 8.52±2.70 7.76±2.54 3.65±0.43

P values:

Group1-Group 2 p=0.016 p=0.003 p<0.001

Group1-Group3 p>0.05 p>0.05 p<0.001

Group2-Group3 p=0.033 p=0.024 p=0.024

Ozlem Alıcı et al.

Pak. J. Pharm. Sci., Vol.24, No.2, April 2011, pp.227-231 229

levels in group 2 were significantly higher compared to

group 1 (10.93±1.89 vs. 6.89±1.31, p=0.003). When

sepsis was induced in rats pretreated with NSO (group 3),

MDA levels were found to be similar to control group

(group 1) (p>0.05) and significantly lower compared to

group 2 (7.76±2.54 vs 10.93±1.89, p<0.024).

Fig. 2: Malondialdehyde levels in group 2 were

significantly higher compared to group 1 (p=0.003). In

group 3, malondialdehyde levels were significantly lower

compared to group 2 (p<0.024).

Tissue superoxide dismutase levels

Tissue SOD levels were determined as a marker of

antioxidant status (fig. 3). SOD levels were highest in

group 1. SOD levels decreased significantly with

induction of sepsis in group 2 (6.92±2.11 vs 1.76±0.46,

p<0.001). However, in rats pretreated with NSO (group

3), SOD levels were higher compared to group 2

(3.65±0.43 vs. 1.76±0.46, p=0.024).

Fig. 3: Superoxide dismutase in group 1 was significantly

higher compared to group 2 (p<0.001). In group 3,

superoxide dismutase levels were higher compared to

group 2 (p=0.024).

DISCUSSION

The results revealed that pretreatment with NSO

significantly reduced serum ET-1 and MDA levels when

sepsis was induced in a rat sepsis model. As well, SOD

levels were found to be significantly higher in the NSO

pretreated sepsis group when compared to the sepsis

group.

Constituents of the Nigella sativa seed are known to

possess potent antioxidant effects (Alenzi et al., 2010;

Terzi et al., 2010). Nigella sativa (family:

Ranunculaceae), which is also commonly known as Black

Seed, Black Cumin, or Habbatul Barakah, have long been

used in traditional medicine in the Arabian Gulf region,

Far East Asia, and Europe. Its healing powers has been

described by The Prophet Mohammad, the Holy bible,

Hippocrates, Dioscorides and by Pliny. It was believed to

be a remedy for all diseases. In the modern era,

pharmacological studies have proved it to have multiple

pharmacological effects among which are its analgesic,

antimicrobial (against a wide range of organisms),

antiinflammatory and antioxidant activities (Coban et al.,

2010; Hamdy and Taha 2009; Helal 2010; Tariq 2008;

Terzi et al., 2010 ). As well, thymoquinone, a constituent

of NSO, has been reported to reduce acute liver

dysfunction when endotoxemia was induced by LPS

administration (Helal 2010).

Endothelin (ET)-1 is a potent vasoconstrictor peptide and

it is involved in the pathogenesis of septic shock.(Forni et

al., 2005). In sepsis, ET-1 levels are thought to correlate

with sepsis severity and, especially with circulatory

dysfunction (Figueras-Aloy et al., 2004; Piechota et al.,

2007). Of remark, there are promising reports that ET-1

receptor antagonists may have beneficial effects on

cardiovascular performance and survival in sepsis models

(Iskit and Guc, 2004; Konrad et al., 2007). Our results

reveal that NSO may have an effect to decrease ET-1

levels which may be beneficial during sepsis.

Oxidative stress has been implicated as one of the causes

of multiple organ dysfunction syndrome during the course

of sepsis and it is the result of systemic inflammatory

response (Andrades et al., 2009a; Novak et al., 2010;

Rinaldi et al., 2009). It is marked by increased levels of

reactive oxygen species (ROS), resulting from both a

decrease in antioxidant levels and from increased

production of ROS (Andrades et al., 2009 a; Ribeiro et

al., 2009). Oxidative stress has multiple deleterious

effects on tissues. One of which is the lipid peroxidation

of cellular membranes, characterised by lipid

hydroperoxides that are decomposed to a variety of end

products including MDA (Okhawa et al., 1979). MDA

levels were found to be increased with sepsis induction,

however pretreatment with NSO resulted in lower levels

of MDA. As well, NSO improved the antioxidant status

Treatment of Nıgella satıva

230 Pak. J. Pharm. Sci., Vol.24, No.2, April 2011, pp.227-231

as shown by increased levels of SOD despite sepsis

induction in the NSO pretreated group.

Enhanced generation of ROS in sepsis is suspected to be

responsible of detrimental consequences and sepsisrelated

endothelium dysfunction (Andrades et al., 2009 a;

Forceville et al., 2009; Kaymak et al., 2008; Rodríguez et

al., 2009; Víctor et al., 2009). Modalities to counteract

toxicity of ROS are being searched for. Major

endogeneous antioxidant systems include glutathione,

vitamins A, C and E and several other enzymes such as

SOD (Ribeiro et al., 2009). SOD is an antioxidant enzyme

that scavenges the superoxide radical and catalyzes it to

hydrogen peroxide and oxygen. Levels of SOD can

provide information on the antioxidant status (Rabus et

al., 2008). NSO may be a new agent to be studied as a

supplement during sepsis as other antioxidants in the form

of selenium, glutamine, eicosapentaenoic acid and

micronutrients that have been shown to improve sepsis

outcomes (Barichello et al., 2007; Berger and Chioléro,

2007).

In conclusion, the results of this study have shown that

pretreatment with NSO decreased the ET-1 levels and

improved antioxidant status as shown by SOD and MDA

levels. Combined with the results of previous studies, our

results indicate that further studies may establish NSO a

role as an adjuvant therapeutic agent in sepsis.

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