Summary
Oksidatif hasar tıkanma sarılığına bağlı ensefalopatide önemli rol oynamaktadır. Çalışmamızın amacı Nigella sativa (NS, çörekotu) yağının safra kanalı tıkanıklığına bağlı oksidan beyin hasarında olası koruyucu etkisinin araştırılmasıdır.Erkek Wistar albino sıçanların safra kanalları bağlandıktan sonra sıçanlara 28 gün süreyle intragastrik olarak NS yağı (1 mg/kg p.o.) ya da serum fizyolojik verilmiştir. Daha sonra Evans mavisi (EB) geçirgenliği yöntemiyle kan beyin bariyeri (KBB) değerlendirilmiştir. Ayrıca beyin dokuları çıkarılarak malondialdehid (MDA), glutatyon (GSH), myeloperoksidaz (MPO) ve Na+,K+-ATPaz aktivitesi ölçülmüştür.
Kronik safra kanalı tıkanıklığı KBB geçirgenliğinde anlamlı bir artışa neden olurken bu etki NS tedavisi ile azalmıştır. Diğer taraftan safra kanallarının bağlanmasıyla azalan beyindeki GSH düzeyi ve Na+,K+-ATPaz aktivitesi NS tedavisi ile neredeyse kontrol düzeylerine yükselmiştir. Beyin MDA düzeyi ve MPO aktivitesindeki artış da NS tedavisiyle önlenmiştir.
Bulgularımız NS tedavisinin safra tıkanıklığına bağlı beyin hasarına karşı koruyucu olduğunu göstermektedir. Dolayısıyla sirozlu hastalarda NS takviyesi hepatik ensefalopatiye karşı fayda sağlayabilir.
Introduction
Obstruction of the common bile duct induces a reproducible model of biliary cirrhosis in rats. Hepatic fibrosis, the main complication of chronic liver disease, is usually initiated by hepatocyte damage, leading to recruitment of inflammatory cells and platelets, activation of Kuppfer cells and subsequent release of cytokines and growth factors[1]. It is known that increased concentration of bile acids induce lipid peroxides, probably related to the stimulation of phagocytic activity in the polymorphonuclear leukocytes and inflammatory cells, which are present after biliary tract obstruction and enhance the tissue injury[2,3]. Several clinical and experimental studies have shown that oxygen free radicals have a role in the pathogenesis of tissue injury in obstructive jaundice[4-7]. Thus free radical ablation for the treatment of cholestatic liver injury has been shown to have beneficial effects in the prevention of fibrosis and oxidative damage following biliary obstruction.In contrast to portacaval anastomosis, bile duct-ligated (BDL) animals have liver failure, developing jaundice, portal hypertension[8], portal-systemic shunting[9], bacterial translocation and immune system dysfunction[10]. BDL rats provides a model of acute-on-chronic hepatic encephalopathy, which reproduces the human neuropathology (Alzheimer Type II astrocytosis) and alterations of brain osmolytes that are characteristic of human Type C hepatic encephalopathy as well as low-grade brain oedema, inflammation and deficits of motor coordination[11,12]. Oxidative stress is one of the important mechanisms of jaundice induced encephalopathy.
Nigella sativa L. (N. sativa) have been widely studied due to its strong traditional claims and beliefs of having therapeutic role in almost every disease process[13-16]. Several therapeutic roles have been asserted thereafter such as antioxidant, anti-inflammatory[17], analgesic, antitumor, antihistaminic[18], oral hypoglycemic[19], hepatoprotective[20,21], nephroprotective[22], neuroprotective[23] and gastroprotective actions[24].
The aim of this study was to examine the possible protective effect of Nigella sativa (NS) oil against the oxidative stress of the brain tissue due to experimental obstructive jaundice in rats.
Methods
Biliary obstruction was performed in male Wistar albino rats by bile duct ligation and scission (BDL). Intragastric NS oil or saline was administered to the BDL rats for 28 days. At the end of the experiment, in the half of the rats the blood brain barrier (BBB) permeability was evaluated by Evans blue (EB) extravasation. Other rats were decapitated and brain tissue samples were obtained for the measurement of malondialdehyde (MDA) and glutathione (GSH) levels, myeloperoxidase (MPO) and Na+,K+-ATPase activities.
Animal model of liver fibrosis and treatment procedure
Male Wistar albino rats (250–300 g) were housed in a room at
a mean constant temperature of 22 ± 2 °C with a 12 hour lightdark
cycle, and free access to standard pellet chow and water.
The study was was conducted according to the ethical guidelines
of Marmara University Animal Care and Use Committee.
Liver fibrosis was induced by biliary obstruction in rats. Rats were anaesthetized (75 mg/kg ketamine and 1 mg/kg chlorpromazine; i.p.) and the common bile duct was exposed and ligated by double ligatures with silk suture. The first ligature was made below the junction of the hepatic ducts and the second ligature was made above the entrance of the pancreatic ducts. Finally, the common bile duct was resected between the double ligatures[25]. In sham-operated control rats, an incision was made in the abdomen which was then closed and given saline only. Each group consists of 12 rats (6 for biochemical studies, 6 for evaluation of edema and permeability). Nigella sativa oil (1 mg/kg p.o.) or saline was administered for 28 days. After 28 days of treatment, rats were killed by decapitation and trunk blood was collected for the biochemical analysis in the serum. Brain tissues were stored at −70 0C for the measurement of malondialdehyde (MDA) and glutathione (GSH) levels, myeloperoxidase (MPO) activity, Na+,K+-ATPase activity, water content and Evans blue extravasation .
Biochemical analysis in the serum
Serum AST, ALT and LDH levels[26] were determined spectrophotometrically
using an automated analyzer. Serum total
bilirubin level was assayed on Bayer Opera Autoanalyzer.
Cytokine determination
Serum TNF-alpha was quantified according to the manufacturer’s
instructions and guidelines using enzyme-linked immunosorbent
assay (ELISA) kits specific for the previously
mentioned rat cytokines (Biosource International, Nivelles,
Belgium). These particular assay kits were selected because of
their high degree of sensitivity, specificity, inter- and intra assay
precision, and small amount of serum sample required to
conduct the assay.
Biochemical analysis in the brain tissue
Malondialdehyde (MDA) and Glutathione (GSH) assays
Tissue samples were homogenized with ice-cold trichloracetic
acid (1 g tissue plus 10 ml 10% TCA) in an Ultra Turrax tissue
homogenizer. The MDA levels were assayed for products of
lipid peroxidation by monitoring thiobarbituric acid reactive
substance formation as described previously[27]. Lipid peroxidation
is expressed in terms of MDA equivalents using an
extinction coefficient of 1.56 x 105 M–1 cm –1 and the results are
expressed as nmol MDA/g tissue. Glutathione measurements
were performed using a modification of the Ellman procedure[28]. Briefly, after centrifugation at 2,000 g for 10 min, 0.5 ml of
supernatant was added to 2 ml of 0.3 mol/l Na2HPO4.2H2O
solution. A 0.2 ml solution of dithiobisnitrobenzoate (0.4 mg/
ml 1% sodium citrate) was added and the absorbance at 412
nm was measured immediately after mixing. Glutathione levels
were calculated using an extinction coefficient of 1.36 x 104
M–1 cm –1. The results are expressed in μmol GSH/g tissue.
Measurement of brain myeloperoxidase (MPO) activity
Tissue samples were homogenized in 50 mM potassium phosphate
buffer (PB, pH 6.0), and centrifuged at 41,400 g (10 min);
pellets were suspended in 50 mM PB containing 0.5 % hexadecyltrimethylammonium
bromide (HETAB). After three freeze
and thaw cycles, with sonication between cycles, the samples
were centrifuged at 41,400 g for 10 min. Aliquots (0.3 ml) were
added to 2.3 ml of reaction mixture containing 50 mM PB, o-dianisidine,
and 20 mM H2O2 solution. One unit of enzyme activity
was defined as the amount of MPO present that caused
a change in absorbance measured at 460 nm for 3 min. MPO
activity was expressed as U/g tissue[29].
Na+,K+-ATPase activity
Since the activity of Na+,K+-ATPase, a membrane-bound enzyme
required for cellular transport, is very sensitive to free
radical reactions and lipid peroxidation, reductions in this activity
can indicate membrane damage indirectly. Measurement
of Na+,K+-ATPase activity is based on the measurement
of inorganic phosphate released by ATP hydrolysis during incubation
of homogenates with an appropriate medium containing
3 mM ATP as a substrate. The total ATPase activity
was determined in the presence of 100 mM NaCl, 5 mM KCl, 6
mM MgCl2, 0.1 mM EDTA, 30 mM Tris HCl (pH 7.4), while the
Mg2+-ATPase activity was determined in the presence of 1mM
ouabain. The difference between the total and the Mg2+-ATPase
activities was taken as a measure of the Na+,K+-ATPase
activity[30]. The reaction was initiated with the addition of the
homogenate (0.1 ml) and a 5-min preincubation period at 37º C was allowed. Following the addition of Na2ATP and a 10-min
re-incubation period, the reaction was terminated by the addition
of ice-cold 6 % perchloric acid. The mixture was then centrifuged
at 3500 g, and Pi in the supernatant fraction was determined
by the method of Fiske and Subarrow[31]. The specific
activity of the enzyme was expressed as nmol Pi mg-1 protein
h-1. The protein concentration of the supernatant was measured
by the Lowry method[32].
Neurological examination
The neurological examination scores were conducted according
to Bederson’s modified neurological examination test[33].
A twenty-point neuroscore was used to assess motor and behavioral
deficits. Briefly, the consciousness, performance in a
smooth climbing platform, extremity tonus, walking and postural
reflexes, circling and response to the nociceptive stimuli
were assessed. For walking and posture, rats were allowed to
move about freely on the floor, while they were observed. In
the circling test, the rats were held gently from the tail, suspended
one meter above the floor, and observed for forelimb
flexion, where normal rats are expected to extend both forelimbs
toward the floor. The rotation degree and time were
measured. Finally, the responses to the nociceptive stimuli
were assessed by tail-immersion test in 560C water. All behavioral
tests were conducted by a ‘blinded’ investigator. The sequence
of testing animals by a given task was randomized for
the animals.
Evans blue assay for the evaluation of blood brain barrier
permeability
To evaluate the blood brain barrier (BBB) integrity, Evans blue
dye (EB) was used as a marker of albumin extravasation[33].
Briefly, EB (2 % in saline, 4 ml/kg) was injected via the jugular
vein at the 48th h of the SAH induction and it was allowed to
remain in circulation for 30 min. Then, chests were opened and
the rats were perfused transcardially with 250 ml of saline at a
pressure of 110 mm Hg for approximately 15 min. After decapitation,
the brain was removed and dissected into cerebral
cortex and cerebellum, which were then weighed separately
for the quantitative measurement of EB-albumin extravasation
. Brain samples were homogenized in 2.5 ml phosphatebuffered
saline and mixed by vortexing for 2 min after the addition
of 2.5 ml of 60 % trichloroacetic acid to precipitate the
protein. Samples were cooled and then centrifuged for 30 min
at 1000 g. The supernatant was measured at 620 nm for the
absorbance of EB using a spectrophotometer (Shimadzu
UV1208, Japan). EB was expressed as μg/mg of brain tissue
against a standard curve.
Evaluation of the brain edema
Brain edema was evaluated by the gravimetric method based
on the measurement of the water content of brain[33]. The
whole brain was weighed and then dried for 48 h at 100 °C,
afterwards re-weighed. The percentage of water was calculated
according to the following formula: % H2O = [(wet weight
− dry weight) / wet weight] ×100.
Statistical analysis
Statistical analysis was carried out using GraphPad Prism 3.0
(GraphPad Software, San Diego; CA; USA). All data were expressed
as means ± S.E.M. Groups of data were compared
with an analysis of variance (ANOVA) followed by Tukey’s
multiple comparison tests. Values of P<0.05 were regarded as
significant.
Results
As shown in Table 1, serum LDH activity, total bilirubin, AST, ALT, and the plasma levels of pro-inflammatory cytokine (TNF-α) in the BDL group were significantly higher (p<0.001) than that of the control group, while treatment of NS abolished these elevations significantly (p<0.01-0.001).The MDA levels, measured as a major degradation product of lipid peroxidation in the brain tissue, were found to be significantly higher in the BDL group (p<0.001) as compared to those of the control group, while treatment with NS abolished these elevations (p<0.05; Figure 1). In accordance with these findings, levels of the major cellular antioxidant GSH in the BDL group was depleted (p<0.05); however, in the NS-treated BDL group, depleted GSH stores were partially replenished with this antioxidant (p<0.01; Figure 1).
Click Here to Zoom |
FIGURE 1: a) Malondialdehyde (MDA) and b) Glutathione (GSH) levels in the brain tissues of control and saline- or Nigella sativa oil (NS)-treated bile duct ligated (BDL) groups (n=6 per group). **p <0.05 compared with control group. + p <0.05, compared with saline-treated BDL group. |
Myeloperoxidase activity, which is accepted as an indicator of neutrophil infiltration, was significantly higher in the brain tissue of the BDL group (p<0.01) than that of the control group (p<0.05, Fig 2). On the other hand, NS treatment in the BDL group significantly decreased brain MPO level (p<0.05) back to the levels of the control group. The activity of Na+,K+ ATPase, indicating the functional transport capacity of the brain cells, was not found to be significantly different between the groups (p>0.05, Figure 2).
Click Here to Zoom |
FIGURE 2: a) Myeloperoxidase (MPO)and b) Na-K-ATPase activity of the brain tissues in control and saline- or Nigella sativa oil (NS)-treated bile duct ligated (BDL) groups (n=6 per group). ** p <0.01 compared with control group. +p <0.05, compared with saline-treated BDL group. |
Brain edema was not significantly different in any of the groups (p>0.05). However, EB content of the brain significantly increased in the BDL group. NS treatment reduced the permeability but this reduction was not significant (p>0.05, Figure 3).
Click Here to Zoom |
FIGURE 3: a) Evans blue (EB) extravasation and b) Water content of the brain tissues of control and saline or Nigella sativa oil (NS) -treated bile duct ligated (BDL) groups (n=6 per group). *p <0.05 compared with control group. |
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