Editor-in-Chief Hatice Kübra Elçioğlu Vice Editors Levent Kabasakal Esra Tatar Online ISSN 2630-6344 Publisher Marmara University Frequency Bimonthly (Six issues / year) Abbreviation J.Res.Pharm. Former Name Marmara Pharmaceutical Journal
Journal of Research in Pharmacy 2013 , Vol 17 , Num 1
The protective effect of Nigella sativa oil in the brain of the biliary obstructed rats
Hale Zerrin Toklu, Tülay Akmandor İnac, Ahmet Özer Şehirli, Göksel Şener
Marmara University, School of Pharmacy Department of Pharmacology, Istanbul, Türkiye DOI : 10.12991/201317392

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).

TABLE 1: Serum total biluribin levels, aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH) activities and TNF-alpha levels in control and saline- or Nigella sativa oil (NS)-treated bile duct ligated (BDL) groups.

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).


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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).


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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).


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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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