The animals were obtained from Atatürk University Medical Experimental Application and Research Center. A total of 18 albino wistar male rats weighing between 250-270 grams (8-10 months) were used in the experiment. Animals were hosted in standard cages with 6 rats per cage and fed ad-libitum in groups. They were maintained in a 12:12-h light–dark (LD) cycle at constant temperature (22°C ± 1°C). Animal experiments were performed in accordance with the National Guidelines for the Use and Care of Laboratory Animals and were approved by the local animal ethics committee of Ataturk University, Erzurum, Turkey (Ethics Committee Number: 77040475-000-E.1700216877, Dated:03.08.2017).
Of the chemical substances used for the experiments, thiopental sodium was provided by IE
Ulagay, Istanbul, Turkey. Liv-52 was provided by Himalaya Drug, Shankar Nagar, Nagpur, Maharashtra, India.
Experimental animals were randomly divided into three groups, with 6 rats in each group treated as follows: liver ischemia/reperfusion (IR), 20 mg/kg Liv-52+ liver ischemia reperfusion (LIR) and sham operation applied to the healthy group (HG).
The surgical interventions on rats were carried out under sterile conditions. Anesthesia was performed by administering 25 mg/kg of intraperitoneal (ip) thiopental sodium and xylazine by inhalation at appropriate intervals. One hour before thiopental sodium anesthesia, Liv-52 was given to the LIR animal group orally by means of a catheter at a dose of 20 mg/kg as given in previous studies. Also, 100 mg/kg Liv-52 studied for isoniazid and rifampicin induced hepatotoxicity. Distilled water as solvent was administered to the IR and HG rat groups with the same method. After the injection of thiopental sodium, the rats were kept for the appropriate surgery period. Surgical intervention was applied after the period when the animals were motionless in supine position was considered to be appropriate. During this process, all the rats were brought to supine position and laparotomy was performed by the 3,5-4 cm long vertical dissection of the abdomen's anterior portion. Later, one hour for ischemia was performed by placing clamps on the hepatic artery, portal vein and bile duct in order to create total hepatic ischemia (excluding the HG group). And after ischemia period six hours of reperfusion was provided. At the end of this period, one blood sample for each rat (each sample replicated three times for biochemical analyzes and averaged) was taken from the tail veins of the animals for the measurement of Alanine aminotransferase (ALT), Aspartate aminotransferase (AST) and Lactate dehydrogenase (LDH) activities. Later, rat groups were killed with high dosage of anesthesia (50 mg/kg i.p. thiopental sodium IE Ulagay-Türkiye) and their liver tissues were removed. Oxidant/antioxidant parameters were determined from the removed tissues and the tissues were examined histopathologically. The biochemical results obtained from the LIR group were compared with the results obtained from IR and HG groups.
In this part, 20 mg of whole liver tissue was weighed for each liver. The samples were homogenized in ice with 2-mL buffers (consisting of 0.5% HDTMAB [0.5% hexadecyltrimethylammonium bromide] pH 6 potassium phosphate buffer for myeloperoxidase analyze, consisting of 1.15% potassium chloride solution for thiobarbituric acid reactions (TBARS) analysis and pH 7.5 phosphate buffer for the superoxide dismutase, total glutathione analysis. Then, they were centrifuged at 4 °C, 10.000xg for 15 minutes. The supernatant part was used as the analysis sample. And each sample replicated three times for biochemical analyzes and averaged. The supernatant was used as the sample for the analysis. The protein concentration of the supernatant was measured with the method described by Bradford.
Serum Alanine aminotransferase, Aspartate aminotransferase and Lactate dehydrogenase measurements
Venous blood samples were collected into tubes without anticoagulant. Serum was separated by centrifugation after clotting and stored at −80 °C until assay. Serum AST and ALT activities as liver function tests, and LDH activity as a marker of tissue injury, were measured spectrophotometrically on a Cobas 8000 (Roche) autoanalyser using commercially available kits (Roche Diagnostics, GmBH, Mannheim, Germany).
The concentrations of liver lipid peroxidation were determined by estimating MDA using the thiobarbituric acid test. The rat livers were rinsed with cold saline. The corpus mucosa was scraped, weighed, and homogenized in 10 ml of 100 g/L KCl. The homogenate (0.5 ml) was added to a solution containing 0.2 ml of 80 g/l sodium lauryl sulfate, 1.5 ml of 200 g/l acetic acid, 1.5 ml of 8 g/L 2-thiobarbiturate, and 0.3 ml distilled water. The mixture was incubated at 98°C for 1 h. Upon cooling, 5 ml of n-butanol:pyridine (15:l) was added. The mixture was vortexed for 1 min and centrifuged for 30 min at 1800xg. The absorbance of the supernatant was measured at 532 nm. The standard curve was obtained by using 1,1,3,3- tetramethoxypropane (1.56 - 3.12 - 6.25 - 12.5 - 25 - 50 - 100 µM) .
The activity of myeloperoxidase (MPO) in the total homogenate was measured according to the method of Wei and Frenkel with some modifications. The sample was weighed and homogenized in 2 ml of 50 mmol/L phosphate buffer containing 0.5% hexadecyltrimethyl ammonium bromide (HDTMAB) and centrifuged at 1200xg for 60 minutes at 4°C. The supernatant was used to determine MPO activity using 1.3 mL 4-aminoantipyrine-2% phenol (25 mM) solution. 25 mmol/L 4-aminoantipyrine–2% phenol solution and 1.5 mL of 0.0005% H2O2 were added and equilibrated for 3–4 minutes. After establishing the basal rate, a 0.2 mL sample suspension was added and quickly mixed. Increases in absorbance at 510 nm for 4 minutes at 0.1-min intervals were recorded. Absorbance was measured at 412 nm using a spectrophotometer.
Superoxide dismutase analysis
Measurements were performed according to the method of Sun et al. When xanthine is converted into uric acid by xanthine oxidase, superoxide dismutase (SOD) forms. Nitro blue tetrazolium (NBT) is added to this reaction, SOD reacts with NBT forming a purple-colored formazan dye. The sample was weighed and homogenized in 2 ml of 20 mmol/L phosphate buffer containing 10 mmol/L EDTA at pH 7.8. The sample was centrifuged at 3600xg for 10 minutes and the brilliant supernatant was used as assay sample. The measurement mixture containing 2450 μL measurement mixture (0.3 mmol/L xanthine, 0.6 mmol/L EDTA, 150μmol/L NBT, 0.4 mol/L Na2CO3, 1 g/l bovine serum albumin), 500 μL supernatant and 50 μL xanthine oxidase (167 U/l) was vortexed. Then it was incubated for 10 min. Formazan occured at the end of the reaction. The absorbance of the purple-colored formazan was measured at 560 nm. As more of the enzyme exists, the least O2− radical that reacting by NBT occurs.
Total glutathione analysis
The amount of GSH in the total homogenate was measured according to the method of Sedlak and Lindsay with some modifications. The sample was weighed and homogenized in 2 mL of 50 mmol/L Tris–HCl buffer containing 20 mmol/L EDTA and 0.2 mmol/L sucrose at pH 7.5. The homogenate was immediately precipitated with 0.1 mL of 25% trichloroacetic acid, the precipitate was removed after centrifugation at 1800xg for 40 min at 4 °C, the supernatant was used to determine GSH level. A total of 1500 μL of measurement buffer (200 mmol/L Tris–HCl buffer containing 0.2 mmol/L EDTA at pH 7.5), 500 μL supernatant, 100 μL DTNB (10 mmol/L) and 7900 μL methanol were added to a tube vortexed, incubated for 30 min in 37°C. 5,5-Dithiobis (2-nitrobenzoic acid) (DTNB) was used as chromogen and it formed a yellow-colored complex with sulfhydry groups. The absorbance was measured at 412 nm using a spectrophotometer (Beckman DU 500, USA). The standard curve was obtained by using reduced glutathione (0.5 - 1 - 2 - 4 - 8 - 16 - 32 µM).
Glutathione peroxidase analysis
GPO activity was determined according to the method of Lawrence and Burk. After tissue homogenization, supernatant was used for GPO measurement. After the KH2PO4, EDTA, GSH, B-NADPH, NaN3, and GR addition, mixture was incubated. As soon as H2O2 was added, and the absorbance was recorded every 15 s for 5 min, at 340 nm.
Glutathione reductase analysis
GSHRd activity was determined spectrophotometrically by measuring the rate of NADPH oxidation at 340 nm according to Carlberg and Mannervik method. After tissue homogenization, supernatant was used for GSHRd measurement. After the NADPH and GSSG addition, and absorbance was measured for 5 min by 30-min intervals at 340 nm spectrophotometric methods.
Glutathione S transferase activity
GST activity was determined by Habig and Jakoby. Briefly, the enzyme's activity was assayed spectrophotometrically at 340 nm in a 4-ml cuvette containing 0.1 M PBS (pH 6.5), 30 mM GSH, 30 mM 1-chloro-2,6-dinitrobenzene, and tissue homogenate.
Liver tissues obtained from the rats were fixed in 10% formalin solution for 24 hours. After routine tissue processing, 4 micron thick sections were obtained from the paraffin blocks and were stained with Hematoxylin&Eosin. All sections were examined under a light microscope (Olympus BX 52, Tokyo, Japan) by two pathologists who do not know which treatment protocol used.
The results obtained from the experiments are depicted as “mean ± standard error” (x ± SEM). Normality of the data was tested with Shapiro-Wilk test. All the parameters showed normal distribution. The significance level of the inter-group difference was identified using one-way ANOVA test. Then, Bonferroni post-hoc test was performed. All statistical analyses were performed using “IBM SPSS Statistics Version 22” program and p<0.05 was considered significant.