The animals were obtained from Atatürk University Medical Experimental Application and Research Center. A total of 18 albino wistar male rats with the weight ranging 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 and 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-Turkey. Liv–52 was provided by Himalaya drug –India.
Experimental animals were randomly divided into three groups, with 6 rats in each group as follows: liver ischemia reperfusion (IR), 20 mg/kg Liv–52+ liver ischemia reperfusion (LIR) and the sham group (HG).
The surgical interventions on rats were carried out under sterile conditions; anesthesia was provided by giving 25 mg/kg of intraperitoneal (ip) thiopental sodium and administering xylazine by inhalation at appropriate intervals. One hour before thiopental sodium anesthesia, a dose of 20 mg/kg of Liv–52 (n–6) was given to the LIR animal group orally with a catheter. Distilled water as solvent was treated to the IR and HG rat groups with the same method. After the injection of thiopental sodium, the rats were kept waiting for the appropriate surgery period to occur. The period when the animals are motionless in the supine position is considered to be an appropriate period for surgical intervention. 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, an hour of ischemia and six hours of reperfusion were performed by placing clamps on the hepatic artery, portal vein and gail ductus in order to create total hepatic ischemia (excluding the HG group). At the end of this period, blood samples were 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, 0.2 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% hexa desil tri methyl ammonium bromide] pH: 6 potassium phosphate buffer for myeloperoxidase analyze, consisting of 1.15% potassium chloride solution for thio barbituric acid reactions (TBARS) analysis and pH: 7.5 phosphate buffer for the superoxide dismutase, total glutathione analysis. Then, they were centrifuged at 4 °C, 10.000 rpm for 15 minutes. The supernatant part was used as the analysis sample.
Serum ALT, AST and LDH 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 thio barbituric 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 4000 rpm. The absorbance of the supernatant was measured at 532 nm. The standard curve was obtained by using 1,1,3,3- tetramethoxypropane.
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 3500 rpm 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 0.0005% 1.5 mL 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.
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. If nitro blue tetrazolium (NBT) is added to this reaction, SOD reacts with NBT and a purple-colored formazan dye occurs. 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 6000 rpm 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. At the end of the reaction, formazan occured. The absorbance of the purple-colored formazan was measured at 560 nm. As more of the enzyme exists, the least O2− radical that reacts with NBT occurs.
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, and the precipitate was removed after centrifugation at 4200 rpm for 40 min at 4 °C and 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 and vortexed and incubated for 30 min in 37°C. 5,5-Dithiobis (2-nitrobenzoic acid) (DTNB) was used as an 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.
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, chronometer was on, and the absorbance at 340 nm was recorded for 5 min every 15 s.
GR 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 GR measurement. After the NADPH and GSSG addition, chronometer was on and absorbance was measured for 5 min by 30-min intervals at 340 nm spectrophotometric methods.
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 the light microscope (Olympus BX 52, Tokyo, Japan) by two pathologists who do not know which treatment protocol is used.
The results obtained from the experiments are depicted as “mean ± standard error " ( x ± SEM). Normality of the data have been 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, Fisher’s post-hoc Bonferroni was performed. All statistical analyses were performed using “IBM SPSS Statistics Version 22” program and p<0.05 was considered significant.