Of 1.5 mg/kg body weight/rat.Inducing hepatic fibrosis in ratsbutanol
Of 1.5 mg/kg body weight/rat.Inducing hepatic fibrosis in ratsbutanol layer was taken after centrifugation at 950 xg for 15 minutes and absorbance was taken for fluorometric measurement at 553 nm with 515-nm excitation.Biochemical analyses in liver tissuesHepatic fibrosis was induced by intraperitoneal of dimethylnitrosamine (DMN) injected to the animals based on10 mg/kg for 3 consecutive days each week over a period of 4 wk according to Asakura et al, [30]. During the experimental period (4 weeks), diet consumed and body weights for rats were recorded twice a week and liver index, which is calculated as percent of liver weight at final body weight [31].Biochemical analyses in the serumAt completion of the experiment, the animals were fasted overnight, anaesthetized with CO 2 and sacrificed to obtain blood samples. Each blood sample was placed in dry clean centrifuge tube, and then centrifuged at 950 xg for 20 min. at 4 to separate the serum. Serum was carefully separated into clean dry Wassermann tubes by using a Pasteur pipette and kept frozen at -20 until analyses. The liver was separated from each rat then washed thoroughly in ice-cold physiological saline [0.9 (w/v) NaCl], and weighed to calculate liver to body weight percentage. Hepatotoxicity was assessed by quantifying the activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), according to [32]. Superoxide dismutase (SOD) content was determined by the xanthine oxidase method.Thiobarbituric acid-reactive substances (TBARS)Liver catalase (CAT) was determined by Goth’s colorimetric method, in which supernatant was incubated in H2O2 substrate and the enzymatic reaction was stopped by the addition of ammonium molybdate. The intensity of the yellow complex formed by molybdate and H2O2 was measured at 405 nm [34]. Superoxide dismutase (SOD) activity was determined by using a measurement method developed by McCord and Fridovich [35]. This method is based on the generation of superoxide radicals produced by xanthenes and xanthenes oxidase, which react with 2-(4-iodophenyl)-3(4-nitrophenol)-5-phenyltetrazolium chloride (INT) to form a red formazan dye. SOD activity was expressed as units per gram protein. Glutathione BRDU site peroxidase (GSH-Px) activity was measured on standard assay conditions in 340 nm (absorbance) at 37 according to the method developed by Paglia and Valentine [36]. In this measurement, GSH-Px catalyzes the oxidation of glutathione by cumene hydroperoxide. Measurements were performed by an autoanalyzer (Biochemistry Dept., Faculty of Veterinary Laboratories, Cairo University) according to PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/27488460 the Randox application procedure. GSH-Px activity was expressed as units per gram protein.Histopathological studiesThe content of serum lipid peroxides was analyzed by using thiobarbituric acid-reactive substances (TBARS) and expressing them as malonaldehyde equivalents using the method of Yagi [33] with reduced proportions. In brief, 20 L serum was added to 2 mL 40 mmol/L H2SO4 (Sigma Pharmaceutical Industries, Cairo, Egypt), then 0.25 mL 10 wt/vol phosphotungstic acid (Sigma Chemical Co. Cairo, Egypt) was added and mixed. The mixture was centrifuged at 950 xg for 15 min., the supernatant was discarded, and the sediment was mixed with 1 mL 40 mmol/L H2SO4 and 0.15 mL 10 wt/vol phosphotungstic acid. The mixture was centrifuged at 950 xg for another 15 min. The sediment was suspended in 2 mL distilled water, and 0.5 mL 0.33 wt/ vol thiobarbituric acid.
