This study was conducted in the Experimental Animal Laboratory of the Faculty of Pharmacy, Al-Ahliyya Amman University, and ethically approved by ethical committee for the care and use of laboratory animals (ethical approval no. AAU-1/14/2017-2018).
Chemicals, reagents, and kits
Arctigenin (Item No. 270652), glutathione (GSH) kit (Item No.703002), superoxide dismutase (SOD) kit (Item No. 706002), and glutathione reductase kit (Item No. 703202) were purchased from (Cayman chemicals, USA). CCl4 (> 99.9%, Item No. 270652), carboxymethylcellulose (CMC) (Item No. C9481), and metphosphoric acid (MPA) (Item No. 79613) from Sigma Aldrich, USA.
Total MMP-2 ELISA kit (Item No. MMP200) and lysis buffer (Item No. 895347) were supplied by RnD Systems, USA. Alanine aminotransferase (ALT) (Item No. 11533), aspartate aminotransferase (AST) (Item No. 11531), alkaline phosphatase (ALP) (Item No.11592), and bilirubin (Item No. 11515) assay kits were purchased from BioSystems S.A., Barcelona (Spain). Silymarin (Legalon® 70 mg) was kindly provided by Chemical Industries Development CID, Egypt.
Animals
A total of 24 male Wistar rats (age 6-8 weeks, weight 210-240 g) were provided from the Jordanian University of Science and Technology (JUST), Irbid, Jordan. All animals were kept under observation in Al-Ahliyya Amman University animal house, for 2 weeks prior to the study with free access to commercial rat diet and water ad libitum. Rats were housed at 22 ± 2 °C with a 12 h light-dark cycle. All animals’ handling and treatment were in adherence to the ARRIVE guidelines.
Experimental design
The rats were randomly divided into 4 equal groups (n = 6 rats). Group A (control) and B (toxic), animals were administered the vehicle daily (1% CMC, 4 mL/kg, i.p.). Group C (standard), rats were daily administered silymarin (200 mg/kg, 4 mL/kg, i.p.) [22]. Group D (treatment), rats were daily administered AG (15 mg/kg, 4 mL/kg, i.p.) [19] (Fig. 1). All animals were treated for 6 weeks. The experimental design was approved by the ethical committee in Al-Ahliyya Amman University.
Induction of hepatotoxicity
A single dose of CCl4 (1 mL/kg, i.p.) was chosen according to Kandil et al. [23]. Diluted CCl4 solution was prepared by dissolving CCl4 in olive oil (1:1) to prevent its evaporation. On the last day of the designated period, animals were overnight fasted before the injection with diluted CCl4 (groups of B, C, and D) or olive oil (2 mL/kg, group A). One hour later, they were provided with food. On the next day, animals were fasted for 4 h, lightly anesthetized then sacrificed by cervical dislocation after taking the blood samples.
Blood samples
Twenty-four hours after CCl4 injection, blood samples were withdrawn by heparinized capillary tubes from a retro-orbital vein under light anesthesia using a piece of cotton immersed in diethyl-ether [24], allowed to clot for 30 min, sera were separated by centrifugation at RCF 1000×g for 10 min. Four aliquots were prepared from each serum and stored at −20 °C until analysis.
Liver tissue specimens
After blood samples collection, animals were sacrificed by cervical dislocation, livers were taken using histological scissors, rinsed with cold saline, dried on a filter paper, and photographed. A portion of each liver was excised, put in 10% formalin solution, and processed as for routine histological evaluation. The remaining part of each liver was stored at −80 °C for later oxidative stress analyses [23].
Liver tissue homogenization
Around 50 mg sample was excised from each liver and homogenized in 1 ml of cell lysis buffer using Teflon homogenizer in ice. The lysate was then cold-centrifuged at RCF 10,000×g for 15 min at 4 °C. Supernatants were distributed into four Eppendorf tubes and stored at −80 °C to be analyzed later.
Histological investigation
Five-micrometer sections were stained with hematoxylin-eosin, examined using a light microscope (Leica). and photographed using MC 170 HD Leica Camera (Switzerland) and LAS EZ software. The histological sections were investigated by 2 of the authors in a blinded fashion.
Serum parameters
Serum ALT, AST, ALP, total bilirubin, and total MMP-2 were analyzed 24 h after induction of hepatotoxicity according to manufacturer instructions.
Oxidative stress
Hepatic total protein was determined in the tissue homogenate according to Lowry method [25], total GSH was assayed according to Eyer et al. [26] method. Briefly, the clear supernatant obtained from the homogenate was first deproteinized using 5% MPA then Ellman’s reagent (5,5′-dithiobis-2-nitrobenzoic acid) was added which is reduced by sulfhydryl group of GSH to yield a yellow color with a maximum absorbance at 405-412 nm. The concentration was expressed as nM/mg tissue.
Superoxide dismutase activity was analyzed according to Spits and Oberley [27] utilizing a tetrazolium salt for the detection of superoxide radicals generated by xanthine oxidase and hypoxanthine. One unit of SOD is defined as the amount of enzyme needed to exhibit 50% dismutation of the superoxide radical.
Glutathione reductase activity was measured by measuring the rate of NADPH oxidation which is accompanied by a decrease in absorbance at 340 nm [28].
Statistical analyses
All descriptive statistics, analyses, and graphics were performed using GraphPad Prism version 6 (GraphPad Software, San Diego. USA). Data passed the Shapiro-Wilk normality test and were expressed in tables as mean, standard deviation, and standard error of the mean. One-way analysis of variance (ANOVA) followed by Tukey-Kramer post-analysis procedure was used to compare the means of all groups. Differences between means were considered statistically significant at P ≤ 0.05.
