2.1. Chemicals and drugs, Protocatechuic acid (PCA) and APAP, were purchased from Sigma-Aldrich chemical company (St. Louis, MO, USA).and other commercial kits were purchased from Navand Salamat Company (Urmia, Iran).
2.2. Animals
48 male ICR mice (20–30 g) were divided into six groups (eight mice in each group) with 8–10 weeks’ old were purchased from the Animals Center of Ahwaz Jundishapur University of Medical Sciences, Iran. They were kept under standard conditions for acclimated with the situation for one-week prior being studied. All procedures were done under the NIH Guide for the Care and Use of Laboratory Animals (National Institutes of Health Publication No. 85–23, revised1985) and approved by the local ethics committee at Dezful University of Medical Sciences (Ethic code: IR.dums.REC.1397.042). The study was carried out in compliance with the ARRIVE guidelines.
2.3. Experimental design
The mice were randomly divided in to 6 groups (8 mice per group).
GroupI (normal control): received an equal volume of vehicle PCA by p.o. for 7days and a single dose of vehicle of A PAP by ip on the7th.
GroupII(APAP): received vehicle of APAP by ip for 6 days then a single dose of APAP (300mg/kg,ip) on the 7th, .
Groups III, IV and V (APAP+PCA): were treated 40, 80, and 160 mg/kg/day of PCA (po) for 7days and a single dose of APAP (300mg/kg/day,ip) on the 7th 1 h after the last dose PCA.
Group VI (PCA control): were received 160mg/kg/day of PCA (po) for 7days and a single dose of vehicle of APAP by,i.p) On 7 th 1 h after the last dose PCA.
All the animals were anaesthetized with Ketamine/ Xylazine and sacrificed after 24 h food deprivation then, their blood samples were collected for the assessment of activities of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) in the serums. Liver samples were rapidly dissected, weighed and divided into 2 parts: one part was fixed in formaldehyde saline (10%) for histopathological evaluation and second part of fresh liver tissue was retained at−20°C for biochemistry assay, including Malondialdehyde (MDA), glutathione (GSH), Catalase (CAT) activity and Superoxide dismutase (SOD) activity.
2.4. Biochemical assays
2.4.1. Serum assessments
Liver function tests activity (AST and ALT) was performed in the serum samples using the Reitman-Frankel and king's methods by an automation analyzer (Hitachi 911, Roche Diagnostics, Basel, Switzerland).
2.4.2. Tissue antioxidant assay
The Malondialdehyde (MDA) level and antioxidant enzyme activity (Gpx, CATand SOD) were evaluated in the homogenate tissue by spectrophotometry using Navand Salamat kit (Urmia, Iran)
2.5. Histological evaluations
In order to observe microscopic changes, the obtained Livers were fixed in 10% paraformaldehyde. Afterward, the tissues were dehydrated by alcohol and xylol and then embedded in paraffin. Thereafter, the 5μ- tissue sections were stained by hematoxylin and eosin (H&E) staining. Finally, the histopathological changes in liver tissue such as apoptotic cells, infiltration of lymphocytes, degeneration, and necrosis were reported by an expert pathologist.
2.6. Statistical analysis
All the data of the present research were expressed as mean ± S.E.M. The group variances were assessed using one-way analysis of variance (ANOVA) followed by performing Tukey’s post hoc analysis by the Graph pad prism 5.04 software package. P < 0.05 was considered as the statistical significance level.