Thymoqunione (TQ) was purchased from Sigma-Aldrich (St. Louis, MO, USA). BCA mega kit (Pierce, Rockford, USA). All assay kits were obtained from BIORAD (England). Primers were obtained from Bioline Inc., (Taunton, MA, USA). Monoclonal antibody, anti- NFkBp were obtained from Bioline Inc. All the chemicals used in the study were of analytical grade.
Male Wister rats weighting 190 ± 30 g were kept for 7 days with free access to water ad libitum and standard rat chow.
All the animal protocol was approved from the University Animal Ethics committee. CEGMR committee licensee # at KASCT. HA-02 J-003.
Rats were randomly divided into three groups each consists of 15 rats. Group 1 served as the normal control group. Animals of this group were treated with normal saline intrapertoneal (ip). Group 2 was injected i.p. with a single dose of CP (12 mg/kg b.w, i.p.) obtained from Sigma (England). Group 3 was orally pre-treated with TQ in polypropylene glycol (500 mg. kg−1. day−1)  for one month, then the animals were injected i.p. with CP 12 mg.kg−1 body weight . TQ treatment was continued for one month. At the end of the experiment, rats were sacrificed, the blood was collected, and serum was separated and stored at −80°C. Serum was used for the determination of serum lactate dehydrogenase (LDH), alanine amino transferase (ALT), γGGT, aspartate amino transferase (AST), total protein (t. Protein) alkaline phosphatase (ALP), albumin, and total bilirubin.
Liver was isolated and rinsed carefully with cold normal saline and divided into four parts. The first part was used to prepare the tissue homogenate in phosphate buffered saline (50 mmol/l, pH 7). The liver homogenate was used for the assay of the activity of superoxide dismutase (SOD), catalase, glutathione peroxidase (GSH-Px), glutathione S transferase (GST), and the levels of reduced glutathione (GSH), malondialdehyde (MDA), NO, IL-1β and TNFα. The second part of the liver was used in the preparation of the tissue extract for electromobility shift assay (EMSA) analysis. Total RNA was extracted from the third part of the liver and the fourth part was used for histopathological examinations.
Assay of ALT, AST, ALP, t. protein, albumin, t. bilirubin, and γGGT
ALT, ALP, AST, albumin, t. protein, γGGT and t. bilirubin in the serum of all rats were assayed using the available kits obtained from Bio Vision Research Products (Avenue, USA). All assays were made according to the instructions of the manufacturer.
Determination of LDH
The method of King  was used for the assay of LDH using kit from BIORAD (England).
Assay of reduced GSH
Reduced GSH was assayed by the colorimetric end point method as described by Sayed  using kit from BIORAD (England).
Determination of the activity of glutathione peroxidase
The method of Paglia and Valentine  was used for the determination of the activity of glutathione peroxidase in the liver homogenate using kit from BIORAD (England). The activity was expressed as U/mg protein.
Assay of GST activity
The activity of GST was determined as described by Moron et al. by using 1-chloro −2, 4- dinitro benzene (CDNB) as an electrophilic substrate using kit from BIORAD (England). The activity was expressed as nmol/mg protein.
Assay of malondialdehyde (MDA)
Malondialdehyde (MDA) was assayed using the method described [20, 21]. The concentration of MDA was calculated and expressed as nmol . mg−1 protein.
Assay of the activity of SOD
The activity of super oxide dismutase was assayed as described  using kit from BIORAD (England). The activity was expressed as U/mg protein.
Assay of the nitric oxide concentration
The formation of NO was determined by indirect method by quantifying the tissue level of nitrite by a calorimetric method by Griess reaction  using kit from BIORAD (England).
Assay of IL-1β and TNF-α
IL-1β and TNF-α levels were assayed using an available ELISA kit from R & D (Berlin, Germany) according to the manufacturer’s instructions . Protein concentration in the liver homogenate was assayed using BCA mega kit (Pierce, Rockford, USA).
The transcripts were amplified in a single reaction containing 1 mg cDNA and 0.5 mM each of the sense and antisense primers for IL-1β ( sense 5′- AT GG CA AC TG TC CC TG AA CT C -3′; antisense 5′- GT CG TT GC TT GT CT CT CC TTG -3′ ), TNFα sense 5′- TCATGCACCACCATCAAGGA-3′ and antisense 5′- GA CA TT CG AG GC TC CA GT GA A -3′ and iNOS sense 5′-CA GC CA AG TA TT CC AA AG CA A −3′ and antisense 5′- AG TC CA GT CC CC TC AC CA A −3′; β- actin sens 5′ GT GC TA TG TT GC TC TA GA CT TC G −3′, antisens 5′ AT GC CA CA GG AT TC CA TA CC −3′ were obtained from BIOLINE Inc., (Taunton, MA, USA).
The cycle consistes of preheating step at 95°C for 5 minute followed by 35 cycles of 90°C for 1 minute, and 60°C for 30 seconds.
Part of the liver was dissolved in TOTEX buffer. The extract was used for determination of the binding activity using NF-κBp65 consensus sequence: 5′-AG TT GA GG GG AC TT TC CC AG GC -3′ which was obtained from BIOLINE Inc. (Taunton, MA, USA). The increase in the expression of NFkBp65 gene was determined by a Phosphor Imager using background subtraction .
Liver tissues were collected from rats, washed carefully by cold normal saline 3 times, then fixed in formalin solution 10%, processed, and embedded in a paraffin film. Sections of 5-μm thick slices of liver were prepared. The sections were stained with H & E. The method was described in Fukuzawa et al. .
The sections of tissues in phosphate-buffered saline (pH = 7.2) were incubated overnight at 4°C with the respective primary monoclonal antibody, and anti- NFkBp dilutions (1:100, 1:50). Immunohistochemical staining was performed by the streptavidin-biotin complex method. All sections were then counterstained with hematoxylin.
All values are expressed as the mean ± SD. Data were evaluated by using SPSS for windows. The one way ANOVA test was used to examine whether there are any significant differences between the treatment groups, and the value of P < 0.05 was considered significant.