Cypermethrin was purchased from arid agriculture center Kohat. Rhizomes were obtained from Curcuma longa L. plant which was purchased from the local farmers field of Bannu, Khyber Pakhtunkhwa, Pakistan. Crude ethanolic extract of Curcuma longa rhizomes was used as CMN. The plant material was identified by taxonomist Dr. Mushtaq Ahmad, Dr. Muhammad Zafar at Herbarium of Pakistan (ISL) Quaid-I-Azam University Islamabad and voucher specimen has not submitted in publicly available herbarium. The enzymes activities of aminotransferases (AST & ALT), alkaline phosphatase (ALP) and the level of urea and creatinine were analyzed by kits that were purchased from med-lab chemicals Islamabad. All the chemicals were of analytical grade. The entire chemicals were dissolved in DMSO and for control group only DMSO was used.
Animals and experimental design
20 white male albino rabbits were used in the present study which were purchased from National Institute of Health (NIH), Islamabad, Pakistan. The selection of male rabbits was according to already published studies. Moreover, to omit the effect of “sex as a biological variable (SABV)” on the study, only one sex is included in the whole study. The title specified the sex of animals (male) as per “Sex And Gender Equity in Research (SAGER)” guidelines . Animals were provided standard environmental and food conditions accordingly. Rabbits were divided into control (received no treatment), CYP-treated, CMN-treated, and combination of both CMN + CYP-treated group. Rabbits were orally administered their respective doses every day for 45 days. There was 1 h difference between the administration of CYP and CMN. The dose selection of all compounds was based on previous published studies with some modifications [2, 3, 14].
CYP-induced rabbit model, animals were divided into four groups and every group with five rabbits:
Group I: Normal control animals
Group II: CYP-induced group (25 mg/kg b.w)
Group III: CMN- treated group (50 mg/kg b.w)
Group IV: CYP- (25 mg/kg b.w) + CMN-treated group (50 mg/kg b.w)
Sampling and tissue preparation
Rabbits were euthanized by a trained technician through cervical dislocation. Blood samples were obtained from the sacrificed rabbits and retained instantly on ice. Heparin was used as an anticoagulant and plasma samples were gained by centrifugation at 1000 rpm for 15 min and stored at -40◦C. Biochemical parameters were investigated in serum while the activity of endogenous antioxidants was assayed in liver and kidney tissues. These tissues were instantly separated and washed with chilled saline solution. The tissues were then distinctly homogenized (10%w/v) in ice cold phosphate buffer (0.01 M, Ph. 7.4) containing 1.5% KCL using homogenizer. The homogenate was centrifuged at 10,000 rpm/20mint at 4С0 and the resultant supernatant was used for the enzyme assay. Histopathological examinations of kidney and liver were performed by H&E (hematoxylin and eosin) staining [2, 3, 15].
Determination of liver and kidney function
The effect of CMN on sensitive biomarkers of liver and kidney injury such as ALT, AST, ALP, bilirubin, urea and creatinine were investigated respectively by using commercially available kits as described previously [8, 15, 16]. Briefly, the blood samples collected at the end of experiment from all the animals and serum was separated after 10 min of centrifugation at 4oc/6000 rpm for analysis as per manufacturer’s protocol.
Determination of antioxidant activities
Estimations of numerous oxidative stress-related biochemical markers were carried out in homogenized liver and kidney tissues as per previous methodology [15, 16]. In brief, liver tissues were homogenized at 4oc/12000 rpm for 30 min and the resultant supernatant was used for estimation of various antioxidant enzymes and proteins. GSH (glutathione) content was evaluated by the method of Ellman et al. [15, 17]. GST (glutathione S transferase) activity was analyzed by using a method established by Warholm et al [17, 18] . CAT (Catalase) activity was assayed by the procedure as described by Aebi et al. [15, 17]. The activity of SOD (superoxide dismutase) was measured as per the method of Sundaram et al. [16, 19]. GPx (Glutathione peroxidase) activity was determined by the method of Necheles et al. [20, 21]. The presence of these antioxidant enzymes and proteins were identified by monitoring the changes in absorbance at specific wavelength using spectrophotometer.
Estimation of lipid peroxidation (malondialdehyde)
Lipid peroxidation (LPO) was estimated in terms of malondialdehyde (MDA) production by the procedure narrated by Utley et al. [16, 22]. The quantity of thiobarbituric acid reactive substances (TBARS) was determined by recording the absorbance of the supernatant at 532 nm by using a microplate reader.
Lipid profile analysis
Total lipids (TL), cholesterol, triglycerides (TG), high-density lipoprotein (HDL), low-density lipoprotein cholesterol (LDL), and very low-density lipid protein (VLDL) were estimated in plasma by the methods as described previously [3, 23, 24].
To notice any changes in total blood count associated with CYP or CMN, blood was collected in anti-coagulated EDTA blood tubes from each rabbit. The non-coagulated blood was then tested for total erythrocyte count (TEC), total leukocyte counts (TLC), packed cells volume (PCV) and hemoglobin (Hb) according to previously described procedures .
For histopathological examination, the kidney and liver of rabbits were dissected. Next, these tissues were embedded in paraffin after routine fixation (in 10 % formalin), decalcification and dehydration (in alcohol). Tissue sections of kidney and liver were prepared at 4-5 μm thickness and stained with H&E after which examined with the help of light microscope as per formerly described methodology for any histopathology appeared during the experimentation period. The histological changes were classified by using various symbols such as (-) showing no changes and (+) showing changes [8, 15].
Determination of body weight variations
Body weights of rabbits were measured initially (day 0) and at the end (day 45) of
experimentation to assess any changes in weight among the groups. The changes were described as a percentage of average weight gain in all the groups as described by Navarro-Alvarez N et al. [15, 25].
Data collected was presented as mean ± SD. Groups were compared for statistical significances by one way ANOVA followed by pairwise comparison. R studio version 3.6.1 was used for analysis of data. Statistical significance was fixed at p < 0.05.