Drug and Chemicals
Cyclophosphamide purchased from Roche chemical company (Grenzach, Germany). Berberine, reduced glutathione, tris–hydrochloric acid, bovine serum albumin (BSA), 2,2′-dinitro-5,5′-dithiodibenzoic acid (DTNB), oxidized glutathione, trichloroacetic acid (TCA), and 2-thiobarbituric acid (TBA) obtained from Sigma St. Louis.
Animal and Study Design
Forty male NMRI mice (25±2 g) were purchased from the Experimental Animal Center Laboratory of Ahvaz Jundishapur University of Medical Sciences. The mice were kept in a room with 25 ± 2°C temperature in polycarbonate cages with a 12/12 cycle. In addition, food and water were available without any limitation. The investigation complies with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication no. 85-23, revised 1996). The Animal Ethics Committee of the Ahvaz Jundishapur University of Medical Sciences approved our research protocol (Ethic code: IR.BHN.REC.1397.033).
Five experimental groups consisting of 8 randomly selected mice each were established:
Control group (I): 0.1 ml of normal saline (vehicle of BBR, p.o.) was administered for a week and one dose of 0.9% normal saline was injected (vehicle of CP, i.p.) on 7th day.
CP group (II): 0.1 ml of normal saline was administered for a week and one dose of CP (200mg/kg, i.p.) was injected on the 7h day.
Group III (CP + BBR 50): BBR (50 mg/kg, p.o.) was administered for a week and one dose of CP (200 mg/kg, i.p.) was injected on the 7th day.
Group IV (CP + BBR 100): BBR (100 mg/kg, p.o.) was administered for a week and one dose of CP (200 mg/kg, i.p.) was injected on the 7th day.
Group V (BBR group): BBR (100 mg/kg, p.o.) was administered for a week and one dose of normal saline 0.9% was injected on the 7th day.
The dose of CP and BBR was selected based on our previous studies and other studies(El-Naggar et al., 2015; Goudarzi et al., 2017). On 8th day, ketamine/xylazine (60/6 mg/kg, i.p) was used for performing anesthetized and then were sacrificed by fast decapitation. The retro-orbital sinus was selected for taking blood samples. All blood samples were centrifuged at 3000g for 10 min and kept at −20 °C until investigations. After removing and washing the kidneys with normal saline, the right kidney was fixed in 10% phosphate-buffer formalin for histological analysis. The left kidney was used for biochemical assays
Tissue homogenization and protein measurement
The left kidney weighed and homogenized (1/ 10 w/v) with a tissue homogenizer (Wisetis HG-150) for 60 minutes in ice-cold phosphate-buffered saline (PBS) solution (10 mM Na2HPO4, 10 mM KH2PO4, 0.9 g NaCl/100 mL, pH 7.4) and the clear supernatant was kept in -70 °C for further investigations. The total protein content in the clear supernatant of the kidney was estimated following the technique of Bradford(Bradford, 1976).
Analysis of serum biochemical parameters
The level of Serum biochemical marker blood urea nitrogen (BUN) and creatinine (Cr) were measured using the kits according to the protocol of the manufacturer (Pars Azmun. Co. Iran), and using an auto-analyzer (BT-TARGA-3000 model). BUN and Cr levels were expressed as mg/dL serum. Assessment of KIM-1 and NGAL levels were done using commercial kits (IBL-America (Immuno-Biological Laboratories).
Assay of MDA level
MDA in the kidney as a marker of lipid peroxidation was assayed via the TBA color reaction by Aust’s technique(Goudarzi et al., 2021; Moore and Roberts, 1998). MDA content was expressed as nmol/mg protein.
Assay of GSH level
GSH in the kidney was measured by a modified version of Ellman’s technique following the observation of a yellow complex with Ellman’s (2, 2′-dinitro-5, 5′-dithiodibenzoic acid Reagent) (Kalantar et al., 2016; Riddles et al., 1979).
Assay of CAT activity
CAT function in the kidney was measured using Abebi’s method(Aebi, 1984) using H2O2 as a substrate. Briefly, H2O2 was mixed and decomposition rate of H2O2 was evaluated through estimating the absorbance alternations at 240 nm for 60 seconds. An unit of catalase (CAT) function was considered as 1 μM of H2O2 that is decomposed in 1 minute and the function of this enzyme was expressed as U/mg protein.
Assay of NO level
Griess’s technique was applied to measure the NO in the kidney along with Griess diazotization reaction following conversion of the nitrate to nitrite by nitrate reductase in the supernatant(Tracey et al., 1990) NO level was expressed as nmol/mg protein.
Assay of SOD and GPx activity
The function of SOD and GPx enzymes was measured by the kits following the protocol of the producer (ZellBio GmbH, Germany), and the function of these enzymes was expressed as U/mg protein.
TNF-α and IL-1β assay
levels of TNF-α and IL-1β cytokines in the kidney was evaluated using ELISA kits, following the guideline published by the producer (R&D Systems, Inc., Minneapolis, MN, USA).
Histopathological assays
In order to perform histological examination, left kidney from all groups was fixed in formalin (10%) for at least 24h and following preparing the tissue, all tissues were put into paraffin. Routine hematoxylin & eosin (H & E stain) were applied to stain 5 μm sections(Wadie et al., 2021). A light microscope in a blind manner (Nikon Labophot, Japan) was used to study six microscopy Slides per animal and Light microscopic assessment was conducted by authors in a blinded manner for assessment of histological alternations like congestion of RBC and inflammatory cell infiltration.
Statistical Analysis
The Graph Pad Prism version 6.01 (Graph Pad Software, USA) were used to conduct all statistical analyses. Parameters within groups were statistically analyzed using the ANOVA, followed by Tukey’s post hoc and P value less than .05 was considered significant. Results were reported as mean ± standard deviations (SD) of number of experiments (n = 8).