Chemicals
cis-Diammineplatinum (II) dichloride (product number: 479306, purity ≥ 99.9%), curcumin from Curcuma longa (product number: c1386, purity ≥ 65%), α-tocopherol (product number: 258024, purity ≥ 95.5%), Bradford assay kits and 10% neutral buffered formalin solution were purchased from Sigma-Aldrich Chemical Company (St. Louis, MO, USA). Thiobarbituric Acid Reactive Substances (TBARS), SOD and catalase assay kits were obtained from Cell Biolabs, Inc (San Diego, CA, USA). RNeasy mini kit, Omniscript RT kit and HotStar Taq DNA polymerase were obtained from Qiagen (Hilden, Germany). All other chemicals were of analytical grade.
Animals
Twenty-five male Wistar rats (Rattus norvegicus), ranking from 180-200 g, were obtained from the Division of Animal House, Faculty of Science, Prince of Songkla University, Thailand. All animal procedures were reviewed and approved by the Animal Ethics Committee, Walailak University (Protocol number: 004/2012) and were conducted according to the Guide for the Care and Use of Laboratory Animals, National Research Council. Rats were maintained in stainless-steel cages under constant conditions of temperature (23 ± 2°C), relative humidity (50-60%) and lighting (12 h light/dark cycles). Animals were provided with a standard commercial rat diet and distilled water. Animals were acclimatized and closely monitored under laboratory conditions for 2 weeks before the commencement of the experiment.
Experimental design and specimen collection
Wistar rats were divided into five groups (5 rats per group). Saline group, rats were treated with a single intraperitoneal (i.p.) injection of 1 ml of normal saline. Cis group, rats were injected with a single dose of cisplatin (20 mg/kg b.w.) via i.p. route. Cis + α-tocopherol group, rats were treated with a single dose of α-tocopherol (250 mg/kg b.w.); Cis + Cur group, rats were treated with a single dose of curcumin (200 mg/kg b.w.); and Cis + α-tocopherol + Cur group, rats were treated with a single dose of combined α-tocopherol (250 mg/kg b.w.) and curcumin (200 mg/kg b.w.), via i.p. route for 24 h prior a single dose injection of cisplatin (20 mg/kg b.w.) [16]. Following 72 h of first injection, rats were anesthetized with thiopental sodium intraperitoneally (50 mg/kg b.w.). The peripheral blood from heart was collected in clot activator tubes. Then, rats were euthanized by anesthetizing thiopental sodium overdose (100 mg/kg b.w.). After opening the abdominal cavity, the liver was harvested and immediately washed in ice-cold isotonic saline.
Determination of serum AST and ALT levels
Blood samples were centrifuged at 3000 rpm for 5 min. Sera were collected and the levels of serum ALT and AST were measured using a Cobas Mira Plus CC Chemistry Analyzer (Switzerland).
Determination of liver MDA levels
The liver tissue was homogenized to give a final concentration of 50 mg/mL in phosphate buffered saline (PBS) containing 1X butylated hydroxytoluene (BHT), homogenized on ice and centrifuged at 10000 × g for 5 min to collect supernatant. In accordance with the protocol of an OxiSelectTM TBARS Assay Kit (Cell Biolabs, Cat No.: STA-330, San Diego, CA, USA), 100 μL of samples or MDA standard was added to separate microcentrifuge tubes, and then 100 μL of the SDS lysis solution was added and mixed thoroughly. The samples were then incubated for 5 min at room temperature, 250 μL of thiobarbituric acid (TBA) reagent was added, and then each tube was closed and incubated at 95°C for 60 min. The tubes were then removed and cooled to room temperature in an ice bath for 5 min. All sample tubes were then centrifuged at 3000 rpm for 15 min, the supernatant was removed from the samples, and then finally 200 μL of samples and MDA standard was transferred to a 96-well microplate compatible with a spectrophotometric plate reader. The absorbance was read at 532 nm.
Determination of liver SOD activity
The liver tissue was homogenized to give a final concentration of 50 mg/ml in cold 1X lysis buffer (containing 10 mM Tris, pH 7.5, 150 mM NaCl and 0.1 mM EDTA), centrifuged at 12000 × g for 10 min and the supernatant was collected for analysis. In accordance with the protocol of an OxiSelect™ SOD Activity Assay Kit (Cell Biolabs, Cat No.: STA-340, San Diego, CA, USA), 20 μL of samples, 5 μL of xanthine solution, 5 μL of chromagen solution, 5 μL of 10X SOD assay buffer and 50 μL of deionized water were added (total volume of 90 μL) to a 96-well microplate, and then 10 μL of pre-diluted 1X xanthine oxidase solution was added to each well. The samples were then mixed well and incubated for 1 h at 37°C. The absorbance was read using the spectrophotometric microplate reader at 490 nm.
Determination of liver catalase activity
The liver tissue was homogenized to give a final concentration of 50 mg/ml in cold PBS with 1mM EDTA, centrifuged at 10000 × g for 15 min at 4°C and the supernatant was collected. In accordance with the protocol of an OxiSelectTM Catalase Activity Assay Kit (Cell Biolabs, Cat No: STA-341, San Diego, CA, USA), 20 μl of the diluted catalase standard or sample and 50 μl of the H2O2 working solution (12 mM) were added to a 96 well microplate. The samples were then mixed well and incubated for 1 min. The reaction was stopped by adding 50 μl of the catalase quencher into each well and mixed thoroughly, 5 μl of each reaction well was transferred to a fresh well, 250 μl of the chromogenic working solution was added to each well. The samples were then mixed well and incubated for 60 min. The absorbance was read using the spectrophotometric microplate reader at 520 nm.
Protein determination
Protein content was estimated by Bradford assay (Sigma, USA) using bovine serum albumin (BSA) as standard.
Liver histology
The liver tissue was preserved in 10% neutral buffered formalin solution for 24 h and washed with 70% ethanol. Tissue was then placed in small metal caskets, stirred by a magnetic stirrer, dehydrated using alcohol series from 70% to 100% alcohol and embedded in paraffin using an embedding machine. Paraffin block was sectioned using a rotary ultra-microtome, distributed onto glass slides and then dried overnight. Slide was observed under a light microscope after being stained with hematoxylin and eosin (H&E) dyes and mounted.
Liver NADPH oxidase gene expression by reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was extracted from the liver tissue by RNeasy mini kit. RNA content and purity were measured by a UV spectrophotometer. RT-PCR was done using the extracted RNA for detection of NADPH oxidase gene. For amplification of the targets gene, reverse transcription and PCR were run in two separate steps. Briefly, Reaction mixture of RT reaction containing 1 μg total RNA, 0.5 μg random primer, 5 × RT buffer, 2.5 mmol/l dNTP, 20 U RNase inhibitor and 200 U MMLV reverse transcriptase in a total volume of 25 μl was incubated at 37°C for 60 min, then heated to 95°C for 5 min to inactivate MMLV. PCR was carried out with 1.5 μl RT products, 10 × PCR buffer (without Mg2+) 2.5 μl, 2.0 μl dNTP (2.5 mmol/l), 2.0 μl MgCl2 (25 mmol/l), 0.5 μl each primer (20 μmol/l) of β-actin, 0.5 μl each primer of gene to be tested (20 μmol/l) and 1 U of Taq DNA polymerase, in a final volume of 25 μl. Thermal cycler conditions were as follows: a first denaturing cycle at 97°C for 5 min, followed by a variable number of cycles of amplification defined by denaturation at 96°C for 1.5 min, annealing for 1.5 min and extension at 72°C for 3 min. A final extension cycle of 72°C for 15 min was included [17]. The primers including:
NADPH oxidase: Forward primer: 5’-GGAAATAGAAAGTTGACTGGCCC -3’
Reverse primer: 5’-GTATGAGTGCCATCCAGAGCAG-3’
Beta actin: Forward primer: 5’TGTTGTCCCTGTATGCCTCT-3’
Reverse primer: 5’-TAATGTCACGCACGATTTCC-3’
The sample was equally loaded on 2% gel agarose, stained with ethidium bromide and visualized by UV transilluminator. The amount of PCR product was quantified using the gel and image analysis software (Syngene, USA).
Statistical analysis
Results were expressed as mean ± standard error of the mean (S.E.M.). Differences between groups were determined by one-way analysis of variance (ANOVA). Post hoc testing was performed for group comparisons using the Least Significant Difference (LSD) test and p < 0.05 was considered significant.