3.1 Experimental Animal
A total of thirty (30), Wistar rats (200 ± 5.00 g) were obtained from the Animal Holding Unit of the Department of Biochemistry, University of Ilorin, Ilorin, Nigeria. The rats were housed in clean cages and well-ventilated house conditions (Temperature: 28.30ºC), (Photo-period; 12hr light, 12hr dark), (Humidity: 45-55%). They were fed with pelletized rats feed (Vital Feed, Grand Cereals, Jos, Nigeria) and clean tap water which were provided before and during administration.
3.2 Exposure of Rats to Radiation and Induction of Cellular Alteration in Rats
Experimental rats were exposed to single dose of 6 grey whole-body gamma radiation by the method described by [28] and modified by. [34] The rats were kept in an improvised carton cage with holes made round the carton for ventilation (carton was used to restricts the movement and ensure uniform exposure of the rats to radiation) instead of the usual radiation exposure coat that was used by. [28]
3.3 Drugs, Chemicals and Assay Kits
Nuclear Factor Kappa-B (Nf-kB) and nuclear related factor-2 (Nrf-2) enzyme linked immunoassay (ELISA) kits were products of Cayman Laboratory Limited, United State of America (USA). All chemicals used were all prepared in all-glass distilled water using standard procedures.
3.4 Preparation Catechin, Epicatechin and Quercetin Solution
The 40 mg/kg body weight solutions of catechin, epicatechin and quercetin were prepared by the method described by [33].
3.5. Table 1: Animal groupings and Administration
Groups
|
Administration
|
Group 1 (Positive)
|
Non-irradiated (NIR)
|
Group 2 (Negative)
|
Irradiated (IR)
|
Group 3 (IR +CAT)
|
IR + 40 mg/kg bwt Catechin
|
Group 4 (IR + EPC)
|
IR + 40 mg/kg bwt Epicatechin
|
Group 5 (IR + QCT)
|
IR + 40 mg/kg bwt Quercetin
|
3.6 .Animal sacrifice and nuclear extract preparation
The rats in Table 1 were sacrificed by cervical dislocation, they were dissected and tissue (liver) were isolated. The 1.14 % KCL washing solution was used to rinse the liver off blood and other stains. About 1 g of the liver were homogenized in ice cold 0.25 M sucrose solution. The homogenate were cold-centrifuged at 12000 rpm. The supernatant were discarded and sediment were retained for the preparation of nuclear extract.
3.7 Preparation of nuclear extract
The nuclear extract (from which Nf-kB and Nrf-2 were produced) was prepared by the method described by [31].
3.8 Determination of Nf-kB and Nrf-2 Status of Irradiated Rats administered with Catechin, epicatechin and quercetin
Nf-kB and Nrf-2 status were described by the method adopted by [35]
3.9. Table 2: Determination of the Antioxidant Enzymes Activities of Irradiated Rats, administered CAT, EPC and QCT
Antioxidant Enzymes
|
Procedures
|
Superoxide Dismutase (SOD)
|
According to method adopted by Haliwell et al. (2021)
|
Catalase (CAT)
|
According to method adopted by Yhong-sur (1989)
|
Glutathione Transferase (GST)
|
According to method adopted by Abolaji (2015)
|
Glutathione Peroxidase (GPx)
|
According to method adopted by Lazekhs et al. (1982b)
|
Malondialdehyde (MDA)
|
According to method adopted by Rice –Evans et al. (1986)
|
3.9.1 Ethical Approval
Ethical approval was obtained from the University of Ilorin Ethical Committee, University of Ilorin. With the ethical approval number obtained as follow UERC/ASN/2018/1409 on the 13th September, 2022. The study was conducted following the guidelines on the care and use of laboratory animals.
3.9 Statistical Analysis
Data were expressed as mean ± SEM of 6 determinations except otherwise stated. All results were statistically analyzed using Duncan Multiple Range Test and complemented with Student’s t-test. Statistical Package for Social Sciences, version 21 (SPSS Inc., Chicago, IL, USA) were used for statistical analyses. Statistical significance was set at 95% confidence interval (Mahajan, 1997).