Thiobarbituric acid (TBA), trichloroacetic acid (TCA), n-butanol, hexadecyltrimethyl ammonium bromide (HETAB), tri (2-pyridyl)-s-triazine (TPTZ), HCl, malondialdehyde (MDA), ferric chloride (FeCL3-6H2O), D-galactose, and vitamin E (Trolox) were purchased from Merck chemical Co. (Germany). Rat specific tumor necrosis factor-α (TNF-α), interlukine-1β (IL-β), interlukine-6 (IL-6), Nuclear Factor-kappaB (NF-κb) ELISA kits were purchased from BenderMed Systems Inc. (Austria). Testosterone and dehydroepiandrosterone ELISA kits were purchased from Dia Metra (Italy). IMOD and Angipars were obtained from Parsrus Research Group (Iran).
Male BALB/c mice (3 months old, 18–22 g) were provided from Tehran University of Medical Sciences (TUMS) animal house. The animals were housed in standard polypropylene cages with wired-net top in a controlled room (temperature 23 ± 1°C, humidity 55 ± 10%, 12-h light–dark cycle and were allowed free access to standard laboratory pellet diet and water during the experiments. All ethical issues on the use of animals were carefully considered and the study protocol was approved by TUMS review board with code number of 90-03-33-15668.
Before starting the main study, a pilot was designed to set up aging model and to get proper doses of treatments. In the main study, fifty mice were randomly divided into five groups, each consisting of 10 animals. D-galactose was dissolved in a measured quantity of mice drinking water. D-galactose was given to four out of five groups of animals at 500 mg/kg D-galactose per 10 ml drinking water for 6 weeks[13, 14]. The fifth group of animals was the sham group which was not given D-galactose. After 2 weeks, the four groups which had been given D-galactose were randomly divided into aging control group (500 mg/kg D-galactose per 10 ml drinking water, for 4 weeks), positive control group (500 mg/kg D-galactose per 10 ml drinking water plus vitamin E 200 mg/kg/day intraperitoneally for 4 weeks) and IMOD treatment group (500 mg/kg D-galactose per 10 ml drinking water plus IMOD 20 mg/kg/day intraperitoneally for 4 weeks), and Angipars treatment group (500 mg/kg D-galactose per 10 ml drinking water plus Angipars 2.1 mg/kg/day by gavage for 4 weeks).
Twenty-four hours after the last drug administration, blood samples were taken of each animal under anesthesia by cardiac puncture. Serum samples were obtained by centrifuging the whole blood at 1000 × g at 4°C for 10 minutes and the supernatants were transferred into several microtubes for separate biochemical assays and maintained at −80°C until the analyses were performed. Biochemical markers including TNF-α, IL-β, IL-6, NF-κb, ferric reducing/total antioxidant power (TAP), lipid peroxidation (LPO) marker and male sex hormones including testosterone and dehydroepiandrosterone-sulfate (DHEA-S) were measured in the serum.
Measurement of LPO in serum of D-galactose-induced aged mice
LPO was measured by the reaction of TBA with MDA and other lipid peroxides. Samples were mixed with TCA (20%) and the precipitate was dispersed in H2SO4 (0.05 M). After addition of TBA (0.2% in sodium sulfate), the sample was heated for 30 min in a boiling water bath. Then LPO adducts were extracted by n-butanol and absorbance was measured at 532 nm as described in details in our previous work. Data were expressed as nM.
Measurement of TNF-α, IL-1β, IL-6 and NF-κb levels in serum of D-galactose-induced aged mice
Quantitative detection of TNF-α, IL-1β, IL-6 and NF-κb levels in serum were performed using an enzyme-linked immunosorbaent assay rat specific ELISA kit. The absorbance of the final colored product was measured in 450 nm as the primary wave length and 620 nm as the reference wave length. TNF-α, IL-1β, IL-6 and NF-κb levels were expressed as pg/mg.
Measurement of TAP in the serum of D-galactose-induced aging mice
Serum TAP was evaluated by measuring the ability to reduce Fe3+ to Fe2+. Interaction of TPTZ with Fe2+ results in formation of a blue color with a maximum absorbance at 593. The whole procedure has been described in our previous study. Data were expressed as mM.
Measurement of testosterone and DHEA-S in the serum of D-galactose-induced aged mice
For determination of testosterone and DHEA-S, we used ELISA kits and did as instructed by the kit brochure. Testosterone and DHEA-S levels were expressed as ng/ml.
Results are expressed as mean ± standard error of the mean (SEM). Data were analyzed by one-way ANOVA followed by Tukey post-hoc test for multiple comparisons to ensure the variances of the data are distributed properly. A p-value less than 0.05 was considered significant.