GA was purchased from Sigma-Aldrich Co (Germany), D-GAL, was obtained from Sigma-Aldrich Co (Germany), and Pentobarbital sodium salt was purchased from Sigma-Aldrich Co (St. Louis, MO, USA).
Animals
The rats were housed in standard cages (12 h light/dark cycle, 25° C ± 2 °C). Moreover, the animals had availability to water and food ad libitum and were handled to minimize the stress of substance administration during the overall experimental term. All experimental protocols were carried out through ethics by the Animal Experiment Committee performed by the Guide for the Care and Use of Laboratory Animals (Ethics Committee permission No. IR.UMSHA.REC.1400.270).
Experimental design
After seven days of acclimatization, male Wistar rats (age: 3 month old; body weight: 300 ±20 g) were randomly divided into four experimental groups (n = 8 in each group):
1. Untreated Control (CONT)
2. Control treated with GA at 25 mg/kg (CONT+GA)
3. Aged rats induced by D-GAL (D-GAL)
4. Aged rats treated with GA at 25 mg/kg (D-GAL + GA)
In animals, aging was induced via D-GAL at 150 mg/kg via intraperitoneally (IP) injection once daily for eight weeks [21]. The animals were administrated with GA (25 mg/kg) orally by gavage co-administrated with D-GAL for eight weeks. Sodium pentobarbital at 60 mg/kg was IP injected into rats after the last administration of D-GAL and GA to anesthetize rats. Blood was immediately collected from the abdominal aorta and serum was separated by centrifugation. Then serum was stored at − 20◦C for various biochemical evaluations [18].
Biochemical evaluations in serum
The cardiac injury was measured through creatine kinase (CK-MB) and lactate dehydrogenase (LDH) evaluations in the serum. The LDH (Pars Azmoon) and CK-MB (Pars Azmoon) amounts were determined by kits. The results were expressed as U/L.
Malondialdehyde and Superoxide dismutase in the heart
Heart tissue was stored at− 20◦C for various biochemical analysis. Following homogenization, the cardiac content of Malondialdehyde (MDA) and Superoxide dismutase (SOD) were measured to detect the amounts of intracellular oxidative stress through a commercial kit (Kiazist, Iran) according to the manufacturer's instructions.
Cardiac hypertrophy index assessment
After heart removing, washed with saline, dried by a cleaned cloth, and then weighed. Cardiac hypertrophy index was considered as the ratio of the Heart Weight (HW)/ Body Weight (BW)(g/g) by the following formula: Cardiac hypertrophy index= Heart Weight(g)/Body Weight (g) × (100)[22].
Histopathological examination of the heart
For structural evaluation, the heart tissues were removed from the animals and were fixed in 10% neutral buffered formalin. The heart tissues were sectioned (4 μm slices) and stained with hematoxylin and eosine (H&E). The samples were surveyed histopathologically through photomicroscope [23].
Gene expression levels of SIRT1, PGC-1α, and TFAM in the heart tissue by real-time PCR
About 30 mg of sample tissues from the apex of hearts were removed and homogenized and total RNA was isolated with Kiazol reagent according to the manufacturer's instructions. Then, Nano drop spectrometer was used to determine the purity and concentration of total RNA at 260 and 280 nm, and optical density amounts were measured. Then,1 μg of total RNA was reverse transcribed using a cDNA synthesis kit in gradient thermal cycler according to the manufacturer's instructions. Real time PCR was performed using (LightCycler96, Germany). Finally, the relative expression of genes was analyzed through the 2-ΔΔCT method. The relative levels of SIRT1, PGC-1α, and TFAM mRNAs were normalized with beta-actin mRNA level as an internal control.
Statistical analysis
Findings were expressed as means ± standard error (SE). Comparisons were done through analysis of variance (ANOVA) followed by post hoc Tukey's test. P value of <0.05 was regarded statistically significant.