4.1. Animal
Purchased C57bl/6j mice (male, 7 month-old, about 30 g) from Pengyue Experiment Animal Breeding Co. LTD. The mice were fed in the experimental animal house which a constant temperature of 23 ± 2 ℃, a 12 h dark/light cycle, and no restriction on access to a standard diet and water was provided. For the care and use of laboratory animals, all of the animal experiments were conducted in accordance with the guide of national institute. The experimental protocols were backed by the ethics board of Liaocheng university.
4.2. Treatment of D-gal induced aging of mice
Divided C57bl/6j mice randomly, after adopted feed 1 weeks, into 5 groups (n = 8). All of the mice but those raised in the group of control received daily intraperitoneal injection of 500 mg/kg D-gal, and the mice which raised in the group of 50, 100 or 200 mg/kg received daily oral administration of corresponding concentrations of compounds dissolved in physiological saline for 10 weeks. The mice were decapitated after the last administration.
4.3. Histopathology analysis
Brain, liver and kidney were taken from decapitated mice, then soaked in formalin, embedded in paraffin, and then sectioned. Staining the sections obtained from brain, liver and kidney with H&E and obtained from brain with Nissl, then those sections stained was photographed by the Olympus microscope (BX53 + DP80).
4.4. Measurement of AST/ALT
According to the manuscription (Applygen), added 24 µL standard solution into the standard well, 4 µL sample and 20 µL buffer solution into the sample well, and 4 µL sample into the reference well. After incubation at 37 ℃ for 30 min, 20 µL chromogen solution was added into all of the wells, and 20 µL buffer solution was added into the reference well. After incubation at 37 ℃ for 20 min, 200 µL stop solution was added into all of the wells. Then the mixture was incubated at room temperature for 10 min and measured at 520 nm by a microplate reader (BiotekSynergy H1).
4.5. Measurement of BUN
According to the manuscription (Nanjing Jiancheng Bioengineering Institute), 1mL of reagent 1 and reagent 2 was added into 20 µL of sample, then heated the mixture at 100 ℃ for 15 min, lower the temperature with cold water, and measured at 520 nm by a microplate reader (BiotekSynergy H1).
4.6. Measurement of CRE
According to the manuscription (Nanjing Jiancheng Bioengineering Institute), 6 µL of sample or standard was added into 180 µL of reagent 1. The mixture was incubated at 37 ℃ for 5 min and measured at 546 nm (A1). Then the mixture was added 60 µL of reagent 2, incubated at 37 ℃ for 5 min, and the absorbance of mixture was measured at 546 nm (A2). ΔA = A2-186*A1/246; the content of CRE (µmol/L) = ΔAsample / ΔAstandard * 442.
4.7. GPx assay
Adjusted room temperature to 25 ℃. 40 µL detection buffer (Beyotime), 10 µL sample and 40 µL detection working solution of GPx were added into 96-well plates. Following to the instruction of reagent kit, the mixture solution were incubated for 15 min, added 10 µL 30 mM peroxide reagent, and continuously measured the absorbance at 340 nm for 5 min by a multiwall plate reader (Biotek Synergy H1). The activity of GPx in sample = (ΔA340/min)/( εµM × L(cm). The εµM of NADPH at A340 nm is 0.00622 µM− 1cm− 1, and the height of liquid in 96-well plates is 0.276 cm.
4.8. T-AOC assay
Detect the total antioxidant capacity in serum and tissue supernatant by T-AOC detection kit (Beyotime). At first, following the instructions of the reagent kit prepared the working solution before 16 h. Added 10 µL sample to 200 µL working solution in 96-well plates. The mixture was incubated for 6 min and measured at 734 nm by a microplate reader (Biotek Synergy H1).
4.9. T-SOD assay
Following the instructions of the reagent kit (Beyotime), added NBT/enzyme working solution, sample and reaction start working solution into 96-well plates, and the mixture was incubated at 37 ℃ for 30 min. Finally, the mixture was measured at 540 nm by a microplate reader (Biotek Synergy H1).
4.10. MDA assay
Added 0.1 mL the supernatant tissue homogenate and 0.2 mL MDA working solution (Beyotime) in 1.5 ml centrifuge tubes, and then mixed. Heated the mixture at 100 ℃ for 15 min, then lower the temperature of the mixture to room temperature using flowing water, and added 200 µL supernatant into 96-well plates after centrifuged for 10 min at 1,000 g at 4 ℃, finally the absorbance was measured at 532 nm by a microplate reader (Biotek Synergy H1).
4.11. CAT assay
Added 10 µL sample and 30 µL CAT detection buffer (Beyotime) into 1.5 mL centrifuge tubes, and then 10 µL 250 mM hydrogen peroxide solution were added for 5 min at 25 ℃. In order to terminate the reaction, 450 µL stop solution were added. Then pipetted 10 µL of the terminated reaction solution to another centrifuge tubes with 40 µL CAT detection buffer. Pipetted 10 µL from the 50 µL mixture system in the previous step and 200 µL chromophoric reagent to 96-well plates, then the mixture was incubated at 25 ℃ for 15 min and measured at 520 nm by a microplate reader (Biotek Synergy H1).
4.12. Western blotting assay
The tissues of sacrificed mice was lysed by radio immunoprecipitation assay (RIPA) buffer (Beyotime), and the protein extracted from the tissues was examined by western blot assay. Primary antibodies: SIRT1 (Wanleibio, WL00599, 1:1000), p53 (Wanleibio, WL01919, 1:1000), p21 (Wanleibio, WL0362, 1:1000), p16 (Wanleibio, WL01418, 1:1000), HO-1 (Wanleibio, WL02400, 1:1000), Nrf2 (Beyotime, AF7623, 1:1000), Caspase-3 (Wanleibio, WL02117, 1:1000), Bax (Cell signaling technology, 2772S, 1:1000), IL-6 (Wanleibio, WL02841, 1:1000), TNF-α (Wanleibio, WL01581, 1:1000), β-actin (Servicebio, GB15003-100, 1:1000).
4.13. Statistical analysis
The data were expressed as the mean ± SD of at least three independent experiments. Statistical significances were analyzed using one-way ANOVA followed by Dunnett’s multiple comparison test. p < 0.05 was considered as statistically significant.