All experiments were conducted following the guidelines of the committee of the Royan Institute (IR. ACECR.ROYAN.REC1398.44). Mice were acclimatized to the Royan animal housing condition for two weeks.
Experimental animal and treatments
Four-week-old male C57BL/6 mice were procured from Royan Institute for Biotechnology (Isfahan, Iran) and were housed in a temperature-controlled facility (24 ± 3 °C and humidity of 65% ±5), under a 12 h light-dark cycle. They were held ad libitum to access water and foods throughout the study. After adaptation, mice with an approximate weight of 14 ± 2 g, were randomly divided into two groups of control (Ctrl) and diabetic mice (DM) that respectively fed with normal diet and (AGE)-rich diet (Abedpoor et al., Unpublished data) which were obtained from Royan Institute for Biotechnology, for 16 weeks. After ensuring the emergence of DM, the diabetic group was divided into seven groups randomly (n=5), as follows: 1. Diabetic mice (DM group), 2. Diabetic mice treated with SPTC (130 mg/kg, DM/SPTC group), 3. Diabetic mice treated with Salvia Officinalis (65 mg/kg, DM/Sal), 4. Diabetic mice treated with metformin (300mg/kg, DM/Met), 5. Diabetic mice with endurance exercise training (DM/EX), 6.Diabetic mice treated with SPTC+metformin (130 mg/kg+300mg/kg respectively, DM/SPTC+Met) 7. Diabetic mice treated with SPTC+exercise training (DM/SPTC+EX).
SPTC (Salvia officinalis 145 mg, Panax ginseng 145 mg, Trigonella foenum-graeceum 65 mg, and 25 mg Cinnamomum zeylanicum) and Salvia were provided as a dried powder. All chemical and herbal drugs were administered as a gavage supplement, five times per week (day 1 up to 5 /each week) for 8 weeks.
During the experiment period, body weight was measured weekly. In addition, calorie intake, and water drinking were monitored daily. At the end of experiment, mice were sacrificed under xylazine and ketamine anesthesia. Blood and tissue samples were collected for further tests. In the Figure 1 we designed the flowchart of our experiments.
Biochemical analyses of plasma glucose, insulin levels, blood ROS, and Total antioxidant capacity
The plasma level of insulin was determined by Ultra-Sensitive Mouse Insulin ELISA Kit (Crystal Chem, US) according to the protocol. Glucose tolerance test (GTT) and fasting blood sugar (FBS) were measured using an Alpha TRAK glucometer and its standard strips (Zoetis, US) as described elsewhere. The blood ROS activity was measured with the OxiSelect Intracellular ROS Assay Kit (Cell Biolabs, USA) according to the manufacturer’s instruction. Total antioxidant capacity of blood was measured by OxiSelect Total Antioxidant Capacity (TAC) Assay Kit (Cell Biolabs, USA).
Exercise training protocol
Endurance exercise was applied as a type of moderate-high intensity exercise on a motorized treadmill with a 0 ° incline during 8 weeks (5 days/week). At first, mice were acclimated to treadmill exercise for two weeks, and after that speed and duration of treadmill training were gradually increased at a rate of 3 m/min from 10 to 25 m/min in the final session for 45 min (~ 70% VO2 max).
Quantitative real-time PCR (qRT-PCR)
Total RNA was isolated from the liver using TRIzol reagent (Thermo Scientific, USA). To remove contaminating genomic DNA, samples were treated with DNaseI (TaKaRa, Japan). mRNA was reverse transcribed with 1 μg of total RNA using the cDNA synthesis kit according to the manufacturer’s instruction (TaKaRa). qRT-PCR was performed with CYBR green (TaKaRa, Japan) on an Applied Biosystems real-time PCR thermal cycler (Thermo Fisher Scientific, Waltham, MA, USA). Evaluation of gene expression was carried out according to the 2-ΔΔct method. Accordingly, relative quantification was calculated according to 18s rRNA as an internal control (Housekeeping). Primers were ordered from micro-gene (Korea), and their sequences are shown in Table 1.
Western blot analysis
Tissue proteins were extracted using TRI reagent, according to the manufacturer protocol. Proteins were resolved by SDS-PAGE (10%) and transferred to PVDF membranes (Bio-Rad, USA). Membranes were blocked with different blocking buffer containing 10% skim milk (Millipore, USA), and 5% TBST. Then, they were respectively incubated with primary antibodies for 1.5 h (Anti-Nrf2 antibody [1:1000, EP1808Y, Abcam, UK], Anti-Glucose Transporter GLUT4 [1:2000, ab188317, Abcam, UK], anti- β actin antibody (1:500, Sigma, USA), anti-Keap1 antibody [ 1:1000, ab119403, Abcam, UK] and secondary antibodies (Goat Anti-Rabbit IgG H&L (HRP) (1:20000, Santa Cruz SC-2301, HRP-conjugated goat antimouse IgG (1:5000, Dako, Japan P0447), for 1 h at room temperature. Blots were detected by an Amersham ECL Advance Western Blotting Detection Kit (GE Healthcare, USA). Image J software (National Institutes of Health, Bethesda, MD, USA) was utilized for quantification of the intensity band.
Immediately after mice sacrificing, tissues were fixed in 10% buffered formalin and embedded in paraffin. Accordingly, fixed tissues were cut into slices with 5 µm thickness. After deparaffinization and hydration, tissue sections were stained with hematoxylin and eosin (H&E) and finally were observed under light microscopy.
ROS generation in mice liver was measured by using 2',7'-Dichlorofluorescin Diacetate (DCFDA) fluorescence method as previously described. Briefly, liver samples (100mg) were homogenized in 1 mL ice-cooled (4 °C) 40 mM Tris–HCl buffer (pH:7.4). After diluting to 0.25 % with the same buffer, the total homogenate of each sample was divided into two equal portions of 2 mL. Approximately 40 μL of 1.25 mM DCFDA was added to one portion, and the same volume of methanol was added to the other portion (Control). After 20 min incubation of all samples in 37 °C, the conversion of DCFH to the fluorescent product DCF was determined at 488 nm excitation and 525 nm emission using BD FACSCalibur Flow Cytometer (Becton Dickinson, USA). The results were evaluated according to DCF fluorescence intensity.
Total antioxidant capacity
Total antioxidant capacity of tissue and drug samples were determined using Ferric Reducing Antioxidant Power (FRAP) Assay Kit (Naxifer™- TAC Capacity Assay Kit, NS-15012, Iran) as described in manufacturer protocol.
The statistical analyses were carried out using GraphPad Prism 8.5 software (GraphPad
Software, San Diego, CA, USA). The paired samples t-test was performed to evaluate the diabetic group compared to the control group. Also, One-way analysis of variance (ANOVA) was used to make comparisons between all treatment groups. All experimental results are presented as mean ± SD. p-value < 0.05 represents significant difference between the samples.