All of the chemicals used in this study, whose manufacturer is not listed in the text, are from Sigma Chemical Company, USA.
Isolation of Human DNA
Blood was drawn from healthy individuals. Blood was collected according to the instructions of the Ministry of Health of the Islamic Republic of Iran (Ethical number: IR.UI.REC.1399.056). Genomic DNA was isolated by optimization of the Proteinase K-Buffer method47, using lysis buffer A (11% sucrose, 1% Triton X-100, 5 mM MgCl2, 10 mM Tris pH 8) and buffer B (10 mM sodium citrate, 1% SDS, 10 mM Tris, 10 mM EDTA pH 8.0), from human blood.
Preparation of glycated and treated DNA whit RA
In vitro glycation of DNA was done and characterized, as previously described48. To summarize, DNA (20 ng/µl final concentration) from the blood of non-diabetic individuals was incubated with 25 mM fructose at 37 °C for 5, 10, and 15 days in the presence or absence of RA (25 µM final concentration) in phosphate buffer saline (PBS). This fructose concentration correlates to the minimum concentration needed for in vitro DNA glycation49. It has also been shown that 25 μM rosmarinic acid reduces the glycosylation of human fibroblasts in vitro50. 0.02 % (w/v) NaN3 was added to the solution and purified into a low protein-binding filter (-GV 0.22 µm filter unit, Millipore) to avoid bacterial contamination. Aliquots were taken from the DNA–fructose solution after each incubation period and extensively dialyzed against the sterile PBS at 4 °C to remove the free fructose molecules. Under the same conditions, pure DNA solution was used as the control sample.
UV-Visible spectroscopy
The UV–vis spectroscopy was used to confirm the structural changes induced in the human blood DNA. The ultraviolet absorption profile of the modified DNA (20 ng/µl final concentration), incubated with 25 mM fructose at 37 °C for 5, 10 and 15 days in the PBS, was recorded in the wavelength range of 200–400 nm on the UV-2100 spectrophotometer in a quartz cuvette with 1 cm path length27.
Agarose gel electrophoresis
To evaluate the effect of glycation (fructation) on DNA, 5µl of native, glycated, and treated DNA was mixed with 1µl of the loading dye 6X buffer. The samples were loaded into the wells of 1.5 % agarose gel and electrophoresed for 1.5 h at 100 v. The gels were stained with ethidium bromide (0.5 mg/ ml), viewed by illumination under UV light, and photographed.
Fluorescence studies
Fluorescence measurements were made on a Cary-Eclipse spectrophotometer (Varian model, Australia). Sample mixtures showing absorbance changes were subjected to the excitation wavelength of 370 nm and the emission wavelengths were recorded at the range of 380-500 nm. Fluorescence spectra of the modified nucleotides and DNA were determined by the subtraction of the background fluorescence of fructose and its possible degradation product during prolonged incubation.
Fluorescence microscopy
Fluorescence microscopic measurements were performed using ThT dye (λex 440 nm, λem 480 nm) to visualize the morphological structure of the inter-strand cross-links formed in the duplex DNA molecules and the inhibitory activity of biophenol51. ThT (32 μM) was mixed with native (at 25 °C) and glycated DNA samples in the absence and presence of biophenols. After 60 min incubation at room temperature, 15 μl of each sample was transferred on cleaned glass slides to be analyzed under fluorescence microscopy. The images were obtained with an Olympus IX71 fluorescence microscope equipped with a digital CCD camera at 20 and 40 objective magnifications.
Zeta Potential measurements
Zeta potential evaluation was done according to the method developed by Chetty and Singh26. To describe briefly, the zeta potential and average particle sizes of DNA, DNA/Fructose, and biophenol–DNA/Fructose were determined by using ZetasizerNano-ZS90 (Malvern Instruments, Ltd.UK). The sample solution of DNA/Fructose (1 mg/mL) and biphenol–DNA/Fructose was placed in the test vessel; the average of three repeats of the measurements was reported. The molar ratio of biophenol to DNA/Fructose in the mixed system was 1:1.
Thermal denaturation (Tm Measurement)
A temperature scan of 30-95 °C at an increment of 1 °C/min was performed with a Shimadzu UV-240 spectrophotometer fitted with a temperature programmer and controller assembly; The thermal denaturation of native, glycated, and RA DNA samples was evaluated under similar conditions. Absorbance change (260 nm) and the melting temperature (Tm) of the samples were reported52.
Experimental animals and samples collection
Male rats (Rattus Norvegicus) with an average weight of 200-220 g were purchased from the Faculty of Pharmacy, University of Isfahan. In the animal room of the Faculty of Science, University of Isfahan., Iran, the animals were housed in a 12-h alternating light-dark cycle at a temperature of 21 ± 2 °C. All experiments were approved by the Ethics Committee of the University of Isfahan, Iran, (Ethical number: IR.UI.REC.1399.056); they were conducted according to the guide for the Internationally Accepted Principles for Animal Use and Care53. The rats were divided into three groups (n=8): (i) Control group, (ii) diabetic group, and (iii) diabetic group treated with RA. Sixteen animals were fasted for 24 h, and diabetes was induced using a single intraperitoneal injection (i.p.) of Streptozotocin (STZ) (45 mg/kg, freshly dissolved in 0.1 M citrate buffer, pH 4). Three days later, fasting blood glucose levels were determined using tail blood. Only rats were considered diabetic if basal blood glucose levels exceeded 250 mg/dl. After confirmation of diabetes, eight rats in the diabetic group received 30 mg/kg RA (mixed in deionized water), once daily by oral gavage for 8 weeks. This concentration of RA shows anti-oxidant and anti-glycate effects in diabetic rats by reducing the formation of advanced glycation end products54. Rats in the control groups were given an equal volume of water. The animals were anesthetized with a mixture of pentobarbital sodium and phenytoin sodium (Euthanasia III) at the end of week 8. Rats were sacrificed when they failed to respond to a toe pinch. The hippocampus tissue samples were isolated and flash frozen in liquid nitrogen; they were preserved at -80°C until further experiments.
Total RNA extraction and reverse transcription.
Total RNA was extracted from the hippocampus tissue samples using Trizol reagent, according to the manufacturer's instructions. The extracted RNA was dissolved in DEPC-treated water. Gel electrophoresis was used to check the quality of the isolated RNA; also, Nanodrop (Thermo fisher-Onec) was used to determine the RNA concentration. Reverse transcription was conducted on 1000 nanograms of the total RNA in a final volume of 20 µl. Under the conditions suggested by the supplier (Thermo Fisher), cDNA was synthesized using the random hexamer primers and MMLV-reverse transcriptase.
Primer design for real-time PCR
The primers were designed using Oligo 7 and Beacon Designer 8 software. For Akt1 (NM_033230.2), Akt2 (NM_017093.1), Akt3 (NM_031575.1), and Beta-actin (NM_031144.3), the specific primers should result in a 156, 127, 107, and 123 bps product, respectively. The primer sequences are listed in Table 1.
Akt genes expression analysis by real-time PCR
For the amplification of Akt, 1 µl cDNA was added to the SYBRGreen Master Mix (Ampliqon) containing the specific primers. Real-time PCR was performed in a Bio-Rad thermocycler. The same total volume (12 µl) and thermal settings were used for all genes: 5 min of pre-incubation at 95 °C and this was followed by 40 cycles of 30 s at 95 °C, 30 s at 58 °C, and 30 s at 72 °C. The melting curve plot was drawn between 55 and 95 °C. Triplicates of each sample were run.
Statistical analysis
All data were presented as means ± standard deviation (SD). Statistical analysis was performed by one-way ANOVA, using GraphPad Prism software, version 7. A Duncan's post-hoc comparison was then made to analyze the sources of significant differences by SAS, version 9.2. A p-value ˂ 0.05 was considered statistically significant.