Synthesis of QCSPIONs
Synthesis of QCSPIONs referred to our previous studies [22,29,47]. Briefly, anhydrous FeCl2, FeCl3 and dextran were liquefied in deionized water. After complete mixing, the pH of the mixture was regulated on 9 using ammonia solution. The mixture was heated at 90ºC for 2 hours in consort with stirring, and then by means of a strong external magnet, the resultant dextran-coated Fe3O4 nanoparticles were collected. After washing with deionized water and ethanol, the nanoparticles dehydrated in an oven at 70ºC overnight. QCSPIONs were prepared by adding QC to dextran-coated Fe3O4 nanoparticles and using EDC/NHS as linkers. Then, synthesized QCSPIONs were sequestered from suspension by a magnet. After washing with deionized water, and acetone and QCSPIONs were dehydrated using freeze drier.
Dextran-coated Fe3O4 nanoparticles and QCSPIONs were characterized by the X-ray diffraction (XRD) and Fourier transform infrared (FTIR) spectrometry. The size and shape of the nanoparticles was determined by a Hitachi S–4700 field emission–scanning electron microscope (FE–SEM), prepared with an energy dispersive X-ray analysis (EDAX) detector.
Experimental induction of diabetes and treatment schedule
Animal maintenance, diabetes induction, and treatment schedule were elaborated in the previous study [22]. Briefly, male Wistar rats were bought from Isfahan Royan Institute, Iran and maintained for two months in animal house with standard condition. Type 1 DM was induced by intrapritoneal injection of 20 mg/kg of streptozotocin (STZ) for 5 consecutive days. Rats divided randomly into five groups including eight rats each (4 animals per cage): control, diabetes, and diabetes treated with SPIONs, QC, and QCSPIONs. All formulations (at the dose of 25 mg/kg) were suspended in deionized water (DI) instantly before administration and gavaged at a daily dose for 35 consecutive days. At the end of the experiment, rats were sacrificed by ketamine-xylazine anesthesia. The hippocampus was removed from the hemispheres and ½ of that immediately frozen in liquid nitrogen and then stored in −70 °C until use. The other half of the hippocampus and other tissues were fixed in 10% formalin and routine paraffin sections (3-4 μm) were preserved for histopathologic evaluation. The ethical respects were done in agreement with the guidelines for the use and care of research laboratory animals (USA National Institute of Health Publication No 80-23, revised 1996) and were permitted by the animal ethics committee organized by University of Isfahan.
Nanoparticles determination in brain
In order to determine the quantitative concentration of SPIONs in the brain as the target tissue in this study, inductive coupled plasma-atomic emission spectrometer (ICP-AES) (ICPS-7500, Shimadzu, Japan) was done. To achieve this purpose, an equal amount of samples was individually exposed to 3 mL nitric acid (HNO3, 65%) overnight. After digestion, resultant mixtures were filtered and iron concentration analyzed using ICP- AES technique and reported as a graph.
RNA isolation
In order to extract RNA, 50-100 mg of tissue was taken from each animal and squashed in a sterile Petri. TRIzol Reagent (Invitrogen, Life Technologies, Grand Island, NY, USA) was used to extract total cellular RNA (including messenger RNA and microRNA). The concentration, purity, and integrity of the RNA samples were assessed by a Nanodrop spectrophotometer (Thermo Fisher Scientific, USA) and denaturing agarose gel electrophoresis. DNA contamination was removed from RNA samples by treating 1 μg of the RNA by 1 unite of RNAase-free DNase (Thermo Fisher Scientific Inc, USA).
Complementary DNA (cDNA) synthesis
To synthesize cDNA, a PrimeScript RT reagent kit (Takara Bio, Ohtsu, Japan) was used. Two μL 5× PrimeScript buffer, 0.5 μL oligo dT primer, 0.5 μL RT enzyme, 0.5 μL of random 6mer and 500 ng DNase- treated total RNA were mixed, and incubated for 15 min at 37°C and 5 s at 85°C, respectively. In order to prepare cDNA from microRNAs, aBON-miR miRNA 1st strand cDNA synthesis kit (Bonyakhteh, Tehran, Iran) was applied. Then, elongations of miRNAs were briefly performed in a polyadenylation reaction with a final volume of 20 μL at 37°C for 30 min. To stop the reaction, miRNAs were incubated at 65°C for 20 min. Then, the cDNA synthesis reaction was accomplished in a final volume of 20 μL by adding 1 μL RT enzyme, 1 μM Bon-RT adaptor, 4 μL 5× RT buffer, 10 μL polyadenylated total RNA, and 2 μL dNTP mix. Finally, reactions were incubated at 75, 25, 42, and 70°C, for 5, 10, 60, and 10 min, respectively.
Real-time PCR
Each synthesized cDNA was applied as a template for a distinct microRNA and mRNA quantitative real-time PCR assay by means of a SYBR Green PCR Master Mix (RealQ Plus 2x, Ampliqon, Odense, Denmark). β-Actin and U78 (SNORD78) were selected as reference genes for normalization mRNA and microRNA expression levels respectively. Both of them are stable and reproducible in tissue and are suitable reference genes for assessment of mRNA and miRNA expression. Forward and universal reverse primer of miRNA-27a were designed and synthesized (Bonyakhteh, Tehran, Iran). The assay was done in a final reaction volume 13 μL containing 1 μL miRNA specific forward and reverse primer, (0.5 μL of each primer), 1 μL cDNA, 6.5 μL QPCR master mix. QPCR reactions were carried out (95 °C for 2 min, and 40 cycles of 95 °C for 5 s and 60 °C for 30 s).
We used Allele ID primer design software version 7.5 (Premier Biosoft, USA) to design all primers for mRNA samples (Table 1). Then checked on the National Center for Biotechnology Information (NCBI) website (www.ncbi. nlm.nih.gov/blast) and purchased from BIONEER (City, Korea). QPCR for mRNAs assay was performed in a final reaction volume of 10 μL containing 5 μL SYBR Green I Master mix, 0.5 μM of 10 pM forward, 0.5 μM of 10 pM reverse primers and 1 μl cDNA. To assay Real-time PCRs the following cycling conditions were chosen: 15 s at 95 °C, followed by 39 cycles at 95 °C for 15 s, 57 °C for 30 s, and 72 °C for 15 s. It should be noted that the primers used for catalase gene expression were 1 μM. The mean threshold cycle (CT) was calculated using a chromo4 (Bio-Rad, USA) device. To compare the expression level of genes between groups the ΔΔCt method was applied.
Total antioxidant capacity (TAC) determination
To measure the total antioxidant capacity of the hippocampus a commercial kit according to the company’s instructions (ZellBio GmbH, Germany) based on the oxidation-reduction colorimetric assay at a wavelength of 490 nm was used. In order to the preparation tissue homogenate, 100 mg of the hippocampus of each animal was homogenized in a phosphate buffer solution (0.1 M, pH = 7.4) by a homogenizer. In order to collect supernatant, centrifugation (at 4,000 RPM, for 20-minutes at room temperature) was performed. Then a TAC assay kit by microplate reader detection at 490 nm was used to measure the TAC index. TAC level was considered as the amount of antioxidant in the sample in comparison to ascorbic acid acts as a standard. The sensitivity of this method is 0.1 mM (100 μmol/L).
Histological examination
To evaluate histopathological alterations, the extracted specimens including hippocampus, liver, kidney, and pancreas were instantaneously fixed in 10% formaldehyde and inserted in paraffin wax. Then samples (3-4 μm) were deparaffinized and imbedded in distilled water. Based on the standard protocols, to assess the architecture of tissues samples were stained with hematoxylin-eosin (H&E), and to access the storage of glycogen and evaluate basement membrane stained with Periodic acid- Schiff (PAS). Finally, histological images were taken by Leitz microscope (Nikon Eclipse E-200, Japan) with 400 times enlargement.
Statistical analysis
For data analyses, we used GraphPad Prism software version 6. Data were analyzed by the one-way ANOVA followed by Tukey’s multiple comparison tests. All values were given as mean ± SEM and p-value fewer than 0.05 was measured as significant.