Ethical statement, study design and allocation
Ethical statement
Approval was obtained from the ethical committee of Faculty of Medicine, Mansoura University (No. R21.05.1328) in accordance with “principles of laboratory animal care NIH publication revised 1985” (Code number: 2020–107). Reporting of all experimental procedures complied with recommendations in ARRIVE guidelines.
Study design and allocation
Randomized, placebo-controlled, blinded animal study was conducted. The sample size was calculated using G power 3.9.1.4 software, to detect a 0.7 effect size between the null hypothesis and the alternative hypothesis with significance level of 0.05 and a power of 0.85, using a one-way ANOVA F-test. Twenty seven male Wistar rats, 100–120 g, were maintained in a controlled temperature (24–26 °C), relative humidity of 60–80% and on a 12-h light–dark cycle for one week acclimatization. Rats were randomly allocated using list randomizer (https://www.random.org/lists) into 3 groups with 9 rats/group as follow; Group1: served as a control, Group 2: represented diabetic rats, and Group 3: denoted as the treated group in which the diabetic rats received intraperitoneal (IP) injection of 100 mg/kg/3 times a week GA (Sigma-Aldrich, St Louis, MO, USA) for 8 weeks33,34.
Diabetes induction
After overnight fasting, rats assigned to groups 2 and 3 were injected with (50 mg/kg/ip) of freshly prepared streptozotocin (STZ) dissolved in citrate buffer, pH 4.5 (STZ, Sigma Chemical Co., St. Louis, MO, USA) while, the control animals in group 1 were injected by an equal volume of the buffer by the same qualified person35. Three days after the STZ injection, animals with stable fasting blood glucose levels at > 250 mg/dl were considered diabetic.
Euthanasia and biopsy collection
After eight weeks of treatment, all rats were anesthetized with Xylazine (5 mg/kg, ADWIA Co. S.A.E 10 of Ramadan city, Egypt) and Ketamine (40 mg/kg, Segmatec Pharmaceutical Industries Co., Egypt) injection into the peritoneum (IP) and euthanized by decapitation (at 8 am to minimize the circadian effect)36,37 and the SMG tissues were collected. The right halves were processed for the histological analysis, and the left halves were snap frozen in liquid nitrogen and kept at − 80 °C until used for oxidative stress estimation, RT-PCR and ELISA techniques.
Histological analysis
The 4 μm sections of paraformaldehyde-fixed and paraffin-embedded SMG tissues were stained with hematoxylin and eosin (H&E). For the semithin sections, tissue biopsies were dehydrated through an ascending series of ethanol (to 100%) and then washed in dry acetone and embedded in epoxy resin then stained with toluidine blue.
Immunohistochemistry (IHC) and image analysis
The protein expression of SIRT1 (Bioss Antibodies, USA, 1:200), ET-1 (Bioss Antibodies, USA, 1:200), AQP1 (Scervicebio Co., USA, 1:1000), AQP4 (Scervicebio Co., USA, 1:1500), AQP5 (ABclonal, USA, 1:200) and autophagy biomarkers LC3 (Abcam, USA, 1:1200), P62 (ABclonal, USA, 1:200) were determined in each group by incubating tissue sections in primary antibodies overnight followed by incubation with secondary antibodies to perform IHC. The visualization of slides was detected using 3,3-Diaminobenzidine (DAB, Abcam, USA), and counterstained with hematoxylin. Then, the sections were analyzed and photographed using an Olympus microscope (Japan) with installed camera. The positive reaction was thresholded and calculated in relation to the surface area using Image J. The data were then decoded and statistically analyzed.
Biochemical analysis of oxidative stress markers
The SMG tissue was homogenized with sodium phosphate buffer, centrifuged, and the supernatant was used for the biochemical analysis. Oxidative stress markers; reduced glutathione (GSH), superoxide dismutase (SOD) and malondialdehyde (MDA) were measured spectrophotometrically38,39.
Assessment of SMG levels of Beclin-1and LC3II
Rat Beclin-1 ELISA Kit (MBS733192) and Rat LC3II ELISA kit (MBS169564) were used for quantitative measurement of Beclin-1and LC3II protein levels in the SMG homogenate according to the manufacturers’ instructions.
Quantitative Assay of LC3 and AQP5 gene expression using RT-PCR
Total RNA was extracted from SMG samples, and then RNA quality and purity were assured. Then cDNA was synthesized from RNA. The cDNA was amplified and used in SYBR Green Based Quantitative Real-Time PCR. For Relative Quantification (RQ) of LC3 gene expression, a primer with Gene Bank Accession No. NM_022867.2, Forward sequence: 5-ACG-GCT-TCC-TGT-ACA-TGG-TC-3 and Reverse sequence: 5-GTG-GGT-GCC-TAC-GTT-CTG-AT was used. And for AQP5, a primer with Gene Bank Accession No. NM_012779.2 was used. The forward primer sequence was 5-GGGCCATCTTGTGGGGATCT-3 and the reverse primer sequence was 5-CCAGTGAGAGGGGCTGAACC-3. The RQ of both genes expression was performed using comparative 2−ΔΔCt method, where the amount of the target genes mRNA were normalized to an endogenous reference gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and relative to a control40.
Statistical analysis
Data were tested for normal distribution by Shapiro–Wilk test. Quantitative data were analyzed using Graph Prism 8 (GraphPad Software, Inc., CA, USA) to test the significance between different groups using analysis of variance (ANOVA) followed by Tukey’s test. Data were presented as mean ± standard error (SE). Significance was inferred at P < 0.05.
