Research and Publication Ethics: All animal experiments were carried out at the Ataturk University Medical Experimental Research Center. The Ataturk University Animal Experiments Local Ethics Committee authorized this work (approval number: 2022–11/224).
Chemicals: Rutin, MLT, and all other chemicals used in the analyses were of analytical quality and purchased from Sigma Chemical Co. (St. Louis, USA).
Experimental Animals: In the study, 35 male Sprague-Dawley rats aged 8 to 10 weeks and weighing 220 to 250 g were employed. Rats were purchased from the Ataturk University Medical Experimental Application and Research Center (Erzurum, Türkiye). The rats were adapted to the environment for 1 week before the application. The ambient temperature was 24±1 °C, and the humidity was 45±5%. A 12-hour light/dark cycle was also incorporated into the rats' environment. Rats were fed standard pellet diet and ad libitum tap water.
Experimental Procedure: Rats were divided into five distinct groups, with seven rats in each. Doses of MLT and RUT given to animals were determined to be referenced according to prospective studies by Gur and Kandemir (3) and Kandemir et al. (9). As illustrated in Table 1, the experimental design contains a control and four experimental groups.
The rats were decapitated under mild sevoflurane anesthesia 24 hours after the last RUT administration (day 29) and their gastric tissues were collected, rinsed with physiological saline, and frozen at -80 °C until biochemical analysis.
Preparation of Tissue Homogenates: The tissues were diluted 1:20 (w/v) with phosphate-buffered saline (PBS; pH 7.4) to generate the gastric tissue homogenate. The resulting mixture was rapidly homogenized with a tissue lysate device (TissueLyser II, Qiagen). The homogenate was centrifuged for 30 minutes at +4 °C and 3000 rpm. The resulting supernatant was used for the analysis.
Determination of Lipid Peroxidation and Antioxidant Enzyme Activities in Gastric Tissue: The level of lipid peroxidation was determined by measuring the amount of malondialdehyde (MDA) using the method established by Placer et al. 33 and expressed as nmol/g tissue. Superoxide dismutase (SOD) activity was determined using a method developed by Sun et al. 34. The findings are presented as U/g protein. The catalase (CAT) activity was measured using a method established by Aebi 35, and the data were expressed as catal/g protein. Glutathione peroxidase (GPx) activity was assessed using a technique created by Lawrence and Burk 36. Results were reported as U/g protein. GSH levels were determined using the method described by Sedlak and Lindsay 37, and the findings were expressed as nmol/g tissue. The analysis of total protein was performed using the method established by Lowry et al. 38 using bovine serum albumin (BSA) as the reference.
Real-Time PCR Analysis: Relative mRNA transcript levels of Bcl-2-associated X protein (Bax), B-cell lymphoma 2 (Bcl-2), cyclooxygenase-2 (COX-2), iNOS, NF-κB and TNFα genes in gastric tissues were analyzed by RT-PCR method. The first step was isolating total RNA from tissues using QIAzol Lysis Reagent (79306; Qiagen). NanoDrop (BioTek Epoch) was used to determine the amount of RNA obtained at the end of the processes. According to the results obtained, total RNA concentrations of all samples were equalized to 1000 ng/mL. At this stage, all procedures were performed in accordance with the manufacturer's instructions. After that, cDNA synthesis was performed using the iScript cDNA Synthesis Kit on the total RNAs collected (Bio-Rad). This procedure was carried out in accordance with the manufacturer's instructions, and the following reaction conditions were established:
1. Priming: 5 minutes at 25 °C
2. Reverse transcription: 20 minutes at 46 °C
3. RT inactivation: 1 minute at 95 °C
In the last step, cDNAs from all groups were mixed with iTaq Universal SYBR Green Supermix (BIORAD) and reaction conditions were set up in a Rotor-Gene Q (Qiagen) device according to the manufacturer's instructions. Primer sequences have been presented in Table 2. Reaction conditions:
1. Polymerase activation and DNA denaturation: 95 °C for 30 seconds,
2. Denaturation: 95 °C for 5 seconds (40 cycles),
3. Annealing/Elongation and Plate Reading: 60 °C for 30 seconds (40 cycles),
4. Melt Curve Analysis: 65-95 °C 2-5 seconds per increase of 0.5 °C
CT values were obtained from the device after the cycle was over. These data were standardized in accordance with β-Actin. According to the 2-ΔΔCT method developed by Livak and Schmittgen 39, normalization was performed.
Statistical Analysis: Shapiro-Wilk test was done to evaluate the samples as the sample number was under 50. Because of the distribution of the data is normal and the number of groups is more than 2, comparison of differences between groups were performed using one-way analysis variance (ANOVA) and Tukey’s multiple comparison test via SPSS software (version 20.0; SPSS, Chicago, IL). Results were shown as mean ± standard deviation. Statistical significance was defined as a P value <0.05.
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