Data of the article "Total Glucosides of Paeony Ameliorates Liver Injury in Type 2 Diabetic Rats by Inhibiting Ferroptosis via the Nrf2/SLC7A11/GPX4 Signaling Pathway"

The dataset presented in this study originates from an experimental investigation into the effects of total glucosides of paeony (TGP) on regulating ferroptosis via the Nrf2/SLC7A11/GPX4 pathway to ameliorate liver injury in type 2 diabetic rats. A total of 60 Sprague-Dawley rats were acclimatized under conditions of 22 ± 2 °C with a 12-hour light/dark cycle. After three days, the rats were randomly assigned for the first time into a control group (n = 8) and a model induction group (n = 52). The control group was fed a standard chow diet, while the model group received a high-fat and high-sugar diet (composition: 62% standard chow, 10% lard, 15% sucrose, 10% egg yolk powder, 2.5% cholesterol, 0.5% sodium cholate). After four weeks, rats in the model group received an intraperitoneal injection of streptozotocin (STZ, 35 mg/kg) to establish the T2DM model [17], whereas the control group received an equivalent volume of citrate buffer (pH = 4.5). At 72 hours post-injection, random blood glucose was measured via tail vein blood. A successful T2DM model was confirmed by three blood glucose readings ≥16.7 mmol/L within 14 days. Ultimately, 40 rats were successfully modeled, yielding a success rate of 76.92%. For the second randomization, the model rats were divided into five groups (n = 8 per group): Model group, Metformin group (180 mg/kg), TGP-L group (50 mg/kg), TGP-M group (100 mg/kg), and TGP-H group (200 mg/kg). The control and model groups were administered an equivalent volume of distilled water via oral gavage once daily. Body weight was measured daily to adjust the administered dosage accordingly. The treatment lasted for four consecutive weeks. During this period, rats in the model groups continued to receive the high-fat and high-sugar diet, while the control group continued on the standard chow diet. The experimental protocol was approved by the Institutional Animal Care and Use Committee.Sample CollectionAfter the final administration, all rats were fasted for 24 hours with free access to water. Fasting blood glucose was measured after 12 hours of this fasting period. Subsequently, rats were anesthetized with an intraperitoneal injection of pentobarbital sodium (30 mg/kg). Body weight (BW) was recorded, and blood was collected from the abdominal aorta. The liver was weighed, and portions of the liver tissue were either fixed in 4% paraformaldehyde or flash-frozen in liquid nitrogen and then stored at -80 °C for later use.Fasting Blood Glucose Measurement in RatsFasting blood glucose (FBG) was measured at 0, 1, 2, 3, and 4 weeks post-drug intervention. After a 12-hour fast (with free access to water), blood was collected from the tail vein, and FBG was measured using a blood glucose meter. If the reading displayed "H1", it was recorded as 33.3 mmol/L.Measurement of Liver Weight and Liver IndexThe liver was rinsed with 4 °C physiological saline, blotted dry with filter paper, and weighed to obtain the liver weight (LW). The liver index (LI) was calculated as: LI = (LW / BW) × 100%.Measurement of Rat TC, TG, LDL-C, HDL-C, ALT, and AST LevelsWhole blood samples were allowed to stand for 2 hours and then centrifuged at 3,000 r/min for 15 minutes at 4 °C. The上层血清 (supernatant serum) was collected. Levels of TC, TG, LDL-C, HDL-C, ALT, and AST in the serum samples were measured using a fully automated biochemical analyzer, strictly following the instructions provided in the reagent kits. These measurements were used to assess the rats' serum lipid levels and liver function.Pathological Sectioning and Staining of Rat Liver TissueHE StainingLiver specimens fixed in 4% paraformaldehyde were dehydrated in a graded ethanol series, cleared by two immersions in xylene, and embedded in molten paraffin. After cooling, the blocks were sectioned. Following deparaffinization, sections were rehydrated in a descending ethanol series, washed with distilled water, and pre-treated with a high-definition staining solution. Subsequently, the sections were stained with hematoxylin and eosin (HE) sequentially. After dehydration, the sections were mounted with neutral balsam. The morphology of the stained liver tissue was observed under a light microscope at both low and high magnifications.Oil Red O StainingFrozen liver tissue samples were dehydrated sequentially in 15% and 30% sucrose solutions at 4 °C, embedded in OCT compound, and sectioned. The frozen sections were fixed, immersed in Oil Red O staining solution twice, differentiated in 60% isopropanol, counterstained with hematoxylin, differentiated again, then immersed in a bluing solution, and finally mounted with glycerol gelatin. The presence of lipid droplets within hepatocytes was observed.Colorimetric Detection of Oxidative Stress Markers (SOD, MDA, GSH) and Fe²⁺ Content in Liver TissueLiver tissue was ground at low temperature and high speed. Levels of SOD, MDA, GSH, and Fe²⁺ were measured according to the instructions provided in the respective reagent kits. These measurements were used to assess hepatic lipid peroxidation and iron accumulation in the rats.Western Blot Detection of Ferroptosis-Related Protein ExpressionFrozen liver tissue was thawed, ground at low temperature and high speed, and lysed with a lysis buffer. After homogenization, the protein concentration was determined using the BCA method. Samples were prepared by adding a loading buffer, followed by gel casting, sample loading, SDS-PAGE electrophoresis, and transfer to PVDF membranes. The membranes were blocked with 5% non-fat milk diluted in TBST Buffer for 2 hours at room temperature. Following the antibody instructions, the membranes were incubated with primary antibodies (Nrf2 1:2000; GPX4 1:5000; SLC7A11 1:2000; TFR1 1:2000; FPN1 1:800) overnight at 4 °C. After washing, the membranes were incubated with secondary antibodies for 2 hours at room temperature, washed again, and then developed using an ECL chemiluminescence solution for imaging. Protein expression levels were quantified using ImageJ 1.6.0 software, and the data were analyzed and graphed using GraphPad Prism 9.4.1 software.qPCR Detection of Ferroptosis-Related Gene ExpressionTotal RNA was extracted from rat liver tissue samples. The purity of each RNA sample was assessed using a micro-volume UV-Vis spectrophotometer. Genomic DNA was removed from the samples to be tested. Reverse transcription was performed according to the qPCR kit instructions, adding the reagents and placing the mixture in a thermal cycler under the following conditions: pre-denaturation (95 °C, 1 min), denaturation (95 °C, 10 s), annealing (55 °C, 30 s) for 45 cycles, and extension (72 °C, 10 s). The relative expression levels of target genes in each group were analyzed using Thermo Fisher's QuantStudio software, calculated using the 2-△△CT method. The full gene sequences were obtained from the NCBI database, and primers were designed using Primer Premier software. All primers were designed and synthesized by Sangon Biotech (Shanghai) Co., Ltd.Data File DescriptionsThis dataset includes one Excel file. File 1, "Master_Data.xlsx", contains 9 worksheets. The tabs represent various indicators, including the raw statistical data for: "FBG", "LI, ALT, AST", "TG, TC, LDL-C, HDL-C", "SOD, MDA, GSH", "Fe2+", "NrF2 mRNA", "SLC7A11 mRNA", "GPX4 mRNA", and "FPN1, TFR1, Nrf2, SLC7A11, GPX4 WB". File 2, "Individual_Figures", contains the individual images for the pathological HE staining, Oil Red O staining, and all other figures presented in the article. These have been named according to their order in the manuscript (e.g., Figure 1 Fasting Blood Glucose FBG). File 3, "Western_Blot_Images", contains the原始条带 (original blot images) for each target protein.