Chemicals and reagents
All chemicals and reagents used were purchased in analytical quality. The purity of the reference substances was >95%. Ginsenoside Rg1 (purity>95%) was purchased from Didakexiang Biological Co., Ltd (Guizhou, China) and dissolved in phosphate buffered saline (PBS) as a stock solution until further use. Fer-1 was purchased from DC Chemicals and used according to the manufacturer’s suggestion.
The following procedures were strictly faithful to 3R principles, and animal care and housing procedures were followed according to the Chinese regulatory requirements. The protocol used in this study was approved by the Ethics Committee of Union Jiangbei Hospital, Huazhong University of Science and Technology (HUST-LA0008). All tested animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health.
Establishment of SAKI model
Healthy SD male rats (250-300 g) were chosen to establish the SAKI animal model. After the SD rats were kept in the laboratory for one week, cecal ligation and perforation were performed to establish a sepsis rat experimental model. All rats were divided into four groups (6 in each group): control group, model group, blank treatment group, Ginsenoside Rg1 treatment group. The Rg1 group was given by intraperitoneal injection of 125 mg/kg/d 24 h before cecal ligation. The blank treatment group was treated with cecal ligation. The same dose of normal saline as that of the Rg1 group was injected intraperitoneally one day before, and the model group was ligated and perforated in the cecum to establish a septic kidney injury rat model. After 1, 3, and 7 days after operation, after anesthesia, venous blood was collected by eyeball removal, rat urine and rat kidney tissue were collected for pathological examination.
Cells from the rat renal tubular epithelial cell and HK-2 cell line were cultured with BEGM Bronchial Epithelial Cell Growth Medium BulletKit (Lonza) or DEME/F12 medium containing 10% fetal bovine serum without antibiotics in a humidified incubator at 37°C with 5% CO2. Stimulation with LPS (1 μg/mL) to HK-2 cells was performed for 24 h prior further examination.
Urinary samples were collected and the concentration of NGAL, KIM-1, and MIOX were measured using the enzyme linked immunosorbent assay (ELISA), according to the manufacturer’s instructions. Serum was isolated after centrifugation at 3000 x g for 15 min. Measurement of IL-18 (ab213909, Abcam), IL-6 (ab234570, Abcam), and TNF-α (ab236712, Abcam) was performed by ELISA kits using pairs of specific mAbs and recombinant cytokine standards obtained from Invitrogen Life Technologies.
The kidney tissues of normal and Rg1 treated groups were collected separately and were stained with PAS (Periodic Acid-Schiff).
Fluorescence-activated cell sorter (FACS) cytometry analysis was performed using the H2O2-activated green fluorescent dye, dihydro-dichloro-fluorescein diacetate (H2DCFDA, Molecular Probes). This dye is readily taken up by cells, cleaved by cellular esterases to produce the noncell permeant H2DCF, which can be activated by ROS giving green fluorescent DCF as an indication of intracellular levels of oxidative stress. Cell suspensions were adjusted to an OD600 of 0.5, pelleted by centrifugation, and resuspended in phosphate buffered saline (PBS) containing 20 mM H2DCFDA. The suspension was incubated for 4 h, diluted 1:100 in PBS, and the fluorescence levels of 50,000 cells were recorded using a FACScalibur cytometer (BD Biosciences). Summit software (Dako Colorado) was used for data analysis.
Evaluation of iron level
To evaluate the ferroptosis level in different groups, iron concentrations in cell lysates were assessed using an Iron Assay Kit (Sigma-Aldrich, Cat #: MAK025) according to the manufacturer’s instructions.
Cell viability assay
Cell viability assay was done according to the the instructions of CCK-8 kit (APExBIO). Briefly, 20 μL of the CCK-8 reagent was added to each well (containing 200 μL of medium) of a 96-well microplate, and the microplate was further incubated at 37℃ for 4 h. Finally, the OD (450 nm) were measured in different groups (n = 3). The cell viability in the control group (without any treatment) was regarded as “100%”, and the relative cell viability of the other groups was calculated, respectively.
Western Blot Analysis
Concentrations of proteins were detected via a BCA protein assay kit (Sigma-Aldrich, Shanghai, China). Equal amounts of protein (20 μg) were loaded in each well and separated by 10% SDS-PAGE and blocked with 5% skimmed milk (BD) dissolved in TBST for 1 h, and then incubated with primary antibodies (anti-Gpx4, ARA70, P53, ERK1/2, Nrf2, p-Akt, GAPDH, 1:1000; Abcam, USA) at 4°C for 12 h. The membranes were washed three times for 7 min and incubated with the appropriate second antibody conjugate (Abcam, USA) or horseradish peroxidase-conjugated protein antibody (Sigma-Aldrich, Shanghai, China) for 1 h at room temperature. Then, the membranes were washed three times and stained with DAB Horseradish Peroxidase (Beyotime, Shanghai, China). The proteins were detected by using gel visualization (Tanon, Shanghai, China). Protein levels were normalized to GAPDH and quantified via densitometry.
Lactate dehydrogenase (LDH) was assessed with Lactate Dehydrogenase Activity Assay Kit (Sigma-Aldrich) according to the manufacturer’s instructions.
Graphpad Prism software version 7 was used for all statistical analyses. The tests are indicated in the figure legends. Measured data are expressed as mean ± standard deviation and analyzed using the Student’s t-test and variations considered significant at p < 0.05.