Cell culture

The human retinal pigment epithelial (RPE) cell line ARPE-19 was purchased from Otwo Biotechnology Co., Ltd. (Shenzhen, China). ARPE-19 cells were cultured in Dulbecco’s modified Eagle medium (DMEM-F12) (ATCC, Manassas, USA) supplemented with 2 mmol/L glutamine, 10% fetal bovine serum and 100 U/mL penicillin/streptomycin and incubated in an incubator at 37 °C and 5% CO2. The cell culture medium was changed every two days. When cells reached 80% confluence, the cells were digested with trypsin and then inoculated in porous plates for subsequent experiments. Before the experiment, the cells were washed once with phosphate buffer (PBS), then ARPE-19 cells were induced with a high glucose concentration (HG, 25 mM) for 48 h, and the control cells were cultured in a medium containing normal glucose (NC, 5.5 mM)(Fan et al. 2022).

Cell transfection

The expression vectors, oe-Sestrin2 and si-Sestrin2 (see supplementary file 1 for the sequences), were designed and synthesized by GeneChem (Shanghai, China). The coding sequences for the mRNAs of Sestrin2 were cloned into pcDNA3.1. Oe-Sestrin2 and si-Sestrin2 were transfected into cells with Lipofectamine®3000 reagent (Invitrogen, CA, USA), and transfection efficiency was measured.

Cell viability test

Cell viability was measured using a CCK-8 assay kit as previously described (Liu et al. 2020). ARPE-19 cells were inoculated into 96-well plates at a density of 1 × 105 cells/well and placed in a 5% CO2 incubator at 37 °C for 24 h. CCK-8 reagent (10 µL/well) was added, and the cells were cultured for 2 h. The 96-well plate was then placed on a microplate reader, and the absorbance of the cells at 450 nm was measured.

Flow cytometry

In this study, flow cytometry was used to measure apoptosis. After cells (EDTA-free 0.25% trypsin-digested) from each treatment group were collected, they were washed with PBS three times and resuspended in 100 µL of buffer. At 25 °C, the cells were coincubated with 5 µL Annexin V-FITC and 5 µL PI (BD Biosciences) for 10 min. Finally, after adding the termination buffer, the apoptosis rate was determined by flow cytometry (BD FACSCalibur, USA).

Reactive oxygen species (ROS) detection

The experimental procedure is described in a previous article (Gu et al. 2019). ROS levels in the cells were determined using an ROS detection kit (Abcam, UK). The cells were mixed with 5 µL of DCFH-DA and incubated at 37 °C for 30 min. The cells were rinsed with fresh medium and imaged under a fluorescence microscope (488 nm excitation).

Malondialdehyde (MDA) detection

As described previously (Bahr et al. 2019), we used the MDA kit (Beyotime, Shanghai, China) for the detection of MDA in sample cells or mouse retinal tissue. The retinal tissues and cells were lysed and then centrifuged at 1600 × g for 10 min, and the supernatant was taken for subsequent determination. Then, 0.1 mL of lysate was added to the centrifuge tube as a blank control. The samples to be tested and the corresponding reagents were added according to the instructions, mixed well, heated for 15 min using a PCR instrument at 100 °C and cooled at 25 °C in a water bath. Subsequently, the samples were centrifuged for 10 min (1000 g, 25 °C), transferred to 200 µL of supernatant in a black 96-well plate, and placed into a 37 °C microplate reader for detection of absorbance (excitation wavelength 532 nm).

Fe2+ detection

In this study, we used an iron assay kit to detect the content of Fe2+ in cells (Fan et al. 2022). The solution and samples were prepared according to the experimental requirements, and the standard and reaction wells were set up (standard wells = 100 µL standard dilution, sample wells were added with 50 µL samples and diluted to 100 µL/well with iron assay buffer). L assay buffer, mixed and incubated (37 °C) for 30 min, 100 µL iron probe was added, mixed and incubated again (37 °C, protected from light) for 60 min and absorbance at 593 nm was measured in a microplate reader.

Western blot

Western blotting was carried out according to previous studies (Fan et al. 2022). In this study, total protein was extracted from cells and mouse retinal tissue using RIPA lysis buffer containing protease and phosphatase inhibitors, and its concentration was determined by BCA (Sangon Biotech, Shanghai, China). The protein sample was mixed with the loading buffer and denatured at 95 °C for 10 min. Then, the same amount of protein (30 µg/lane) was separated using 10% SDS‒PAGE electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked with 5% BSA in TBS in the presence of Tween-20 (0.05%) for 1 h. Subsequently, primary antibodies (the antibodies were purchased from Abcam, and the antibody dilution concentration was 1:1000) were added: Sestrin2, GPX4 (1:2000), FTH1, xCT, HO-1, cleaved-caspase3, BAX, BCL-2, ATF4, CHOP (Thermo Fisher, USA), XBP-1, GRP78, p-GRP78, LC3BII/I (1:2000), Beclin1 (1:2000), and P62, and incubated overnight at 4 °C. The next day, the primary antibody was removed, and the proteins were washed three times with membrane wash buffer for 5 min each. Secondary antibody (1:1000, Abcam, UK) was added and incubated for 2 h at 4 °C, and TBST buffer was used to wash the PVDF membranes. The control protein was β-actin. Subsequently, chemiluminescent reagents were added, and the bands were analyzed for grayscale values using ImageJ software.

Immunofluorescence staining

ARPE-19 cells were fixed with 4% paraformaldehyde for 30 min, permeated with 0.5% Triton X-100 for 20 min, and then blocked with 5% bovine serum albumin at room temperature for 1 h. Cells were incubated with primary antibodies against GPX4 (1:100) and LC3 (1:200) overnight at 4 °C, washed three times with PBS, and then incubated with fluorescein-labeled secondary antibodies (1:1000) for 2 h. The cells were washed with PBS twice, DAPI (Invitrogen, CA, USA) was added, and the cells were incubated for 15 min in the dark for re-staining. Finally, the slides were sealed with anti-fluorescence quencher. The cells were observed by fluorescence microscopy (Eclipse 80i, Nikon, Japan).

Laboratory animals

In this study, 70 female C57BL/6 mice aged 7–8 weeks (purchased from the Animal Experimental Center of Kunming Medical University) were selected as experimental animals. Mice were given free access to food and water under SPF conditions, a temperature of 22–26℃, a relative humidity of 52–58%, and a light-dark cycle of 12 h/12 h. After one week of adaptation, the experiment was carried out. The mice were randomly divided into the NC group (the control group ate a normal diet without any treatment; n = 10), DM group (diabetic group, intraperitoneal injection of STZ into mice induced diabetes; n = 15), DM + oe-Sestrin2 group (2 µg oe-Sestrin2 was injected into diabetic mice intravitreally; n = 15), DM + oe-Sestrin2 + erastin group (DM + oe-Sestrin2-treated mice were intraperitoneally injected with 20 mg/kg of the ferroptosis activator erastin three times a week for 4 weeks (Menon et al. 2022); n = 15) and DM + oe-Sestrin2 + 3-MA group (DM + oe-Sestrin2-treated mice were intraperitoneally injected with 15 mg/kg of the autophagy inhibitor 3-methyladenine (3-MA) three times a week for 4 weeks (Bo et al. 2020); n = 15). Diabetes was induced by intraperitoneal injection of 60 mg/kg streptozotocin (STZ) for 5 consecutive days (Suvas et al. 2020; Zhang et al. 2009). Blood glucose was monitored on the 7th day, and the model was established when blood glucose was ≥ 16.7 mmol/L, and used for treatment experiments in the STZ-treated groups. Two months after the successful establishment of the diabetes model, the mice were euthanized by intraperitoneal injection of pentobarbital sodium, and the eyeballs of the mice were collected for subsequent experiments. All animal experimental protocols were approved by the Animal Ethics Committee of Kunming Medical University (approval number: kmmu20211334).

HE staining

After killing the mice, the eyeballs of the mice were quickly removed and immersed in FAS eyeball fixation solution (Servicebio, Wuhan, China) for 24 h. The sections were then dehydrated with 75%, 85%, 90% and 95% alcohol and anhydrous ethanol. After clearing in xylene, tissues were embedded in paraffin wax and sectioned at 4 μm. Sections were dewaxed with xylene, re-hydrated with graded ethanol, and washed once with distilled water. The samples were stained with hematoxylin for 1 min, rinsed with tap water, differentiated with 1% hydrochloric acid for 10 s, hydrated with 1% ammonia for 5 s, and stained with eosin for 2 min. Finally, the tissues were dehydrated and cleared, sealed with neutral gum, observed and photographed under an optical microscope (BX53, Olympus, Japan).

TUNEL staining

After xylene dewaxing and gradient alcohol hydration, mouse retinal sections were incubated at 37 °C with 100 µL of protease for 30 min. Then, 50 µL of TdT enzymatic reaction solution was incubated at 37 °C for 1 h away from light. After washing with PBS 3 times, the sections were incubated with 50 µL streptavidin-TRITC solution at 37 °C for 30 min away from light. After washing with PBS three times, the nuclei were re-stained with DAPI staining solution, and the apoptotic cells were observed by fluorescence microscopy after the slides was sealed.

Statistics and analysis

The data were analyzed and plotted using GraphPad Prism 8. The experimental data are presented as the mean ± standard deviation. Student’s t-test was used for comparisons between two groups, one-way analysis of variance (ANOVA) and Tukey’s post hoc tests were used for comparisons among multiple groups with P < 0.05 considered as statistically significant.