The rat intestinal epithelial cell line No. 6 (IEC-6) was provided by iCell Bioscience Inc (Shanghai, China). The cells were cultured in Dulbecco’s Modified Eagle Medium (Procell, Wuhan, China) supplemented with 10% fetal bovine serum (Procell) and 0.1 U/mL insulin (MedChemExpress, New Jersey, USA) in a humidified atmosphere at 37°C with 5% CO₂.

Cell viability was measured using cell counting kit-8 (CCK-8) solution (Bimake, Houston, Texas, USA) following instructions of the manufacturer. IEC-6 cells were seeded (1 × 10⁴ cells) into 96-well plates containing 100 μL complete medium per well, treated with RSL3 (MedChemExpress). Ferrostatin-1 (Fer-1) (MedChemExpress) was used as the positive control. Then, to each well, 10 μL CCK-8 solution was added and incubated at 37°C for 2 h in a dark place. The absorbance level was measured at 450 nm using a microplate reader (Molecular Devices, USA). The cell viability was calculated based on the control group.

The compound library, which included 66 natural small molecule compounds were purchased from APExBIO Technology (Houston, Texas, USA). The IEC-6 cells (1 × 10⁴ cells) were seeded into 96-well plates in 100 μL complete medium per well, which were treated with 0.5 μM RSL3 and 2.5 μM natural small molecule compounds for 48 h. Fer-1 was used as the positive control. The cell viability was measured by the CCK-8 assay.

Total RNA was extracted from IEC-6 cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The RNA concentration was detected by measuring the absorbance at 260 and 280 nm using a microplate reader (Molecular Devices, San Jose, USA). cDNA was synthesized using TransScript® One-step cDNA synthesis kit (Transgen, Beijing, China) from 1 µg total RNA for detection of genes coding for GSH-dependent peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11), and qRT-PCR reactions were amplificated by SYBR Green qPCR SuperMix kit (Transgen). The qRT-PCR analysis for expression of the miR-17-92 cluster, including miR-17, miR-18a, miR-19a, miR-19b, miR-20a, and miR-92a was conducted with the miRNA 1st strand cDNA synthesis kit (Accurate Biotechnology, Beijing, China) and amplified by SYBR Green Pro Taq HS qPCR Kit II (Accurate Biotechnology). A total of 40 cycles were set for the qPCR reaction, including 95°C, 30 s for denaturation, 95°C, 5 s for annealing, and 60°C, 30 s for the extension. Cycle threshold (Ct) values of genes were determined by Applied Biosystems QuantStudio 5 (Thermo Fisher, Waltham, USA). β-actin was used as the internal reference of GPX4 and SLC7A11. U6 was used as the internal reference of the miR-17-92 cluster. The relative level of mRNA and miRNA expressions were calculated using the 2^−∆∆CT method. Primers of the miR-17-92 cluster, U6, GPX4, SLC7A11, and β-actin were designed and synthesized by Sangon Biotech (Beijing, China) and their sequences are mentioned in Table 1.

IEC-6 cells were harvested and counted, then every well of 6-well plates was seeded with 5 × 10⁵ cells and cultured for 6 h with or in the absence of RSL3, and the concentration of Tan IIA was set at 5 and 2.5 µM. Cells were digested with trypsin, collected by centrifuge, labeled with 2′,7′-Dichlorofluorescein diacetate (DCFH-DA) probe (Elabscience, Wuhan, China), at 10 µM concentration, and incubated at 37°C in dark for 30 min. Finally, cells were labeled with 7-aminoactinomycin D (7-AAD) viability staining solution (BioLegend, San Diego, CA, USA). Dead cells were excluded from the collected cells by the APC channel and the ROS level was detected by the FITC channel using CytoFLEX (Beckman Coulter, CA, USA).

After IEC-6 cells were treated with RSL3, Fer-1, and Tan IIA, cells were fixed with methanol for 2 min. Then, the cells were washed twice with phosphate-buffered saline (PBS) and stained at room temperature for 20 min by Giemsa solution (Solarbio, Beijing, China). Finally, the Giemsa solution was discarded and the cells were washed with PBS. The cell morphology was observed and photographed by an optical microscope (Leica, Germany).

IEC-6 cells were cultured with RSL3 and Tan IIA for 24 h, then cells were fixed with TEM fixative solution (Servicebio, Wuhan, China). The TEM preparation and cell morphology observation were conducted by Servicebio Technology.

IEC-6 cells were cultured with RSL3 and Tan IIA and lysed with RIPA (Meilunbio, Dalian, China) containing 1% phenylmethanesulfonyl fluoride (Meilunbio) on ice. Protein concentration was measured by BCA Protein Assay Kit (Solarbio). A total of 20 µg protein was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Yeasen, Shanghai, China) and transferred to a polyvinylidene fluoride membrane (Millipore, Germany). Then membranes were blocked with 5% defatted milk for 1 h and labeled with primary antibodies (β-actin, ABclonal, Wuhan, China; SLC7A11, ABclonal; GPX4, Cell Signaling, Danvers, Massachusetts, USA) overnight at 4°C. Subsequently, membranes were incubated with goat anti-rabbit IgG H&L (horseradish peroxidase) secondary antibody (ZSGB-Bio, Beijing, China) and visualized by adding enhanced chemiluminescence solution (Yeasen). The protein bands were analyzed by the ProteinSimple instrument (Bio-Techne, Minnesota, USA).

Data were analyzed with GraphPad Prism version 9.0 software (La Jolla, CA, USA). All results were expressed as mean ± SD. Differences between the two groups were analyzed by paired t-test. p-value < 0.05 was considered as statistical significance.

RSL3 ((1S,3R)-RSL3) is an inhibitor of GPX4 (ferroptosis activator) and induces ferroptotic death in numerous cell types. To set up a ferroptosis model of intestinal epithelial cells, we treated IEC-6 cells with different concentrations of RSL3 and observed the RSL3-induced cell death. CCK-8 assay revealed that RSL3 treatment, even at low concentrations of 0.125 μM, significantly reduced cell viability of IEC-6 cells. Ferrostatin-1 (Fer-1), a specific ferroptosis inhibitor, could significantly rescue RSL3-induced cell death (Fig. 1A) and Giemsa staining of RSL3-treated cells revealed typical ferroptotic morphological changes, such as cell volume shrinkage, cell membrane disruption, the cell morphology (Fig. 1B). Ferroptosis is characterized by the production of plasma membrane phospholipid peroxide-induced by ROS in iron-mediated Fenton reactions. RSL3 treatment caused a rapid generation of ROS in IEC-6 cells, which could be reversed by Fer-1. Thus, RSL3-induced IEC-6 cell death is a typical ferroptosis model of epithelial cells (Fig. 1C).

To identify the inhibitors of RSL3-induced ferroptosis, we screened a small natural product library from APExBIO with 66 compounds in IEC-6 cells. Cell viability was measured using CCK-8 in IEC-6 cells following treatment with 2.5 μM natural product compounds for 48 h. Twelve compounds were screened to protect the IEC-6 cells from RSL3-induced cell ferroptosis (Fig. 2). Among these, Tan IIA was shown to significantly reverse RSL3-induced ferroptosis.

Then we treated IEC-6 cells with different concentrations of Tan IIA in the presence of RSL3. CCK-8 assay and Giemsa staining revealed that Tan IIA could rescue RSL3-induced cell death at a concentration of 0.5 μM. Tan IIA could not stimulate the growth of IEC-6 cells even at a high concentration of 5 μM, whereas Tan IIA significantly protected IEC-6 cells from RSL3-induced ferroptosis even at a low concentration of 0.5 μM (Fig. 3A). Fig. 3B shows the morphological characteristics of RSL3-treated IEC-6 cells in the presence or absence of Tan IIA by Giemsa staining. Furthermore, examination of microstructures of cells by TEM (Fig. 3C) revealed that RSL3-induced cell mitochondrial shrinkage and membrane disruption, the inner cristae of mitochondria were reduced or absent. Thus, both Fer-1 and Tan IIA protected the IEC-6 cells from ferroptosis. Moreover, Tan IIA decreased the ROS level in RSL3-induced IEC-6 cells (Fig. 3D).

Ferroptosis suppressors such as GPX4, SLC7A11, and miR-17-92 have been proven to inhibit ferroptosis of endothelial cells (Bai et al., 2020; Xiao et al., 2019). The expression of these genes in RSL3-treated IEC-6 cells in the presence of Tan IIA was further investigated. GPX4, SLC7A11 and miR-17-92 function as RSL3 response genes in IEC-6 cells. The expressions of GPX4 and SLC7A11 are shown in Fig. 4A, and miR-17-92 cluster expressions at 24, 48, and 72 h are shown in Fig. 4B. RSL3 treatment significantly induced the expression of these genes within 24 h. Treatment of IEC-6 cells with 5 μM Tan IIA significantly upregulated GPX4 and SLC7A11 until 48 and 72 h, and also partly upregulated miR-17-92 expression. Moreover, Tan IIA treatment upregulated the levels of GPX4 and SLC7A11 proteins (Figs. 4C and 4D). Therefore, GPX4 and SLC7A11 might mediate the suppression of epithelial cell ferroptosis induced by Tan IIA.