Animals

Hepatocyte-specific ERRFI1 knockout (ERRFI1-HKO) mice were generated by mating ERRFI1-flox mice (Cyagen Biosciences) with Albumin (Alb)-enhancer/promoter-driven Cre transgenic mice (GemPharmatech Co., Ltd.). C57BL/6J wild-type (WT) mice (Liaoning Changsheng biotechnology Co., Ltd.) and ERRFI1-HKO mice were housed in a specific pathogen-free facility (temperature of 24 ± 2 °C, humidity between 30 and 70%) with alternating 12:12 h light-dark cycles and were allowed access to food and water ad libitum.

Mouse model of hepatic IR

All animal experiments were approved by the Animal Care and Use Committee of The First Affiliated Hospital of Jinzhou Medical University. The experiments were conducted in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. Following anesthesia, 8- to 10-week-old mice were incised at the ventral midline to expose the liver hilum. A noninvasive vascular clamp was employed to interrupt the hepatic artery/portal venous blood supply to the left and middle lobes. After 90 min of ischemia, the clamp was removed to restore hepatic blood flow, and the abdomen was closed with sutures. To maintain body temperature throughout the surgical procedures, mice were placed supine on a heating pad under a heat lamp. No hepatic blood flow blockage was performed in Sham mice. Blood samples and ischemic hepatic tissues were collected at 6 h of reperfusion.

Biochemical analysis

Blood samples were centrifuged to obtain serum. The degree of hepatic injury was detected by the serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) using the ALT Activity Assay Kit (ab105134; Abcam, Cambridge, UK) and the AST Activity Assay Kit (ab138878; Abcam).

The levels of glutathione (GSH) and malondialdehyde (MDA) in liver tissues and hepatocytes were measured using commercial kits (A006, A003; Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions.

Hematoxylin and eosin (H&E) staining

Liver tissues were fixed with paraformaldehyde (Sinopharm Chemical Reagent Beijing Co., Ltd., Beijing, China), and dehydrated with gradient alcohol. Afterwards, the tissues were embedded in paraffin (M060590, MREDA) and sectioned into 5-µm slices. The slices were deparaffinized with xylene (Sinopharm Chemical Reagent Beijing Co., Ltd.) and then rehydrated in gradient alcohol. The sections were stained with hematoxylin (Sigma, St Louis, MO, USA), incubated in hydrochloric acid solution (Sinopharm Chemical Reagent Beijing Co., Ltd.), and stained with eosin (Xiya Reagent, Linyi, China). After dehydration and sealing with neutral gum (Solarbio Science & Technology, Co., Ltd., Beijing, China), the sections were observed under a microscope (OLYMPUS, Tokyo, Japan) at a magnification of 200×. The histological severity of hepatic injury was graded using Suzuki’s method. The Suzuki criteria are as follows: congestion (none = 0, minimal = 1, mild = 2, moderate = 3, severe = 4), vacuolization (none = 0, minimal = 1, mild = 2, moderate = 3, severe = 4) and necrosis (none = 0, single cell necrosis = 1, < 30% = 2, 30–60% = 3, > 60% = 4); scores for each parameter ranged from 0 to 4, with a maximum score of 12.

Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining

For liver tissues, the sections were dewaxed and rehydrated. For cell slides, the slides were fixed with paraformaldehyde. A TUNEL Cell Apoptosis Detection Kit (G1504; Servicebio, Wuhan, China) was employed to determine the apoptotic cells. The images were photographed using a microscope (OLYMPUS) at a magnification of 400×.

Western blot analysis

Total proteins from liver tissues and hepatocytes were extracted by RIPA lysis buffer (AS1004; Aspen Biological, Wuhan, China). The protein concentration in the supernatants was measured using a BCA protein assay kit (AS1086, Aspen Biological). Proteins were loaded on sodium dodecyl sulfate-polyacrylamide gels and transferred to PVDF membranes. After blocking, membranes were incubated with primary antibodies overnight at 4℃, followed by horseradish peroxidase-conjugated secondary antibodies (Aspen Biological). Finally, protein bands were visualized by an enhanced chemiluminescence kit (AS1059, Aspen Biological). The following primary antibodies were used: antibodies for ERRFI1 (sc-137,154; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Bax (#2772; Cell Signaling Technology, Danvers, MA, USA), Bcl-2 (ab182858, Abcam), cleaved caspase-3 (AF7022; Affinity Biosciences, Cincinnati, OH, USA), caspase-3 (AF6311, Affinity Biosciences), GRB2 (ab32037, Abcam), and GAPDH (ab181602, Abcam).

ROS measurement

ROS production was evaluated using the ROS sensitive fluorescent probe DCFH-DA (S0033; Beyotime Institute of Biotechnology, Haimen, China). Liver homogenate or hepatocytes were incubated with DCFH-DA probe at 37℃ for 20 min. After washing with serum-free solution three times, cell samples were analyzed using a fluorescence microplate reader.

Ferrous iron (Fe2+) measurement

An Iron Assay Kit (ab83366, Abcam) was employed to measure the iron concentration in liver homogenate or cell lysates. Briefly, assay buffer was added to liver homogenate or cell lysates and incubated for 30 min at 37℃, then iron probe was added and incubated for 60 min at 37℃. The absorbance at 593 nm was measured using a microplate reader. An iron standard curve was constructed using known concentrations of iron solutions. The iron concentration was normalized to the protein concentration.

Real-time PCR analysis

RNAiso Plus (9109; Takara, Tokyo, Japan) was employed to isolate total RNA from liver tissues or hepatocytes, and the PrimeScript™ RT reagent Kit (RR047A, Takara) was used to synthesize cDNA. Quantitative real-time PCR was carried out with SYBR Green PCR Master Mix (QPK-201; TOYOBO, Osaka, Japan). The relative gene-expression was normalized to the RNA level of the housekeeping gene β-actin, and the data were calculated according to the 2−ΔΔCt method. The primer sequences were as follows: mouse ACSL4: Forward 5’-CTTCCTCTTAAGGCCGGGAC-3’, Reverse 5’-TGCCATAGCGTTTTTCTTAGATTT-3’; mouse SLC7A11: Forward 5’-TTGCAAGCTCACAGCAATTC-3’, Reverse 5’-AGGGCAACCCCATTAGACTT-3’; mouse GPX4: Forward 5’-TGCATCGTCACCAACGTGGC-3’, Reverse 5’-CTTCACCACGCAGCCGTTCT-3’; human ACSL4: Forward 5’-GGAATGACAGGCCAGTGTGA-3’, Reverse 5’-TAGCACATGAGCCAAAGGCA-3’; human SLC7A11: Forward 5’-GGGCATGTCTCTGACCATCT-3’, Reverse 5’-TCCCAATTCAGCATAAGACAAA-3’; human GPX4: Forward 5’-CGATACGCTGAGTGTGGTTTGC-3’, Reverse 5’-CATTTCCCAGGATGCCCTTG-3’; human ERRFI1: Forward 5’-CTGGAGCAGTCGCAGTGAG-3’, Reverse 5’-GCCATTCATCGGAGCAGATTTG-3’; and human GRB2: Forward 5’-CCATCGCCAAATATGACTTCAAA-3’, Reverse 5’-CTTCGTTCAAAACCTTGAGGATGT-3’.

Immunohistochemical staining

Liver sections were incubated with 3% H2O2 (Sinopharm Chemical Reagent Beijing Co., Ltd.) to block endogenous peroxidase. Then the sections were incubated with primary antibodies diluted in bovine serum albumin (BSA; Biofroxx, Einhausen, Germany) overnight at 4℃. Primary antibodies were: 8-OHdG (ab48508; Abcam), ACSL4 antibody (22401-1-AP; Proteintech Group, Wuhan, China), SLC7A11 antibody (26864-1-AP, Proteintech Group), and GPX4 antibody (67763-1-Ig, Proteintech Group). After washing with PBS, the sections were incubated with the goat anti-rabbit/mouse IgG secondary antibodies (Aspen Biological) at 37℃ for 30 min. Subsequently, the sections were stained with 3,3’-diaminobenzidine (DAB; ZLI-9019; ZSGB-Bio, Beijing, China) and counterstained with hematoxylin (Sigma). The sections were subjected to dehydration and sealing, and were observed under a microscope (OLYMPUS) at a magnification of 400×.

Cell culture and treatments

The normal human liver cell line L-02 was purchased from iCell Bioscience Inc (Shanghai, China). L-02 cells were cultured in RPMI-1640 medium (iCell Bioscience Inc) containing 20% fetal bovine serum (FBS) and 1% penicillin-streptomycin mixture and maintained in a humidified incubator with 5% CO2 at 37℃.

For cell treatments, L-02 cells were treated with 10 µM MG132 (S1748, Beyotime Institute of Biotechnology) for 4 h, or treated with 100 µg/mL cycloheximide (CHX; ab120093, Abcam) for 0, 3, 6, or 9 h.

Cell transfection

Specific shRNA oligonucleotides targeting ERRFI1 mRNA and nontargeting version of shRNA were inserted into the pRNA-H1.1 plasmid to construct sh-ERRFI1 and sh-NC vectors. The PCR-amplified coding sequence of GRB2 was cloned into the pCDNA3.1(+) plasmid to generate GRB2 overexpression plasmid. L-02 cells were transfected with the above vectors using Lipofectamine 3000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.

Oxygen-glucose deprivation and reoxygenation (OGD/R) model

For oxygen-glucose deprivation experiments, normal culture medium was replaced with glucose-free RPMI-1640 medium. Cells in this group were incubated in a hypoxic environment with 5% CO2, 1% O2, and 94% N2 in a 37℃ incubator for 4 h. For reoxygenation, the glucose-free medium was then replaced with normal culture medium, and the cells were cultured under normal conditions (95% air, 5% CO2) for 12 h.

CCK-8 assay

L-02 cells were seeded in 96-well plates at a density of 5 × 103 cells/well. After OGD/R treatment, 10 µL CCK8 reagent (C0042, Beyotime Institute of Biotechnology) was added to each well and incubated at 37℃ for 4 h. The absorbance was detected at 450 nm using a microplate reader (Bio-Rad; Hercules, CA, USA).

Determination of lipid peroxidation

To determine OGD/R-induced lipid peroxidation in vitro, L-02 cells with different treatment were collected. After washing with PBS, 10 µM C11-BODIPY 581/591 (Invitrogen; Carlsbad, CA, USA) was added to cell medium and incubated at 37℃ for 1 h. The nuclei were labeled with Hoechst 33,258 (Solarbio Science & Technology, Co., Ltd.). Fluorescence intensity was measured using flow cytometry (BD; Franklin Lakes, NJ, USA).

Immunofluorescence staining

To perform immunofluorescence staining, cell slides were prepared. The slides were fixed with 4% paraformaldehyde. After being washed with PBS, the slides were incubated with primary antibody against GPX4 (67763-1-Ig), ACSL4 antibody (22401-1-AP), and SLC7A11 antibody (26864-1-AP) overnight at 4℃. The slides were then washed, and incubated with the secondary antibody at 37℃ for 40 min. Nuclear staining was conducted using 4’,6-diamidino-2-phenylindole (DAPI, Sigma) at room temperature for 30 min in the dark. The slides were observed under a fluorescence microscope (OLYMPUS) at a magnification of 400×.

Propidium iodide (PI) staining

Propidium iodide (PI; P8080, Solarbio Science & Technology, Co., Ltd.) was dissolved in DMSO at a final concentration of 1.5 mM. L-02 cells with different treatment were fixed with 4% paraformaldehyde and stained with PI dye in the dark for 10 min. After removal of PI dye and being washed with PBS, the cells were covered with antifade mounting medium and observed under a fluorescence microscope (OLYMPUS) at a magnification of 200×.

Co-immunoprecipitation (Co-IP)

Cells were lysed with lysis buffer on ice. Then cell lysates were adjusted to equal amounts of protein and immunoprecipitated with GRB2 antibody (ab32037, Abcam) at 4℃ overnight. Rabbit IgG control antibody (#3423; Cell Signaling Technology, Danvers, MA, USA) was used as a negative control. The immunocomplex was pulled down with Protein A/G PLUS-Agarose (sc-2003, Santa Cruz Biotechnology). Immunoprecipitates were subjected to SDS-PAGE, and coimmunoprecipitated proteins were detected by immunoblotting with GRB2 antibody (ab32037, Abcam) or ERRFI1 (sc-137,154, Santa Cruz Biotechnology).

Statistical analysis

Data are expressed as the mean ± standard deviation (SD). Analysis was performed using GraphPad Prism 9.0. Data were analyzed using Student’s t test and one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test, and a p value < 0.05 was considered statistically significant.