Animals and models

Male 8–12 weeks old wild-type (WT) mice (Cyagen, Suzhou, China) on the C57BL/6 background were used in this experiment. YAP1flox/flox mice were crossbred with Alb-Cre mice to generate liver-specific YAP1-deficient mice (termed as YAP1fl/fl Alb-Cre) [17]. Tamoxifen (Sanbio #13258) was intraperitoneally injected on five consecutive days (16 mg/kg in oil), followed by a 3-week wash out period. The control group mice were Yapflox/flox. Control mice and YAP1-deficient mice were randomly distributed into sham and CLP groups. All experiments were executed following the criteria of the NIH and authorized by Animal Ethics Committee of Wuhan University (No. 2021187).

According to previous researches [18], The sepsis-associated liver injury model mice were established by cecal ligation and puncture (CLP). After anaesthetized with an intraperitoneal injection of pentobarbital sodium (50 mg/kg), the abdomen was sterilized with povidone iodine, then about 2 cm midline incision was conducted in the mice’s lower abdomen. After that, the cecum was exposed and ligated with a 3-0 silk. We punctured the cecum with a 21-gauge needle twice. After gently squeezing the cecum to push the feces into the abdominal cavity, the cecum was put back to the original location followed by suturing the peritoneum layer by layer. Volume resuscitation was performed immediately after surgery with 30 ml/kg ringer saline. Only abdominal incision was performed on the WT mice, exposed for 5 min and then closed in layers. The mice enjoyed free access of food and water and were monitored until been sacrificed at 24 h post-operation.

Liver function and pro-inflammatory factors assessment

Plasma levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were analyzed using the commercial ELISA kits (R&D Systems, USA) following the manufacturer’s instructions. TNF-α, IL-1β and IL-6 in liver tissue were detected using the Duoset ELISA system (R&D systems, MN) according to the manufacturer’s instructions.

Transmission electron microscopy (TEM)

The left liver lobe was resected and fixed in glutaraldehyde, then the samples were dehydrated with a cascade ethanol series and cut into ultrathin slices. The slices were dyed with lead citrate and uranyl acetate respectively, in turn, observed using a HT-7500 transmission electron microscope (Hitachi. Co., Tokyo, Japan).

Histological analysis

The left-liver samples were isolated and fixed with 4% paraformaldehyde. Following paraffin embedding and slicing, hematoxylin–eosin (HE), macrophages (CD68) (AbD Serotec, Raleigh, NC), neutrophils (Ly6G) (BD Biosciences, San Jose, CA), was utilized to stain liver samples. The degree of liver damage was assessed by two independent technicians who were unknown the experimental group protocols in line with the recently published criterion [19]. The histological photographs were observed by optical microscopy (Nikon, Japan).

Immunohistochemical stain (IHC stain)

Liver tissues were paraffin fixed and incubated overnight at 37 °C. Then, these liver sections were deparaffinized and hatched with 3% hydrogen peroxide for 15 min. The slices were heated at microwave treatment and then naturally cooled for 40 min. Then the samples were incubated with anti-NCOA4 and anti-SLC7A11. Lastly, homologous fluorescent or biotin-labeled secondary antibodies were incubated with the liver tissues for observing protein expression.

Primary hepatocytes culture and treatments

Mice were killed and perfused with 75% alcohol for 2–3 s. For the isolation of hepatocytes, livers were then taken and minced, then placed in a dish containing PBS. Then, the buffer was replaced with 0.1% collagenase I solution (Sigma, C0130) in HBSS (containing 4 mM CaCl2, 0.8 mM MgSO4) at 37 °C for 10 min. Freed from the liver, hepatocytes were collected and filtered through a sterile 100 µm pore size nylon mesh, then wash 2–3 times at 4 °C for 4 min. Hepatocytes were cultured in Williams’ E medium (Invitrogen, Carlsbad, CA) added with 100 lg/mL streptomycin, 10% fetal bovine serum, 100 U/mL penicillin, 0.5 IU/mL insulin, and 10 lg/mL dexamethasone. LPS (1 µg/mL, Sigma, Aldrich, USA) was given to generate cell injury models in vitro for 24 h.

Cells transfection

Human liver LO2 cells (American Type Culture Collection [ATCC], Manassas, VA, USA, USA) were cultured with fetal bovine serum (10%) in DMEM (Gibco, USA) and arranged at an incubator containing 5% CO2 with suitable temperature (37 °C). YAP1 overexpression (YAP1 OE) clone lentiviral particle (supplied by Genechem Co. LTD Shanghai, China) were transfected to LO2 cells to constitute the stable YAP1 OE cell lines. Transfection of YAP1 was accomplished referring to the working instructions, cells transfected with scramble were used as negative controls. LO2 cell were transfected with siRNA specific for YAP1 (Shanghai GenePharma Co. China) by using Lipofectamine 2000 (Thermo Fisher Scientific, USA) according to the manufacturer’s instruction. Following expansion and maintenance, stable LO2 cells YAP1 OE or YAP1−/− were selected to detection. LPS (1 µg/mL, Sigma, Aldrich, USA) was given to generate hepatocyte injury models in vitro for 24 h.

Cell viability assay

As previously illustrated, cell viability was examined by the CCK-8 assay kit (Beyotim, Shanghai, China) [20].

Determination of ROS

LO2 cells were suspended by 0.25% trypsin and subsequently centrifuged at 1500 rpm (4 °C, 5min).The cells were incubated with the 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) fluorescent probe (D6883, Sigma-Aldrich, MO, USA) in serum-free medium following the manufacturer’s protocols. The level of ROS in LO2 cells was acquired by a TE-2000 fluorescent microscope (Nikon, Tokyo, Japan) at an excitation (Ex) wavelength of 485 nm and emission (Em) wavelength of 530 nm. After BODIPY-581/591-C11 fluorescence probe was added into cell culture, a flow cytometry (BD Accuri C6 plus, BD Biosciences, USA) and FlowJo software were adopted aiming to detect the lipid ROS level.

The ROS level of liver tissues was evaluated with the dihydroethidium (DHE) fluorescent probe (D7008, Sigma-Aldrich, MO, USA). The already frozen sections were incubated with 50 μm DHE away from light at 37 °C for 30 min. To obtain the cell images, we used Nikon TE-2000 fluorescent microscope (Tokyo, Japan) at (Ex/Em) 525 nm/610 nm.

Determination of mitochondria ROS

LO2 cells were seeded in 24-well lucifugal plate, then incubated with 5 mmol/L MitoSOX™ reagent solution for 10 min. The cells were washed two times by PBS, the level of mitochondria ROS was assessed by a fluorescence microscope (TE-2000, Nikon, Co., Tokyo, Japan).

Detection of related indicators

The levels of ferrous iron (Fe2+), malondialdehyde (MDA) and glutathione (GSH) in LO2 cells and liver tissues lysate were detected by the iron assay kit (MAK025, Sigma-Aldrich, MO, USA), MDA assay kit (Beyotime, China) and GSH assay kit (Beyotime, Shanghai, China) according to the relevant manufacturer’s protocols.

Western blot

We extracted the lysate from liver tissues or cells in a RIPA buffer and protein contents were quantified by the BCA protein assay kit (Beyotime, China). Protein samples (50 µg) from each group underwent the 10% SDS-PAGE gel electrophoresis and then transferred to a PVDF membrane. After blocked with nonfat dry milk (5%), these blots were hatched with the primary antibodies all night at 4 °C. GPX4, ACSL4, SFXN1, LC3, NCOA4, FTH1, SLC7A11, YAP1or β-actin. Moreover, the blots were cleaned in TBST and hatched with the secondary antibody at 37 °C lasting 120 min. An ECL kit was applied to detect the bands, which were assayed by the Image J software.

Distribution of ferrous iron in the Mito Tracker

The MitoTracker Green fluorescent probe (Beyotime, China) was firstly added to the LO2 cells for 30 min, then the previous solution was replaced by fresh PBS. Subsequently, the prewarming Ferro Orange working solution was used to treat the cells kept in lucifuge place for 30 min, and PBS was used to wash the medium. The cell photographs were captured by a confocal microscope (TCS-SP2, Leica, German) to acquire the mitochondrial ferrous iron and green fluorescent lysosomes at Ex and Em wavelengths of 488 nm and 510–550 nm, respectively.

Ferritin and LAMP2

LO2 Cells were fixed as pre-described and immunostained by anti-ferritin (Abcam, ab75973, UK) and anti-LAMP2 (Proteintech, 66301-1-Ig, USA). Then, samples were incubated with secondary antibodies or DAPI. LO2 cells were photographed using Confocal laser scanning microscopy and fluorescence microscopy (TE-2000, Nikon, Co., Tokyo, Japan). Quantification of the ferritin and lysosomes in each hole cells were analyzed and calculated by software Image-pro Plus 6.0.

Co-localization of LC3 and ferritin

LO2 cells in 4 groups were transfected with LC3-GFP plasmid, while the autophagosome in cells presented green fluorescence puncta. Following the manipulation manuals, we firstly blocked the cells with 0.1% BSA (A2153, Sigma-Aldrich, USA) for 1 h, then applied the primary and secondary antibodies to the medium. After using anti-Ferritin Heavy chain1, LO2 cells were analyzed via Confocal laser scanning microscopy and evaluated by fluorescence microscopy (Nikon TE-2000, Tokyo, Japan).

Co-immunoprecipitation (IP) assay

The concentration of protein in LO2 cells was assessed by BCA protein (Beyotime, Shanghai, China) assay kit. 1 mg total protein lysates extracted from each group were utilized for immunoprecipitation (IP). The specimen was hatched with rabbit polyclonal IgG control antibody. After rotating the lysates lasting 4 h at 4 °C, a total volume of 25 µL resuspended of protein A/G plus Agarose was mixed with the lysates and the mixture solution continued rotating lasting 2 h again. The immunoprecipitation buffer was washed and denatured, then the elution proteins were immunoblotted with the antibody (anti-NCOA4, anti-Ferritin), the co-IP assay was performed.

RNA sequence analysis

First, RNA was extracted from CLP or normal liver tissue. Bioanalyzer 2100 system was used to detect RNA purity. Ribo-off rRNA Depletion Kit (N406-02, Vazyme, China) and MGIEasy RNA Library Prep Kit (1000006385, MGI, China) were used as to generate sequencing libraries in terms of manufacturer’s instructions. Then, after purifying the PCR products by the AMPureXP system, the library quality was analyzed (Agilent Bioanalyzer 2100 system). Finally, the index codes were clustered by Cluster Kit of HiSeq 4000 PE (Illumina, USA), the sequenced library preparations were performed for 150 cycles on the former platform. Total library constructions and sequencing were implemented at BGI Wuhan. Hierarchical clustered heatmap and Genomes enrichment analysis were adopted to detect the differential expression of both groups. Differentially expressed genes (DEGs) were displayed for log2 Fold Change > 1 and adjusted p value < 0.05.

Statistical analysis

The Prism 8.0 software (GraphPad, San Diego, CA) was used to analyze the data presented as Means ± SD. Comparisons between two groups were performed with a two-tailed unpaired t-test. Discrepancies among four groups were checked by one-way ANOVA analysis. Welch correction was applied when necessary. P < 0.05 was defined as statistically significant.