C57BL/6 mice (Beijing Huafukang Biotechnology Co., Ltd.) were housed under a standard 12 h light/dark cycle with ad libitum access to food and water. The mice were fed a high-fat diet (HFD, 60% fat) for 12 weeks to induce hepatic steatosis. All animal protocols were approved by the Animal Ethics Committee of Qingdao University (approval no. 20260401C57BL/6452026 0601066) and conducted in accordance with NIH guidelines (publication no. 88-23, revised 1996).

AML12 cells (The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences; https://www.cellbank.org.cn/search-detail.php?id=893) were cultured in RPMI 1640 medium (GUMD-B303; HyCyte™; Suzhou Haixing Biotechnology Co., Ltd.) supplemented with 10% foetal bovine serum (FCP-X310; HyCyte™; Suzhou Haixing Biotechnology Co., Ltd.) and 1% penicillin-streptomycin at 37°C in a humidified 5% CO₂ atmosphere. To establish an in vitro MASLD model, AML12 cells were treated with a 750 μM free fatty acid mixture composed of palmitic acid (PA; 12 mM) and oleic acid (OA; 12 mM) at a 1:2 molar ratio at 37°C for 24 h. This specific concentration and OA-enriched ratio was selected based on a previously established protocol to robustly induce hepatic lipid accumulation while minimising lipotoxicity (25).

For hypoxia/reoxygenation (H/R) experiments, the cells were subjected to 6 h of hypoxia (37°C, 1% O₂, 5% CO₂) in a gas-controlled incubator, followed by standard 12 h of reoxygenation (37°C, 21% O₂, 5% CO₂) for the indicated durations.

HY-149394 (cat. no. HY-149394), HY-10249A (cat. no. HY-10249A), HY-18749 (cat. no. HY-18749), MG132 (cat. no. HY-13259) and chloroquine (CQ; cat. no. HY-17589A) were all purchased from MedChemExpress. For in vitro experiments (cell H/R experiments), cells were treated with HY-149394 (50 μM), HY-10249A (5 μM), HY-18749 (10 μM), MG132 (20 μM) or CQ (50 μM) for 10 h at 37°C. In the H/R model, these compounds were added at the onset of reoxygenation (following 6 h of hypoxia) and maintained throughout the 12 h reoxygenation period. For in vivo experiments, mice were administered HY-149394 (10 mg/kg), HY-10249A (55 mg/kg) or HY-18749 (10 mg/kg) via intraperitoneal injection at 1 h prior to the induction of ischaemia.

For lipid accumulation analysis, cells cultured in confocal dishes or tissue sections were washed with PBS and fixed with 4% paraformaldehyde for 30 min at room temperature. After washing with PBS, the samples were stained with Oil Red O working solution (cat: BL941A; Biosharp Life Sciences) for 20 min at room temperature, followed by differentiation with 60% isopropanol for 5 sec. The samples were then washed with distilled water, counterstained with hematoxylin (cat: BL702B; Biosharp Life Sciences) for 10 min at room temperature and examined under a CKX53 cell culture microscope (Olympus Corporation).

The intracellular ROS level was determined using DCFH-DA (cat: S0033S, Beyotime Biotechnology). After overnight seeding of the cells, the cells were subjected to H/R treatment at 37°C. Subsequently, the cells were washed with PBS and incubated with 10 μM DCFH-DA in serum-free medium at 37°C in the dark for 20 min. After three washes with PBS, the fluorescence signal was detected by fluorescence microscopy.

Male C57BL/6 mice (aged 8 weeks, weighing 22-25 g) were randomly allocated to experimental groups. Following an overnight fast (~12 h) with ad libitum access to water, the mice were anaesthetised via intraperitoneal injection of pentobarbital sodium (50 mg/kg). Subcutaneous injection of buprenorphine (0.1 mg/kg) was administered for postoperative analgesia. During the surgical procedure, core body temperature was continuously monitored and maintained at 37°C using a feedback-controlled heating pad.

A standard 70% hepatic I/R model was established. After midline laparotomy, systemic heparinisation was achieved via intravenous injection of heparin (100 U/kg). The blood supply to the left and median liver lobes was occluded using an atraumatic microvascular clamp. Successful occlusion was immediately confirmed by visual blanching (pale discolouration) of the targeted liver lobes.

After 60 min of ischaemia, the clamp was removed to initiate reperfusion, which was confirmed by the restoration of colour in the ischaemic lobes. Sham-operated mice underwent identical surgical procedures, including laparotomy and vessel dissection, but without vascular occlusion. The 6 h reperfusion time points were selected based on preliminary experiments and previous literature (25), establishing these time points as the peak of acute liver injury and inflammatory responses in this model.

At the end of the designated reperfusion period (6 h), mice were euthanized with an intraperitoneal overdose of pentobarbital (150 mg/kg) for collection of blood and liver tissue samples. To minimize potential bias, all subsequent histological and biochemical assessments were performed by investigators blinded to the experimental groups.

Mice were intravenously injected via the tail vein with 100 μl of recombinant adenovirus encoding Orm1 (the adenovirus delivers shRNA targeting Orm1) (1.0×10¹² viral particles/ml (GIMRA Genomics; Shanghai GenePharma Co., Ltd.) or an equal volume of empty control adenovirus (NC). At 3 days postinjection, the mice were subjected to the aforementioned hepatic I/R procedure. shRNA primers can be found in Table SI.

The overexpression plasmid (100 μg/μl, GIMRA Genomics; Shanghai GenePharma Co., Ltd.) and transfection reagent (Lipofectamine™ 3000; Invitrogen; Thermo Fisher Scientific, Inc.) were mixed and incubated at room temperature for 15 min. The mixture was then added dropwise to the cultured cells and incubated at 37°C for 48 h (Table SI). The mixture was then added dropwise to the cultured cells and incubated at 37°C for 48 h (Table SI), after which the cells were harvested and subjected to subsequent experiments.

Serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) were measured using an automatic chemistry analyser (AU5800; Olympus Corporation) and serum concentrations of triglyceride (TG) was determined using the TG assay kit (cat. no. A110; Nanjing Jiancheng Bioengineering Institute).

Liver tissue samples were fixed in 4% paraformaldehyde at room temperature for 48 h and then embedded in paraffin for immunohistochemical analysis. Following deparaffinisation in xylene and rehydration through a graded series of ethanol (100, 95, 80, and 70%), antigen retrieval was carried out by heating the sections in citrate buffer (pH 6.0) at 95°C for 20 min, the tissue sections were blocked and permeabilised with a solution containing 3% bovine serum albumin (cat. no. IA0910; Beijing Solarbio Science & Technology Co., Ltd.) at room temperature for 2 h and 0.4% Triton X-100 (cat. no. T8200; Beijing Solarbio Science & Technology Co., Ltd.) at room temperature for 1 h. The sections were then incubated with the corresponding primary antibodies (Orm1; 1:100; cat. no. 16439-1-AP; Proteintech Group, Inc.) overnight (12 h) at 4°C, followed by incubation (2 h) at room temperature with the appropriate secondary antibodies (Anti-rabbit IgG H+L; 1:5,000; cat. no. RGAM009; Proteintech Group, Inc.). Antibody details can be found in Table SII.

At room temperature, AML12 cells cultured in confocal dishes were fixed with 4% paraformaldehyde (30 min), permeabilised with 0.1% Triton X-100 (2 h) and blocked with 5% goat serum (30 min) (cat: BL1097A; Biosharp Life Sciences) for immunofluorescence analysis. Following an overnight incubation with specific primary antibodies for Orm1 (1:400; cat. no. 16439-1-AP; Proteintech Group, Inc.) and Prdx1 (1:200; cat. no. 15816-1-AP; Proteintech Group, Inc.) at 4°C, the cells were then labelled with Alexa Fluor 488-(1:100; cat no. SA00013-2; Proteintech Group, Inc.) and 594-(1:100; cat. no. SA00013-3; Proteintech Group, Inc.) conjugated secondary antibodies at room temperature for 2 h and counterstained with DAPI (cat no. 28718-90-3; Beijing Solarbio Science & Technology Co., Ltd.) at room temperature for 10 min. Images were acquired using a Leica STELLARIS 5 (Leica Microsystems GmbH) confocal laser scanning microscope. Antibody details can be found in Table SII.

Hoechst/PI staining was performed to assess cell apoptosis and necrosis. AML12 cells were seeded in 12-well plates and subjected to the indicated treatments. The staining was performed according to the manufacturer's protocol (cat. no. C1056; Beyotime Biotechnology), and images were captured using a fluorescence microscope.

Total RNA was extracted from frozen samples and cultured cells using TRIzol reagent (cat. no. 15596018; Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol (n=3 per group). RNA quality was assessed using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Inc.), 1% agarose gel electrophoresis and an Agilent 5300 Fragment Analyzer (Agilent Technologies, Inc.) to determine RIN/RQN values; only samples with ≥1 μg total RNA, ≥30 ng/μl concentration, RQN >6.5 and OD260/280 of 1.8-2.2 were used. Library construction and paired-end (PE150) sequencing were performed by Shanghai Majorbio Bio-Pharm Technology Co., Ltd. using an Illumina NovaSeq 6000 platform with a NovaSeq 6000 SP reagent kit (100 cycles; cat. no. 2002746; Illumina, Inc.), with the final library loaded at 1-20 pM as quantified by Qubit 4.0 and Agilent 5300 (Agilent Technologies, Inc.). Differentially expressed genes (DEGs) were identified using the DESeq2 package (version 1.36.0; https://bioconductor.org/packages/DESeq2/) in R software (version 4.2.1; RStudio, Inc.), with significance thresholds of |log₂ fold change|>1 and adjusted P-value (false discovery rate) <0.05. Kyoto encyclopaedia of genes and genomes (KEGG; https://www.kegg.jp/kegg/pathway.html) pathway enrichment analysis was performed using the Majorbio Cloud Platform (Shanghai Majorbio Bio-Pharm Technology Co., Ltd.; https://cloud.majorbio.com)

Gels were silver-stained using a Fast Silver Stain Kit (cat. no. P0017S; Beyotime Biotechnology) according to the manufacturer's instructions. Briefly, gels were fixed (50% ethanol/10% acetic acid, 20 min at room temperature), washed with 30% ethanol (10 min at room temperature) and Milli-Q water (10 min at room temperature), sensitized (2 min at room temperature), stained with 1x silver solution (10 min at room temperature) and developed until protein bands were visible at room temperature. The reaction was stopped, and gels were washed and stored in Milli-Q water at room temperature.

Cells were lysed using NP-40 lysis buffer (cat. no. P0013F; Beyotime Biotechnology). For immunoprecipitation, the lysates were incubated overnight at 4°C with specific antibodies for Orm1 (1:50; cat. no. 16439-1-AP; Proteintech Group, Inc.) or control IgG (1:50; cat. no. A7028, Beyotime Biotechnology). Protein A/G magnetic beads (20 μl; cat. no. P1791; Beyotime Biotechnology) were then added to the lysates and incubated for an additional 2 h at room temperature with gentle rotation. After extensive washing, bead-bound immunocomplexes were resolved by SDS-PAGE, and the subsequent immunoblotting procedure was performed as described in the 'Western blot analysis' section. Gel bands of interest were excised and sent to Shanghai Majorbio Bio-Pharm Technology Co., Ltd. for LC-MS/MS analysis. Samples were reduced with 10 mM TCEP (37°C, 60 min), alkylated with 40 mM IAM (room temperature, 40 min in the dark), precipitated with acetone and digested with trypsin (1:40, m/m) at 37°C overnight. Peptides were desalted on a C18 column and quantified with a NanoDrop One (cat: 840-317400, Thermo Fisher Scientific, Inc.). A total of 400 ng of peptides was loaded onto the column for each LC-MS/MS run. Liquid chromatography-mass spectrometry was performed on a Vanquish Neo HPLC system (Thermo Fisher Scientific, Inc.) coupled to a timsTOF HT mass spectrometer (Bruker Corporation) with Compass HyStar software (Bruker Corporation). Separation was conducted on a homemade column (15 cm ×100 μm, 1.7 μm) over 8 min at a flow rate of 300 nl/min using mobile phases A (2% acetonitrile/0.1% formic acid) and B (80% acetonitrile/0.1% formic acid). Data were acquired in positive ionisation mode with a capillary voltage of 1.6 kV, a nitrogen drying gas temperature of 180°C, an ion mobility range of 0.7-1.3 V·sec/cm², and a mass scan range of 300-1,500 m/z in data-independent acquisition mode. As Co-IP magnetic bead samples were used, no additional quality control was performed prior to mass spectrometry.

Total protein was extracted using RIPA lysis buffer (Beyotime Biotechnology). Protein concentration was determined using a BCA protein assay kit (cat. no. PC0020; Beijing Solarbio Science & Technology Co., Ltd.) according to the manufacturer's instructions. Following quantification, the protein concentrations were adjusted accordingly, and equal amounts of protein (20 μg per lane) were loaded into SDS-polyacrylamide gels with varying concentrations (8%, 10% or 12%). These gel samples were subsequently subjected to electrophoresis, and the proteins obtained were then transferred onto polyvinylidene fluoride membranes. The membrane was incubated with specific primary antibodies overnight at 4°C, followed by incubation with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies (1:50,000; cat no. HA1023; HUABIO) for 2 h at room temperature. The primary antibodies used in the present study included anti-Orm1 (1:5,000; cat. 16439-1-AP; Proteintech Group, Inc.), anti-Bcl2 (1:1,000; cat. no. 3498 T; Cell Signaling Technology), anti-Bax (1:1,000; cat. no. 2772 T; Cell Signaling Technology), anti-Pi3k (1:1,000; cat. no. HA722522; HUABIO), anti-Akt (1:2,000; cat. no. HA721870; HUABIO), anti-P-Pi3k (1:2,000; cat. no. HA721673; HUABIO), anti-P-Akt (1:1,000; cat. no. HA724447; HUABIO) and anti-β-actin (1:5,000; cat. no. AB0035; Abways) antibodies. The protein bands were visualised using an enhanced chemiluminescence detection system (Tanon Science and Technology Co., Ltd.). The details of relevant antibodies are presented in Table SII.

Total RNA was extracted using an RNA rapid extraction kit (Suzhou Xinsaimei Biotechnology Co., Ltd.) according to the manufacturer's instructions. Reverse transcription was performed using a PrimeScript RT kit (cat. no. 639505; Takara Bio, Inc.) according to the manufacturer's instructions. Briefly, genomic DNA was removed by incubation with gDNA Eraser at 42°C for 2 min, followed by reverse transcription at 37°C for 15 min and inactivation at 85°C for 5 sec. RT-qPCR was performed using TransStart^® Green qPCR SuperMix UDG (cat. no. AQ111; TransGen Biotech Co., Ltd.), which contains SYBR Green I as the fluorescent dye. Each 20 μl reaction mixture contained 10 μl of 2x TransStart^® Green qPCR SuperMix UDG, 0.4 μl of forward primer (10 μM), 0.4 μl of reverse primer (10 μM), 0.4 μl of Passive Reference Dye (50x; added according to the qPCR instrument used), an appropriate amount of template and nuclease-free water to a final volume of 20 μl. The thermocycling conditions were as follows: 50°C for 2 min (UDG incubation), 94°C for 10 min (UDG inactivation), followed by 40-45 cycles of 94°C for 5 sec, 60°C for 15 sec and 72°C for 10 sec, followed by a dissociation stage. Fluorescence signals were acquired during the annealing or extension step, with acquisition times adjusted according to the manufacturer's instructions for the specific instrument. β-actin (Proteintech Group, Inc.) was used as the endogenous control gene. Relative gene expression was calculated using the 2^−ΔΔCq method (26). The relevant primers are detailed in Table SIII.

A CCK-8 was obtained from Suzhou Xinsaimei Biotechnology Co., Ltd. AML12 cells were seeded into 96-well plates at a density of 1×10⁴ cells per well and cultured in complete medium for 12 h. Following the indicated treatments and H/R exposure, 10 μl of CCK-8 solution was added to each well. After the cells were incubated for an additional 2 h at 37°C, the absorbance at 450 nm was measured using a microplate reader.

Liver tissues were fixed in 4% paraformaldehyde for 1 h at room temperature, embedded in paraffin and cut into 5-μm-thick sections. Following standard deparaffinisation and rehydration, the sections were permeabilised with proteinase K (20 μg/ml) for 20 min at 37°C and washed twice with PBS. TUNEL staining was performed using a TUNEL apoptosis detection kit (cat. no. G4890; Beijing Solarbio Science & Technology Co., Ltd.) according to the manufacturer's instructions. Briefly, sections were incubated with 50 μl of TUNEL reaction mixture containing TdT enzyme and FITC-labelled dUTP at 37°C for 60 min in the dark. After washing with PBS, nuclei were counterstained with DAPI (10 μg/ml) for 5-10 min at room temperature in the dark. Sections were then mounted with an anti-fade mounting medium and images were captured using a fluorescence microscope (Olympus Corporation) with excitation at 450-500 nm and emission at 515-565 nm.

To assess protein stability, cells were treated with the protein synthesis inhibitor CHX (50 μM; cat. no. HY-12320; MedChemExpress) for the indicated durations (37°C; 0, 2, 4 and 8 h). After treatment, the cells were collected and lysed in RIPA buffer supplemented with protease inhibitors. The lysates were sonicated and centrifuged at 12,000 × g, for 15 min at 4°C, and the resulting supernatants were collected. Protein samples were then mixed with loading buffer, denatured, resolved by SDS-PAGE, and analysed by western blotting (as aforementioned) using specific antibodies to evaluate protein expression levels over time.

In the present study, the GSE193764 dataset was obtained from the Gene Expression Omnibus (GEO) database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE193764). This dataset includes 12 pretransplant and 12 posttransplant samples from rats with steatotic livers. Differential expression analysis was performed using R software (version 4.2.1; RStudio, Inc.). The resulting DEGs were subsequently validated using the GSE279693 dataset (7), where genes showing consistent expression trends with our dataset were considered validated. For single-cell RNA-seq data (CRA004061) (27), which were downloaded from the Genome Sequence Archive of the National Genomics Data Center (https://ngdc.cncb.ac.cn/gsa/), dimensionality reduction and gene-to-cell mapping analyses were conducted using Seurat v4 (https://satijalab.org/seurat/).

Sample sizes for in vivo experiments (n=4 per group) were determined based on our preliminary studies and similar publications in the field to ensure adequate statistical power (28). The animals were randomly allocated to different experimental groups. To minimise bias, histological evaluations and signal quantifications were performed by investigators who were blinded to the group assignments. Preestablished exclusion criteria included surgical mortality before the designated endpoint/severe infection; no other data points or samples were excluded. All in vitro experiments were performed with at least three independent biological replicates, with specific replicate numbers detailed in the respective figure legends.

Statistical analysis and data visualisation were performed using GraphPad Prism (version 9.0; Dotmatics). Data are presented as the mean ± standard deviation (SD). Before applying the parametric tests, the assumption of normality was assessed using the Shapiro-Wilk test, and the homogeneity of variance was evaluated using the Brown-Forsythe test or Levene's test.

Comparisons between two independent groups were performed using an unpaired two-tailed Student's t test for normally distributed data with equal variances. Welch's t test was applied when the assumption of equal variance was violated, and the Mann-Whitney U test was used for non-normally distributed data. Experiments involving three or more groups (such as multiarm rescue experiments and inhibitor/agonist treatments), data were analysed using one-way analysis of variance (ANOVA) followed by Tukey's post hoc test. Two-way ANOVA followed by Bonferroni post hoc correction was employed to analyse data with two independent variables. The Kruskal-Wallis test followed by Dunn's post hoc test was utilised for non-parametric data across multiple groups.

Representative images were selected from independent experiments with results approximating the group mean. P<0.05 was considered to indicate a statistically significant difference.

The rat transcriptomic dataset GSE193764 was analysed to identify key molecular drivers of I/R in steatotic livers. Differential expression analysis revealed 1,551 DEGs, including 1,112 upregulated and 439 downregulated genes (Table SIV). Notably, Orm1 emerged as a significantly upregulated candidate gene following transplantation (Figs. 1A and S1). To further validate these findings, the mouse dataset GSE279693 was examined, which confirmed a consistent and significant increase in Orm1 expression during I/R in steatotic livers (Fig. 1B). Given this marked upregulation, the cellular source of Orm1 was further investigated using the CRA004061 single-cell dataset. Dimensionality reduction and cluster analysis revealed that Orm1 was predominantly expressed in hepatocytes (Fig. 1C-F). This cell type specificity is consistent with the findings of previous reports identifying hepatocytes as the primary site of Orm1 synthesis (29).

To validate Orm1 expression, a mouse model of hepatic I/R in steatotic livers was employed (Fig. 2A). After 12 weeks of HFD feeding, H&E staining revealed moderate hepatic steatosis, characterised by the presence of lipid droplets (Fig. 2B), in line with prior reports (30). Serum and liver tissue TG levels were significantly greater in HFD-fed mice compared with normal diet-fed mice (Fig. 2C and D), and their body weights were also significantly greater (Fig. S2A).

HFD-fed mice were then subjected to 1 h of ischaemia followed by 6 h of reperfusion. Western blotting and RT-qPCR analysis demonstrated that I/R significantly upregulated Orm1 expression in steatotic liver tissues (Fig. 2E and F). Immunohistochemical analysis further confirmed elevated Orm1 levels after I/R, as reflected by increased immunohistochemical scores (Fig. 2G and H). In parallel, primary hepatocytes isolated from HFD-fed mice exhibited steatotic degeneration and were subjected to 6 h of hypoxia followed by 6 h of reoxygenation. Consistent with the aforementioned findings, both western blotting and RT-qPCR revealed a marked increase in Orm1 expression following H/R (Fig. S2B and C).

Collectively, these findings suggest that the upregulation of Orm1 expression represents an endogenous compensatory protective response to I/R in steatotic livers.

Mice with specific Orm1 knockdown were generated using a recombinant adenovirus expressing shRNA targeting Orm1 (Adv-shOrm1). These mice were then subjected them to hepatic I/R to investigate the functional role of Orm1 in steatotic liver I/R (Fig. 3A). Western blotting and RT-qPCR confirmed that Orm1 expression was significantly lower in the liver tissues of Adv-shOrm1-treated mice than in those of control mice (Fig. 3B and C). Liver injury was assessed by measuring serum levels of AST and ALT, as well as by histologically evaluating of necrosis. Notably, Orm1 knockdown resulted in significantly elevated serum AST and ALT levels (Fig. 3D and E). Moreover, H&E staining revealed that the area of necrotic tissue was markedly larger in the Adv-shOrm1 group than in the Adv-shNC group (Fig. 3F). Given that apoptosis plays a critical role in the progression of hepatic I/R (31), the effect of Orm1 knockdown on hepatocyte apoptosis was further examined. TUNEL staining revealed a significant increase in the number of apoptotic cells in the livers of Adv-shOrm1 mice (Fig. 3G). Consistent with these findings, western blot analysis revealed that, compared with the control treatment, the knockdown of Orm1 led to upregulated expression of the pro-apoptotic protein Bax and downregulated expression of the anti-apoptotic protein Bcl-2 (Fig. 3H). These results indicate that Orm1 deficiency exacerbates liver injury and apoptosis following steatotic liver I/R.

An in vitro model of steatotic hepatocytes was established by treating AML12 cells with a PA:OA mixture to further clarify the functional role of Orm1 in hepatic I/R accompanied by steatosis. The oil red staining results revealed that the liver cells stimulated by the PA:OA mixture exhibited accumulation of lipids within the cells (Fig. S3A). Following successful modelling, an Orm1 overexpression plasmid was constructed to overexpress Orm1 in steatotic hepatocytes. Western blotting and RT-qPCR revealed that the expression of Orm1 was significantly increased in liver cells (Fig. S3B and C). Compared with the control, Orm1 overexpression significantly reduced the expression of the pro-apoptotic protein Bax and increased the expression of the anti-apoptotic protein Bcl-2 (Fig. S3D). Furthermore, overexpression of Orm1 markedly enhanced cell viability and decreased the number of PI-positive cells following H/R injury (Fig. S3E and F). These results suggest that Orm1 protects steatotic hepatocytes against apoptosis and cell death under ischaemic stress.

RNA-seq was performed on liver tissues from Orm1 knockdown and wild-type mice following I/R to investigate the role of Orm1 in steatotic liver I/R. A total of 3,327 DEGs were identified, comprising 1,809 upregulated and 1,518 downregulated genes (Fig. 4A). KEGG pathway enrichment analysis revealed the top 20 signalling pathways ranked by P-value, among which the 'PI3K-Akt signalling pathway' was significantly enriched (Fig. 4B). Western blot analysis revealed that Orm1 knockdown markedly reduced the phosphorylation levels of Pi3k and Akt (Figs. 4C and S4A). Conversely, Orm1 overexpression in steatotic liver cells significantly increased phosphorylation levels of Pi3k and Akt (Figs. 4D and S4B).

The involvement of the Pi3k/Akt pathway was subsequently verified in vivo using mice treated with specific Adv-shOrm1. After 1 h of treatment with HY-18749 (an Akt agonist), the role of the Pi3k/Akt pathway was assessed in hepatic I/R induced by Orm1 knockdown in the steatotic liver. HY-18749 significantly reversed the detrimental effects of Orm1 knockdown following hepatic I/R (Fig. 5A-E). ALT and AST levels were significantly decreased (Fig. 5B and C), and the area of necrotic tissue was significantly reduced (Fig. 5D and E). Rescue experiments were performed using an Akt inhibitor (HY-10249A) to investigate the role of phosphorylated Pi3k and Akt in Orm1-mediated hepatic I/R in the steatotic liver. The results showed that treatment with an Akt inhibitor significantly aggravated liver injury, which was alleviated by Orm1 overexpression. CCK-8 and Hoechst/PI staining assays further revealed that, compared with the vector group, the Akt inhibitor markedly reduced the viability of steatotic liver cells (Fig. S5A and B). Hence, these findings suggest that Orm1 regulates hepatic I/R in the steatotic liver by modulating the activity of the Pi3k/Akt signalling pathway.

A combined immunoprecipitation-mass spectrometry (IP-MS) approach was used to identify novel Orm1-interacting proteins to further elucidate the mechanism by which Orm1 regulates Pi3k/Akt signalling. Prdx1 was ranked highest among the differentially enriched proteins associated with the Pi3k/Akt pathway when Orm1 antibody immunoprecipitates were compared with IgG controls (Fig. 5F and G and Table SV) (32,33). To validate the MS data, Co-IP assays were performed to confirm the physical interaction between Orm1 and Prdx1 (Fig. 5H). Additionally, confocal immunofluorescence microscopy revealed substantial colocalization of endogenous Orm1 and Prdx1 in AML12 cells (Fig. 5I). To further substantiate the physical interaction between Orm1 and Prdx1, an exogenous Co-IP assay was performed. Plasmids encoding Flag-tagged Orm1 (Flag-Orm1) and HA-tagged Prdx1 (HA-Prdx1) were co-transfected into cells. Subsequent immunoprecipitation using anti-Flag magnetic beads successfully co-precipitated HA-Prdx1. Notably, no HA signal was detected in the immunoprecipitates from the negative control cells transfected with HA-Prdx1 alone (Fig. S3G). This robust interaction between the exogenously tagged proteins, alongside the endogenous Co-IP and mass spectrometry data, validates the specific physical association between Orm1 and Prdx1.

Next, the cells were treated with the Prdx1-specific inhibitor HY-149394 to explore the functional role of Prdx1 in I/R. CCK-8 and Hoechst/PI staining revealed that, compared with DMSO treatment, Prdx1 inhibition significantly reduced hepatocyte viability and increased cell death (Fig. 6A and B). Furthermore, Prdx1 inhibition markedly suppressed the phosphorylation of Pi3k and Akt, upregulated the expression of the proapoptotic molecule Bax and downregulated the expression of the antiapoptotic protein Bcl-2 (Figs. 6C and S4C). Next, it was investigated whether altered Orm1 expression could concomitantly affect Prdx1 expression. Prdx1 protein levels were upregulated upon Orm1 overexpression and downregulated upon Orm1 knockdown (Fig. 6D and E); however, no significant changes were observed at the mRNA level (Fig. S5C), suggesting posttranscriptional or posttranslational regulation. These results indicate that Prdx1 activation protects hepatocytes against I/R through the Pi3k/Akt pathway and that Orm1 may regulate hepatocyte survival via the modulation of Prdx1 protein stability or activity.

Previous studies have demonstrated that Prdx1 undergoes constitutive ubiquitination (34,35) and that endogenous protein degradation primarily occurs through the ubiquitin-proteasome and lysosomal pathways. CHX chase assays were performed to assess Prdx1 degradation dynamics. Orm1 overexpression significantly attenuated Prdx1 degradation in AML12 cells (Fig. 6F and G). To elucidate the degradation pathway, the cells were treated with the proteasome inhibitor MG132 or the lysosome inhibitor CQ. MG132 treatment restored Prdx1 levels in Orm1-knockdown cells, whereas CQ had no significant effect (Fig. 6H), indicating that Prdx1 is degraded primarily via the proteasomal pathway. Furthermore, cell-based ubiquitination assays revealed that Orm1 overexpression markedly reduced Prdx1 ubiquitination (Fig. 6I), suggesting that Orm1 protects Prdx1 from ubiquitin-mediated proteasomal degradation.

The role of Prdx1 in I/R of steatotic liver was further investigated. CCK-8 and Hoechst/PI staining assays revealed that the protective effect of Orm1 overexpression on hepatocyte injury was significantly reversed by a Prdx1 inhibitor (Fig. 7A and B), indicating that Prdx1 mediates the hepatoprotective effects of Orm1 in I/R. Subsequent in vivo experiments demonstrated that administration of an Akt agonist alleviated hepatic I/R induced by Prdx1 inhibition. Compared with the control group, which received the Akt agonist, the experimental group showed improved liver function (Fig. 7C and D). H&E staining revealed a reduction in the area of necrosis in the liver (Fig. 7E), along with a decrease in the proportion of TUNEL-positive cells (Fig. 7F). Overall, these findings indicate that overexpression of Orm1 alleviates hepatic I/R through the Prdx1-Pi3k-Akt axis. Prior to the subsequent experiments, the transfection efficiency of sh-Prdx1 was confirmed by RT-qPCR and western blotting (Fig. S5D and E). Furthermore, considering that oxidative stress is one of the core changes during cell and tissue H/R, the cells were subjected to H/R treatment. The results revealed that overexpression of Orm1 reduced the H/R-induced ROS production and (Figs. S6A-D) and that inhibition of Prdx1 expression reversed the protective effect of Orm1 overexpression (Fig. S6A-D). Subsequently, Prdx1-knockdown cells were treated with an AKT agonist, and the results revealed that the AKT agonist reversed the cell damage and ROS production caused by Prdx1 (Fig. S6A-D).