HMOX1 Inhibits Ferroptosis in Non-Alcoholic Fatty Liver Disease

HMOX1 Inhibits Ferroptosis in Non-Alcoholic Fatty Liver Disease | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article HMOX1 Inhibits Ferroptosis in Non-Alcoholic Fatty Liver Disease Li Yao, Jun Zou, Xin-ze Qiu, Jing-rong Liang, Meng-bin Qin, Jin-xiu Zhang, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5035306/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background & Aims: This research seeks to elucidate the significance of ferroptosis-related genes in the diagnosis, prognosis, and treatment of non-alcoholic fatty liver disease (NAFLD). Methods: Key genes pertinent to NAFLD were identified using the GEO database. The role of Heme oxygenase-1 (HMOX1) in NAFLD was validated via immunohistochemical analysis of hepatic tissues. Mice on a methionine-choline-deficient (MCD) diet were administered Hemin, followed by the collection of serum and liver samples for biochemical and histopathological examinations. HL7702 cells were transfected with a plasmid to elevate HMOX1 expression, then treated with oleic acid (OA) to induce lipid accumulation, and subsequently with erastin and AZD1480. A series of assays measured iron levels, reactive oxygen species, lipid peroxidation, and mitochondrial damage. Western blotting analysis was employed to elucidate the underlying molecular mechanisms. Results: HMOX1 is crucial in the pathogenesis of NAFLD, evidenced by its decreased expression in patient liver tissues. Mice on an MCD diet exhibited significant hepatic steatosis, along with elevated levels of ALT, AST, TG, LDL, Fe 2+ , MDA, and ROS, and reduced levels of HMOX1 and GSH. Notably, Hemin effectively ameliorated NAFLD and prevented ferroptosis. Cellular analysis revealed activation of the JAK/STAT pathway in NAFLD. Upregulation of HMOX1 reduced OA-induced lipid peroxidation, inhibited ferroptosis, and suppressed the JAK/STAT pathway. Erastin negated the protective effects of HMOX1 overexpression. Moreover, the JAK/STAT pathway inhibitor AZD1480, which had the opposite effect with erastin, suppressed ferroptosis and ameliorated NAFLD. Conclusions: This study elucidates that HMOX1 suppresses ferroptosis by inhibiting the JAK/STAT pathway in NAFLD. Heme oxygenase-1 Nonalcoholic fatty liver disease Ferroptosis Bioinformatics analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1 INTRODUCTION Non-alcoholic fatty liver disease (NAFLD) is a chronic hepatic condition characterized by lipid metabolism disruptions, closely associated with insulin resistance and genetic factors ( 1 ). NAFLD is regarded as the hepatic manifestation of metabolic syndrome, encompassing simple fat accumulation (steatosis), non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and hepatocellular carcinoma. The prevalence of NAFLD has escalated to 25% in Western populations ( 2 ), reaching up to 80% among individuals with obesity ( 3 ). NAFLD significantly contributes to advanced liver disease and represents a substantial public health challenge. Contemporary research indicates that NAFLD is associated with a myriad of factors, including inflammation, oxidative stress, insulin resistance, lipid metabolism disorders, obesity, and endoplasmic reticulum stress ( 4 , 5 ). The "multiple-hit hypothesis" is posited as the central mechanism driving NAFLD pathogenesis. The initial insult involves hepatic steatosis induced by insulin resistance, leading to hepatic fat accumulation and impaired fatty acid export. Subsequent insults result in increased reactive oxygen species (ROS) production, culminating in oxidative stress and inflammation ( 6 ). Furthermore, oxidative stress contributes to mitochondrial dysfunction ( 7 ), often characterized by lipid peroxidation ( 8 ). ROS targets polyunsaturated fatty acid double bonds, producing lipid peroxides such as 4-hydroxy-2-nonenal (4-HNE) and malondialdehyde (MDA), which subsequently cause cellular damage ( 6 , 9 ). Ferroptosis, a novel form of programmed cell death, is driven by iron-dependent lipid peroxidation ( 10 ). Mitochondria undergoing ferroptosis exhibit distinctive morphological changes, including reduced size, increased membrane density, diminished or absent mitochondrial cristae, and ruptured outer membranes ( 11 , 12 ), differentiating it from apoptosis, necrosis, and autophagy both morphologically and mechanistically. Abnormal iron metabolism and lipid peroxidation, stemming from iron-dependent depletion of glutathione (GSH) and inactivation of glutathione peroxidase 4 (GPX4), along with the accumulation of polyunsaturated fatty acid-containing phospholipids (PUFA-PLs), act as catalysts for ferroptosis ( 13 ). This mode of cell death is implicated in various human diseases, including neurodegenerative disorders ( 14 , 15 ), cardiovascular diseases ( 16 ), cancer ( 17 ), and notably, liver diseases ( 18 ). Emerging evidence suggests that ferroptosis plays a crucial role in the pathological progression of NAFLD. During NAFLD development, oxidative stress induced by reactive oxygen species (ROS) is identified as the primary catalyst for cellular ferroptosis ( 19 , 20 ). The liver, as the central organ for iron storage and lipid metabolism, is particularly susceptible to ferroptosis. Inhibition of ferroptosis holds the potential for enhancing liver function and mitigating liver damage ( 21 , 22 ). While initial studies on ferroptosis predominantly targeted cancer, specific ferroptosis-related genes (FRGs) have emerged as valuable biomarkers for diagnosing, prognosticating, and treating various cancers ( 23 – 25 ). Nevertheless, the potential application of FRGs in NAFLD management remains elusive. This study utilized NAFLD datasets from the Gene Expression Omnibus (GEO) and FRGs from GeneCards to identify reliable differentially expressed genes (DEGs) associated with ferroptosis in NAFLD. Heme oxygenase-1 (HMOX1) was identified as a crucial biomarker in our study, and its ability to inhibit the ferroptotic process in NAFLD was validated. Additionally, the underlying mechanisms were explored through both in vivo and in vitro experiments. 2 MATERIALS AND METHODS 2.1Detection of Key DEGs Associated with Ferroptosis The microarray dataset GSE89632, associated with NAFLD, was sourced from the GEO database. FRGs were initially filtered from the GeneCards website using the term “ferroptosis.” Subsequent data analysis was performed using the R project (version 4.0.3). Probe names in the gene expression profiles were first transformed into gene names based on the platform's annotation files. DEGs were then identified using the “limma” package, with an adjusted p-value of less than 0.05 and a fold change exceeding |log2FoldChange| of 1.5. The R package “umap” (v0.2.3.1) was used to perform Uniform Manifold Approximation and Projection (UMAP). Critical DEGs associated with ferroptosis were determined using an online Venn diagram tool ( http://bioinformatics.psb.ugent.be/webtools/Venn/ ). 2.2 PPI network construction and analysis of modules The STRING database was employed to analyze protein-protein interactions (PPI) and identify interactions among target proteins. Interaction scores above 0.4 were visualized in the PPI network using Cytoscape software (v3.9.0), where genes were depicted as nodes and their interactions as edges. Clustering analysis was performed using the molecular complex detection (MCODE) algorithm to identify key modules within the PPI network. Each module was associated with a specific biological function, with statistical significance set at p < 0.05. 2.3 Functional enrichment analysis The “clusterProfiler” package ( 26 ) in R was employed to perform enrichment analyses for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) related to ferroptosis-related DEGs. GO analysis was divided into three categories: biological processes (BP), cellular components (CC), and molecular functions (MF), essential for investigating biological roles. KEGG analysis was utilized to identify potential pathways. The STRING online platform ( https://string-db.org/ ) facilitated the protein-protein interaction (PPI) network analysis for ferroptosis-related DEGs. The results from STRING were subsequently imported into Cytoscape (version 3.8.2), where the “cytoHubba” plugin was used to extract key subnetworks. Hub genes were identified by selecting the ten genes with the highest scores using the maximum correlation criterion (MCC algorithm) within the subnetworks. Gene-set enrichment analysis (GSEA) was employed to classify genes based on their differential expression between two samples and to compare entire genome expression profiles against predefined gene sets ( 27 , 28 ). In the GSEA performed via WebGestalt, gene sets containing fewer than three or more than 2,000 genes were excluded by default. The KEGG analysis was based on WebGestalt's GSEA results. 2.4 Reagents and chemicals The antibodies anti-GPX4 (10701-1-AP) and anti-HMOX1 (14432-1-AP) were sourced from Proteintech, Wuhan, China. Erastin (HY-15673), AZD1480 (HY-10193), and Hemin (HY-19424) were provided by MCE, New Jersey, USA. Lipofectamine 2000 (11668-027) was procured from Invitrogen, California, USA, and oleic acid (0108484-500ml) from Aladdin, Shanghai, China. Kits for ALT (C009-2-1), AST (C010-2-1), TG (A110-1-1), and LDL (A113-1-1) were obtained from Nanjing Jiancheng Bioengineering Institute, Nanjing, China. GSH, Fe 2+ , MDA, and ROS ELISA kits were acquired from Mlbio, Shanghai, China. Dihydroethidium (S0063) was purchased from Beyotime, Shanghai, China. Additionally, anti-JAK2 (A7694), anti-P-JAK2 (AP0374), anti-STAT1 (A12075), anti-P-STAT1 (AP0135), anti-STAT3 (A1192), and anti-P-STAT3 (AP0474) were procured from Abclonal, Wuhan, China. 2.5 Liver biopsy The study involved 31 liver tissue samples from patients with NAFLD and 30 from individuals with normal liver function. Liver biopsies were conducted on 61 patients using either the percutaneous method with a Menghini needle or through surgery. Liver tissues from the normal group were obtained from patients who underwent surgery for noncancerous liver conditions. The biopsy samples underwent standard staining procedures and were examined by a single pathologist blinded to patients' clinical and laboratory data. NAFLD/NASH was assessed using the NAFLD Activity Score (NAS), evaluating steatosis (0–3), lobular inflammation (0–3), and hepatocellular ballooning (0–2), and the SAF score system, which includes steatosis (0–3), activity (lobular inflammation [0–2] + hepatocellular ballooning [0–2]), and fibrosis (0–4). 2.6 Histological and immunohistochemical analyses Fresh liver tissue was embedded in paraffin and sectioned into 4 µm thick slices for periodic acid–Schiff (PAS) staining. Monoclonal antibodies anti-HMOX1 (1:200 dilution, Proteintech) and anti-GPX4 (1:500 dilution, Proteintech) were used for immunohistochemical staining. Images were captured using the Olympus Microscope Camera System (Tokyo, Japan). Protein expression was analyzed by examining five randomly selected fields per slide. Immunohistochemical scoring assessed both staining intensity and the proportion of positive cells. Staining intensity was categorized as follows: 0 for no staining, 1 for light yellow, 2 for yellow, and 3 for brown. The proportion of positively stained cells was scored as follows: less than 5% scored 0, 5–25% scored 1, 26–50% scored 2, and greater than 50% scored 3. The final score for each sample was determined by multiplying these two scores: a score of 0–2 was denoted as (−), 3–4 as (+), and 5 or higher as (++) ( 29 ). 2.7 Animals and treatments Approval for all animal experiments was obtained from the Animal Ethics Committee at Guangxi Medical University. Male C57BL/6J mice, aged 6–8 weeks, were sourced from The Center for Comparative Medicine at Yangzhou University, China. The mice were maintained on a 12-hour light-dark cycle at 22 ± 2°C, with 55% humidity, and provided ad libitum access to food and water. A one-week acclimatization period was allowed before the experiment, during which the mice were fed either a normal chow diet (NCD) or a methionine/choline-deficient diet (MCD) for 8 weeks. The mice were randomly divided into three groups, each with five individuals: ( 1 ) control group - NCD diet; ( 2 ) MCD group - MCD diet; ( 3 ) MCD + Hemin group - MCD diet with intraperitoneal (i.p.) injections of the HMOX1 inducer hemin (30 µmol/kg) three times weekly. After the 8-week experimental period, all animals were fasted overnight before being euthanized. Liver tissue and serum samples were collected for further analysis. The experimental protocols complied with the guidelines of the Guangxi Committee for Care and Use of Laboratory Animals and were approved by the Animal Experimentation Ethics Committee at Guangxi Medical University. 2.8 Cell Culture and Transient Transfection and Oleic acidinduced steatosis HL7702 cell lines, provided by the Cell Bank of the Chinese Academy of Sciences in Shanghai, China, were cultured in Dulbecco's Modified Eagle's Medium (DMEM) with high glucose, supplemented with 10% FBS. Incubation was conducted at 37°C with 5% CO2. Transient transfection was performed in 6-well plates using plasmids from GenePharma, Shanghai, China, and Lipofectamine 2000 from Invitrogen, California, USA. After six hours, the serum-free medium was replaced with a complete medium, and cells were incubated for an additional 48 hours before collection. Total RNA was extracted using a Trizol reagent from Biosharp, Wuhan, China, and converted to cDNA with a reverse transcription kit from YE SEN, Shanghai, China. Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction (qRT-PCR) was conducted on a Roche instrument from Shanghai, China, and relative gene expression levels were calculated using the 2-ΔΔCt method. Primers were designed with Primer 5.0 software, with sequences as follows: HMOX1 – forward: 5′-AAGACTGCGTTCCTGCTCAAC-3′, reverse: 5′-AAAGCCCTACAGCAACTGTCG-3′; β-actin – forward: 5′-ACGTGGACATCCGCAAAG-3′, reverse: 5′-TGGAAGGTGGACAGCGAGGC-3′. cDNA amplification started with an initial step at 95°C for 5 minutes, followed by 40 cycles of 10 seconds at 95°C and 30 seconds at 60°C. HL7702 cells were plated in 6-well plates at a density of 2×10⁵ cells per well and treated with 1 mM OA for 24 hours. Post-treatment, cells were analyzed using ELISA, Western blotting, flow cytometry, and transmission electron microscopy (TEM). 2.9 Histological and Biochemical Analyses and ROS Assessment Fresh liver stored at -80°C was sectioned into 4 µm slices, then heated, dried, fixed with 4% paraformaldehyde, and stained with oil red O (Servicebio, China) in the dark for 10 minutes. Subsequently, sections were stained with hematoxylin (Servicebio, China) for 5 minutes following differentiation with 60% isopropanol. Levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides (TG), low-density lipoprotein (LDL), GSH, MDA, and Fe 2+ were determined using ELISA kits according to the manufacturer's instructions. Hepatic ROS levels were assessed with an ROS assay kit (Mlbio, China) following the manufacturer’s guidelines. Intracellular ROS levels were evaluated using flow cytometry with Dihydroethidium (Beyotime, Nanjing Jiancheng Biotechnology Institute, China). 2.10 Western blotting analysis Proteins from liver tissues and HL7702 cells were extracted using RIPA lysis buffer and PMSF. Protein concentrations were determined using the BCA Protein Assay Reagent (Biosharp, China). A total of 30 µg of protein was separated on a 10% SDS–PAGE gel and transferred onto nitrocellulose membranes. The membranes were blocked with 5% BSA in 1× TBST at room temperature for 1 hour, followed by overnight incubation with primary antibodies at 4°C. Protein bands were visualized by incubating with horseradish peroxidase-conjugated secondary antibodies and imaged using the One-piece chemiluminescence imager (BIO-RAD, USA). 2.11 Transmission electron microscopy The liver was washed twice in PBS and then sliced. Cells were collected until the sediment reached the size of a mung bean. The cells were fixed in an electron microscope holder at room temperature for 2 hours and then stored at 4°C. Morphological alterations in the mitochondria were visualized using a transmission electron microscope (TEM). 2.12 Statistical Analysis Mean values were reported with standard deviation (SD). Continuous variables were compared using Student’s t-test, and categorical variables were evaluated using chi-square or Fisher’s exact test. Group differences were analyzed through ANOVA. Data analysis was performed using R software (version 4.1.0) and SPSS (version 19.0). Statistical significance was defined as p < 0.05 (two-tailed). 3 RESULTS 3.1 HMOX1 Is an Essential Ferroptosis-Related DEG Identified From NAFLD Datasets 3.1.1 Identification of Ferroptosis-Related DEGs in NAFLD The microarray dataset related to NAFLD, GSE89632, was retrieved from the GEO database. Clinical characteristics of NAFLD within the GSE89632 dataset are presented in Table A.1 . Differential expression analysis was conducted using the “limma” package on RNA samples from whole blood of NAFLD patients and normal controls. A total of 1,995 DEGs were identified, with 1,121 downregulated (green dots in Fig. 1A) and 874 upregulated (red dots in Fig. 1A). Ferroptosis-related genes were subsequently extracted from the GeneCards platform using the keyword “ferroptosis.” The left count (1,908 genes) corresponds to DEGs specific to GSE89632, the middle count (87 genes) to ferroptosis-related DEGs, and the right count (1,853 genes) to unique ferroptosis genes (Fig. 1D). The heatmap in Fig. 1B shows the differential expression of genes between NAFLD and control groups in the GSE89632 dataset, with 20 upregulated and 20 downregulated genes. UMAP analysis of DEGs is presented in Fig. 1C. Figure 1D categorizes the 87 ferroptosis-related DEGs, including 56 downregulated and 31 upregulated genes in NAFLD, as detailed in Table A.2 . Within this subset, 2 genes serve as markers, 17 function as drivers, and 25 act as suppressors, as specified in Table A.3 . Notably, HMOX1 is implicated as both a driver and suppressor gene according to Table A.3 . Figure 1 Overview of Ferroptosis-Related DEGs in NAFLD and Controls: (A) Volcano plot of genes differentially expressed between NAFLD and controls in the GSE89632 dataset. Blue nodes represent down-regulation in NAFLD; red nodes represent up-regulation; and gray nodes represent no significant difference from controls; (B) Heatmap of DEGs in NAFLD. The heatmap shows the top 20 upregulated (red, positive value) and downregulated (blue, negative value) DEGs in these samples; (C) UMAP analysis of DEGs in NAFLDL; (D) Venn diagram of the ferroptosis-related DEGs. The Venn diagram indicates 87 co-DEGs (i.e., ferroptosis-related DEGs) from the intersection of the ferroptosis dataset (1940 genes) with DEGs in NAFLD above. 3.1.2 Enrichment Analysis of Ferroptosis-Related DEGs and PPI Network Analysis The KEGG pathway analysis (Fig. 2A) identified the top ten enriched pathways, including the Micro-RNA tumor signaling pathway, ferroptosis, HIF-1 signaling pathway, and JAK/STAT signaling pathway. The GO-BP analysis (Fig. 2B) highlighted pathways such as cellular response to chemical stimulus and response to organic substance. The GO-CC analysis (Fig. 2C) indicated significant enrichment in the cytosol. The GO-MF enrichment analysis (Fig. 2D) revealed key findings related to enzyme binding and oxidoreductase activity. The PPI network construction (Fig. 2E) displayed 51 nodes and 175 edges, with 36 of the 87 genes lacking interactions and thus not forming molecular networks. The network settings were adjusted to a default cutoff (interaction score > 0.4) using the STRING database. Hub genes were identified using the Cytoscape CytoHubba plug-in, selecting the top ten genes with the highest scores according to the MCC algorithm (HMOX1, JUN, MYC, STAT3, IL6, FOXO1, ERBB2, PTGS2, ATF3, CDKN1A) (Fig. 2F). Venn diagram analysis (Fig. 3A) identified a single co-DEG (HMOX1) shared between the ferroptosis pathway genes in KEGG (4 genes) and the hub genes in NAFLD (10 genes). GSEA analysis results (top five NES and P values < 0.05) showed enrichment in pathways such as JAK/STAT signaling, LEISHMANIA INFECTION, COMPLEMENT AND COAGULATION CASCADES, and CYTOKINE-CYTOKINE RECEPTOR INTERACTION (Fig. 3B). Figure 2 Enrichment Analysis of Ferroptosis-Related DEGs and PPI Network Analysis. (A) Top 10 KEGG pathways; (B) Top 10 GO biological processes pathways; (C) Top 10 GO cellular component pathways; (D) Top 10 GO molecular function pathways; (E) PPI network of ferroptosis-related DEGs; (F) Subnetwork of hub genes from the PPI network. Node color reflects the degree of connectivity (red represents a higher degree). Figure 3 Identification of the Hub Gene and GSEA. (A) Venn diagram of the hub genes and the genes in the ferroptosis pathway in KEGG. The Venn diagram indicates one co-DEG (HMOX1) from the intersection of the ferroptosis pathway genes in KEGG (4 genes) with the hub genes in NAFLD; (B) GSEA analysis of the ferroptosis-related DEGs. 3.2 Expression and Clinical Feature of HMOX1 and GPX4 in NAFLD Tissues HMOX1 and Glutathione Peroxidase 4 (GPX4) expression levels in NAFLD tissues were significantly lower than those in normal liver tissues (Fig. 4 and Table 1 ). Additionally, HMOX1 expression strongly correlates with both BMI and GPX4 expression in NAFLD tissues (Table 2 ,Table A.4 and Table A.5 ). Table 1 HMOX 1 expression in NAFLD and normal liver tissues. N HMOX1 χ χ 2 P + - Normal 30 28 2 42.690 <0.001 NAFLD 31 3 28 Table 2 Correlation between HMOX 1 expression and GPX4 in NAFLD. Expression GPX4 Correlation p - + HMOX1 - 23 2 0.712 <0.001 + 1 5 HMOX1,Heme oxygenase-1;GPX4,Glutathione peroxidase 4. Figure 4 Aberrant Expression of HMOX1 and GPX4 in NAFLD. Immunohistochemical staining of HMOX1 and GPX4 in NAFLD and normal liver tissues (magnification, ×200, **** p < 0.0001 vs. normal group). Data are presented as mean ± SD of three replicates. 3.3 HMOX1 Alleviated Hepatic Inflammation in NAFLD Mice given an MCD diet for 8 weeks exhibited macrovesicular steatosis, ballooning degeneration, disordered lobule structure in Zone 3, focal necrosis, and inflammatory infiltration (Fig. 5A). Treatment with the HMOX1 inducer hemin significantly improved the severity of steatosis and inflammation (Fig. 5A, 5B). Additionally, hemin-induced HMOX1 expression resulted in a notable reduction in serum levels of ALT, AST, TG, and LDL (Fig. 5C-F) compared to mice fed the MCD diet alone. These findings suggest that HMOX1 induction alleviates liver damage in MCD diet-induced steatohepatitis. Figure 5 HMOX1 Improves NAFLD by Inhibition of Ferroptosis in Mice. (A-B) HE and Oil-red-O staining of liver tissue (magnification, ×200). (C-F) Blood biochemical analysis of mice. (G-J) HMOX1 improves oxidative stress and reduces lipid peroxidation (MDA, GSH, ROS, and Fe 2+ levels). Data are presented as mean ± SD, *** P < 0.001 vs. the control group or the MCD group. 3.4 HMOX1 Inhibited Ferroptosis in NAFLD Oxidative stress resulting from lipid peroxidation plays a crucial role in the development of ferroptosis. To investigate the impact of HMOX1 regulation on ferroptosis in NAFLD pathogenesis, hepatic Fe 2+ levels, MDA as a lipid peroxidation marker, ROS, and GSH levels were assessed in each mouse group. The findings revealed that the MCD diet significantly increased hepatic Fe 2+ , MDA, and ROS levels while reducing hepatic GSH levels. Conversely, HMOX1 induction by hemin significantly decreased Fe 2+ , MDA, and ROS levels and increased hepatic GSH levels compared to MCD diet-fed mice (Fig. 5G-J). Additionally, HMOX1 and GPX4 expression was notably decreased in the MCD group but increased upon hemin induction (Fig. 6A-C). Ferroptosis in the liver was observed using transmission electron microscopy. Mice fed an MCD diet displayed signs of ferroptosis, including smaller mitochondria with reduced or absent cristae, increased density, and occasional outer membrane rupturing. Hemin treatment improved mitochondrial ridge numbers and outer membrane integrity (Fig. 6D). Figure 6 HMOX1 Inhibits Ferroptosis in Mice. (A-C) Western blotting analysis of HMOX1 and GPX4. (D) Transmission electron microscopy showing ferroptosis in mice. 3.5 The Effects of HMOX1 on JAK/STAT Signaling Pathway in NAFLD. To investigate the impact of HMOX1 on the JAK/STAT signaling pathway, the activation of JAK/STAT pathways was analyzed in HL7702 cells. In NAFLD, the levels of p-JAK2, p-STAT1, and p-STAT3 were elevated at the cellular level (Fig. 7A, 7D, 7E, 7F). Activation of the JAK/STAT pathway was observed in NAFLD cells, and the overexpression of HMOX1 (OE-HMOX1) mitigated OA-induced lipid peroxidation(Fig. 7G-I), inhibited ferroptosis(Fig. 8G), reduced ROS generation(Fig. 8A-F), and suppressed the JAK/STAT pathway (Fig. 7A, 7D, 7E, 7F). Conversely, the ferroptosis inducer erastin reversed the effects of OE-HMOX1 (Fig. 7, Fig. 8). Additionally, the JAK/STAT pathway inhibitor AZD1480, which had the opposite effect with erastin, suppressed ferroptosis and ameliorated NAFLD (Fig. 7, Fig. 8). These results suggest that HMOX1 may attenuate JAK/STAT signaling to prevent ferroptosis in NAFLD. Figure 7 HMOX1 Regulates the JAK/STAT Signaling Pathway and Ferroptosis in NAFLD In Vitro. (A-F) Protein levels of the JAK/STAT signaling pathway and GPX4 were detected by Western blotting analysis. β-Actin was used as the loading control. (G-I) HMOX1 improves oxidative stress in vitro (GSH, MDA, and Fe 2+ levels). Data are presented as mean ± SD; *** P < 0.001 vs. the control group. Figure 8 HMOX 1 Inhibits Lipid Peroxidation and Ferroptosis via Regulation of the JAK/STAT Signaling Pathway in NAFLD In Vitro. (A-F) ROS levels in cells measured by flow cytometry. (G) Transmission electron microscopy showing ferroptosis in cells. Data are presented as mean ± SD; *** P < 0.001 vs. the control group. 4 DISCUSSION The precise mechanisms underlying the development of NAFLD are not yet fully understood. Recent studies have identified a connection between NAFLD and redox imbalance. Prolonged oxidative stress disrupts the equilibrium between ROS and the body's antioxidant defenses in liver cells, leading to lipid peroxidation, insulin resistance, damage to cellular organelles, and activation of hepatic stellate cells ( 30 ). Elevated levels of superoxide can stimulate iron-containing compounds to release Fe 2+ , which plays a critical role in the catalytic subunit of lipoxygenase, promoting lipid peroxidation ( 31 ). During the progression of NAFLD, ROS significantly advances the condition to non-alcoholic steatohepatitis (NASH) ( 32 ). ROS-induced lipid peroxidation is a key factor in the development of NAFLD. Ferroptosis, a type of cell death reliant on iron and characterized by lipid peroxidation, is recognized as a pathological characteristic of NAFLD ( 33 ). Glutathione (GSH), the primary intracellular antioxidant and a cofactor for GSH peroxidase 4 (GPX4), helps shield cells from oxidative harm, thereby preventing ferroptosis. This study identified key genes associated with ferroptosis in NAFLD and confirmed the presence of ferroptosis in MCD diet-induced steatohepatitis. In the MCD liver, a significant increase in Fe 2+ production, ROS levels, and the lipid peroxidation marker MDA was observed, accompanied by a notable decrease in GPX4 and GSH levels. Additionally, under MCD conditions, there was a reduction in mitochondrial size, diminished ridges, increased density, and occasional rupture of the outer membrane, consistent with the known morphological characteristics of mitochondria affected by ferroptosis. HMOX1 acts as a stress-induced enzyme that breaks down heme into carbon monoxide, iron, and biliverdin, playing a crucial role in counteracting oxidative processes ( 34 ). Numerous studies have shown that HMOX1 exhibits anti-inflammatory and anti-apoptotic properties in various obesity-induced metabolic syndromes ( 35 – 37 ). In cases of hepatocyte injury, HMOX1 activation can trigger an adaptive stress response, protecting hepatocytes from oxidative damage ( 38 ) , ( 39 ). The data in this study highlight the significance of HMOX1 in regulating FRGs, demonstrating a notable reduction in NAFLD in both animal models and cell cultures. Specifically, mice fed the MCD diet exhibited decreased HMOX1 expression and elevated levels of ALT and AST. However, hemin treatment significantly decreased ALT, AST, TG, LDL, and lipid peroxidation levels compared to the MCD group. These results align with previous research indicating that higher HMOX1 expression is associated with less severe NAFLD progression in animal models ( 40 , 41 ). Furthermore, hemin administration reversed the decrease in GSH and GPX4 levels induced by the MCD diet, while also enhancing mitochondrial ridges and improving mitochondrial outer membrane integrity. The findings from both animal and cell studies support the conclusion that HMOX1 alleviates NAFLD and inhibits ferroptosis. NAFLD is regulated by various transcription factors and associated signaling pathways. The JAK/STAT signaling pathway is a widely expressed intracellular signal transduction pathway that plays a crucial role in several essential biological processes, including cell proliferation, differentiation, apoptosis, immune regulation, and adipogenesis ( 42 ). JAKs interact noncovalently with cytokine receptors, leading to receptor phosphorylation and the recruitment of one or more STAT proteins. Once phosphorylated, STATs form dimers and translocate into the nucleus to modulate specific genes. Current research on this pathway in relation to disease and drug development has primarily focused on inflammatory and neoplastic conditions ( 43 , 44 ). The research data indicated that blocking hepatic STAT3 activation can prevent liver fibrosis caused by NAFLD and act as a protective signal against lipotoxicity. This study confirmed the activation of the JAK/STAT pathway in OA-induced NAFLD cells and observed improved outcomes after treatment with AZD1480. The results support the role of STAT3 as a crucial pro-inflammatory signal in NAFLD, regulating liver inflammation. Another study highlighted the importance of the IL-22-mediated JAK1/STAT3/BAX signaling pathway in reducing NAFLD progression ( 45 ). Jung et al.'s research using a NASH-associated HCC model demonstrated that inhibiting STAT3 with a small molecule conferred resistance to NASH-related damage, thereby reducing liver fibrosis ( 46 ). Tron et al. found that HMOX1 SBE3 triggers HMOX1 gene expression via the IL-6-induced JAK/STAT pathway ( 47 ). Yu et al. also showed that IFNγ treatment increased the sensitivity of adrenocortical carcinoma cells to erastin-induced ferroptosis through the JAK/STAT pathway ( 48 ). This study revealed that targeted upregulation of HMOX1 suppressed the JAK/STAT signaling pathway, thereby inhibiting ferroptosis in NAFLD. Additionally, treatment with the ferroptosis inducer erastin reversed the effects of HMOX1, further improving NAFLD and suppressing ferroptosis. Ultimately, inhibiting the JAK/STAT pathway led to better NAFLD outcomes, suggesting that HMOX1 exerts its anti-ferroptosis effects by modulating the JAK/STAT pathway to alleviate NAFLD. As previously discussed, diet-induced steatohepatitis via MCD and steatosis through OA can lead to the downregulation of HMOX1 and the activation of the JAK/STAT signaling pathway. However, the impact of HMOX1 regulation on the JAK/STAT signaling pathway has been infrequently addressed in NAFLD. This research introduces a novel discovery regarding the relationship between HMOX1 and the JAK/STAT signaling pathway in NAFLD, offering a new avenue for investigating the disease's pathogenesis. Targeting the JAK/STAT signaling pathway for inhibition may serve as a potential therapeutic strategy for NAFLD prevention. Nevertheless, this study has some limitations. First, because the clinical liver tissue specimens are difficult to obtain, the clinical sample size used for analysis is relatively small. Second, the anti-ferroptotic function of HMOX1 was not assessed using HMOX1 +/+ or HMOX1 −/− mice to conduct a more convincing validation. Future research should aim to elucidate the precise mechanisms linking HMOX1 and the JAK/STAT signaling pathway in NAFLD. In summary, this research highlights the crucial role of HMOX1 in the development of NAFLD and provides new evidence that HMOX1 suppresses ferroptosis by modulating the JAK/STAT signaling pathway in NAFLD. These findings suggest a novel therapeutic approach for treating NAFLD. Declarations E THICAL APPROVAL AND CONSENT TO PARTICIPATE This study was approved by the Medical Ethics Committee of The Second Affiliated Hospital of Guangxi Medical University (Guangxi, China), and written informed consent was obtained from all patients. All animal experiments complied with the Policy of Guangxi Medical University on the Care and Use of Laboratory Animals. F UNDING This research was supported in part by grants from the Natural Science Foundation of China (grant no. 81760516), the Natural Science Foundation of Guangxi, China (grant no. 2019GXNSFAA185030), the Science Foundation of The Second Affiliated Hospital of Guangxi Medical University (No. EFYKY202013), and the Self-raised Foundation of Guangxi Zhuang Autonomous Region, China (No. Z-A20220664). CONFLICT OF INTEREST The authors declare no competing interests. AUTHOR CONTRIBUTIONS Li Yao, Xin-ze Qiu, Jun Zou, Meng-bin Qin, Jin-xiu Zhang, and Jie-an Huang developed the original hypothesis and supervised the experimental design. Li Yao and Jing-rong Liang performed in vitro and in vivo experiments. Li Yao, Jun Zou, Peng Peng, and Jing-rong Liang participated in the clinical specimen collection. Li Yao and Xin-ze Qiu analyzed the data and performed statistical analysis. Li Yao, Xin-ze Qiu, and Jun Zou wrote and revised the manuscript. Jie-an Huang was the guarantor and corresponding author. All authors read and approved the final manuscript. DATA AVAILABILITY STATEMENT The datasets used and/or analyzed during the current study are available upon reasonable request. References Zhang H, Zhang E, Hu H. Role of Ferroptosis in Non-Alcoholic Fatty Liver Disease and Its Implications for Therapeutic Strategies. Biomedicines. 2021;9(11). Younossi ZM, Koenig AB, Abdelatif D, Fazel Y, Henry L, Wymer M. Global epidemiology of nonalcoholic fatty liver disease-Meta-analytic assessment of prevalence, incidence, and outcomes. Hepatology (Baltimore, Md). 2016;64(1):73-84. Ait Boudaoud A, Rives-Lange C, Perregaux JF, Radu A, Messager-Josipowicz D, Barsamian C, et al. [Nutritional Management of Nonalcoholic fatty liver disease (NAFLD)]. Presse medicale (Paris, France : 1983). 2019;48(12):1496-501. Chen Z, Tian R, She Z, Cai J, Li H. Role of oxidative stress in the pathogenesis of nonalcoholic fatty liver disease. 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Sumida Y, Niki E, Naito Y, Yoshikawa T. Involvement of free radicals and oxidative stress in NAFLD/NASH. Free radical research. 2013;47(11):869-80. Lei P, Bai T, Sun Y. Mechanisms of Ferroptosis and Relations With Regulated Cell Death: A Review. Frontiers in physiology. 2019;10:139. Dixon SJ, Lemberg KM, Lamprecht MR, Skouta R, Zaitsev EM, Gleason CE, et al. Ferroptosis: an iron-dependent form of nonapoptotic cell death. Cell. 2012;149(5):1060-72. Hagström H, Nasr P, Bottai M, Ekstedt M, Kechagias S, Hultcrantz R, et al. Elevated serum ferritin is associated with increased mortality in non-alcoholic fatty liver disease after 16 years of follow-up. Liver international : official journal of the International Association for the Study of the Liver. 2016;36(11):1688-95. Nguyen T, Nioi P, Pickett CB. The Nrf2-antioxidant response element signaling pathway and its activation by oxidative stress. The Journal of biological chemistry. 2009;284(20):13291-5. Araujo JA, Zhang M, Yin F. Heme oxygenase-1, oxidation, inflammation, and atherosclerosis. Frontiers in pharmacology. 2012;3:119. Ndisang JF. Role of heme oxygenase in inflammation, insulin-signalling, diabetes and obesity. Mediators of inflammation. 2010;2010:359732. Tu TH, Joe Y, Choi HS, Chung HT, Yu R. Induction of heme oxygenase-1 with hemin reduces obesity-induced adipose tissue inflammation via adipose macrophage phenotype switching. Mediators of inflammation. 2014;2014:290708. Sodhi K, Puri N, Favero G, Stevens S, Meadows C, Abraham NG, et al. Fructose Mediated Non-Alcoholic Fatty Liver Is Attenuated by HO-1-SIRT1 Module in Murine Hepatocytes and Mice Fed a High Fructose Diet. PloS one. 2015;10(6):e0128648. Wang RQ, Nan YM, Wu WJ, Kong LB, Han F, Zhao SX, et al. Induction of heme oxygenase-1 protects against nutritional fibrosing steatohepatitis in mice. Lipids in health and disease. 2011;10:31. Salley TN, Mishra M, Tiwari S, Jadhav A, Ndisang JF. The heme oxygenase system rescues hepatic deterioration in the condition of obesity co-morbid with type-2 diabetes. PloS one. 2013;8(11):e79270. Hinds TD, Jr., Sodhi K, Meadows C, Fedorova L, Puri N, Kim DH, et al. Increased HO-1 levels ameliorate fatty liver development through a reduction of heme and recruitment of FGF21. Obesity (Silver Spring, Md). 2014;22(3):705-12. Bolli R, Dawn B, Xuan YT. Role of the JAK-STAT pathway in protection against myocardial ischemia/reperfusion injury. Trends in cardiovascular medicine. 2003;13(2):72-9. O'Shea JJ, Plenge R. JAK and STAT signaling molecules in immunoregulation and immune-mediated disease. Immunity. 2012;36(4):542-50. O'Shea JJ, Schwartz DM, Villarino AV, Gadina M, McInnes IB, Laurence A. The JAK-STAT pathway: impact on human disease and therapeutic intervention. Annual review of medicine. 2015;66:311-28. Zhu J, Zhou M, Zhao X, Mu M, Cheng M. Blueberry, combined with probiotics, alleviates non-alcoholic fatty liver disease via IL-22-mediated JAK1/STAT3/BAX signaling. Food & function. 2018;9(12):6298-306. Jung KH, Yoo W, Stevenson HL, Deshpande D, Shen H, Gagea M, et al. Multifunctional Effects of a Small-Molecule STAT3 Inhibitor on NASH and Hepatocellular Carcinoma in Mice. Clinical cancer research : an official journal of the American Association for Cancer Research. 2017;23(18):5537-46. Tron K, Samoylenko A, Musikowski G, Kobe F, Immenschuh S, Schaper F, et al. Regulation of rat heme oxygenase-1 expression by interleukin-6 via the Jak/STAT pathway in hepatocytes. Journal of hepatology. 2006;45(1):72-80. Yu X, Zhu D, Luo B, Kou W, Cheng Y, Zhu Y. IFNγ enhances ferroptosis by increasing JAK‑STAT pathway activation to suppress SLCA711 expression in adrenocortical carcinoma. Oncology reports. 2022;47(5). Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5035306","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":356969073,"identity":"f0af700e-ba4c-4a7a-9ff4-9dba7186c2b0","order_by":0,"name":"Li Yao","email":"","orcid":"","institution":"the Second Affiliated Hospital of Guangxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Yao","suffix":""},{"id":356969074,"identity":"1339382d-bef7-43f2-8fac-1b93bf657130","order_by":1,"name":"Jun Zou","email":"","orcid":"","institution":"the Second Affiliated Hospital of Guangxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jun","middleName":"","lastName":"Zou","suffix":""},{"id":356969075,"identity":"3ecf0944-606d-4281-83b9-e2e9a647da61","order_by":2,"name":"Xin-ze Qiu","email":"","orcid":"","institution":"the Second Affiliated Hospital of Guangxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xin-ze","middleName":"","lastName":"Qiu","suffix":""},{"id":356969076,"identity":"e2956960-f14b-4779-bb39-7aa9b855d5a2","order_by":3,"name":"Jing-rong Liang","email":"","orcid":"","institution":"the Second Affiliated Hospital of Guangxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jing-rong","middleName":"","lastName":"Liang","suffix":""},{"id":356969077,"identity":"433bdd4a-e67e-4e7d-9a6d-7cdddb66194e","order_by":4,"name":"Meng-bin Qin","email":"","orcid":"","institution":"the Second Affiliated Hospital of Guangxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Meng-bin","middleName":"","lastName":"Qin","suffix":""},{"id":356969078,"identity":"70f395dd-954b-41d6-99c8-564b0877b31d","order_by":5,"name":"Jin-xiu Zhang","email":"","orcid":"","institution":"the Second Affiliated Hospital of Guangxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Jin-xiu","middleName":"","lastName":"Zhang","suffix":""},{"id":356969079,"identity":"10f0feae-25fb-4cce-9591-a86e83328c11","order_by":6,"name":"Peng Peng","email":"","orcid":"","institution":"the Second Affiliated Hospital of Guangxi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Peng","middleName":"","lastName":"Peng","suffix":""},{"id":356969080,"identity":"7331ee87-1325-48cf-acee-5aa3212f068d","order_by":7,"name":"Jie-an Huang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAt0lEQVRIiWNgGAWjYBACA4YDYFqOjb39AGlajPl4ziQQqwUCEudJOBjgVQkH5oyHH7/mzTmc3ibBkMDwo2IbYS2WDcfMLGduO5zbJt14gLHnzG0iHHbggJnBR5AWmQMJzIxtRGk5/s0gcdvhdDaJBANitZwxfgC0JYEkLWWMM7elG7YBA/kgcX65cXzzZ95t1vLy7e0HH/yoIEILg8QBNgkY+wAR6oGAv4H5A3EqR8EoGAWjYMQCABNBQzbtfTAtAAAAAElFTkSuQmCC","orcid":"","institution":"the Second Affiliated Hospital of Guangxi Medical University","correspondingAuthor":true,"prefix":"","firstName":"Jie-an","middleName":"","lastName":"Huang","suffix":""}],"badges":[],"createdAt":"2024-09-05 05:15:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5035306/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5035306/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":67197310,"identity":"c4f459d9-d908-4980-9396-d65ba5e8f0d6","added_by":"auto","created_at":"2024-10-22 09:19:01","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":773241,"visible":true,"origin":"","legend":"\u003cp\u003eOverview of Ferroptosis-Related DEGs in NAFLD and Controls: (A) Volcano plot of genes differentially expressed between NAFLD and controls in the GSE89632 dataset. Blue nodes represent down-regulation in NAFLD; red nodes represent up-regulation; and gray nodes represent no significant difference from controls; (B) Heatmap of DEGs in NAFLD. The heatmap shows the top 20 upregulated (red, positive value) and downregulated (blue, negative value) DEGs in these samples; (C) UMAP analysis of DEGs in NAFLDL; (D) Venn diagram of the ferroptosis-related DEGs. The Venn diagram indicates 87 co-DEGs (i.e., ferroptosis-related DEGs) from the intersection of the ferroptosis dataset (1940 genes) with DEGs in NAFLD above.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5035306/v1/ef869673163a15dfb3bbcdf6.png"},{"id":67196432,"identity":"841c1b70-9c3a-4873-bb4d-73f857178f23","added_by":"auto","created_at":"2024-10-22 09:11:01","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1458001,"visible":true,"origin":"","legend":"\u003cp\u003eEnrichment Analysis of Ferroptosis-Related DEGs and PPI Network Analysis. (A) Top 10 KEGG pathways; (B) Top 10 GO biological processes pathways; (C) Top 10 GO cellular component pathways; (D) Top 10 GO molecular function pathways; (E) PPI network of ferroptosis-related DEGs; (F) Subnetwork of hub genes from the PPI network. Node color reflects the degree of connectivity (red represents a higher degree).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5035306/v1/4a53285c72cdd3f00d151b33.png"},{"id":67196434,"identity":"22cc368d-b3cf-4c79-adad-516006f7a0a9","added_by":"auto","created_at":"2024-10-22 09:11:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":631535,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of the Hub Gene and GSEA. (A) Venn diagram of the hub genes and the genes in the ferroptosis pathway in KEGG. The Venn diagram indicates one co-DEG (HMOX1) from the intersection of the ferroptosis pathway genes in KEGG (4 genes) with the hub genes in NAFLD; (B) GSEA analysis of the ferroptosis-related DEGs.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5035306/v1/276b69add27ae0ea20f78956.png"},{"id":67196436,"identity":"3f283934-3768-4a2a-b553-52742b983c35","added_by":"auto","created_at":"2024-10-22 09:11:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":2434669,"visible":true,"origin":"","legend":"\u003cp\u003eAberrant Expression of HMOX1 and GPX4 in NAFLD. Immunohistochemical staining of HMOX1 and GPX4 in NAFLD and normal liver tissues (magnification, ×200, ****\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.0001 vs. normal group). Data are presented as mean ± SD of three replicates.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5035306/v1/ef17795378dfda5fd1ff68b8.png"},{"id":67197311,"identity":"8a75a4a9-d285-4e56-97e5-e7fa06ba08e5","added_by":"auto","created_at":"2024-10-22 09:19:01","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1434487,"visible":true,"origin":"","legend":"\u003cp\u003eHMOX1 Improves NAFLD by Inhibition of Ferroptosis in Mice. (A-B) HE and Oil-red-O staining of liver tissue (magnification, ×200). (C-F) Blood biochemical analysis of mice. (G-J) HMOX1 improves oxidative stress and reduces lipid peroxidation (MDA, GSH, ROS, and Fe\u003csup\u003e2+\u003c/sup\u003e levels). Data are presented as mean ± SD, ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 vs. the control group or the MCD group.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-5035306/v1/4cc5e7a0d0676472e5217ca6.png"},{"id":67196441,"identity":"49d817cc-792a-4c6f-ab02-ef05ee990d67","added_by":"auto","created_at":"2024-10-22 09:11:01","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":3310989,"visible":true,"origin":"","legend":"\u003cp\u003eHMOX1 Inhibits Ferroptosis in Mice. (A-C) Western blottinganalysis of HMOX1 and GPX4. (D) Transmission electron microscopy showing ferroptosis in mice.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-5035306/v1/36cd44a78b4eb5d101c55485.png"},{"id":67196438,"identity":"0ba7dad2-af13-4884-8739-764cc1d01d7f","added_by":"auto","created_at":"2024-10-22 09:11:01","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":612588,"visible":true,"origin":"","legend":"\u003cp\u003eHMOX1 Regulates the JAK/STAT Signaling Pathway and Ferroptosis in NAFLD In Vitro. (A-F) Protein levels of the JAK/STAT signaling pathway and GPX4 were detected by Western blotting analysis. β-Actin was used as the loading control. (G-I) HMOX1 improves oxidative stress in vitro (GSH, MDA, and Fe\u003csup\u003e2+\u003c/sup\u003e levels). Data are presented as mean ± SD; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 vs. the control group.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-5035306/v1/fbc444d015e57b4dffd41588.png"},{"id":67196439,"identity":"58d2d704-807b-4eea-8b02-ef0439318d3d","added_by":"auto","created_at":"2024-10-22 09:11:01","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":2302114,"visible":true,"origin":"","legend":"\u003cp\u003eHMOX 1 Inhibits Lipid Peroxidation and Ferroptosis via Regulation of the JAK/STAT Signaling Pathway in NAFLD In Vitro. (A-F) ROS levels in cells measured by flow cytometry. (G) Transmission electron microscopy showing ferroptosis in cells. Data are presented as mean ± SD; ***\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.001 vs. the control group.\u003c/p\u003e","description":"","filename":"Figure8.png","url":"https://assets-eu.researchsquare.com/files/rs-5035306/v1/90c7bb8c93b6ec3cd68e3fa0.png"},{"id":69844152,"identity":"d85b8eef-348b-4cf8-a8ac-9d10e8589b61","added_by":"auto","created_at":"2024-11-25 19:01:50","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":15120124,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5035306/v1/28f69028-f05c-4acc-8ac1-18741551a012.pdf"},{"id":67197312,"identity":"8a6d5e5f-f635-4e58-a403-c8176498280d","added_by":"auto","created_at":"2024-10-22 09:19:02","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":18588,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementTables.docx","url":"https://assets-eu.researchsquare.com/files/rs-5035306/v1/15f7017e828809e21e8a9dd1.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003e\u003cstrong\u003eHMOX1 Inhibits Ferroptosis in Non-Alcoholic Fatty Liver Disease\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"1 INTRODUCTION","content":"\u003cp\u003eNon-alcoholic fatty liver disease (NAFLD) is a chronic hepatic condition characterized by lipid metabolism disruptions, closely associated with insulin resistance and genetic factors (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). NAFLD is regarded as the hepatic manifestation of metabolic syndrome, encompassing simple fat accumulation (steatosis), non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and hepatocellular carcinoma. The prevalence of NAFLD has escalated to 25% in Western populations (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e), reaching up to 80% among individuals with obesity (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). NAFLD significantly contributes to advanced liver disease and represents a substantial public health challenge.\u003c/p\u003e \u003cp\u003eContemporary research indicates that NAFLD is associated with a myriad of factors, including inflammation, oxidative stress, insulin resistance, lipid metabolism disorders, obesity, and endoplasmic reticulum stress (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). The \"multiple-hit hypothesis\" is posited as the central mechanism driving NAFLD pathogenesis. The initial insult involves hepatic steatosis induced by insulin resistance, leading to hepatic fat accumulation and impaired fatty acid export. Subsequent insults result in increased reactive oxygen species (ROS) production, culminating in oxidative stress and inflammation (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). Furthermore, oxidative stress contributes to mitochondrial dysfunction (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e), often characterized by lipid peroxidation (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). ROS targets polyunsaturated fatty acid double bonds, producing lipid peroxides such as 4-hydroxy-2-nonenal (4-HNE) and malondialdehyde (MDA), which subsequently cause cellular damage (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Ferroptosis, a novel form of programmed cell death, is driven by iron-dependent lipid peroxidation (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Mitochondria undergoing ferroptosis exhibit distinctive morphological changes, including reduced size, increased membrane density, diminished or absent mitochondrial cristae, and ruptured outer membranes (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e), differentiating it from apoptosis, necrosis, and autophagy both morphologically and mechanistically. Abnormal iron metabolism and lipid peroxidation, stemming from iron-dependent depletion of glutathione (GSH) and inactivation of glutathione peroxidase 4 (GPX4), along with the accumulation of polyunsaturated fatty acid-containing phospholipids (PUFA-PLs), act as catalysts for ferroptosis (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). This mode of cell death is implicated in various human diseases, including neurodegenerative disorders (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e), cardiovascular diseases (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e), cancer (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e), and notably, liver diseases (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eEmerging evidence suggests that ferroptosis plays a crucial role in the pathological progression of NAFLD. During NAFLD development, oxidative stress induced by reactive oxygen species (ROS) is identified as the primary catalyst for cellular ferroptosis (\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). The liver, as the central organ for iron storage and lipid metabolism, is particularly susceptible to ferroptosis. Inhibition of ferroptosis holds the potential for enhancing liver function and mitigating liver damage (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eWhile initial studies on ferroptosis predominantly targeted cancer, specific ferroptosis-related genes (FRGs) have emerged as valuable biomarkers for diagnosing, prognosticating, and treating various cancers (\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Nevertheless, the potential application of FRGs in NAFLD management remains elusive. This study utilized NAFLD datasets from the Gene Expression Omnibus (GEO) and FRGs from GeneCards to identify reliable differentially expressed genes (DEGs) associated with ferroptosis in NAFLD. Heme oxygenase-1 (HMOX1) was identified as a crucial biomarker in our study, and its ability to inhibit the ferroptotic process in NAFLD was validated. Additionally, the underlying mechanisms were explored through both in vivo and in vitro experiments.\u003c/p\u003e"},{"header":"2 MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1Detection of Key DEGs Associated with Ferroptosis\u003c/h2\u003e \u003cp\u003eThe microarray dataset GSE89632, associated with NAFLD, was sourced from the GEO database. FRGs were initially filtered from the GeneCards website using the term \u0026ldquo;ferroptosis.\u0026rdquo; Subsequent data analysis was performed using the R project (version 4.0.3). Probe names in the gene expression profiles were first transformed into gene names based on the platform's annotation files. DEGs were then identified using the \u0026ldquo;limma\u0026rdquo; package, with an adjusted p-value of less than 0.05 and a fold change exceeding |log2FoldChange| of 1.5. The R package \u0026ldquo;umap\u0026rdquo; (v0.2.3.1) was used to perform Uniform Manifold Approximation and Projection (UMAP). Critical DEGs associated with ferroptosis were determined using an online Venn diagram tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.psb.ugent.be/webtools/Venn/\u003c/span\u003e\u003cspan address=\"http://bioinformatics.psb.ugent.be/webtools/Venn/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 PPI network construction and analysis of modules\u003c/h2\u003e \u003cp\u003eThe STRING database was employed to analyze protein-protein interactions (PPI) and identify interactions among target proteins. Interaction scores above 0.4 were visualized in the PPI network using Cytoscape software (v3.9.0), where genes were depicted as nodes and their interactions as edges. Clustering analysis was performed using the molecular complex detection (MCODE) algorithm to identify key modules within the PPI network. Each module was associated with a specific biological function, with statistical significance set at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Functional enrichment analysis\u003c/h2\u003e \u003cp\u003eThe \u0026ldquo;clusterProfiler\u0026rdquo; package (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e) in R was employed to perform enrichment analyses for Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) related to ferroptosis-related DEGs. GO analysis was divided into three categories: biological processes (BP), cellular components (CC), and molecular functions (MF), essential for investigating biological roles. KEGG analysis was utilized to identify potential pathways. The STRING online platform (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://string-db.org/\u003c/span\u003e\u003cspan address=\"https://string-db.org/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e) facilitated the protein-protein interaction (PPI) network analysis for ferroptosis-related DEGs. The results from STRING were subsequently imported into Cytoscape (version 3.8.2), where the \u0026ldquo;cytoHubba\u0026rdquo; plugin was used to extract key subnetworks. Hub genes were identified by selecting the ten genes with the highest scores using the maximum correlation criterion (MCC algorithm) within the subnetworks. Gene-set enrichment analysis (GSEA) was employed to classify genes based on their differential expression between two samples and to compare entire genome expression profiles against predefined gene sets (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e). In the GSEA performed via WebGestalt, gene sets containing fewer than three or more than 2,000 genes were excluded by default. The KEGG analysis was based on WebGestalt's GSEA results.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Reagents and chemicals\u003c/h2\u003e \u003cp\u003eThe antibodies anti-GPX4 (10701-1-AP) and anti-HMOX1 (14432-1-AP) were sourced from Proteintech, Wuhan, China. Erastin (HY-15673), AZD1480 (HY-10193), and Hemin (HY-19424) were provided by MCE, New Jersey, USA. Lipofectamine 2000 (11668-027) was procured from Invitrogen, California, USA, and oleic acid (0108484-500ml) from Aladdin, Shanghai, China. Kits for ALT (C009-2-1), AST (C010-2-1), TG (A110-1-1), and LDL (A113-1-1) were obtained from Nanjing Jiancheng Bioengineering Institute, Nanjing, China. GSH, Fe\u003csup\u003e2+\u003c/sup\u003e, MDA, and ROS ELISA kits were acquired from Mlbio, Shanghai, China. Dihydroethidium (S0063) was purchased from Beyotime, Shanghai, China. Additionally, anti-JAK2 (A7694), anti-P-JAK2 (AP0374), anti-STAT1 (A12075), anti-P-STAT1 (AP0135), anti-STAT3 (A1192), and anti-P-STAT3 (AP0474) were procured from Abclonal, Wuhan, China.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Liver biopsy\u003c/h2\u003e \u003cp\u003eThe study involved 31 liver tissue samples from patients with NAFLD and 30 from individuals with normal liver function. Liver biopsies were conducted on 61 patients using either the percutaneous method with a Menghini needle or through surgery. Liver tissues from the normal group were obtained from patients who underwent surgery for noncancerous liver conditions. The biopsy samples underwent standard staining procedures and were examined by a single pathologist blinded to patients' clinical and laboratory data. NAFLD/NASH was assessed using the NAFLD Activity Score (NAS), evaluating steatosis (0\u0026ndash;3), lobular inflammation (0\u0026ndash;3), and hepatocellular ballooning (0\u0026ndash;2), and the SAF score system, which includes steatosis (0\u0026ndash;3), activity (lobular inflammation [0\u0026ndash;2]\u0026thinsp;+\u0026thinsp;hepatocellular ballooning [0\u0026ndash;2]), and fibrosis (0\u0026ndash;4).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Histological and immunohistochemical analyses\u003c/h2\u003e \u003cp\u003eFresh liver tissue was embedded in paraffin and sectioned into 4 \u0026micro;m thick slices for periodic acid\u0026ndash;Schiff (PAS) staining. Monoclonal antibodies anti-HMOX1 (1:200 dilution, Proteintech) and anti-GPX4 (1:500 dilution, Proteintech) were used for immunohistochemical staining. Images were captured using the Olympus Microscope Camera System (Tokyo, Japan). Protein expression was analyzed by examining five randomly selected fields per slide. Immunohistochemical scoring assessed both staining intensity and the proportion of positive cells. Staining intensity was categorized as follows: 0 for no staining, 1 for light yellow, 2 for yellow, and 3 for brown. The proportion of positively stained cells was scored as follows: less than 5% scored 0, 5\u0026ndash;25% scored 1, 26\u0026ndash;50% scored 2, and greater than 50% scored 3. The final score for each sample was determined by multiplying these two scores: a score of 0\u0026ndash;2 was denoted as (\u0026minus;), 3\u0026ndash;4 as (+), and 5 or higher as (++) (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Animals and treatments\u003c/h2\u003e \u003cp\u003e Approval for all animal experiments was obtained from the Animal Ethics Committee at Guangxi Medical University. Male C57BL/6J mice, aged 6\u0026ndash;8 weeks, were sourced from The Center for Comparative Medicine at Yangzhou University, China. The mice were maintained on a 12-hour light-dark cycle at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, with 55% humidity, and provided ad libitum access to food and water. A one-week acclimatization period was allowed before the experiment, during which the mice were fed either a normal chow diet (NCD) or a methionine/choline-deficient diet (MCD) for 8 weeks. The mice were randomly divided into three groups, each with five individuals: (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e) control group - NCD diet; (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e) MCD group - MCD diet; (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) MCD\u0026thinsp;+\u0026thinsp;Hemin group - MCD diet with intraperitoneal (i.p.) injections of the HMOX1 inducer hemin (30 \u0026micro;mol/kg) three times weekly. After the 8-week experimental period, all animals were fasted overnight before being euthanized. Liver tissue and serum samples were collected for further analysis. The experimental protocols complied with the guidelines of the Guangxi Committee for Care and Use of Laboratory Animals and were approved by the Animal Experimentation Ethics Committee at Guangxi Medical University.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Cell Culture and Transient Transfection and Oleic acidinduced steatosis\u003c/h2\u003e \u003cp\u003eHL7702 cell lines, provided by the Cell Bank of the Chinese Academy of Sciences in Shanghai, China, were cultured in Dulbecco's Modified Eagle's Medium (DMEM) with high glucose, supplemented with 10% FBS. Incubation was conducted at 37\u0026deg;C with 5% CO2. Transient transfection was performed in 6-well plates using plasmids from GenePharma, Shanghai, China, and Lipofectamine 2000 from Invitrogen, California, USA. After six hours, the serum-free medium was replaced with a complete medium, and cells were incubated for an additional 48 hours before collection. Total RNA was extracted using a Trizol reagent from Biosharp, Wuhan, China, and converted to cDNA with a reverse transcription kit from YE SEN, Shanghai, China. Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction (qRT-PCR) was conducted on a Roche instrument from Shanghai, China, and relative gene expression levels were calculated using the 2-ΔΔCt method. Primers were designed with Primer 5.0 software, with sequences as follows: HMOX1 \u0026ndash; forward: 5\u0026prime;-AAGACTGCGTTCCTGCTCAAC-3\u0026prime;, reverse: 5\u0026prime;-AAAGCCCTACAGCAACTGTCG-3\u0026prime;; β-actin \u0026ndash; forward: 5\u0026prime;-ACGTGGACATCCGCAAAG-3\u0026prime;, reverse: 5\u0026prime;-TGGAAGGTGGACAGCGAGGC-3\u0026prime;. cDNA amplification started with an initial step at 95\u0026deg;C for 5 minutes, followed by 40 cycles of 10 seconds at 95\u0026deg;C and 30 seconds at 60\u0026deg;C. HL7702 cells were plated in 6-well plates at a density of 2\u0026times;10⁵ cells per well and treated with 1 mM OA for 24 hours. Post-treatment, cells were analyzed using ELISA, Western blotting, flow cytometry, and transmission electron microscopy (TEM).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Histological and Biochemical Analyses and ROS Assessment\u003c/h2\u003e \u003cp\u003eFresh liver stored at -80\u0026deg;C was sectioned into 4 \u0026micro;m slices, then heated, dried, fixed with 4% paraformaldehyde, and stained with oil red O (Servicebio, China) in the dark for 10 minutes. Subsequently, sections were stained with hematoxylin (Servicebio, China) for 5 minutes following differentiation with 60% isopropanol. Levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglycerides (TG), low-density lipoprotein (LDL), GSH, MDA, and Fe\u003csup\u003e2+\u003c/sup\u003e were determined using ELISA kits according to the manufacturer's instructions. Hepatic ROS levels were assessed with an ROS assay kit (Mlbio, China) following the manufacturer\u0026rsquo;s guidelines. Intracellular ROS levels were evaluated using flow cytometry with Dihydroethidium (Beyotime, Nanjing Jiancheng Biotechnology Institute, China).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Western blotting analysis\u003c/h2\u003e \u003cp\u003eProteins from liver tissues and HL7702 cells were extracted using RIPA lysis buffer and PMSF. Protein concentrations were determined using the BCA Protein Assay Reagent (Biosharp, China). A total of 30 \u0026micro;g of protein was separated on a 10% SDS\u0026ndash;PAGE gel and transferred onto nitrocellulose membranes. The membranes were blocked with 5% BSA in 1\u0026times; TBST at room temperature for 1 hour, followed by overnight incubation with primary antibodies at 4\u0026deg;C. Protein bands were visualized by incubating with horseradish peroxidase-conjugated secondary antibodies and imaged using the One-piece chemiluminescence imager (BIO-RAD, USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.11 Transmission electron microscopy\u003c/h2\u003e \u003cp\u003eThe liver was washed twice in PBS and then sliced. Cells were collected until the sediment reached the size of a mung bean. The cells were fixed in an electron microscope holder at room temperature for 2 hours and then stored at 4\u0026deg;C. Morphological alterations in the mitochondria were visualized using a transmission electron microscope (TEM).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Statistical Analysis\u003c/h2\u003e \u003cp\u003eMean values were reported with standard deviation (SD). Continuous variables were compared using Student\u0026rsquo;s t-test, and categorical variables were evaluated using chi-square or Fisher\u0026rsquo;s exact test. Group differences were analyzed through ANOVA. Data analysis was performed using R software (version 4.1.0) and SPSS (version 19.0). Statistical significance was defined as p\u0026thinsp;\u0026lt;\u0026thinsp;0.05 (two-tailed).\u003c/p\u003e \u003c/div\u003e"},{"header":"3 RESULTS","content":"\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1 HMOX1 Is an Essential Ferroptosis-Related DEG Identified From NAFLD Datasets\u003c/h2\u003e\n \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\n \u003ch2\u003e3.1.1 Identification of Ferroptosis-Related DEGs in NAFLD\u003c/h2\u003e\n \u003cp\u003eThe microarray dataset related to NAFLD, GSE89632, was retrieved from the GEO database. Clinical characteristics of NAFLD within the GSE89632 dataset are presented in Table \u003cspan class=\"InternalRef\"\u003eA.1\u003c/span\u003e. Differential expression analysis was conducted using the \u0026ldquo;limma\u0026rdquo; package on RNA samples from whole blood of NAFLD patients and normal controls. A total of 1,995 DEGs were identified, with 1,121 downregulated (green dots in Fig. 1A) and 874 upregulated (red dots in Fig. 1A). Ferroptosis-related genes were subsequently extracted from the GeneCards platform using the keyword \u0026ldquo;ferroptosis.\u0026rdquo; The left count (1,908 genes) corresponds to DEGs specific to GSE89632, the middle count (87 genes) to ferroptosis-related DEGs, and the right count (1,853 genes) to unique ferroptosis genes (Fig. 1D). The heatmap in Fig. 1B shows the differential expression of genes between NAFLD and control groups in the GSE89632 dataset, with 20 upregulated and 20 downregulated genes. UMAP analysis of DEGs is presented in Fig. 1C. Figure 1D categorizes the 87 ferroptosis-related DEGs, including 56 downregulated and 31 upregulated genes in NAFLD, as detailed in Table \u003cspan class=\"InternalRef\"\u003eA.2\u003c/span\u003e. Within this subset, 2 genes serve as markers, 17 function as drivers, and 25 act as suppressors, as specified in Table \u003cspan class=\"InternalRef\"\u003eA.3\u003c/span\u003e. Notably, HMOX1 is implicated as both a driver and suppressor gene according to Table \u003cspan class=\"InternalRef\"\u003eA.3\u003c/span\u003e.\u003c/p\u003e\n \u003cp\u003eFigure 1 Overview of Ferroptosis-Related DEGs in NAFLD and Controls: (A) Volcano plot of genes differentially expressed between NAFLD and controls in the GSE89632 dataset. Blue nodes represent down-regulation in NAFLD; red nodes represent up-regulation; and gray nodes represent no significant difference from controls; (B) Heatmap of DEGs in NAFLD. The heatmap shows the top 20 upregulated (red, positive value) and downregulated (blue, negative value) DEGs in these samples; (C) UMAP analysis of DEGs in NAFLDL; (D) Venn diagram of the ferroptosis-related DEGs. The Venn diagram indicates 87 co-DEGs (i.e., ferroptosis-related DEGs) from the intersection of the ferroptosis dataset (1940 genes) with DEGs in NAFLD above.\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec18\" class=\"Section3\"\u003e\n \u003ch2\u003e3.1.2 Enrichment Analysis of Ferroptosis-Related DEGs and PPI Network Analysis\u003c/h2\u003e\n \u003cp\u003eThe KEGG pathway analysis (Fig.\u0026nbsp;2A) identified the top ten enriched pathways, including the Micro-RNA tumor signaling pathway, ferroptosis, HIF-1 signaling pathway, and JAK/STAT signaling pathway. The GO-BP analysis (Fig.\u0026nbsp;2B) highlighted pathways such as cellular response to chemical stimulus and response to organic substance. The GO-CC analysis (Fig.\u0026nbsp;2C) indicated significant enrichment in the cytosol. The GO-MF enrichment analysis (Fig.\u0026nbsp;2D) revealed key findings related to enzyme binding and oxidoreductase activity. The PPI network construction (Fig.\u0026nbsp;2E) displayed 51 nodes and 175 edges, with 36 of the 87 genes lacking interactions and thus not forming molecular networks. The network settings were adjusted to a default cutoff (interaction score\u0026thinsp;\u0026gt;\u0026thinsp;0.4) using the STRING database. Hub genes were identified using the Cytoscape CytoHubba plug-in, selecting the top ten genes with the highest scores according to the MCC algorithm (HMOX1, JUN, MYC, STAT3, IL6, FOXO1, ERBB2, PTGS2, ATF3, CDKN1A) (Fig.\u0026nbsp;2F). Venn diagram analysis (Fig.\u0026nbsp;3A) identified a single co-DEG (HMOX1) shared between the ferroptosis pathway genes in KEGG (4 genes) and the hub genes in NAFLD (10 genes). GSEA analysis results (top five NES and P values\u0026thinsp;\u0026lt;\u0026thinsp;0.05) showed enrichment in pathways such as JAK/STAT signaling, LEISHMANIA INFECTION, COMPLEMENT AND COAGULATION CASCADES, and CYTOKINE-CYTOKINE RECEPTOR INTERACTION (Fig.\u0026nbsp;3B).\u003c/p\u003e\n \u003cp\u003eFigure 2 Enrichment Analysis of Ferroptosis-Related DEGs and PPI Network Analysis. (A) Top 10 KEGG pathways; (B) Top 10 GO biological processes pathways; (C) Top 10 GO cellular component pathways; (D) Top 10 GO molecular function pathways; (E) PPI network of ferroptosis-related DEGs; (F) Subnetwork of hub genes from the PPI network. Node color reflects the degree of connectivity (red represents a higher degree).\u003c/p\u003e\n \u003cp\u003eFigure 3 Identification of the Hub Gene and GSEA. (A) Venn diagram of the hub genes and the genes in the ferroptosis pathway in KEGG. The Venn diagram indicates one co-DEG (HMOX1) from the intersection of the ferroptosis pathway genes in KEGG (4 genes) with the hub genes in NAFLD; (B) GSEA analysis of the ferroptosis-related DEGs.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2 Expression and Clinical Feature of HMOX1 and GPX4 in NAFLD Tissues\u003c/h2\u003e\n \u003cp\u003eHMOX1 and Glutathione Peroxidase 4 (GPX4) expression levels in NAFLD tissues were significantly lower than those in normal liver tissues (Fig. 4 and Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Additionally, HMOX1 expression strongly correlates with both BMI and GPX4 expression in NAFLD tissues (Table \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e,Table \u003cspan class=\"InternalRef\"\u003eA.4\u003c/span\u003e and Table\u0026nbsp;\u003cspan class=\"InternalRef\"\u003eA.5\u003c/span\u003e).\u003c/p\u003e\n \u003cdiv class=\"gridtable\"\u003e\u0026nbsp;\u003ctable id=\"Tab4\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eHMOX 1 expression in NAFLD and normal liver tissues.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"7\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\u0026nbsp;\u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eN\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eHMOX1\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u0026chi; \u0026chi;\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003eP\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNormal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e42.690\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eNAFLD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e28\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cdiv class=\"gridtable\"\u003e\n \u003cdiv align=\"left\" class=\"colspec\"\u003e\u003cbr\u003e\u003c/div\u003e\u0026nbsp;\u003ctable id=\"Tab5\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eCorrelation between HMOX 1 expression and GPX4 in NAFLD.\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003ccolgroup cols=\"5\"\u003e\u003c/colgroup\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eExpression\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" colspan=\"2\"\u003e\n \u003cp\u003eGPX4\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003eCorrelation\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\" rowspan=\"2\"\u003e\n \u003cp\u003e\u003cem\u003ep\u003c/em\u003e\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\" colspan=\"3\"\u003e\n \u003cp\u003eHMOX1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e-\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e0.712\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e\u0026lt;0.001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003ctd align=\"left\"\u003e\u0026nbsp;\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eHMOX1,Heme oxygenase-1;GPX4,Glutathione peroxidase 4.\u003c/p\u003e\n \u003cp\u003eFigure 4 Aberrant Expression of HMOX1 and GPX4 in NAFLD. Immunohistochemical staining of HMOX1 and GPX4 in NAFLD and normal liver tissues (magnification, \u0026times;200, ****\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.0001 vs. normal group). Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD of three replicates.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3 HMOX1 Alleviated Hepatic Inflammation in NAFLD\u003c/h2\u003e\n \u003cp\u003eMice given an MCD diet for 8 weeks exhibited macrovesicular steatosis, ballooning degeneration, disordered lobule structure in Zone 3, focal necrosis, and inflammatory infiltration (Fig.\u0026nbsp;5A). Treatment with the HMOX1 inducer hemin significantly improved the severity of steatosis and inflammation (Fig.\u0026nbsp;5A, 5B). Additionally, hemin-induced HMOX1 expression resulted in a notable reduction in serum levels of ALT, AST, TG, and LDL (Fig.\u0026nbsp;5C-F) compared to mice fed the MCD diet alone. These findings suggest that HMOX1 induction alleviates liver damage in MCD diet-induced steatohepatitis.\u003c/p\u003e\n \u003cp\u003eFigure 5 HMOX1 Improves NAFLD by Inhibition of Ferroptosis in Mice. (A-B) HE and Oil-red-O staining of liver tissue (magnification, \u0026times;200). (C-F) Blood biochemical analysis of mice. (G-J) HMOX1 improves oxidative stress and reduces lipid peroxidation (MDA, GSH, ROS, and Fe\u003csup\u003e2+\u003c/sup\u003e levels). Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD, ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs. the control group or the MCD group.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\n \u003ch2\u003e3.4 HMOX1 Inhibited Ferroptosis in NAFLD\u003c/h2\u003e\n \u003cp\u003eOxidative stress resulting from lipid peroxidation plays a crucial role in the development of ferroptosis. To investigate the impact of HMOX1 regulation on ferroptosis in NAFLD pathogenesis, hepatic Fe\u003csup\u003e2+\u003c/sup\u003e levels, MDA as a lipid peroxidation marker, ROS, and GSH levels were assessed in each mouse group. The findings revealed that the MCD diet significantly increased hepatic Fe\u003csup\u003e2+\u003c/sup\u003e, MDA, and ROS levels while reducing hepatic GSH levels. Conversely, HMOX1 induction by hemin significantly decreased Fe\u003csup\u003e2+\u003c/sup\u003e, MDA, and ROS levels and increased hepatic GSH levels compared to MCD diet-fed mice (Fig.\u0026nbsp;5G-J). Additionally, HMOX1 and GPX4 expression was notably decreased in the MCD group but increased upon hemin induction (Fig.\u0026nbsp;6A-C). Ferroptosis in the liver was observed using transmission electron microscopy. Mice fed an MCD diet displayed signs of ferroptosis, including smaller mitochondria with reduced or absent cristae, increased density, and occasional outer membrane rupturing. Hemin treatment improved mitochondrial ridge numbers and outer membrane integrity (Fig.\u0026nbsp;6D).\u003c/p\u003e\n \u003cp\u003eFigure 6 HMOX1 Inhibits Ferroptosis in Mice. (A-C) Western blotting analysis of HMOX1 and GPX4. (D) Transmission electron microscopy showing ferroptosis in mice.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n \u003ch2\u003e3.5 The Effects of HMOX1 on JAK/STAT Signaling Pathway in NAFLD.\u003c/h2\u003e\n \u003cp\u003eTo investigate the impact of HMOX1 on the JAK/STAT signaling pathway, the activation of JAK/STAT pathways was analyzed in HL7702 cells. In NAFLD, the levels of p-JAK2, p-STAT1, and p-STAT3 were elevated at the cellular level (Fig.\u0026nbsp;7A, 7D, 7E, 7F). Activation of the JAK/STAT pathway was observed in NAFLD cells, and the overexpression of HMOX1 (OE-HMOX1) mitigated OA-induced lipid peroxidation(Fig.\u0026nbsp;7G-I), inhibited ferroptosis(Fig.\u0026nbsp;8G), reduced ROS generation(Fig.\u0026nbsp;8A-F), and suppressed the JAK/STAT pathway (Fig.\u0026nbsp;7A, 7D, 7E, 7F). Conversely, the ferroptosis inducer erastin reversed the effects of OE-HMOX1 (Fig.\u0026nbsp;7, Fig.\u0026nbsp;8). Additionally, the JAK/STAT pathway inhibitor AZD1480, which had the opposite effect with erastin, suppressed ferroptosis and ameliorated NAFLD (Fig.\u0026nbsp;7, Fig.\u0026nbsp;8). These results suggest that HMOX1 may attenuate JAK/STAT signaling to prevent ferroptosis in NAFLD.\u003c/p\u003e\n \u003cp\u003eFigure 7 HMOX1 Regulates the JAK/STAT Signaling Pathway and Ferroptosis in NAFLD In Vitro. (A-F) Protein levels of the JAK/STAT signaling pathway and GPX4 were detected by Western blotting analysis. \u0026beta;-Actin was used as the loading control. (G-I) HMOX1 improves oxidative stress in vitro (GSH, MDA, and Fe\u003csup\u003e2+\u003c/sup\u003e levels). Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD; ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs. the control group.\u003c/p\u003e\n \u003cp\u003eFigure 8 HMOX 1 Inhibits Lipid Peroxidation and Ferroptosis via Regulation of the JAK/STAT Signaling Pathway in NAFLD In Vitro. (A-F) ROS levels in cells measured by flow cytometry. (G) Transmission electron microscopy showing ferroptosis in cells. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD; ***\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.001 vs. the control group.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4 DISCUSSION","content":"\u003cp\u003eThe precise mechanisms underlying the development of NAFLD are not yet fully understood. Recent studies have identified a connection between NAFLD and redox imbalance. Prolonged oxidative stress disrupts the equilibrium between ROS and the body's antioxidant defenses in liver cells, leading to lipid peroxidation, insulin resistance, damage to cellular organelles, and activation of hepatic stellate cells (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e). Elevated levels of superoxide can stimulate iron-containing compounds to release Fe\u003csup\u003e2+\u003c/sup\u003e, which plays a critical role in the catalytic subunit of lipoxygenase, promoting lipid peroxidation (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). During the progression of NAFLD, ROS significantly advances the condition to non-alcoholic steatohepatitis (NASH) (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e). ROS-induced lipid peroxidation is a key factor in the development of NAFLD. Ferroptosis, a type of cell death reliant on iron and characterized by lipid peroxidation, is recognized as a pathological characteristic of NAFLD (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e). Glutathione (GSH), the primary intracellular antioxidant and a cofactor for GSH peroxidase 4 (GPX4), helps shield cells from oxidative harm, thereby preventing ferroptosis.\u003c/p\u003e \u003cp\u003eThis study identified key genes associated with ferroptosis in NAFLD and confirmed the presence of ferroptosis in MCD diet-induced steatohepatitis. In the MCD liver, a significant increase in Fe\u003csup\u003e2+\u003c/sup\u003e production, ROS levels, and the lipid peroxidation marker MDA was observed, accompanied by a notable decrease in GPX4 and GSH levels. Additionally, under MCD conditions, there was a reduction in mitochondrial size, diminished ridges, increased density, and occasional rupture of the outer membrane, consistent with the known morphological characteristics of mitochondria affected by ferroptosis.\u003c/p\u003e \u003cp\u003eHMOX1 acts as a stress-induced enzyme that breaks down heme into carbon monoxide, iron, and biliverdin, playing a crucial role in counteracting oxidative processes (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Numerous studies have shown that HMOX1 exhibits anti-inflammatory and anti-apoptotic properties in various obesity-induced metabolic syndromes (\u003cspan additionalcitationids=\"CR36\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). In cases of hepatocyte injury, HMOX1 activation can trigger an adaptive stress response, protecting hepatocytes from oxidative damage (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e)\u003csup\u003e,\u003c/sup\u003e(\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e). The data in this study highlight the significance of HMOX1 in regulating FRGs, demonstrating a notable reduction in NAFLD in both animal models and cell cultures. Specifically, mice fed the MCD diet exhibited decreased HMOX1 expression and elevated levels of ALT and AST. However, hemin treatment significantly decreased ALT, AST, TG, LDL, and lipid peroxidation levels compared to the MCD group. These results align with previous research indicating that higher HMOX1 expression is associated with less severe NAFLD progression in animal models (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). Furthermore, hemin administration reversed the decrease in GSH and GPX4 levels induced by the MCD diet, while also enhancing mitochondrial ridges and improving mitochondrial outer membrane integrity. The findings from both animal and cell studies support the conclusion that HMOX1 alleviates NAFLD and inhibits ferroptosis.\u003c/p\u003e \u003cp\u003eNAFLD is regulated by various transcription factors and associated signaling pathways. The JAK/STAT signaling pathway is a widely expressed intracellular signal transduction pathway that plays a crucial role in several essential biological processes, including cell proliferation, differentiation, apoptosis, immune regulation, and adipogenesis (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). JAKs interact noncovalently with cytokine receptors, leading to receptor phosphorylation and the recruitment of one or more STAT proteins. Once phosphorylated, STATs form dimers and translocate into the nucleus to modulate specific genes. Current research on this pathway in relation to disease and drug development has primarily focused on inflammatory and neoplastic conditions (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe research data indicated that blocking hepatic STAT3 activation can prevent liver fibrosis caused by NAFLD and act as a protective signal against lipotoxicity. This study confirmed the activation of the JAK/STAT pathway in OA-induced NAFLD cells and observed improved outcomes after treatment with AZD1480. The results support the role of STAT3 as a crucial pro-inflammatory signal in NAFLD, regulating liver inflammation. Another study highlighted the importance of the IL-22-mediated JAK1/STAT3/BAX signaling pathway in reducing NAFLD progression (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e). Jung et al.'s research using a NASH-associated HCC model demonstrated that inhibiting STAT3 with a small molecule conferred resistance to NASH-related damage, thereby reducing liver fibrosis (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e). Tron et al. found that HMOX1 SBE3 triggers HMOX1 gene expression via the IL-6-induced JAK/STAT pathway (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e). Yu et al. also showed that IFNγ treatment increased the sensitivity of adrenocortical carcinoma cells to erastin-induced ferroptosis through the JAK/STAT pathway (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). This study revealed that targeted upregulation of HMOX1 suppressed the JAK/STAT signaling pathway, thereby inhibiting ferroptosis in NAFLD. Additionally, treatment with the ferroptosis inducer erastin reversed the effects of HMOX1, further improving NAFLD and suppressing ferroptosis. Ultimately, inhibiting the JAK/STAT pathway led to better NAFLD outcomes, suggesting that HMOX1 exerts its anti-ferroptosis effects by modulating the JAK/STAT pathway to alleviate NAFLD.\u003c/p\u003e \u003cp\u003eAs previously discussed, diet-induced steatohepatitis via MCD and steatosis through OA can lead to the downregulation of HMOX1 and the activation of the JAK/STAT signaling pathway. However, the impact of HMOX1 regulation on the JAK/STAT signaling pathway has been infrequently addressed in NAFLD. This research introduces a novel discovery regarding the relationship between HMOX1 and the JAK/STAT signaling pathway in NAFLD, offering a new avenue for investigating the disease's pathogenesis. Targeting the JAK/STAT signaling pathway for inhibition may serve as a potential therapeutic strategy for NAFLD prevention. Nevertheless, this study has some limitations. First, because the clinical liver tissue specimens are difficult to obtain, the clinical sample size used for analysis is relatively small. Second, the anti-ferroptotic function of HMOX1 was not assessed using HMOX1\u003csup\u003e+/+\u003c/sup\u003e or HMOX1\u003csup\u003e\u0026minus;/\u0026minus;\u003c/sup\u003e mice to conduct a more convincing validation. Future research should aim to elucidate the precise mechanisms linking HMOX1 and the JAK/STAT signaling pathway in NAFLD.\u003c/p\u003e \u003cp\u003eIn summary, this research highlights the crucial role of HMOX1 in the development of NAFLD and provides new evidence that HMOX1 suppresses ferroptosis by modulating the JAK/STAT signaling pathway in NAFLD. These findings suggest a novel therapeutic approach for treating NAFLD.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eE\u003cstrong\u003eTHICAL\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;APPROVAL AND CONSENT TO PARTICIPATE\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was approved by the Medical Ethics Committee of The Second Affiliated Hospital of Guangxi Medical University (Guangxi, China), and written informed consent was obtained from all patients. All animal experiments complied with the Policy of Guangxi Medical University on the Care and Use of Laboratory Animals.\u003c/p\u003e\n\u003cp\u003eF\u003cstrong\u003eUNDING\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported in part by grants from the Natural Science Foundation of China (grant no. 81760516), the Natural Science Foundation of Guangxi, China (grant no. 2019GXNSFAA185030), the Science Foundation of The Second Affiliated Hospital of Guangxi Medical University (No. EFYKY202013), and the Self-raised Foundation of Guangxi Zhuang Autonomous Region, China (No.\u0026nbsp;Z-A20220664).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCONFLICT OF INTEREST\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLi Yao, Xin-ze Qiu, Jun Zou, Meng-bin Qin, Jin-xiu Zhang, and Jie-an Huang developed the original hypothesis and supervised the experimental design. Li Yao and Jing-rong Liang performed in vitro and in vivo experiments. Li Yao, Jun Zou, Peng Peng, and Jing-rong Liang participated in the clinical specimen collection. Li Yao and Xin-ze Qiu analyzed the data and performed statistical analysis. Li Yao, Xin-ze Qiu, and Jun Zou wrote and revised the manuscript. Jie-an Huang was the guarantor and corresponding author. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDATA AVAILABILITY STATEMENT\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eZhang H, Zhang E, Hu H. Role of Ferroptosis in Non-Alcoholic Fatty Liver Disease and Its Implications for Therapeutic Strategies. Biomedicines. 2021;9(11).\u003c/li\u003e\n\u003cli\u003eYounossi ZM, Koenig AB, Abdelatif D, Fazel Y, Henry L, Wymer M. 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Oncology reports. 2022;47(5).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Heme oxygenase-1, Nonalcoholic fatty liver disease, Ferroptosis, Bioinformatics analysis","lastPublishedDoi":"10.21203/rs.3.rs-5035306/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5035306/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground \u0026amp; Aims: \u003c/strong\u003eThis research seeks to elucidate the significance of ferroptosis-related genes in the diagnosis, prognosis, and treatment of non-alcoholic fatty liver disease (NAFLD).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods: \u003c/strong\u003eKey genes pertinent to NAFLD were identified using the GEO database. The role of Heme oxygenase-1 (HMOX1) in NAFLD was validated via immunohistochemical analysis of hepatic tissues. Mice on a methionine-choline-deficient (MCD) diet were administered Hemin, followed by the collection of serum and liver samples for biochemical and histopathological examinations. HL7702 cells were transfected with a plasmid to elevate HMOX1 expression, then treated with oleic acid (OA) to induce lipid accumulation, and subsequently with erastin and AZD1480. A series of assays measured iron levels, reactive oxygen species, lipid peroxidation, and mitochondrial damage. Western blotting analysis was employed to elucidate the underlying molecular mechanisms.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eHMOX1 is crucial in the pathogenesis of NAFLD, evidenced by its decreased expression in patient liver tissues. Mice on an MCD diet exhibited significant hepatic steatosis, along with elevated levels of ALT, AST, TG, LDL, Fe\u003csup\u003e2+\u003c/sup\u003e, MDA, and ROS, and reduced levels of HMOX1 and GSH. Notably, Hemin effectively ameliorated NAFLD and prevented ferroptosis. Cellular analysis revealed activation of the JAK/STAT pathway in NAFLD. Upregulation of HMOX1 reduced OA-induced lipid peroxidation, inhibited ferroptosis, and suppressed the JAK/STAT pathway. Erastin negated the protective effects of HMOX1 overexpression. Moreover, the JAK/STAT pathway inhibitor AZD1480, which had the opposite effect with erastin, suppressed ferroptosis and ameliorated NAFLD.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eThis study elucidates that HMOX1 suppresses ferroptosis by inhibiting the JAK/STAT pathway in NAFLD.\u003c/p\u003e","manuscriptTitle":"HMOX1 Inhibits Ferroptosis in Non-Alcoholic Fatty Liver Disease","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-10-22 09:10:56","doi":"10.21203/rs.3.rs-5035306/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"bb6c5a18-5d90-4618-b378-2724a54a9f4f","owner":[],"postedDate":"October 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-12-25T15:08:11+00:00","versionOfRecord":[],"versionCreatedAt":"2024-10-22 09:10:56","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5035306","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5035306","identity":"rs-5035306","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}