The Role and Expression Patterns of Cuproptosis in Pulmonary Ischemia-Reperfusion Injury: An Experimental Study

The Role and Expression Patterns of Cuproptosis in Pulmonary Ischemia-Reperfusion Injury: An Experimental Study | 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 Article The Role and Expression Patterns of Cuproptosis in Pulmonary Ischemia-Reperfusion Injury: An Experimental Study Zicheng Wang, Hanqun Liu, Wenxing Du, Zhe Wu, Wenjie Jiao This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6375611/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 10 You are reading this latest preprint version Abstract Objective : To investigate the mechanistic role of cuproptosis in acute lung ischemia-reperfusion injury (LIRI). Methods : Microarray datasets GSE127003 and GSE9634 were retrieved from the Gene Expression Omnibus (GEO) database to identify and analyze differentially expressed cuproptosis-related genes (CRGs). To validate the bioinformatics findings, 24 male Sprague-Dawley (SD) rats were randomly allocated into three groups: Control, ischemia-reperfusion (I/R), and I/R with ammonium tetrathiomolybdate (ATTM) intervention (I/R+ATTM). The I/R+ATTM group received ATTM pretreatment for copper chelation prior to surgery. An in situ lung I/R injury model was established, and femoral venous blood was collected intraoperatively, while cardiac blood and left lung tissues were harvested postoperatively.Macroscopic evaluation assessed pulmonary hemorrhage, congestion, and edema. Hematoxylin-eosin (H&E) staining was performed for histopathological examination and lung injury scoring. Serum levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) were quantified using ELISA. Ceruloplasmin (Cp) levels were measured via a rat-specific assay kit. Western blotting analyzed pulmonary expression of ferredoxin 1 (FDX1), lipoic acid synthetase (LIAS), dihydrolipoyl transacetylase (DLAT), DLAT oligomers, dihydrolipoamide succinyltransferase (DLST), succinate dehydrogenase complex subunit B (SDHB), lipoylated DLAT (Lip-DLAT), and lipoylated DLST (Lip-DLST). Immunohistochemistry (IHC) localized FDX1, Lip-DLAT, and Lip-DLST expression in lung tissues. Results : Differential gene analysis revealed significant alterations in CRG RNA expression during lung I/R (P < 0.05). Histopathological assessment demonstrated severe injury in the I/R group, moderate in I/R+ATTM, and minimal in Controls (P < 0.01). Serum TNF-α, IL-1β, and IL-6 levels in I/R+ATTM were elevated versus Controls (P < 0.0001) but reduced compared to I/R (P < 0.05). ATTM intervention significantly decreased serum Cp (P < 0.001). Pulmonary FDX1, LIAS, DLAT, DLST, SDHB, Lip-DLAT, and Lip-DLST expression in I/R+ATTM was lower than I/R (P < 0.05) but remained higher than Controls (P < 0.05). DLAT oligomers increased versus Controls (P < 0.05) but were suppressed relative to I/R (P < 0.0001). IHC confirmed cytoplasmic localization of FDX1 and lipoylated proteins, with I/R+ATTM showing intermediate expression between I/R and Controls (P < 0.05). Conclusion : In a rat model of in situ lung ischemia-reperfusion, cuproptosis exacerbates acute lung injury by modulating key protein effectors, while copper chelation partially mitigates this pathological progression. Health sciences/Diseases Health sciences/Medical research Health sciences/Pathogenesis acute lung injury ischemia reperfusion cuproptosis FDX1 lipoylation Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION Since the 21st century, lung transplantation has become the treatment of choice for patients with end-stage lung diseases, including idiopathic pulmonary fibrosis, advanced lung cancer, and severe chronic obstructive pulmonary disease[ 1 ]. As an inevitable step in lung transplantation, ischemia-reperfusion frequently leads to acute lung ischemia-reperfusion injury (LIRI), which can progress to acute respiratory distress syndrome (ARDS) in severe cases. This complication has emerged as a critical determinant of perioperative survival rates[ 1 , 2 ]. The underlying mechanisms involve oxidative stress, inflammatory responses, and regulated cell death pathways, collectively impairing transplant outcomes[ 3 , 4 ]. Consequently, mitigating the detrimental effects of ischemia-reperfusion injury remains a major focus in clinical research. In March 2022, a groundbreaking study by researchers at the Broad Institute of MIT and Harvard, published in Science, identified a novel form of regulated cell death—cuproptosis. This copper-dependent cell death mechanism is initiated by elevated intracellular copper levels and mediated through ferredoxin 1 (FDX1), which facilitates the binding of copper to lipoylated proteins in the tricarboxylic acid (TCA) cycle. This interaction induces lipoylated protein aggregation, downregulates iron-sulfur cluster (Fe-S) proteins, and triggers proteotoxic stress, ultimately leading to cell death[ 5 ]. Emerging evidence implicates cuproptosis in the pathogenesis of neurodegenerative disorders[ 6 ], cardiovascular diseases[ 7 ], and various cancers[ 8 , 9 ]. Given its involvement in copper redox reactions, reactive oxygen species (ROS) generation, and mitochondrial dysfunction, cuproptosis has also been implicated in ischemia-reperfusion injuries affecting the heart and brain[ 10 , 11 ]. While preliminary studies have reported altered expression of cuproptosis-related biomarkers (NFE2L2, NLRP3, LIPT1, and MTF1) in allograft LIRI models[ 12 ], the role of the FDX1-mediated pathway—the central mechanism of cuproptosis—remains unexplored in LIRI. This study employs a multi-modal approach to elucidate the role of cuproptosis in lung ischemia-reperfusion injury (LIRI). First, we performed comprehensive bioinformatics analysis of publicly available RNA-Seq datasets to identify differentially expressed cuproptosis-related genes. Building upon these computational findings, we established a rat model of in situ pulmonary warm ischemia-reperfusion injury to systematically investigate the spatiotemporal expression patterns of key cuproptosis biomarkers at both whole-organism and tissue levels. To functionally validate these observations, we implemented therapeutic intervention using the copper chelator ammonium tetrathiomolybdate (ATTM) to specifically inhibit cuproptosis pathways in our experimental model. This integrated methodology enables us to: (1) characterize cuproptosis-related molecular signatures in LIRI, (2) evaluate the therapeutic potential of copper chelation, and (3) identify novel targets for clinical intervention in transplantation-associated ischemia-reperfusion injury. MATERIALS Data collection and organization from the GEO database Datasets GSE 127003 and GSE 9634 were downloaded from the GEO database. GSE 127003 includes data from 92 samples, comprising RNA-Seq data from lung tissues of 46 patients at the end of cold ischemia and 46 patients after 2 hours of cold ischemia and reperfusion during lung transplantation. GSE 9634 contains 12 samples, including lung RNA-Seq data from 6 rats in the sham operation group, as well as lung RNA-Seq data from rats 30 minutes (n=3) and 3 hours (n=3) after 2 hours of in-situ thermal ischemia followed by reperfusion. Cuproptosis-related Genes We screened and extracted 12 cuproptosis-related genes (CRGs) from the existing literature[13-15], including 9 positively correlated genes: ferredoxin 1 (FDX1), lipoyltransferase 1 (LIPT1), lipoic acid synthetase (LIAS), dihydrolipoamide succinyltransferase (DLST), dihydrolipoamide dehydrogenase (DLD), dihydrolipoamide acetyltransferase (DLAT), ATPase copper-transporting beta (ATP7B), pyruvate dehydrogenase alpha 1 (PDHA1), pyruvate dehydrogenase E1 subunit beta (PDHB), and 3 negatively correlated genes: cyclin-dependent kinase inhibitor 2A (CDKN2A), glutaminase (GLS), and metal regulatory transcription factor 1 (MTF1). Animals Eight healthy adult male Sprague-Dawley (SD) rats (4-5 weeks old) and sixteen SD rats (7-8 weeks old) were procured from Jinan Pengyue Experimental Animal Breeding Co., Ltd. (License No. SCXK(LU)2014-0007). The animals were housed in the SPF-grade animal facility of the Central Laboratory at the Affiliated Hospital of Qingdao University. Following a one-week acclimatization period, the experiments were initiated.This study was conducted in strict compliance with animal welfare guidelines and was approved by the Animal Ethics Committee of Qingdao University. This study was designed and reported in accordance with the ARRIVE Guidelines to ensure comprehensive reporting of animal experiments. Major Reagents and Instruments The primary reagents and instruments used in this study are detailed in Table 1. Table 1. Names and Manufacturers of Major Reagents and Instruments. Category Item Manufacturer Reagents IL-6 Detection Kit Elabscience Biotechnology, China IL-1β Detection Kit Elabscience Biotechnology, China TNF-α Detection Kit Elabscience Biotechnology, China BCA Protein Concentration Assay Kit Elabscience Biotechnology, China DAB Chromogenic Kit Maixin Biotech, China FDX1 Primary Antibody Abmart Scientific, China LIAS Primary Antibody Abmart Scientific, China SDHB Primary Antibody Abmart Scientific, China DLST Primary Antibody Abmart Scientific, China lipoic acid Antibody Abcam, UK Enzyme-Labeled Goat Anti-Rabbit Secondary Antibody for Immunohistochemistry Abcam, UK Horseradish Peroxidase (HRP)-Labeled Goat Anti-Rabbit Secondary Antibody Servicebio Technology, China Ultra-Sensitive ECL Substrate Solution Yaenzyme Biotechnology, China Rapid Stripping Buffer Yaenzyme Biotechnology, China Protein-Free Rapid Blocking Buffer Yaenzyme Biotechnology, China Multicolor Prestained Protein Marker Yaenzyme Biotechnology, China RIPA Tissue Protein Lysis Buffer Yaenzyme Biotechnology, China Phenylmethanesulfonyl fluoride（PMSF） Yaenzyme Biotechnology, China Na3VO4 Yaenzyme Biotechnology, China Ammonium tetrathiomolybdate (ATTM) Aladdin Biochemical Technology，China Rat ceruloplasmin (Cp) assay kit Shanghai Jianglai Biotechnology，China 4-20% High-Resolution Gradient Precast Gel Yeasen Biotechnology, China Denaturing Protein Precast Gel Buffer Powder Yeasen Biotechnology, China 5× Protein Loading Buffer Beyotime Biotechnology, China PVDF Membrane Solarbio Science & Technology, China Polyoxyethylene (20) Sorbitan Monolaurate (Tween 20) Solarbio Science & Technology, China Absolute Ethanol Fuyu Fine Chemical, China Methanol Fuyu Fine Chemical, China 1× TBST Buffer Servicebio, China 1× PBS Buffer Servicebio, China 10× Citrate Antigen Retrieval Solution (pH 6.0) Biossci Biotechnology, China 10× EDTA Antigen Retrieval Solution (pH 9.0) Biossci Biotechnology, China Hematoxylin Staining Solution Biossci Biotechnology, China Hematoxylin Differentiation Solution Biossci Biotechnology, China Hematoxylin Bluing Solution Biossci Biotechnology, China Normal Goat Serum Boster Biological Technology, China Instruments Vortex Mixer Thermo Fisher Scientific, USA Benchtop Centrifuge Thermo Fisher Scientific, USA General Laboratory Electrophoresis Power Supply Junyi Oriental Electrophoresis Equipment, China Protein Vertical Electrophoresis Tank ‌ Bio-Rad Laboratories , USA Laboratory Chemiluminescence Imaging System GeneGnome, USA Multifunctional Microplate Reader TECAN, Switzerland Small Animal Ventilator HUAYON, China Tissue Dehydrator Zhongwei Electronic Instrument, China Tissue Embedding Machine Zhongwei Electronic Instrument, China Pathology Microtome Thermo Fisher Scientific,USA Upright Microscope Olympus Corporation, Japan Methods Differential gene expression analysis in GEO database We downloaded the gene expression and sample information files for GSE 127003 and GSE 9634 from the GEO database and used the R programming language to organize and transform the data. We then intersected the data with CRGs to extract RNA-Seq data associated with the selected genes. Gene expression differences were statistically analyzed and visualized using GraphPad Prism 9.0 software. A p-value of less than 0.05 was considered statistically significant. ATTM Intervention Rat Model Following a 1-week acclimatization period, SD rats were administered ammonium tetrathiomolybdate (ATTM) via oral gavage for 3 consecutive weeks. A 1 mg/mL ATTM solution was prepared in double-distilled water and administered daily at a dosage of 30 mg/kg body weight using a rodent feeding needle. Special care was taken to perform the gavage slowly while monitoring for any signs of oronasal reflux to prevent aspiration. Throughout the intervention period, the animals maintained normal access to food and water ad libitum. Subsequent experiments were conducted following the completion of this 3-week ATTM treatment protocol[16]. Lung ischemia-reperfusion injury model The pulmonary ischemia-reperfusion injury (IRI) model was established in healthy adult male Sprague-Dawley rats (250–300 g) according to previously described methods[17, 18]. Following anesthesia induction with intraperitoneal injection of 2% sodium pentobarbital (60 mg/kg) and confirmation of adequate anesthetic depth (assessed by loss of pedal reflex), the animals were positioned in dorsal recumbency and prepared for aseptic surgery. The surgical site was shaved and disinfected before performing a midline cervical incision to expose the trachea. Tracheotomy was performed, followed by endotracheal intubation connected to a rodent ventilator (Harvard Apparatus) with initial settings of 60 breaths/min and a tidal volume of 10 mL/kg. A left thoracotomy was performed by carefully dissecting the intercostal muscles along the left sternal border between the second and third ribs. After achieving hemostasis, venous access was established via the left femoral vein for blood collection into heparinized Eppendorf tubes, followed by systemic heparinization with heparinized saline (62 IU/mL, 3 mL/kg). Following a 10-min circulation period, the intercostal incision was reopened to expose the left pulmonary hilum using sterile cotton-tipped applicators. The left pulmonary hilum was clamped with microvascular clips during peak lung inflation, with successful occlusion confirmed by the absence of left lung ventilation and loss of distal vascular pulsation. Ventilator parameters were adjusted to 50 breaths/min and a tidal volume of 8 mL/kg during the 90-min ischemic period. Reperfusion was initiated by removing the vascular clips, and ventilator settings were immediately restored to baseline (60 breaths/min, 10 mL/kg tidal volume). After 2 h of reperfusion, terminal procedures were performed, including cardiac blood collection via heparinized syringe, pulmonary artery perfusion with 20 mL saline, and left lung tissue harvest. The animals were then euthanized by CO₂ asphyxiation in accordance with institutional animal care guidelines. Core body temperature was maintained throughout the procedure using a heating pad, and anesthetic depth was continuously monitored to ensure animal welfare. Experimental Animal Grouping 1.Control Group: Eight randomly selected 7-8-week-old rats underwent anesthesia, tracheotomy, intubation, heparinization, thoracotomy, and left pulmonary hilum exposure without ischemia induction. The animals were ventilated for 3.5 hours before sample collection and euthanasia. 2.Ischemia-Reperfusion (I/R) Group: Eight randomly selected 7-8-week-old rats were subjected to the complete pulmonary ischemia-reperfusion injury model as described above, followed by sample collection and euthanasia. 3.ATTM Intervention (I/R+ATTM) Group: Eight randomly selected 4-5-week-old rats first received ATTM pretreatment to establish the intervention model, then underwent the same pulmonary ischemia-reperfusion procedure as the I/R group before terminal sample collection and euthanasia. HE staining was used to observe the lung histological characteristics After dewatering, embedding, dewaxing, and hydration, lung tissues from rats in the Control、I/R and I/R+ATTM groups were subjected to hematoxylin and eosin (HE) staining. The histological characteristics of the rat lungs were observed under a microscope. Two pathologists, blinded to the group assignments, independently evaluated alveolar congestion, edema, hemorrhage, neutrophil infiltration, alveolar wall thickness, and hyaline formation in lung injury[19]. Determination of serum inflammatory factors in Rats Cardiac blood samples were collected from rats in all three experimental groups into heparinized sterile centrifuge tubes (Eppendorf tubes) and allowed to clot at room temperature for 2 hours. Following centrifugation at 1,000 × g for 15 minutes at 4°C, the supernatant serum was carefully collected. Serum concentrations of TNF-α, IL-1β, and IL-6 were quantified using enzyme-linked immunosorbent assay (ELISA) in 96-well microplates pre-coated with specific capture antibodies. The assay procedure involved sequential addition of test samples, standards, biotin-conjugated detection antibodies, and horseradish peroxidase (HRP)-labeled avidin (ABC) in duplicate wells. After standardized incubation and washing steps, color development was achieved using 3,3',5,5'-tetramethylbenzidine (TMB) substrate. Optical density (OD) values were measured at 450 nm using a microplate reader, and cytokine concentrations were calculated using ELISACalc software. This standardized protocol ensures accurate and reproducible quantification of inflammatory mediators through implementation of duplicate measurements and strict adherence to established ELISA procedures. Determination of Serum Ceruloplasmin in Rats Femoral venous blood samples from rats in all three experimental groups were collected in heparinized sterile Eppendorf tubes and allowed to clot at room temperature for 2 hours. The samples were then centrifuged at 1,000 × g for 15 min at 4 °C to separate the serum. Serum ceruloplasmin (Cp) levels were quantified using a rat-specific Cp ELISA kit according to the manufacturer's protocol. Briefly, 96-well microplates pre-coated with rat Cp capture antibody were used for the assay. Samples and standards were added to the wells in duplicate, followed by sequential incubation with biotinylated detection antibody and horseradish peroxidase (HRP)-conjugated avidin (ABC). After appropriate incubation and washing steps, the reaction was developed with 3,3',5,5'-tetramethylbenzidine (TMB) substrate. Optical density (OD) values were measured at 450 nm using a microplate reader, and the average Cp concentration for each sample was calculated using ELISA analysis software (ELISACalc). Western Blot (WB) was used to detect the expression of cuproptpsis related protein Partial left lung tissues from rats in all three experimental groups were immersed in PBS buffer for 10 minutes before homogenization in protein lysis buffer containing PMSF and phosphatase inhibitors. Following 30 minutes of incubation on ice, the homogenates were centrifuged at 12,000 rpm for 10 minutes at 4°C to collect the supernatant. Protein concentrations were determined using the BCA protein assay to ensure consistent loading amounts across samples. Equal amounts of protein samples from the three groups were separated by electrophoresis, transferred onto membranes, and blocked before overnight incubation at 4°C with primary antibodies against FDX1, LIAS, DLST, DLAT, SDHB, and lipoic acid. After thorough washing, the membranes were incubated with goat anti-rabbit secondary antibody at room temperature for 2 hours. Protein bands were finally visualized using enhanced chemiluminescence (ECL) reagent and detected with a chemiluminescence imaging system. Immunohistochemical (IHC) staining was used to detect the expression of FDX1 and lipoacylated protein Lung tissue from three rat groups was collected for paraffin sectioning. After dewaxing, hydration, antigen retrieval, and incubation at room temperature for 25 minutes, the antigens were blocked using blocking serum. Subsequently, primary antibodies against FDX1 and lipoic acid were added and incubated at 4°C overnight. After washing, goat anti-rabbit IgG secondary antibody was applied. Finally, DAB staining solution was added, and the slides were observed under a microscope. After hematoxylin counterstaining, dehydration, and sealing, the expressions of FDX1 and lipoacylated proteins were examined under a microscope. Statistical Analysis Statistical analysis was performed using GraphPad Prism 9.0 software. Normality and homogeneity of variance tests were conducted for each data set. For data that followed a normal distribution with homogeneity of variance, a two-sample t-test was used, and the results were expressed as mean ± standard deviation (x̄ ± s). For data that did not conform to normal distribution, the Mann-Whitney U test was applied, and the results were presented as median (first quartile (Q1), third quartile (Q3)). If the data showed significant differences in components, log transformation was applied to improve the data distribution. A P value of < 0.05 was considered statistically significant. RESULTS Analysis of differential expression gene of cuproptpsis The results of differential expression analysis of CRGs RNA-Seq data in the cold ischemia group (n = 46) and the reperfusion group 2 hours after lung transplantation (n = 46) from GSE127003 showed that, compared with the cold ischemia group, the expression of the cuproptosis-related gene FDX1 and the cuproptosis inhibitor gene MTF1 were significantly increased in the reperfusion group (P < 0.001). Conversely, the expressions of lipoic acid pathway genes LIAS, DLD, pyruvate dehydrogenase (PDH) complex-related genes DLAT, PDHA1, PDHB, and the copper transporter gene ATP7B were significantly decreased (P < 0.01) (FIG. 1A). In the rat lung ischemia-reperfusion experiment (GSE9634), the ischemia reperfusion groups at 30 minutes (n = 3) and 3 hours (n = 3) were combined into a single ischemia-reperfusion group. Compared with the control group (n = 6), the expressions of FDX1, DLD, SLC31A1, and PDHA1 were significantly increased in the ischemia-reperfusion group (n = 6) (P < 0.05). In contrast, the expression of the cuproptosis-related suppressor gene GLS was significantly decreased (P < 0.01) (FIG. 1B). The expression results of CRGs in each group are shown in Table 2. Table 2. Expression analysis of CRGs in GSE127003 and GSE9634. Table 2A presents the expression analysis results of CRGs in the lung transplantation cold ischemia group (n = 46) and the reperfusion group (n = 46). The data are presented in their raw format, with results including mean ± standard deviation and P-value. Table 2B presents the expression analysis results of CRGs in GSE9634, comparing the rat control group (n = 6) and the ischemia-reperfusion group (n = 6). The data are presented as log10 transformations, with results including the median (Q1, Q3) and P-value. A Gene cold ischemia group reperfusion group P-value FDX1 403.21±72.92 471.58±95.15 0.000167 LIAS 238.64±69.86 159.24±57.32 <0.000001 DLST 1353.85±98.24 1379.2±111.45 0.101771 DLAT 320.52±53.6 272.14±53.48 0.000038 DLD 267.37±27.05 249.11±26.27 0.000840 PDHB 2130.5±241.88 1718.12±229.88 <0.000001 ATP7B 119.7±28.24 101.32±24.29 0.000808 SLC31A1 778.62±159.58 790.92±154.01 0.259903 PDHA1 772.41±76.17 725.76±73.01 0.001772 MTF1 262.84±27.47 312.94±37.52 <0.000001 GLS 350.91±69.91 346.74±68.33 0.260265 CDKN2A 44.38±6.45 46.15±8.11 0.101771 B Gene control group I/R group P-value FDX1 2.950(2.935,2.952) 3.296(3.244,3.357) 0.0022 LIAS 2.528(2.514,2.547) 2.480(2.398,2.516) 0.1797 DLST 2.977(2.917,3.027) 3.007(2.979,3.042) 0.5887 DLD 2.772(2.702,2.790) 2.863(2.847,2.911) 0.0260 PDHB 3.165(3.154,3.200) 3.185(3.154,3.196) >0.9999 ATP7B 1.703(1.326,1.889) 1.702(1.629,1.796) 0.9372 SLC31A1 2.108(2.043,2.157) 2.278(2.198,2.364) 0.0260 PDHA1 3.423(3.411,3.433) 3.572(3.523,3.597) 0.0043 MTF1 1.893(1.815,1.922) 1.938(1.907,1.965) 0.1320 GLS 2.722(2.716,2.755) 2.607(2.570,2.640) 0.0043 CDKN2A 1.272(1.248,1.436) 1.287(0.930,1.306) >0.9999 Histopathological and Inflammatory Cytokine Profiles in Rat Pulmonary Tissues H&E staining revealed distinct pathological alterations in lung tissues across experimental groups. Compared to the Control group, the I/R group exhibited characteristic features of acute lung injury, including severe alveolar congestion, edema, hemorrhage, prominent neutrophil infiltration, alveolar wall thickening, and hyaline membrane formation. In contrast, the I/R+ATTM group demonstrated marked attenuation of these pathological changes, with significantly reduced alveolar congestion, hemorrhage, edema, and hyaline membrane formation compared to the I/R group. Notably, alveolar wall thickness was not significantly increased, and neutrophil infiltration was substantially diminished in the I/R+ATTM group (FIG. 2A). Quantitative analysis of lung injury scores demonstrated that the I/R+ATTM group exhibited significantly higher scores than the Control group (P<0.0001), yet remained significantly lower than the I/R group (P<0.01) (FIG. 2B). Compared to the Control group, serum levels of inflammatory cytokines (TNF-α, IL-6, and IL-1β) were significantly elevated in both the I/R and I/R+ATTM groups (P<0.0001). However, ATTM intervention markedly reduced these inflammatory cytokine levels in the ischemia-reperfusion model when compared to the I/R group (P<0.05) (FIG. 2C), indicating that the rat model of acute lung injury induced by lung ischemia and reperfusion was successfully established. Serum Ceruloplasmin (Cp) Levels in Experimental Rats The I/R+ATTM group demonstrated a significant 40% reduction in serum Cp levels compared to both Control and I/R groups (P<0.001) (FIG. 2D). This pronounced suppression of Cp expression, a key copper transport protein, provides biochemical confirmation of successful establishment of the ATTM-mediated copper-deficient model in rats. Expression of Cuproptosis-Related Proteins in Rat Lung Tissues Western blot analysis revealed:(1) The I/R group showed significantly decreased expression of FDX1, LIAS, DLAT, DLST, SDHB, Lip-DLAT, and Lip-DLST (P<0.05), while DLAT-Oligomers expression was increased (P<0.05) compared to Controls (FIG. 3A-B).(2) ATTM intervention significantly upregulated FDX1, LIAS, DLAT, DLST, SDHB, Lip-DLAT, and Lip-DLST expression versus I/R group (P<0.05), though remaining below Control levels (P<0.05). DLAT-Oligomers were markedly reduced compared to I/R (P<0.0001) but remained elevated versus Controls (P<0.05) (FIG. 3C-D). Expression of FDX1 and lipoylprotein in Rat Lung Tissues Immunohistochemical staining demonstrated that both FDX1 and lipoylated proteins (Lip-DLAT and Lip-DLST) were predominantly localized in the cytoplasm of rat pulmonary tissue cells. Quantitative evaluation revealed significantly higher expression levels of these proteins in the Control group compared to both I/R and I/R+ATTM groups (P < 0.05). Notably, ATTM intervention partially restored the expression of FDX1 and lipoylated proteins in the I/R+ATTM group relative to the I/R group (P<0.05), though remaining below Control levels (P<0.05) (FIG. 4). DISCUSSION In this study, based on RNA-Seq data from the GEO database, we observed significant changes in the expression of key genes associated with cuproptosis during rat lung warm ischemia-reperfusion, including FDX1, the copper transporter SLC31A1, the pyruvate dehydrogenase (PDH) complex-related gene PDHA1, and the cuproptosis suppressor gene GLS. Similarly, in human lung transplantation under cold ischemia-reperfusion conditions, not only FDX1 but also genes in the lipoic acid pathway (e.g., LIAS, DLD), PDH complex-related genes (e.g., DLAT), and cuproptosis suppressor genes (e.g., MTF1) exhibited significant alterations. These findings align with previous studies demonstrating that cuproptosis involves the oligomerization of lipoylated proteins and a reduction in Fe-S cluster proteins[ 13 ], suggesting that cuproptosis may play a role in lung ischemia-reperfusion injury. Furthermore, in the GSE127003 dataset, compared to the cold ischemia group, the expression of cuproptosis-promoting genes was significantly reduced in the 2-hour reperfusion group, while the expression of suppressor genes increased. This may be attributed to the exacerbation of cuproptosis during reperfusion, which is associated with acute lung injury and subsequent modulation of cuproptosis-related gene (CRG) expression. However, due to the lack of lung histopathology and protein-level data, it cannot be ruled out that lung injury may improve during reperfusion, leading to the normalization of CRG expression. Therefore, the underlying mechanisms require further investigation. Notably, compared to human lung transplantation, some cuproptosis-promoting CRGs showed significantly increased expression in rat lung ischemia-reperfusion models, and the differentially expressed CRGs varied between species. These discrepancies may be due to the smaller sample size in the rat study (n = 12), differences in ischemia methods (warm vs. cold ischemia), and variations in control group standards (ischemia group vs. sham operation group). These factors highlight the need for further studies to clarify the role of cuproptosis in lung ischemia-reperfusion injury across different experimental conditions. In recent years, with the continuous increase in the number of lung transplantation surgeries, in-depth research into the pathophysiological mechanisms of lung ischemia-reperfusion injury (LIRI) has become particularly important. Currently, the core pathogenesis of LIRI is believed to involve multiple pathological processes, including but not limited to oxidative stress, inflammatory responses, calcium overload, microcirculatory dysfunction, apoptosis, necrosis, mitochondrial dysfunction, and endoplasmic reticulum stress[ 20 ]. According to Tsvetkov et al., the accumulation of copper ions and the catalytic role of FDX1 are the direct causes of the reduction in Fe-S cluster proteins, impaired synthesis of lipoylated proteins, and the aggregation of lipoylated proteins, ultimately leading to proteotoxic stress[ 5 ]. Among these, FDX1, as both a key regulator of cuproptosis and an Fe-S cluster protein, is also affected by cuproptosis, resulting in decreased expression[ 5 ]. LIAS can directly bind to FDX1 to regulate the lipoylation process of proteins and, as an Fe-S cluster protein, its expression is downregulated[ 21 , 22 ]. SDHB catalyzes the oxidation of succinate to fumarate in the tricarboxylic acid (TCA) cycle, and its expression decreases during cuproptosis as an Fe-S cluster protein[ 23 , 24 ]. The downregulation of SDHB can lead to mitochondrial dysfunction and hypoxia-induced injury[ 25 ]. To further investigate the changes in cuproptosis-related proteins during LIRI, we employed a rat hilar clamp-reperfusion model to simulate acute lung injury induced by ischemia-reperfusion during lung transplantation[ 17 ]. We ultimately observed that during pulmonary ischemia-reperfusion, alongside morphological changes in lung injury, the expression of Fe-S cluster proteins such as FDX1, SDHB, and LIAS, as well as lipoylated proteins (Lip-DLAT and Lip-DLST), significantly decreased, while lipoylated proteins (DLAT) aggregated extensively. These findings are consistent with the process of cuproptosis, where abnormal intracellular accumulation of Cu²⁺ leads to the catalytic generation of cuprous ions (Cu⁺) by the key cuproptosis protein FDX1, followed by the binding of Cu⁺ to lipoylated proteins (e.g., Lip-DLAT, Lip-DLST)[ 5 ]. This suggests a potential association between cuproptosis and LIRI. ATTM, as a widely used copper chelator, forms a stable, insoluble complex upon contact with Cu²⁺, thereby reducing Cu²⁺ levels in the body[ 26 ]. This is manifested by a significant decrease in serum ceruloplasmin (Cp) levels. In our experiments, ATTM intervention significantly reduced serum Cp levels in rats[ 16 ], confirming the effectiveness of the intervention and the successful establishment of a low-copper rat model. In previous studies on ischemia-reperfusion injury, it was found that copper ions can participate in the Fenton reaction, where the redox cycling between Cu²⁺ and Cu⁺ continuously catalyzes the generation of hydroxyl radicals (·OH) from H₂O₂[ 27 ], exacerbating oxidative stress in tissues and organs. Additionally, copper ions directly target components of the TCA cycle, binding to thiol groups in mitochondria and inhibiting the activity of respiratory chain complexes, leading to reduced ATP production and further aggravating metabolic dysfunction under ischemic and hypoxic conditions[ 11 ]. Building on this, researchers have found that reducing copper ion concentrations in myocardial ischemia-reperfusion injury significantly decreases infarct size and improves cardiac function[ 28 ]. Similarly, in models of cerebral ischemia-reperfusion injury, downregulating FDX1 expression with disulfiram (DSF) suppresses oxidative stress, alleviates neuroinflammation, and protects mitochondrial function[ 29 ]. In our experiment, after using the copper chelator ATTM to reduce Cp levels in rats, we observed increased expression of Fe-S cluster proteins such as FDX1 and lipoylated proteins compared to post-ischemia-reperfusion levels, along with reduced aggregation of lipoylated proteins. Concurrently, pathological changes in lung tissue, such as neutrophil infiltration, alveolar wall thickening, and pulmonary interstitial edema, were significantly ameliorated after ischemia-reperfusion. This further demonstrates that the cuproptosis mechanism, mediated by FDX1, participates in and promotes the development of LIRI, while inhibiting cuproptosis can significantly improve tissue changes during LIRI. This targeted therapeutic strategy holds significant clinical value, offering new hope for the treatment of lung ischemia-reperfusion injury (LIRI). Similar to other studies, this research also has several limitations:1. Small Sample Size: The GSE9634 dataset from the GEO database used in this study included only three rats in the experimental group. The small sample size may increase heterogeneity and reduce the reliability of the results. Although additional validation experiments were conducted to supplement the findings, potential biases may still exist. Future studies should expand the sample size to enhance the robustness and generalizability of the results.2. Limited Exploration of Ischemia and Reperfusion Phases: This study focused on the changes in cuproptosis mechanisms during in situ lung ischemia-reperfusion injury but did not separately investigate the ischemia and reperfusion phases. It remains unclear whether cuproptosis exhibits differential expression during these two stages, warranting further investigation.3. Preclinical Model Limitations: The findings of this study are primarily based on animal models, and their clinical translation requires further validation. Future research should include human studies or clinical trials to confirm the relevance of these findings in a clinical setting. CONCLUSION In summary, cuproptosis, as a novel form of cell death, participates in the pathogenesis of LIRI, and the inhibition of cuproptosis significantly ameliorates LIRI. This conclusion enriches our understanding of the pathological mechanisms underlying acute lung ischemia-reperfusion injury and provides new insights for its treatment. Declarations Author Contribution Z.C. Wang and W.J. Jiao wrote the main manuscript text and designed experiments, W.X. Du and H.Q. Liu provided experimental technical support, Z. Wu provided data analysis support, . All authors reviewed the manuscript. Data Availability The datasets analyzed in this study are publicly available in the Gene Expression Omnibus repository, with accession numbers GSE127003 and GSE9634. These data can be accessed at: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE127003 and https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE9634. References T.F. Chen-Yoshikawa, Ischemia-Reperfusion Injury in Lung Transplantation, Cells (6) (2021). O. Pak, A. Sydykov, D. Kosanovic, R.T. Schermuly, N. Weissmann, Lung Ischaemia–Reperfusion Injury: The Role of Reactive Oxygen Species, Pulmonary Vasculature Redox Signaling in Health and Disease2017. V.E. Laubach, A.K. Sharma, Mechanisms of lung ischemia-reperfusion injury, Current Opinion in Organ Transplantation 21(3) (2016) 246. M. Capuzzimati, O. Hough, M. Liu, Cell death and ischemia-reperfusion injury in lung transplantation, J Heart Lung Transplant 41(8) (2022) 1003-1013. T. Peter, C. Shannon, P. Boryana, D. Margaret, V. Ana, A. Mai, R. Jordan, J. Lena, H. Ranad, S.R. D, E.J. K, F. Evgeni, K. Mustafa, C.S. M, L. Svetlana, K. Naama, S. Sandro, G.T. R, Copper induces cell death by targeting lipoylated TCA cycle proteins, Science (New York, N.Y.) 375(6586) (2022) 1254-1261. P. Maher, Potentiation of glutathione loss and nerve cell death by the transition metals iron and copper: Implications for age-related neurodegenerative diseases, Free Radic Biol Med 115 (2018) 92-104. X. Chen, Q. Cai, R. Liang, D. Zhang, X. Liu, M. Zhang, Y. Xiong, M. Xu, Q. Liu, P. Li, P. Yu, A. Shi, Copper homeostasis and copper-induced cell death in the pathogenesis of cardiovascular disease and therapeutic strategies, Cell Death Dis 14(2) (2023) 105. J. Xie, Y. Yang, Y. Gao, J. He, Cuproptosis: mechanisms and links with cancers, Mol Cancer 22(1) (2023) 46. S. Zhang, Q. Huang, T. Ji, Q. Li, C. Hu, Copper homeostasis and copper-induced cell death in tumor immunity: implications for therapeutic strategies in cancer immunotherapy, Biomark Res 12(1) (2024) 130. S.R. Powell, D. Hall, A. Shih, Copper loading of hearts increases postischemic reperfusion injury, Circ Res 69(3) (1991) 881-5. Q. Guo, M. Ma, H. Yu, Y. Han, D. Zhang, Dexmedetomidine enables copper homeostasis in cerebral ischemia/reperfusion via ferredoxin 1, Ann Med 55(1) (2023) 2209735. J. Qin, X. Xiao, S. Li, N. Wen, K. Qin, H. Li, J. Wu, B. Lu, M. Li, X. Sun, Identification of cuproptosis-related biomarkers and analysis of immune infiltration in allograft lung ischemia-reperfusion injury, Front Mol Biosci 10 (2023) 1269478. P. Tsvetkov, S. Coy, B. Petrova, M. Dreishpoon, A. Verma, M. Abdusamad, J. Rossen, L. Joesch-Cohen, R. Humeidi, R.D. Spangler, J.K. Eaton, E. Frenkel, M. Kocak, S.M. Corsello, S. Lutsenko, N. Kanarek, S. Santagata, T.R. Golub, Copper induces cell death by targeting lipoylated TCA cycle proteins, Science 375(6586) (2022) 1254-1261. H. Liu, Pan-cancer profiles of the cuproptosis gene set, Am J Cancer Res 12(8) (2022) 4074-4081. G. Zhang, J. Sun, X. Zhang, A novel Cuproptosis-related LncRNA signature to predict prognosis in hepatocellular carcinoma, Sci Rep 12(1) (2022) 11325. H. Wei, B. Frei, J.S. Beckman, W.J. Zhang, Copper chelation by tetrathiomolybdate inhibits lipopolysaccharide-induced inflammatory responses in vivo, Am J Physiol Heart Circ Physiol 301(3) (2011) H712-20. G. Matute-Bello, C.W. Frevert, T.R. Martin, Animal models of acute lung injury, AJP Lung Cellular and Molecular Physiology 295(3) (2008) L379-99. Y.Z. Bai, Y. Yokoyama, W. Li, Y. Terada, D. Kreisel, R.G. Nava, Murine Left Pulmonary Hilar Clamp Model of Lung Ischemia Reperfusion Injury, J Vis Exp (206) (2024). W. Gao, T. Jiang, Y.H. Liu, W.G. Ding, C.C. Guo, X.G. Cui, Endothelial progenitor cells attenuate the lung ischemia/reperfusion injury following lung transplantation via the endothelial nitric oxide synthase pathway, J Thorac Cardiovasc Surg 157(2) (2019) 803-814. B.J. Zimmerman, D.N. Granger, Mechanisms of reperfusion injury, Am J Med Sci 307(4) (1994) 284-92. M.B. Dreishpoon, N.R. Bick, B. Petrova, D.M. Warui, A. Cameron, S.J. Booker, N. Kanarek, T.R. Golub, P. Tsvetkov, FDX1 regulates cellular protein lipoylation through direct binding to LIAS, J Biol Chem 299(9) (2023) 105046. Q. Zhao, T. Qi, The implications and prospect of cuproptosis-related genes and copper transporters in cancer progression, Front Oncol 13 (2023) 1117164. B. Moosavi, X.L. Zhu, W.C. Yang, G.F. Yang, Genetic, epigenetic and biochemical regulation of succinate dehydrogenase function, Biol Chem 401(3) (2020) 319-330. N. Armstrong, C.M. Storey, S.E. Noll, K. Margulis, M.H. Soe, H. Xu, B. Yeh, L. Fishbein, E. Kebebew, B.E. Howitt, R.N. Zare, J. Sage, J.P. Annes, SDHB knockout and succinate accumulation are insufficient for tumorigenesis but dual SDHB/NF1 loss yields SDHx-like pheochromocytomas, Cell Rep 38(9) (2022) 110453. Y. Liu, C. Xue, H. Lu, Y. Zhou, R. Guan, J. Wang, Q. Zhang, T. Ke, M. Aschner, W. Zhang, W. Luo, Hypoxia causes mitochondrial dysfunction and brain memory disorder in a manner mediated by the reduction of Cirbp, Sci Total Environ 806(Pt 3) (2022) 151228. C.F. Mills, T.T. El-Gallad, I. Bremner, Effects of molybdate, sulfide, and tetrathiomolybdate on copper metabolism in rats, J Inorg Biochem 14(3) (1981) 189-207. M. Nagai, N.H. Vo, L. Shin Ogawa, D. Chimmanamada, T. Inoue, J. Chu, B.C. Beaudette-Zlatanova, R. Lu, R.K. Blackman, J. Barsoum, K. Koya, Y. Wada, The oncology drug elesclomol selectively transports copper to the mitochondria to induce oxidative stress in cancer cells, Free Radic Biol Med 52(10) (2012) 2142-50. N. Chen, L. Guo, L. Wang, S. Dai, X. Zhu, E. Wang, Sleep fragmentation exacerbates myocardial ischemia‒reperfusion injury by promoting copper overload in cardiomyocytes, Nat Commun 15(1) (2024) 3834. S. Yang, X. Li, J. Yan, F. Jiang, X. Fan, J. Jin, W. Zhang, D. Zhong, G. Li, Disulfiram downregulates ferredoxin 1 to maintain copper homeostasis and inhibit inflammation in cerebral ischemia/reperfusion injury, Sci Rep 14(1) (2024) 15175. Additional Declarations No competing interests reported. Supplementary Files WB1DLAT.pdf WB1DLATOLI.pdf WB1DLST.pdf WB1FDX1.pdf WB1LIAS.pdf WB1LIPDLAT.pdf WB1LIPDLST.pdf WB1SDHB.pdf WB1ACTIN.pdf WB21ACTIN.pdf WB21DLAT.pdf WB21DLATOLI.pdf WB21DLST.pdf WB21LIPDLAT.pdf WB21LIPDLST.pdf WB21SDHB.pdf WB22ACTIN.pdf WB22FDX1.pdf WB22LIAS.pdf Cite Share Download PDF Status: Under Revision Version 1 posted Editorial decision: Revision requested 10 Nov, 2025 Reviews received at journal 07 Oct, 2025 Reviewers agreed at journal 25 Sep, 2025 Reviews received at journal 22 Aug, 2025 Reviewers agreed at journal 12 Aug, 2025 Reviewers invited by journal 05 May, 2025 Editor assigned by journal 05 May, 2025 Editor invited by journal 10 Apr, 2025 Submission checks completed at journal 07 Apr, 2025 First submitted to journal 04 Apr, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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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-6375611","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":453521109,"identity":"bec987c2-8c28-49b1-847d-19a33365a699","order_by":0,"name":"Zicheng Wang","email":"","orcid":"","institution":"Affiliated Hospital of Qingdao University","correspondingAuthor":false,"prefix":"","firstName":"Zicheng","middleName":"","lastName":"Wang","suffix":""},{"id":453521110,"identity":"e8396a48-35e7-41bf-b450-37b48ccad7a7","order_by":1,"name":"Hanqun Liu","email":"","orcid":"","institution":"Affiliated Hospital of Qingdao University","correspondingAuthor":false,"prefix":"","firstName":"Hanqun","middleName":"","lastName":"Liu","suffix":""},{"id":453521111,"identity":"5fbdebad-fc61-4cec-a9a4-b594630901b0","order_by":2,"name":"Wenxing Du","email":"","orcid":"","institution":"Affiliated Hospital of Qingdao University","correspondingAuthor":false,"prefix":"","firstName":"Wenxing","middleName":"","lastName":"Du","suffix":""},{"id":453521112,"identity":"d720afb7-c7c0-4b33-821e-b6418987362a","order_by":3,"name":"Zhe Wu","email":"","orcid":"","institution":"Affiliated Hospital of Qingdao University","correspondingAuthor":false,"prefix":"","firstName":"Zhe","middleName":"","lastName":"Wu","suffix":""},{"id":453521113,"identity":"ecd42c03-ac1c-4557-976d-299773559e7e","order_by":4,"name":"Wenjie Jiao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA40lEQVRIiWNgGAWjYBACxmYQyWPDwHCAB8SSYGBgJ05LGlyLBAMzcZYdhmlhIKyFuZ354WMemfOyfTdyDz78ucOijr+ZgfFzAV6HsRkb8/DcNp55Iy/ZmPeMhITEYQZm6Rn4/WImncNzO3HDjRwzacY2oF8OM7Ax8+DVwv4NqOUcWIvkT6AWecJaeEC2HABrkeAFajEgQkux8R+eZOOZZ94Yg/wiufEwY7M0Pi2G/cc3PpzZYyfbdzzHEBhidfxyx5sPfsarpQFkVQ8DYwPYTiQSJ5AHkz+IUzwKRsEoGAUjFAAAW0lHIxYtBN4AAAAASUVORK5CYII=","orcid":"","institution":"Affiliated Hospital of Qingdao University","correspondingAuthor":true,"prefix":"","firstName":"Wenjie","middleName":"","lastName":"Jiao","suffix":""}],"badges":[],"createdAt":"2025-04-04 11:08:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6375611/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6375611/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82562280,"identity":"00d7d347-d0a9-44b9-b7b1-bcd94558035c","added_by":"auto","created_at":"2025-05-13 01:44:00","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":459831,"visible":true,"origin":"","legend":"\u003cp\u003eDifferential gene expression analysis of CRGs in GSE127003 and GSE9634. Figure 1A presents the differential expression analysis results of 12 CRGs in GSE127003, comparing the lung transplantation cold ischemia group and the 2-hour post-ischemia reperfusion group. The data are presented in their original format. Figure 1B shows the differential expression analysis results of 11 CRGs in GSE9634, comparing the rat control group and the ischemia-reperfusion group. The data are presented as log10 transformations. *P \u0026lt; 0.05, **P \u0026lt; 0.01, ***P \u0026lt; 0.001, ****P \u0026lt; 0.0001.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-6375611/v1/8760c9f0649f745b82b276f3.jpeg"},{"id":82562869,"identity":"effcd61a-e87f-4223-ba06-e5bc19bfebdb","added_by":"auto","created_at":"2025-05-13 01:52:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":938284,"visible":true,"origin":"","legend":"\u003cp\u003eHistopathological evaluation, lung injury scoring, serum inflammatory cytokine levels, and serum ceruloplasmin (Cp) levels in Control, I/R, and I/R+ATTM groups.FIG. 2A displays representative H\u0026amp;E-stained lung tissue sections from Control, I/R, and I/R+ATTM groups (×100 and ×200 magnification). FIG. 2B presents quantitative analysis of pathological lung injury scores [expressed as median (interquartile range)] among the three experimental groups. FIG. 2C shows serum levels of TNF-α, IL-1β, and IL-6 [expressed as mean ± standard deviation] in Control, I/R, and I/R+ATTM groups. FIG. 2D illustrates serum ceruloplasmin concentrations [expressed as mean ± standard deviation] across all three groups.*P\u0026lt;0.05,**P\u0026lt;0.01,***P\u0026lt;0.001,****P\u0026lt;0.0001.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-6375611/v1/534cff920244b4ce53feddcd.png"},{"id":82560533,"identity":"d44465c3-cc72-4420-8cd5-ef490f75d2d6","added_by":"auto","created_at":"2025-05-13 01:36:01","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":227901,"visible":true,"origin":"","legend":"\u003cp\u003eExpression levels of cuproptosis-related proteins in lung tissues from Control, I/R, and I/R+ATTM groups.FIG. 3A displays representative Western blot bands of target proteins in Control versus I/R groups. FIG. 3B presents quantitative analysis of protein expression (Control vs I/R). FIG. 3C shows Western blot results comparing all three experimental groups (Control, I/R, and I/R+ATTM). FIG. 3D provides quantitative analysis across all groups.Data are expressed as mean ± standard deviation(ｘ±ｓ). Statistical significance is indicated as:\u003cbr\u003e\n*P \u0026lt; 0.05; **P \u0026lt; 0.01; ***P \u0026lt; 0.001; ****P \u0026lt; 0.0001\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-6375611/v1/b6d09119a71a49e1d71534ba.png"},{"id":82560545,"identity":"b51e9f55-aa84-47a2-b60f-677ecb9efca2","added_by":"auto","created_at":"2025-05-13 01:36:01","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1482576,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemical staining of cuproptosis-related proteins in lung tissues from Control, I/R, and I/R+ATTM groups.FIG. A and C display representative IHC staining patterns for FDX1 and lipoylated proteins (Lip-DLAT and Lip-DLST) at ×100 and ×200 magnification. 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01:44:01","extension":"pdf","order_by":18,"title":"","display":"","copyAsset":false,"role":"supplement","size":512513,"visible":true,"origin":"","legend":"","description":"","filename":"WB22LIAS.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6375611/v1/578aa685741943b93c0b388c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Role and Expression Patterns of Cuproptosis in Pulmonary Ischemia-Reperfusion Injury: An Experimental Study","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eSince the 21st century, lung transplantation has become the treatment of choice for patients with end-stage lung diseases, including idiopathic pulmonary fibrosis, advanced lung cancer, and severe chronic obstructive pulmonary disease[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. As an inevitable step in lung transplantation, ischemia-reperfusion frequently leads to acute lung ischemia-reperfusion injury (LIRI), which can progress to acute respiratory distress syndrome (ARDS) in severe cases. This complication has emerged as a critical determinant of perioperative survival rates[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. The underlying mechanisms involve oxidative stress, inflammatory responses, and regulated cell death pathways, collectively impairing transplant outcomes[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Consequently, mitigating the detrimental effects of ischemia-reperfusion injury remains a major focus in clinical research.\u003c/p\u003e \u003cp\u003eIn March 2022, a groundbreaking study by researchers at the Broad Institute of MIT and Harvard, published in Science, identified a novel form of regulated cell death\u0026mdash;cuproptosis. This copper-dependent cell death mechanism is initiated by elevated intracellular copper levels and mediated through ferredoxin 1 (FDX1), which facilitates the binding of copper to lipoylated proteins in the tricarboxylic acid (TCA) cycle. This interaction induces lipoylated protein aggregation, downregulates iron-sulfur cluster (Fe-S) proteins, and triggers proteotoxic stress, ultimately leading to cell death[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Emerging evidence implicates cuproptosis in the pathogenesis of neurodegenerative disorders[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], cardiovascular diseases[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], and various cancers[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Given its involvement in copper redox reactions, reactive oxygen species (ROS) generation, and mitochondrial dysfunction, cuproptosis has also been implicated in ischemia-reperfusion injuries affecting the heart and brain[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. While preliminary studies have reported altered expression of cuproptosis-related biomarkers (NFE2L2, NLRP3, LIPT1, and MTF1) in allograft LIRI models[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], the role of the FDX1-mediated pathway\u0026mdash;the central mechanism of cuproptosis\u0026mdash;remains unexplored in LIRI.\u003c/p\u003e \u003cp\u003eThis study employs a multi-modal approach to elucidate the role of cuproptosis in lung ischemia-reperfusion injury (LIRI). First, we performed comprehensive bioinformatics analysis of publicly available RNA-Seq datasets to identify differentially expressed cuproptosis-related genes. Building upon these computational findings, we established a rat model of in situ pulmonary warm ischemia-reperfusion injury to systematically investigate the spatiotemporal expression patterns of key cuproptosis biomarkers at both whole-organism and tissue levels. To functionally validate these observations, we implemented therapeutic intervention using the copper chelator ammonium tetrathiomolybdate (ATTM) to specifically inhibit cuproptosis pathways in our experimental model. This integrated methodology enables us to: (1) characterize cuproptosis-related molecular signatures in LIRI, (2) evaluate the therapeutic potential of copper chelation, and (3) identify novel targets for clinical intervention in transplantation-associated ischemia-reperfusion injury.\u003c/p\u003e"},{"header":"MATERIALS","content":"\u003cp\u003eData collection and organization from the GEO database\u003c/p\u003e\n\u003cp\u003eDatasets GSE 127003 and GSE 9634 were downloaded from the GEO database. GSE 127003 includes data from 92 samples, comprising RNA-Seq data from lung tissues of 46 patients at the end of cold ischemia and 46 patients after 2 hours of cold ischemia and reperfusion during lung transplantation. GSE 9634 contains 12 samples, including lung RNA-Seq data from 6 rats in the sham operation group, as well as lung RNA-Seq data from rats 30 minutes (n=3) and 3 hours (n=3) after 2 hours of in-situ thermal ischemia followed by reperfusion.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eCuproptosis-related Genes\u003c/p\u003e\n\u003cp\u003eWe screened and extracted 12 cuproptosis-related genes (CRGs) from the existing literature[13-15], including 9 positively correlated genes: ferredoxin 1 (FDX1), lipoyltransferase 1 (LIPT1), lipoic acid synthetase (LIAS), dihydrolipoamide succinyltransferase (DLST), dihydrolipoamide dehydrogenase (DLD), dihydrolipoamide acetyltransferase (DLAT), ATPase copper-transporting beta (ATP7B), pyruvate dehydrogenase alpha 1 (PDHA1), pyruvate dehydrogenase E1 subunit beta (PDHB), and 3 negatively correlated genes: cyclin-dependent kinase inhibitor 2A (CDKN2A), glutaminase (GLS), and metal regulatory transcription factor 1 (MTF1).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAnimals\u003c/p\u003e\n\u003cp\u003eEight healthy adult male Sprague-Dawley (SD) rats (4-5 weeks old) and sixteen SD rats (7-8 weeks old) were procured from Jinan Pengyue Experimental Animal Breeding Co., Ltd. (License No. SCXK(LU)2014-0007). The animals were housed in the SPF-grade animal facility of the Central Laboratory at the Affiliated Hospital of Qingdao University. Following a one-week acclimatization period, the experiments were initiated.This study was conducted in strict compliance with animal welfare guidelines and was approved by the Animal Ethics Committee of Qingdao University. This study was designed and reported in accordance with the ARRIVE \u0026nbsp; Guidelines to ensure comprehensive reporting of animal experiments.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMajor Reagents and Instruments\u003c/p\u003e\n\u003cp\u003eThe primary reagents and instruments used in this study are detailed in\u0026nbsp;Table 1.\u003c/p\u003e\n\u003cp\u003eTable 1. Names and Manufacturers of Major Reagents and Instruments.\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003ctd\u003e\n \u003cp\u003eCategory\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003c/table\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eItem\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eManufacturer\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eReagents\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eIL-6 Detection Kit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eElabscience Biotechnology, China\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eIL-1\u0026beta;\u0026nbsp;Detection Kit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eElabscience Biotechnology, China\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTNF-\u0026alpha;\u0026nbsp;Detection Kit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eElabscience Biotechnology, China\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eBCA Protein Concentration Assay Kit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eElabscience Biotechnology, China\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eDAB Chromogenic Kit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMaixin Biotech, China\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFDX1 Primary Antibody\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAbmart Scientific, China\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eLIAS Primary Antibody\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAbmart Scientific, China\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSDHB Primary Antibody\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAbmart Scientific, China\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eDLST Primary Antibody\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAbmart Scientific, China\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003elipoic acid Antibody\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAbcam, UK\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eEnzyme-Labeled Goat Anti-Rabbit Secondary Antibody for Immunohistochemistry\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAbcam, UK\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHorseradish Peroxidase (HRP)-Labeled Goat Anti-Rabbit Secondary Antibody\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eServicebio Technology, China\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eUltra-Sensitive ECL Substrate Solution\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eYaenzyme\u0026nbsp;Biotechnology, China\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eRapid Stripping Buffer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eYaenzyme\u0026nbsp;Biotechnology, China\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eProtein-Free Rapid Blocking Buffer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eYaenzyme\u0026nbsp;Biotechnology, China\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMulticolor Prestained Protein Marker\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eYaenzyme\u0026nbsp;Biotechnology, China\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eRIPA Tissue Protein Lysis Buffer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eYaenzyme\u0026nbsp;Biotechnology, China\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePhenylmethanesulfonyl fluoride（PMSF）\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eYaenzyme\u0026nbsp;Biotechnology, China\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNa3VO4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eYaenzyme\u0026nbsp;Biotechnology, China\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAmmonium tetrathiomolybdate (ATTM)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAladdin Biochemical Technology，China\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eRat ceruloplasmin (Cp) assay kit\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eShanghai Jianglai Biotechnology，China\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e4-20% High-Resolution Gradient Precast Gel\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eYeasen Biotechnology, China\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eDenaturing Protein Precast Gel Buffer Powder\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eYeasen Biotechnology, China\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5\u0026times; Protein Loading Buffer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eBeyotime Biotechnology, China\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePVDF Membrane\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSolarbio Science \u0026amp; Technology, China\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePolyoxyethylene (20) Sorbitan Monolaurate (Tween 20)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSolarbio Science \u0026amp; Technology, China\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eAbsolute Ethanol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFuyu Fine Chemical, China\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMethanol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFuyu Fine Chemical, China\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1\u0026times; TBST Buffer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eServicebio, China\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e1\u0026times; PBS Buffer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eServicebio, China\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10\u0026times; Citrate Antigen Retrieval Solution (pH 6.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eBiossci Biotechnology, China\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10\u0026times; EDTA Antigen Retrieval Solution (pH 9.0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eBiossci Biotechnology, China\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHematoxylin Staining Solution\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eBiossci Biotechnology, China\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHematoxylin Differentiation Solution\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eBiossci Biotechnology, China\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHematoxylin Bluing Solution\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eBiossci Biotechnology, China\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eNormal Goat Serum\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eBoster Biological Technology, China\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eInstruments\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eVortex Mixer\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eThermo Fisher Scientific, USA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eBenchtop Centrifuge\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eThermo Fisher Scientific, USA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGeneral Laboratory Electrophoresis Power Supply\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eJunyi Oriental Electrophoresis Equipment, China\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eProtein Vertical Electrophoresis Tank\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026zwnj;\u003ca href=\"https://www.baidu.com/s?rsv_dl=re_dqa_generate\u0026sa=re_dqa_generate\u0026wd=Bio-Rad%20Laboratories\u0026rsv_pq=c21e9db800093886\u0026oq=BIORAD\u0026rsv_t=c4a7i4upou4QRJQfA6eCJijmAxaBuIPcaieXtgtPfFoUmAjc4/U+s20+osIqJ4P1Ys0BDbU\u0026tn=15007414_18_dg\u0026ie=utf-8\" target=\"https://www.baidu.com/_blank\"\u003eBio-Rad Laboratories\u003c/a\u003e, USA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eLaboratory Chemiluminescence Imaging System\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eGeneGnome, USA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMultifunctional Microplate Reader\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTECAN, Switzerland\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSmall Animal Ventilator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eHUAYON, China\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTissue Dehydrator\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eZhongwei Electronic Instrument, China\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eTissue Embedding Machine\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eZhongwei Electronic Instrument, China\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003ePathology Microtome\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eThermo Fisher Scientific,USA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eUpright Microscope\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eOlympus Corporation, Japan\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eMethods\u003c/p\u003e\n\u003cp\u003eDifferential gene expression analysis in GEO database\u003c/p\u003e\n\u003cp\u003eWe downloaded the gene expression and sample information files for GSE 127003 and GSE 9634 from the GEO database and used the R programming language to organize and transform the data. We then intersected the data with CRGs to extract RNA-Seq data associated with the selected genes. Gene expression differences were statistically analyzed and visualized using GraphPad Prism 9.0 software. A p-value of less than 0.05 was considered statistically significant.\u003c/p\u003e\n\u003cp\u003eATTM Intervention Rat Model\u003c/p\u003e\n\u003cp\u003eFollowing a 1-week acclimatization period, SD rats were administered ammonium tetrathiomolybdate (ATTM) via oral gavage for 3 consecutive weeks. A 1 mg/mL ATTM solution was prepared in double-distilled water and administered daily at a dosage of 30 mg/kg body weight using a rodent feeding needle. Special care was taken to perform the gavage slowly while monitoring for any signs of oronasal reflux to prevent aspiration. Throughout the intervention period, the animals maintained normal access to food and water ad libitum. Subsequent experiments were conducted following the completion of this 3-week ATTM treatment protocol[16].\u003c/p\u003e\n\u003cp\u003eLung ischemia-reperfusion injury model\u003c/p\u003e\n\u003cp\u003eThe pulmonary ischemia-reperfusion injury (IRI) model was established in healthy adult male Sprague-Dawley rats (250\u0026ndash;300 g) according to previously described methods[17, 18]. Following anesthesia induction with intraperitoneal injection of 2% sodium pentobarbital (60 mg/kg) and confirmation of adequate anesthetic depth (assessed by loss of pedal reflex), the animals were positioned in dorsal recumbency and prepared for aseptic surgery. The surgical site was shaved and disinfected before performing a midline cervical incision to expose the trachea. Tracheotomy was performed, followed by endotracheal intubation connected to a rodent ventilator (Harvard Apparatus) with initial settings of 60 breaths/min and a tidal volume of 10 mL/kg.\u003c/p\u003e\n\u003cp\u003eA left thoracotomy was performed by carefully dissecting the intercostal muscles along the left sternal border between the second and third ribs. After achieving hemostasis, venous access was established via the left femoral vein for blood collection into heparinized Eppendorf tubes, followed by systemic heparinization with heparinized saline (62 IU/mL, 3 mL/kg). Following a 10-min circulation period, the intercostal incision was reopened to expose the left pulmonary hilum using sterile cotton-tipped applicators. The left pulmonary hilum was clamped with microvascular clips during peak lung inflation, with successful occlusion confirmed by the absence of left lung ventilation and loss of distal vascular pulsation. Ventilator parameters were adjusted to 50 breaths/min and a tidal volume of 8 mL/kg during the 90-min ischemic period.\u003c/p\u003e\n\u003cp\u003eReperfusion was initiated by removing the vascular clips, and ventilator settings were immediately restored to baseline (60 breaths/min, 10 mL/kg tidal volume). After 2 h of reperfusion, terminal procedures were performed, including cardiac blood collection via heparinized syringe, pulmonary artery perfusion with 20 mL saline, and left lung tissue harvest. The animals were then euthanized by CO₂ asphyxiation in accordance with institutional animal care guidelines. Core body temperature was maintained throughout the procedure using a heating pad, and anesthetic depth was continuously monitored to ensure animal welfare.\u003c/p\u003e\n\u003cp\u003eExperimental Animal Grouping\u003c/p\u003e\n\u003cp\u003e1.Control Group: Eight randomly selected 7-8-week-old rats underwent anesthesia, tracheotomy, intubation, heparinization, thoracotomy, and left pulmonary hilum exposure without ischemia induction. The animals were ventilated for 3.5 hours before sample collection and euthanasia.\u003c/p\u003e\n\u003cp\u003e2.Ischemia-Reperfusion (I/R) Group: Eight randomly selected 7-8-week-old rats were subjected to the complete pulmonary ischemia-reperfusion injury model as described above, followed by sample collection and euthanasia.\u003c/p\u003e\n\u003cp\u003e3.ATTM Intervention (I/R+ATTM) Group: Eight randomly selected 4-5-week-old rats first received ATTM pretreatment to establish the intervention model, then underwent the same pulmonary ischemia-reperfusion procedure as the I/R group before terminal sample collection and euthanasia.\u003c/p\u003e\n\u003cp\u003eHE staining was used to observe the lung histological characteristics\u003c/p\u003e\n\u003cp\u003eAfter dewatering, embedding, dewaxing, and hydration, lung tissues from rats in the Control、I/R and I/R+ATTM groups were subjected to hematoxylin and eosin (HE) staining. The histological characteristics of the rat lungs were observed under a microscope. Two pathologists, blinded to the group assignments, independently evaluated alveolar congestion, edema, hemorrhage, neutrophil infiltration, alveolar wall thickness, and hyaline formation in lung injury[19].\u003c/p\u003e\n\u003cp\u003eDetermination of serum inflammatory factors in Rats\u003c/p\u003e\n\u003cp\u003eCardiac blood samples were collected from rats in all three experimental groups into heparinized sterile centrifuge tubes (Eppendorf tubes) and allowed to clot at room temperature for 2 hours. Following centrifugation at 1,000 \u0026times; g for 15 minutes at 4\u0026deg;C, the supernatant serum was carefully collected. Serum concentrations of TNF-\u0026alpha;, IL-1\u0026beta;, and IL-6 were quantified using enzyme-linked immunosorbent assay (ELISA) in 96-well microplates pre-coated with specific capture antibodies. The assay procedure involved sequential addition of test samples, standards, biotin-conjugated detection antibodies, and horseradish peroxidase (HRP)-labeled avidin (ABC) in duplicate wells. After standardized incubation and washing steps, color development was achieved using 3,3\u0026apos;,5,5\u0026apos;-tetramethylbenzidine (TMB) substrate. Optical density (OD) values were measured at 450 nm using a microplate reader, and cytokine concentrations were calculated using ELISACalc software. This standardized protocol ensures accurate and reproducible quantification of inflammatory mediators through implementation of duplicate measurements and strict adherence to established ELISA procedures.\u003c/p\u003e\n\u003cp\u003eDetermination of Serum Ceruloplasmin in Rats\u003c/p\u003e\n\u003cp\u003eFemoral venous blood samples from rats in all three experimental groups were collected in heparinized sterile Eppendorf tubes and allowed to clot at room temperature for 2 hours. The samples were then centrifuged at 1,000 \u0026times; g for 15 min at 4 \u0026deg;C to separate the serum. Serum ceruloplasmin (Cp) levels were quantified using a rat-specific Cp ELISA kit according to the manufacturer\u0026apos;s protocol. Briefly, 96-well microplates pre-coated with rat Cp capture antibody were used for the assay. Samples and standards were added to the wells in duplicate, followed by sequential incubation with biotinylated detection antibody and horseradish peroxidase (HRP)-conjugated avidin (ABC). After appropriate incubation and washing steps, the reaction was developed with 3,3\u0026apos;,5,5\u0026apos;-tetramethylbenzidine (TMB) substrate. Optical density (OD) values were measured at 450 nm using a microplate reader, and the average Cp concentration for each sample was calculated using ELISA analysis software (ELISACalc).\u003c/p\u003e\n\u003cp\u003eWestern Blot (WB) was used to detect the expression of cuproptpsis related protein\u003c/p\u003e\n\u003cp\u003ePartial left lung tissues from rats in all three experimental groups were immersed in PBS buffer for 10 minutes before homogenization in protein lysis buffer containing PMSF and phosphatase inhibitors. Following 30 minutes of incubation on ice, the homogenates were centrifuged at 12,000 rpm for 10 minutes at 4\u0026deg;C to collect the supernatant. Protein concentrations were determined using the BCA protein assay to ensure consistent loading amounts across samples. Equal amounts of protein samples from the three groups were separated by electrophoresis, transferred onto membranes, and blocked before overnight incubation at 4\u0026deg;C with primary antibodies against FDX1, LIAS, DLST, DLAT, SDHB, and lipoic acid. After thorough washing, the membranes were incubated with goat anti-rabbit secondary antibody at room temperature for 2 hours. Protein bands were finally visualized using enhanced chemiluminescence (ECL) reagent and detected with a chemiluminescence imaging system.\u003c/p\u003e\n\u003cp\u003eImmunohistochemical (IHC) staining was used to detect the expression of FDX1 and lipoacylated protein\u003c/p\u003e\n\u003cp\u003eLung tissue from three rat groups was collected for paraffin sectioning. After dewaxing, hydration, antigen retrieval, and incubation at room temperature for 25 minutes, the antigens were blocked using blocking serum. Subsequently, primary antibodies against FDX1 and lipoic acid were added and incubated at 4\u0026deg;C overnight. After washing, goat anti-rabbit IgG secondary antibody was applied. Finally, DAB staining solution was added, and the slides were observed under a microscope. After hematoxylin counterstaining, dehydration, and sealing, the expressions of FDX1 and lipoacylated proteins were examined under a microscope.\u003c/p\u003e\n\u003cp\u003eStatistical Analysis\u003c/p\u003e\n\u003cp\u003eStatistical analysis was performed using GraphPad Prism 9.0 software. Normality and homogeneity of variance tests were conducted for each data set. For data that followed a normal distribution with homogeneity of variance, a two-sample t-test was used, and the results were expressed as mean \u0026plusmn; standard deviation (x̄ \u0026plusmn; s). For data that did not conform to normal distribution, the Mann-Whitney U test was applied, and the results were presented as median (first quartile (Q1), third quartile (Q3)). If the data showed significant differences in components, log transformation was applied to improve the data distribution. A P value of \u0026lt; 0.05 was considered statistically significant.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003eAnalysis of differential expression gene of cuproptpsis\u003c/p\u003e\n\u003cp\u003eThe results of differential expression analysis of CRGs RNA-Seq data in the cold ischemia group (n = 46) and the reperfusion group 2 hours after lung transplantation (n = 46) from GSE127003 showed that, compared with the cold ischemia group, the expression of the cuproptosis-related gene FDX1 and the cuproptosis inhibitor gene MTF1 were significantly increased in the reperfusion group (P \u0026lt; 0.001). Conversely, the expressions of lipoic acid pathway genes LIAS, DLD, pyruvate dehydrogenase (PDH) complex-related genes DLAT, PDHA1, PDHB, and the copper transporter gene ATP7B were significantly decreased (P \u0026lt; 0.01) (FIG. 1A). In the rat lung ischemia-reperfusion experiment (GSE9634), the ischemia reperfusion groups at 30 minutes (n = 3) and 3 hours (n = 3) were combined into a single ischemia-reperfusion group. Compared with the control group (n = 6), the expressions of FDX1, DLD, SLC31A1, and PDHA1 were significantly increased in the ischemia-reperfusion group (n = 6) (P \u0026lt; 0.05). In contrast, the expression of the cuproptosis-related suppressor gene GLS was significantly decreased (P \u0026lt; 0.01) (FIG. 1B). The expression results of CRGs in each group are shown in Table 2.\u003c/p\u003e\n\u003cp\u003eTable 2. Expression analysis of CRGs in GSE127003 and GSE9634. Table 2A presents the expression analysis results of CRGs in the lung transplantation cold ischemia group (n = 46) and the reperfusion group (n = 46). The data are presented in their raw format, with results including mean \u0026plusmn; standard deviation and P-value. Table 2B presents the expression analysis results of CRGs in GSE9634, comparing the rat control group (n = 6) and the ischemia-reperfusion group (n = 6). The data are presented as log10 transformations, with results including the median (Q1, Q3) and P-value.\u003c/p\u003e\n\u003cp\u003eA\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003eGene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 168px;\"\u003e\n \u003cp\u003ecold ischemia group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 123px;\"\u003e\n \u003cp\u003ereperfusion group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 119px;\"\u003e\n \u003cp\u003eP-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003eFDX1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 168px;\"\u003e\n \u003cp\u003e403.21\u0026plusmn;72.92\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e471.58\u0026plusmn;95.15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 119px;\"\u003e\n \u003cp\u003e0.000167\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003eLIAS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 168px;\"\u003e\n \u003cp\u003e238.64\u0026plusmn;69.86\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e159.24\u0026plusmn;57.32\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 119px;\"\u003e\n \u003cp\u003e\u0026lt;0.000001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003eDLST\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 168px;\"\u003e\n \u003cp\u003e1353.85\u0026plusmn;98.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e1379.2\u0026plusmn;111.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 119px;\"\u003e\n \u003cp\u003e0.101771\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003eDLAT\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 168px;\"\u003e\n \u003cp\u003e320.52\u0026plusmn;53.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e272.14\u0026plusmn;53.48\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 119px;\"\u003e\n \u003cp\u003e0.000038\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003eDLD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 168px;\"\u003e\n \u003cp\u003e267.37\u0026plusmn;27.05\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e249.11\u0026plusmn;26.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 119px;\"\u003e\n \u003cp\u003e0.000840\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003ePDHB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 168px;\"\u003e\n \u003cp\u003e2130.5\u0026plusmn;241.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e1718.12\u0026plusmn;229.88\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 119px;\"\u003e\n \u003cp\u003e\u0026lt;0.000001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003eATP7B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 168px;\"\u003e\n \u003cp\u003e119.7\u0026plusmn;28.24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e101.32\u0026plusmn;24.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 119px;\"\u003e\n \u003cp\u003e0.000808\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003eSLC31A1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 168px;\"\u003e\n \u003cp\u003e778.62\u0026plusmn;159.58\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e790.92\u0026plusmn;154.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 119px;\"\u003e\n \u003cp\u003e0.259903\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003ePDHA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 168px;\"\u003e\n \u003cp\u003e772.41\u0026plusmn;76.17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e725.76\u0026plusmn;73.01\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 119px;\"\u003e\n \u003cp\u003e0.001772\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003eMTF1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 168px;\"\u003e\n \u003cp\u003e262.84\u0026plusmn;27.47\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e312.94\u0026plusmn;37.52\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 119px;\"\u003e\n \u003cp\u003e\u0026lt;0.000001\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003eGLS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 168px;\"\u003e\n \u003cp\u003e350.91\u0026plusmn;69.91\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e346.74\u0026plusmn;68.33\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 119px;\"\u003e\n \u003cp\u003e0.260265\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003eCDKN2A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 168px;\"\u003e\n \u003cp\u003e44.38\u0026plusmn;6.45\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 123px;\"\u003e\n \u003cp\u003e46.15\u0026plusmn;8.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 119px;\"\u003e\n \u003cp\u003e0.101771\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eB\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003eGene\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 159px;\"\u003e\n \u003cp\u003econtrol group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 132px;\"\u003e\n \u003cp\u003eI/R group\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 38px;\"\u003e\u0026nbsp;\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 119px;\"\u003e\n \u003cp\u003eP-value\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003eFDX1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 159px;\"\u003e\n \u003cp\u003e2.950(2.935,2.952)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e3.296(3.244,3.357)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 119px;\"\u003e\n \u003cp\u003e0.0022\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003eLIAS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 159px;\"\u003e\n \u003cp\u003e2.528(2.514,2.547)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e2.480(2.398,2.516)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 119px;\"\u003e\n \u003cp\u003e0.1797\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003eDLST\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 159px;\"\u003e\n \u003cp\u003e2.977(2.917,3.027)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e3.007(2.979,3.042)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 119px;\"\u003e\n \u003cp\u003e0.5887\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003eDLD\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 159px;\"\u003e\n \u003cp\u003e2.772(2.702,2.790)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e2.863(2.847,2.911)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 119px;\"\u003e\n \u003cp\u003e0.0260\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003ePDHB\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 159px;\"\u003e\n \u003cp\u003e3.165(3.154,3.200)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e3.185(3.154,3.196)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 119px;\"\u003e\n \u003cp\u003e\u0026gt;0.9999\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003eATP7B\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 159px;\"\u003e\n \u003cp\u003e1.703(1.326,1.889)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e1.702(1.629,1.796)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 119px;\"\u003e\n \u003cp\u003e0.9372\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003eSLC31A1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 159px;\"\u003e\n \u003cp\u003e2.108(2.043,2.157)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e2.278(2.198,2.364)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 119px;\"\u003e\n \u003cp\u003e0.0260\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003ePDHA1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 159px;\"\u003e\n \u003cp\u003e3.423(3.411,3.433)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e3.572(3.523,3.597)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 119px;\"\u003e\n \u003cp\u003e0.0043\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003eMTF1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 159px;\"\u003e\n \u003cp\u003e1.893(1.815,1.922)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e1.938(1.907,1.965)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 119px;\"\u003e\n \u003cp\u003e0.1320\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003eGLS\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 159px;\"\u003e\n \u003cp\u003e2.722(2.716,2.755)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e2.607(2.570,2.640)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 119px;\"\u003e\n \u003cp\u003e0.0043\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 106px;\"\u003e\n \u003cp\u003eCDKN2A\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 159px;\"\u003e\n \u003cp\u003e1.272(1.248,1.436)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd style=\"width: 132px;\"\u003e\n \u003cp\u003e1.287(0.930,1.306)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 38px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"bottom\" style=\"width: 119px;\"\u003e\n \u003cp\u003e\u0026gt;0.9999\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eHistopathological and Inflammatory Cytokine Profiles in Rat Pulmonary Tissues\u003c/p\u003e\n\u003cp\u003eH\u0026amp;E staining revealed distinct pathological alterations in lung tissues across experimental groups. Compared to the Control group, the I/R group exhibited characteristic features of acute lung injury, including severe alveolar congestion, edema, hemorrhage, prominent neutrophil infiltration, alveolar wall thickening, and hyaline membrane formation. In contrast, the I/R+ATTM group demonstrated marked attenuation of these pathological changes, with significantly reduced alveolar congestion, hemorrhage, edema, and hyaline membrane formation compared to the I/R group. Notably, alveolar wall thickness was not significantly increased, and neutrophil infiltration was substantially diminished in the I/R+ATTM group (FIG. 2A). Quantitative analysis of lung injury scores demonstrated that the I/R+ATTM group exhibited significantly higher scores than the Control group (P\u0026lt;0.0001), yet remained significantly lower than the I/R group (P\u0026lt;0.01) (FIG. 2B). Compared to the Control group, serum levels of inflammatory cytokines (TNF-\u0026alpha;, IL-6, and IL-1\u0026beta;) were significantly elevated in both the I/R and I/R+ATTM groups (P\u0026lt;0.0001). However, ATTM intervention markedly reduced these inflammatory cytokine levels in the ischemia-reperfusion model when compared to the I/R group (P\u0026lt;0.05) (FIG. 2C), indicating that the rat model of acute lung injury induced by lung ischemia and reperfusion was successfully established.\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSerum Ceruloplasmin (Cp) Levels in Experimental Rats\u003c/p\u003e\n\u003cp\u003eThe I/R+ATTM group demonstrated a significant 40% reduction in serum Cp levels compared to both Control and I/R groups (P\u0026lt;0.001) (FIG. 2D). This pronounced suppression of Cp expression, a key copper transport protein, provides biochemical confirmation of successful establishment of the ATTM-mediated copper-deficient model in rats.\u003c/p\u003e\n\u003cp\u003eExpression of Cuproptosis-Related Proteins in Rat Lung Tissues\u003c/p\u003e\n\u003cp\u003eWestern blot analysis revealed:(1) The I/R group showed significantly decreased expression of FDX1, LIAS, DLAT, DLST, SDHB, Lip-DLAT, and Lip-DLST (P\u0026lt;0.05), while DLAT-Oligomers expression was increased (P\u0026lt;0.05) compared to Controls (FIG. 3A-B).(2) ATTM intervention significantly upregulated FDX1, LIAS, DLAT, DLST, SDHB, Lip-DLAT, and Lip-DLST expression versus I/R group (P\u0026lt;0.05), though remaining below Control levels (P\u0026lt;0.05). DLAT-Oligomers were markedly reduced compared to I/R (P\u0026lt;0.0001) but remained elevated versus Controls (P\u0026lt;0.05) (FIG. 3C-D).\u003c/p\u003e\n\u003cp\u003eExpression of FDX1 and lipoylprotein in Rat Lung Tissues\u003c/p\u003e\n\u003cp\u003eImmunohistochemical staining demonstrated that both FDX1 and lipoylated proteins (Lip-DLAT and Lip-DLST) were predominantly localized in the cytoplasm of rat pulmonary tissue cells. Quantitative evaluation revealed significantly higher expression levels of these proteins in the Control group compared to both I/R and I/R+ATTM groups (P \u0026lt; 0.05). Notably, ATTM intervention partially restored the expression of FDX1 and lipoylated proteins in the I/R+ATTM group relative to the I/R group (P\u0026lt;0.05), though remaining below Control levels (P\u0026lt;0.05) (FIG. 4).\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn this study, based on RNA-Seq data from the GEO database, we observed significant changes in the expression of key genes associated with cuproptosis during rat lung warm ischemia-reperfusion, including FDX1, the copper transporter SLC31A1, the pyruvate dehydrogenase (PDH) complex-related gene PDHA1, and the cuproptosis suppressor gene GLS. Similarly, in human lung transplantation under cold ischemia-reperfusion conditions, not only FDX1 but also genes in the lipoic acid pathway (e.g., LIAS, DLD), PDH complex-related genes (e.g., DLAT), and cuproptosis suppressor genes (e.g., MTF1) exhibited significant alterations. These findings align with previous studies demonstrating that cuproptosis involves the oligomerization of lipoylated proteins and a reduction in Fe-S cluster proteins[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], suggesting that cuproptosis may play a role in lung ischemia-reperfusion injury.\u003c/p\u003e \u003cp\u003eFurthermore, in the GSE127003 dataset, compared to the cold ischemia group, the expression of cuproptosis-promoting genes was significantly reduced in the 2-hour reperfusion group, while the expression of suppressor genes increased. This may be attributed to the exacerbation of cuproptosis during reperfusion, which is associated with acute lung injury and subsequent modulation of cuproptosis-related gene (CRG) expression. However, due to the lack of lung histopathology and protein-level data, it cannot be ruled out that lung injury may improve during reperfusion, leading to the normalization of CRG expression. Therefore, the underlying mechanisms require further investigation.\u003c/p\u003e \u003cp\u003eNotably, compared to human lung transplantation, some cuproptosis-promoting CRGs showed significantly increased expression in rat lung ischemia-reperfusion models, and the differentially expressed CRGs varied between species. These discrepancies may be due to the smaller sample size in the rat study (n\u0026thinsp;=\u0026thinsp;12), differences in ischemia methods (warm vs. cold ischemia), and variations in control group standards (ischemia group vs. sham operation group). These factors highlight the need for further studies to clarify the role of cuproptosis in lung ischemia-reperfusion injury across different experimental conditions.\u003c/p\u003e \u003cp\u003eIn recent years, with the continuous increase in the number of lung transplantation surgeries, in-depth research into the pathophysiological mechanisms of lung ischemia-reperfusion injury (LIRI) has become particularly important. Currently, the core pathogenesis of LIRI is believed to involve multiple pathological processes, including but not limited to oxidative stress, inflammatory responses, calcium overload, microcirculatory dysfunction, apoptosis, necrosis, mitochondrial dysfunction, and endoplasmic reticulum stress[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. According to Tsvetkov et al., the accumulation of copper ions and the catalytic role of FDX1 are the direct causes of the reduction in Fe-S cluster proteins, impaired synthesis of lipoylated proteins, and the aggregation of lipoylated proteins, ultimately leading to proteotoxic stress[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Among these, FDX1, as both a key regulator of cuproptosis and an Fe-S cluster protein, is also affected by cuproptosis, resulting in decreased expression[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. LIAS can directly bind to FDX1 to regulate the lipoylation process of proteins and, as an Fe-S cluster protein, its expression is downregulated[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. SDHB catalyzes the oxidation of succinate to fumarate in the tricarboxylic acid (TCA) cycle, and its expression decreases during cuproptosis as an Fe-S cluster protein[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The downregulation of SDHB can lead to mitochondrial dysfunction and hypoxia-induced injury[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTo further investigate the changes in cuproptosis-related proteins during LIRI, we employed a rat hilar clamp-reperfusion model to simulate acute lung injury induced by ischemia-reperfusion during lung transplantation[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. We ultimately observed that during pulmonary ischemia-reperfusion, alongside morphological changes in lung injury, the expression of Fe-S cluster proteins such as FDX1, SDHB, and LIAS, as well as lipoylated proteins (Lip-DLAT and Lip-DLST), significantly decreased, while lipoylated proteins (DLAT) aggregated extensively. These findings are consistent with the process of cuproptosis, where abnormal intracellular accumulation of Cu\u0026sup2;⁺ leads to the catalytic generation of cuprous ions (Cu⁺) by the key cuproptosis protein FDX1, followed by the binding of Cu⁺ to lipoylated proteins (e.g., Lip-DLAT, Lip-DLST)[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. This suggests a potential association between cuproptosis and LIRI.\u003c/p\u003e \u003cp\u003eATTM, as a widely used copper chelator, forms a stable, insoluble complex upon contact with Cu\u0026sup2;⁺, thereby reducing Cu\u0026sup2;⁺ levels in the body[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. This is manifested by a significant decrease in serum ceruloplasmin (Cp) levels. In our experiments, ATTM intervention significantly reduced serum Cp levels in rats[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], confirming the effectiveness of the intervention and the successful establishment of a low-copper rat model. In previous studies on ischemia-reperfusion injury, it was found that copper ions can participate in the Fenton reaction, where the redox cycling between Cu\u0026sup2;⁺ and Cu⁺ continuously catalyzes the generation of hydroxyl radicals (\u0026middot;OH) from H₂O₂[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], exacerbating oxidative stress in tissues and organs. Additionally, copper ions directly target components of the TCA cycle, binding to thiol groups in mitochondria and inhibiting the activity of respiratory chain complexes, leading to reduced ATP production and further aggravating metabolic dysfunction under ischemic and hypoxic conditions[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eBuilding on this, researchers have found that reducing copper ion concentrations in myocardial ischemia-reperfusion injury significantly decreases infarct size and improves cardiac function[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Similarly, in models of cerebral ischemia-reperfusion injury, downregulating FDX1 expression with disulfiram (DSF) suppresses oxidative stress, alleviates neuroinflammation, and protects mitochondrial function[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In our experiment, after using the copper chelator ATTM to reduce Cp levels in rats, we observed increased expression of Fe-S cluster proteins such as FDX1 and lipoylated proteins compared to post-ischemia-reperfusion levels, along with reduced aggregation of lipoylated proteins. Concurrently, pathological changes in lung tissue, such as neutrophil infiltration, alveolar wall thickening, and pulmonary interstitial edema, were significantly ameliorated after ischemia-reperfusion. This further demonstrates that the cuproptosis mechanism, mediated by FDX1, participates in and promotes the development of LIRI, while inhibiting cuproptosis can significantly improve tissue changes during LIRI. This targeted therapeutic strategy holds significant clinical value, offering new hope for the treatment of lung ischemia-reperfusion injury (LIRI).\u003c/p\u003e \u003cp\u003eSimilar to other studies, this research also has several limitations:1. Small Sample Size: The GSE9634 dataset from the GEO database used in this study included only three rats in the experimental group. The small sample size may increase heterogeneity and reduce the reliability of the results. Although additional validation experiments were conducted to supplement the findings, potential biases may still exist. Future studies should expand the sample size to enhance the robustness and generalizability of the results.2. Limited Exploration of Ischemia and Reperfusion Phases: This study focused on the changes in cuproptosis mechanisms during in situ lung ischemia-reperfusion injury but did not separately investigate the ischemia and reperfusion phases. It remains unclear whether cuproptosis exhibits differential expression during these two stages, warranting further investigation.3. Preclinical Model Limitations: The findings of this study are primarily based on animal models, and their clinical translation requires further validation. Future research should include human studies or clinical trials to confirm the relevance of these findings in a clinical setting.\u003c/p\u003e"},{"header":"CONCLUSION","content":"\u003cp\u003eIn summary, cuproptosis, as a novel form of cell death, participates in the pathogenesis of LIRI, and the inhibition of cuproptosis significantly ameliorates LIRI. This conclusion enriches our understanding of the pathological mechanisms underlying acute lung ischemia-reperfusion injury and provides new insights for its treatment.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eZ.C. Wang and W.J. Jiao wrote the main manuscript text and designed experiments, W.X. Du and H.Q. Liu provided experimental technical support, Z. Wu provided data analysis support, . All authors reviewed the manuscript.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe datasets analyzed in this study are publicly available in the Gene Expression Omnibus repository, with accession numbers GSE127003 and GSE9634. These data can be accessed at: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE127003 and https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE9634.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eT.F. Chen-Yoshikawa, Ischemia-Reperfusion Injury in Lung Transplantation, Cells (6) (2021).\u003c/li\u003e\n\u003cli\u003eO. Pak, A. Sydykov, D. Kosanovic, R.T. Schermuly, N. Weissmann, Lung Ischaemia\u0026ndash;Reperfusion Injury: The Role of Reactive Oxygen Species, Pulmonary Vasculature Redox Signaling in Health and Disease2017.\u003c/li\u003e\n\u003cli\u003eV.E. Laubach, A.K. 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Wang, Sleep fragmentation exacerbates myocardial ischemia‒reperfusion injury by promoting copper overload in cardiomyocytes, Nat Commun 15(1) (2024) 3834.\u003c/li\u003e\n\u003cli\u003eS. Yang, X. Li, J. Yan, F. Jiang, X. Fan, J. Jin, W. Zhang, D. Zhong, G. Li, Disulfiram downregulates ferredoxin 1 to maintain copper homeostasis and inhibit inflammation in cerebral ischemia/reperfusion injury, Sci Rep 14(1) (2024) 15175.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"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":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"acute lung injury, ischemia reperfusion, cuproptosis, FDX1, lipoylation","lastPublishedDoi":"10.21203/rs.3.rs-6375611/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6375611/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective\u003c/strong\u003e: To investigate the mechanistic role of cuproptosis in acute lung ischemia-reperfusion injury (LIRI).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e: Microarray datasets GSE127003 and GSE9634 were retrieved from the Gene Expression Omnibus (GEO) database to identify and analyze differentially expressed cuproptosis-related genes (CRGs). To validate the bioinformatics findings, 24 male Sprague-Dawley (SD) rats were randomly allocated into three groups: Control, ischemia-reperfusion (I/R), and I/R with ammonium tetrathiomolybdate (ATTM) intervention (I/R+ATTM). The I/R+ATTM group received ATTM pretreatment for copper chelation prior to surgery. An in situ lung I/R injury model was established, and femoral venous blood was collected intraoperatively, while cardiac blood and left lung tissues were harvested postoperatively.Macroscopic evaluation assessed pulmonary hemorrhage, congestion, and edema. Hematoxylin-eosin (H\u0026amp;E) staining was performed for histopathological examination and lung injury scoring. Serum levels of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) were quantified using ELISA. Ceruloplasmin (Cp) levels were measured via a rat-specific assay kit. Western blotting analyzed pulmonary expression of ferredoxin 1 (FDX1), lipoic acid synthetase (LIAS), dihydrolipoyl transacetylase (DLAT), DLAT oligomers, dihydrolipoamide succinyltransferase (DLST), succinate dehydrogenase complex subunit B (SDHB), lipoylated DLAT (Lip-DLAT), and lipoylated DLST (Lip-DLST). Immunohistochemistry (IHC) localized FDX1, Lip-DLAT, and Lip-DLST expression in lung tissues.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e: Differential gene analysis revealed significant alterations in CRG RNA expression during lung I/R (P \u0026lt; 0.05). Histopathological assessment demonstrated severe injury in the I/R group, moderate in I/R+ATTM, and minimal in Controls (P \u0026lt; 0.01). Serum TNF-α, IL-1β, and IL-6 levels in I/R+ATTM were elevated versus Controls (P \u0026lt; 0.0001) but reduced compared to I/R (P \u0026lt; 0.05). ATTM intervention significantly decreased serum Cp (P \u0026lt; 0.001). Pulmonary FDX1, LIAS, DLAT, DLST, SDHB, Lip-DLAT, and Lip-DLST expression in I/R+ATTM was lower than I/R (P \u0026lt; 0.05) but remained higher than Controls (P \u0026lt; 0.05). DLAT oligomers increased versus Controls (P \u0026lt; 0.05) but were suppressed relative to I/R (P \u0026lt; 0.0001). IHC confirmed cytoplasmic localization of FDX1 and lipoylated proteins, with I/R+ATTM showing intermediate expression between I/R and Controls (P \u0026lt; 0.05).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e: In a rat model of in situ lung ischemia-reperfusion, cuproptosis exacerbates acute lung injury by modulating key protein effectors, while copper chelation partially mitigates this pathological progression.\u003c/p\u003e","manuscriptTitle":"The Role and Expression Patterns of Cuproptosis in Pulmonary Ischemia-Reperfusion Injury: An Experimental Study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-13 01:35:54","doi":"10.21203/rs.3.rs-6375611/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-10T08:47:08+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-10-07T08:44:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"61595573684205777050233666147640856422","date":"2025-09-25T10:58:43+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-23T01:19:45+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"113623731462536258603555202650030592746","date":"2025-08-12T13:03:02+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-05-06T03:01:03+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-05-06T02:59:41+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-04-10T10:46:43+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-07T12:07:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Scientific Reports","date":"2025-04-04T10:53:56+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"scientific-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"scirep","sideBox":"Learn more about [Scientific Reports](http://www.nature.com/srep/)","snPcode":"","submissionUrl":"","title":"Scientific Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Scientific Reports","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"6b89b1c5-9348-4188-941f-1a6ddf9df184","owner":[],"postedDate":"May 13th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":48229564,"name":"Health sciences/Diseases"},{"id":48229565,"name":"Health sciences/Medical research"},{"id":48229566,"name":"Health sciences/Pathogenesis"}],"tags":[],"updatedAt":"2025-11-10T08:54:00+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-13 01:35:54","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6375611","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6375611","identity":"rs-6375611","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}