{"paper_id":"325d07d7-8ead-48c9-86cb-0ecf5f91c7b9","body_text":"Mitochondrial transplantation attenuates alveolar epithelial cell dysfunction and reduces disruption of tight junction proteins to alleviate lung ischaemia-reperfusion injury | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Mitochondrial transplantation attenuates alveolar epithelial cell dysfunction and reduces disruption of tight junction proteins to alleviate lung ischaemia-reperfusion injury Guangdong Weng, Jie Zhao, Xiedong Zhu, Yao Chen, ChengXin Zhang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6752547/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 26 Nov, 2025 Read the published version in Journal of Translational Medicine → Version 1 posted 4 You are reading this latest preprint version Abstract Objective The aim of this study was to investigate the therapeutic effect of exogenous mitochondrial transplantation (MT) on lung ischemia-reperfusion injury (LI/RI) and to investigate the effect of mitochondrial transplantation on alveolar epithelial cell function as well as on the ultrastructural changes of the alveolar epithelial barrier ultrastructural changes. Methods The therapeutic effect of mouse liver-derived mitochondrial transplantation on LI/RI was assessed by constructing a hypoxia-reoxygenation model of mouse alveolar epithelial cells (MLE-12 cells) and a lung ischaemia-reperfusion injury model in C57BL/6 male mice, which simulated the pathological process of LI/RI. Results The study results showed that MT exhibited significant therapeutic potential in LI/RI. In vitro and in vivo experiments revealed that MT significantly improved lung tissue injury by reducing oxidative stress, inflammatory response, apoptosis, and necrosis. Meanwhile, MT could alleviate alveolar epithelial cell dysfunction, reduce the disruption of tight junction proteins, and protect the alveolar epithelial barrier, thereby mitigating LI/RI. Conclusion This study confirmed in a lung ischemia-reperfusion injury model that MT treatment can repair the structural damage of the alveolar barrier caused by ischemia-reperfusion by targeting and regulating the expression of tight junction proteins in alveolar epithelial cells, providing a new perspective for elucidating the functional targets of MT treatment in protecting the alveolar barrier. Mitochondrial transplantation Lung ischaemia-reperfusion injury Tight junction proteins Repair of lung damage Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1. Introduction Lung ischaemia-reperfusion injury (LI/RI) is a common and challenging pathological process in clinical practice, frequently occurring in scenarios such as lung transplantation, cardiopulmonary bypass surgery in cardiac surgery, and blood flow restoration after severe trauma, which seriously threatens patients' life safety and prognosis [1-3] . Despite the continuous advancement of medical technology and the development of treatment methods for LI/RI, no breakthrough progress has been made yet. The pathophysiological mechanism of LI/RI is complex, in which mitochondrial damage plays a key role [4] . During the lung ischaemia phase, tissue cells suffer from hypoxia, leading to impairment of the mitochondrial electron transport chain function and obstruction of electron transport. This results in electron leakage, which reacts with oxygen to generate reactive oxygen species (ROS) such as superoxide anions [5] . During reperfusion, ROS are explosively produced, while the activity of antioxidant enzymes in mitochondria decreases, failing to promptly clear excessive ROS, thereby triggering lipid peroxidation and damaging the structure and function of the mitochondrial membrane [6] . At the same time, intracellular calcium homeostasis is disrupted, and mitochondria take up excessive calcium ions, activating the mitochondrial permeability transition pore, leading to the loss of mitochondrial membrane potential, inhibition of respiratory chain complex activity, and interference with the oxidative phosphorylation process [7] . In addition, the release of inflammatory factors also affects mitochondrial function through multiple pathways [8] . These factors collectively lead to changes in mitochondrial morphology, respiratory dysfunction, mitochondrial deoxynucleotide damage, and ultimately trigger cell apoptosis, exacerbation of inflammatory responses, and impairment of lung function. Mitochondrial transplantation (MT), an innovative therapeutic approach for mitochondrial dysfunction and related disorders, has emerged as a focal area of research in recent years [9] . The core principle of MT lies in delivering exogenous healthy mitochondria into impaired cells to replace or supplement dysfunctional organelles, thereby restoring cellular bioenergetics and physiological homeostasis [10]. Accumulating evidence demonstrates that mitochondria isolated from diverse cellular sources can be internalized by any cell type in vitro and can migrate to target tissues via local or systemic administration in vivo. Notably, intravenously transplanted mitochondria exhibit tropism for tissues with mitochondrial damage, enabling preferential accumulation in affected sites [10,11] .As preclinical evidence for mitochondrial transfer expands, transplantation of intact, functional mitochondria has gained traction as a therapeutic strategy for multiple diseases. Using autologous, allogeneic, or xenogeneic mitochondrial sources, MT has demonstrated efficacy in preclinical models of ischemia-reperfusion injury, neurodegenerative diseases, and inflammatory conditions by enhancing ATP production, promoting cell survival, dampening inflammatory cascades, and mitigating oxidative stress [12,13] . Despite these advancements, the therapeutic potential of exogenous MT in lung ischemia-reperfusion injury (LI/RI) remains underexplored. Here, we postulated that MT could ameliorate alveolar epithelial dysfunction and preserve tight junction integrity to mitigate LI/RI. We tested this hypothesis using in vitro models with MLE-12 alveolar epithelial cells and in vivo experiments in C57BL/6 mice. 2. Materials and methods 2.1 Isolation, Extraction, and Identification of Mitochondria Fresh liver tissues from 6-week-old male C57BL/6 mice were used as the source for mitochondrial extraction, and mitochondria were isolated using previously described methods [14] . The extracted mitochondria were resuspended in 0.3 mL of respiration buffer (250 mmol/L sucrose, 20 mmol/L K+-HEPES buffer, pH 7.2, 0.5 mmol/L K+-EGTA, pH 8.0), quantified by flow cytometry, stored on ice at 4°C, and used within 2 hours. For ultrastructural imaging by transmission electron microscopy (TEM), isolated mitochondria were fixed in 2.5% glutaraldehyde followed by 1% osmium tetroxide, dehydrated in acetone, embedded in resin, and ultrathin sections (50 nm) were prepared, transferred to 200-mesh copper grids, stained with uranyl acetate and lead citrate, and imaged using a TEM (LEO906, Zeiss). ATP content was measured using a luciferin/luciferase-based luminometer system (Sigma-Aldrich) in the presence of adenosine diphosphate (ADP). Mitochondrial membrane potential was assessed using a JC-1 staining kit (Beyotime, Shanghai, China), and mitochondrial viability was evaluated by staining with MitoTracker Green FM (200 nM, Beyotime) and MitoTracker Red CMXRos (150 nM, Beyotime), followed by confocal microscopy (Carl Zeiss AG, Germany) for image analysis. 2.2. Animal Models and Grouping All animals were purchased from the Animal Experiment Center of Anhui Medical University, including 8–12-week-old male C57BL/6 mice used as experimental subjects and 6-week-old male C57BL/6 mice used for mitochondrial isolation. Animal studies were performed with approval from the Animal Research Ethics and Use Committee of Anhui Medical University (Approval No.: LLSC: 20241781). Thirty-two male C57BL/6 mice (8–12 weeks old) were randomly divided into four groups: Control group (n=8), receiving only normal saline; I/RI group (n=8), receiving only normal saline; I/RI+Buf group (n=8), receiving blank respiratory buffer without mitochondria; and MT group (n=8), receiving mitochondrial treatment. A mouse model of lung ischemia/reperfusion injury (LI/RI) was established using the pulmonary hilum ligation method [15] . Mice were anesthetized by intraperitoneal injection of tribromoethanol (250–400 mg/kg), followed by tracheal intubation with a 20G tracheal catheter and connection to a small animal ventilator set to positive pressure ventilation mode at a frequency of 100–120 breaths/min and tidal volume of 0.8–1 mL. The ventilator inlet was connected to a small animal anesthesia machine, and 1%–1.5% isoflurane was administered via oxygen flow to maintain anesthesia. Mice were given heparin (20 U/kg) before ischemia. A left thoracotomy was performed by incising the fourth intercostal space to expose the left pulmonary hilum, and a 7-0 polypropylene suture was placed around the hilum. Ischemia was induced by ligating the hilum with a slipknot. After 1 hour of ischemia, the slipknot was released to initiate reperfusion. Control mice underwent thoracotomy without hilum ligation and received mechanical ventilation for 1 hour. At the start of reperfusion, the MT group was injected with 0.3 mL of respiratory buffer containing extracted mitochondria (1 × 10⁸ mitochondria suspended in 0.3 mL buffer) via the jugular vein, while other groups received corresponding treatments. Mice were sacrificed by over-anesthesia 4 hours after reperfusion, and lung tissues and other specimens were collected for subsequent analysis. 2.3. Cell Culture and Model Construction Mouse alveolar epithelial MLE-12 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were maintained in a 37°C incubator with 5% CO₂, grown in Dulbecco’s Modified Eagle Medium (11965, Gibco, USA) containing 10% fetal bovine serum (0500, Gibco, USA), penicillin (100 IU/mL), and streptomycin (100 μg/mL). Cells were divided into four groups: NC group, I/RI group, IRI+Buf group, and MT group. For the hypoxia/reoxygenation model, when cells reached the logarithmic growth phase, they were washed with PBS and switched to serum-free and glucose-free medium, then cultured in a hypoxic incubator (37°C, 5% CO₂, 1% O₂, 94% N₂) for 2 hours. Afterward, the medium was replaced with normal culture medium, and cells were reoxygenated in a standard incubator (37°C, 5% CO₂, 95% O₂) for 1 hour [16] . Corresponding treatments were added during medium replacement: PBS for NC and I/RI groups, blank buffer for I/RI+Buf group, and mitochondrial-containing buffer (100 mitochondria per cell) for MT group. Cells were harvested 4 hours after treatment for subsequent analysis. 2.4. Intracellular Localization of Mitochondrial Transplantation Before mitochondrial treatment, exogenous donor mitochondria were stained with MitoTracker Red CMXRos (150 nM, Beyotime, Shanghai, China), then co-cultured with MLE-12 cells after model establishment. Endogenous mitochondria were stained with MitoTracker Green FM (200 nM, Beyotime). After 2 hours of co-culture, cells were washed with PBS, and nuclei were stained with DAPI followed by PBS washing. Intracellular co-localization of mitochondria was observed under a confocal microscope (Carl Zeiss AG, Germany). 2.5. Cellular MitoSOX, ROS, JC-1, and Edu Proliferation Staining Mitochondrial superoxide production was measured using MitoSOX Red Mitochondrial Superoxide Indicator (Beyotime, Shanghai, China). Total intracellular reactive oxygen species (ROS) were detected using dihydroethidium (DHE, Beyotime) and 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA, Beyotime). Mitochondrial membrane potential was assessed using a JC-1 staining kit (Beyotime). Cell proliferation was evaluated using a 5-ethynyl-2'-deoxyuridine (EdU) assay kit. Four hours after treatment, cells were stained according to the manufacturers' instructions. Fluorescence intensity was measured using a fluorescence microscope (Leica, Germany). 2.6. Flow Cytometry Analysis Apoptosis of MLE-12 cells in each group was assessed using an Annexin V staining kit (Beyotime, Shanghai, China). Briefly, cells were washed with 1× binding buffer and stained with FITC-conjugated Annexin V and propidium iodide (PI) for 15 minutes according to the manufacturer’s instructions. Samples were immediately analyzed by flow cytometry, and data were processed using FlowJo software. 2.7. In Vivo Localization of Mitochondrial Transplantation in Mice Isolated mitochondria were resuspended in prewarmed (37°C) staining solution containing MitoTracker Red CMXRos (150 nM) probe in PBS and incubated in the dark for 30 minutes. After removing the staining solution, labeled mitochondria were washed twice with PBS, quantified by flow cytometry, and kept on ice until transplantation. The labeled mitochondria were resuspended in 0.3 mL of fresh prewarmed buffer (1×10⁸ mitochondria) and administered via jugular vein injection for MT treatment. Mice were euthanized at 1, 4, 8, and 24 hours post-administration, and lung tissues were excised, frozen in liquid nitrogen, and prepared into 10-μm-thick cryosections. Tissue cell nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI), and images were observed and analyzed using a fluorescence microscope (Leica, Germany). 2.8. Lung Histopathology, Injury Scoring, and Wet/Dry Weight Ratio Left lung tissues were fixed in 4% paraformaldehyde, paraffin-embedded, sectioned, and stained with hematoxylin and eosin (H&E). Slides were scanned and analyzed using a slide scanner. Lung injury was assessed using a five-point scoring system (0–4 points) evaluating parameters including alveolar and interstitial inflammation, edema, hemorrhage, necrosis, atelectasis, and hyaline membrane formation: 0 = no injury; 1 = injury in 25% of the area; 2 = 50% injury; 3 = 75% injury; 4 = total injury. The wet/dry (W/D) weight ratio was used to evaluate pulmonary edema. The left lung was weighed immediately after harvesting (wet weight), then dried in a vacuum oven at 80°C for 24 hours to obtain the dry weight. The W/D ratio was calculated as follows: W/D ratio= Dry weight/Wet weight. 2.9. TUNEL Staining and DHE Staining of Lung Tissues Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were performed on paraffin-embedded lung tissue sections using a TUNEL in situ cell death detection kit according to the manufacturer’s instructions. Negative and positive control sections were prepared with labeling solution only, and cell nuclei were counterstained with DAPI. Frozen lung tissue sections were stained with a dihydroethidium (DHE) staining kit to detect reactive oxygen species (ROS). Nuclei were stained with DAPI, and fluorescence intensity in different tissue samples was detected using a fluorescence microscope (Leica, Germany). 2.10. Tissue Immunohistochemistry Lung tissue paraffin sections were baked overnight at 37°C, then incubated with 0.2% Triton X-100 in PBS for 10 minutes and blocked with 5% bovine serum albumin at room temperature for 1 hour. The sections were incubated overnight with primary antibodies including rabbit anti-BAX antibody (1:200, Abcam, Cambridge), rabbit anti-BCL-2 antibody (1:200, Abcam, Cambridge), rabbit anti-Occludin-1 antibody (1:200, Abcam, Cambridge), and rabbit anti-ZO-1 antibody (1:200, Abcam, Cambridge). After washing, fluorescein isothiocyanate-conjugated anti-rabbit IgG (1:200, Beyotime, Shanghai, China) was used as the secondary antibody for counterstaining. The sections were re-stained with hematoxylin and eosin (H&E), washed, covered with cover slips, and fixed. Expression of BAX, BCL-2, Occludin, and ZO-1 proteins in lung tissues was analyzed via a slide scanner to determine their levels. 2.11. Transmission Electron Microscopy (TEM) of Lung Tissues Lung tissues were fixed and cut into 1 mm-thick slices, then placed in 2% paraformaldehyde/2% glutaraldehyde electron microscopy fixative and fixed overnight at 4°C. After thorough washing, samples were post-fixed with 1% osmium tetroxide, dehydrated, embedded, and sectioned into ultrathin slices (70 nm) using an ultramicrotome. Sections were stained with lead citrate and uranyl acetate and observed under a transmission electron microscope (Leica, Germany). 2.12. Western Blotting Analysis Protein expression in lung tissues and MLE-12 cells was detected by Western blotting. After homogenization of lung tissues or MLE-12 cells, protein concentrations in supernatants were measured using a BCA protein assay kit (Beyotime, Shanghai, China). Equal amounts of protein were loaded onto sodium dodecyl sulfate-polyacrylamide gels, transferred to nylon membranes, and incubated with primary antibodies: rabbit monoclonal anti-IL-1β (1:1000, Abcam, Cambridge), rabbit monoclonal anti-TNF-α (1:1000, CST, USA), rabbit monoclonal anti-IL-6 (1:1000, Abcam, Cambridge), rabbit monoclonal anti-BAX (1:1000, Abcam, Cambridge), rabbit monoclonal anti-BCL-2 (1:1000, Abcam, Cambridge), rabbit monoclonal anti-Caspase-3 (1:1000, Abcam, Cambridge), rabbit monoclonal anti-Occludin (1:1000, Abcam, Cambridge), and rabbit monoclonal anti-ZO-1 (1:1000, Abcam, Cambridge). Membranes were then incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibodies. Bands were detected by chemiluminescence, and band optical density was quantified by densitometry using ImageJ software. Experiments were repeated three times. 2.13. RT-qPCR Analysis Total RNA was extracted from lung tissues and MLE-12 cells using TRIzol reagent (Takara, Shiga, Japan) according to the manufacturer’s instructions. cDNA synthesis was performed following the manufacturer’s protocol. qPCR reactions were conducted in a 10-μL system using SYBR-Green Supermix (Invitrogen Life Technologies). All reactions were performed in triplicate, with β-actin serving as the internal reference. Primer sequences used in RT-qPCR are listed in Table 1. Table 1 Sequences of RT-PCR primers Species Gene Forward Reverse Mouse IL-1β 5'-TTCCTTGTGCAAGTGTCTGAAG-3' 5'-ACTGTCAAAAGGTGGCATTT-3' TNF-α 5'-TTCTCATTCCTGCTTGTGG-3' 5'-ACTTGGTGGTTTGCTACG-3' IL-6 5'-CTGCAAGAGACTTCCATCCAG-3' 5'-AGTGGTATAGACAGGTCTGTTGG-3' BAX 5'-TTTCATCCAGGATCGAGCAG-3' 5'-AATCATCCTCTGCAGCTCCA-3' BCL-2 5'-GACTTTGCAGAGATGTCCAG-3' 5'-TCAGGTACTCAGTCATCCAC-3' Caspase-3 5'-ACTGGAATGTCAGCTCGCAA-3' 5'-GCAGTAGTCGCCTCTGAAGA-3' Occludin 5'-CCTTCTGCTTCATCGCTTCCTTA-3' 5'-CGTCGGGTTCACTCCCATTAT-3' ZO-1 5'-GATAGTTTGGCAGCAAGAGATGGTA-3' 5'-AGGTCAGGGACGTTCAGTAAGGTAG-3' 2.14. Statistical analysis of data All experiments were repeated at least three times independently; data are expressed as mean and standard error. All statistics were calculated using GraphPad Prism 8 (Graphpad, San Diego, CA, USA) using unpaired t-tests or one-way ANOVA with multiple comparisons tests. Flowjo (software version 7.6.1) was used to analyse flow cytometry data.TEM and fluorescence microscopy images were processed by Image J software. 3. Results 3.1 Characterisation of isolated mitochondria TEM results showed that the isolated mitochondria exhibited intact morphology with well-preserved mitochondrial cristae and membrane structures, indicating high purity (Fig. 1A). Measurement of ATP content in mitochondria revealed that adding different concentrations of ADP to the isolated mitochondria increased ATP production in a dose-dependent manner, confirming their normal functional activity (Fig. 1B). Flow cytometry analysis using the JC-1 kit demonstrated that the isolated mitochondria maintained normal membrane potential levels (Fig. 1C). Confocal microscopy images of MitoTracker Green FM and MitoTracker Red CMXRos staining showed that the isolated mitochondria had high viability and purity, further verifying their normal mitochondrial function (Fig. 1D). Collectively, these results indicate that the isolated mitochondria possess high viability, intact functionality, and high purity, making them suitable for mitochondrial transplantation (MT) treatment. 3.2. Uptake of Isolated Mitochondria into MLE-12 Cells To investigate whether exogenous healthy mitochondria can enter damaged MLE-12 cells and exert protective effects, we initially investigated the feasibility of mitochondrial transplantation into MLE-12 cells. Mitochondria were isolated from liver tissues of 6-week-old healthy C57/BL6 male mice and stained with MitoTracker Red CMXRos, while endogenous mitochondria in MLE-12 cells were pre-stained with MitoTracker Green FM. After incubating the isolated mitochondria with I/RI-modeled MLE-12 cells for 2 hours, confocal live imaging was performed to observe mitochondrial uptake. Confocal images showed that fluorescently labeled exogenous mitochondria were successfully internalized into I/RI-damaged MLE-12 cells via simple co-incubation (Fig. 2). 3.3. Mitochondrial Transplantation Reduces Apoptosis and Necrosis in MLE-12 Cells, Alleviates Inflammatory Responses and Oxidative Stress Inflammatory responses and oxidative stress are not only core mechanisms of cellular damage after I/RI but also key pathological processes driving cell apoptosis and necrosis. To investigate the effects of MT treatment on alveolar epithelial MLE-12 cells after I/RI injury, relevant assays were performed 4 hours post-treatment. Results showed that protein and mRNA expression levels of pro-apoptotic protein BAX and apoptotic execution protein Caspase-3 were significantly increased in MLE-12 cells of the I/RI group, while these apoptosis-related molecules were significantly decreased in the MT treatment group (P < 0.05) (Fig. 3A). Flow cytometry and EdU proliferation fluorescence assays further confirmed that MT treatment significantly reduced cell apoptosis and necrosis (Figs. 3B, C). Additionally, MT treatment significantly inhibited the expression of I/RI-induced inflammatory cytokines (such as TNF-α, IL-1β, and IL-6) (P < 0.05) (Fig. 3D). Further studies revealed that MT treatment exerted protective effects by alleviating oxidative stress and restoring mitochondrial function. ROS and MitoSOX staining results showed that MT treatment significantly reduced intracellular ROS levels; meanwhile, JC-1 staining indicated that MT treatment promoted the recovery of mitochondrial membrane potential (Figs. 3E, F). Collectively, exogenous MT treatment significantly alleviated I/RI-induced apoptosis and necrosis in alveolar epithelial MLE-12 cells by inhibiting inflammatory responses, reducing oxidative stress, and restoring mitochondrial function. 3.4. In Vivo Uptake and Localization of Exogenous Mitochondrial Transplantation in Mice To investigate the biodistribution characteristics of exogenous mitochondria in mice, in vivo localization studies were performed using fluorescent labeling techniques. In a single-group animal experiment, fluorescent microscopy was used for dynamic tracking of transplanted exogenous mitochondria in key organs and tissues of LI/RI mice. Extracted mitochondria were fluorescently labeled with MitoTracker Red CMXRos (150 nM) and administered via jugular vein injection into mice. Lung, heart, liver, kidney, and spleen tissues were collected at 1, 4, 8, and 24 hours post-injection for frozen section analysis. Results showed that transplanted mitochondria were detected in the lung, heart, liver, kidney, and spleen as early as 1 hour after MT administration (Fig. 4), with the highest mitochondrial content observed in the lungs and heart. Additionally, transplanted mitochondria persisted in lung tissue within 24 hours, though tissue fluorescence signals gradually weakened over time. 3.5. MT Treatment Alleviates Pathological Damage in Lung Tissues of Mice Caused by I/RI In H&E-stained pathological sections of lung tissues, the I/RI group exhibited obvious pathological changes, microscopically showing extensive inflammatory cell infiltration and aggregation in various parts of the lung tissue, along with significant alveolar and interstitial edema (Fig. 5A). MT treatment alleviated I/RI-induced lung injury at the histopathological level: lung injury scoring showed that the IRI group had a higher score and more severe lung injury, while the mitochondrial treatment group had a significantly lower score than the I/RI group (P < 0.01) (Fig. 5B); lung tissue wet/dry ratio results also supported this conclusion, as the IRI group showed a higher wet/dry ratio due to pulmonary edema, while the mitochondrial treatment group had a significantly lower wet/dry ratio (P < 0.01), indicating that MT treatment effectively reduced lung tissue edema (Fig. 5C). These results indicate that MT treatment can improve alveolar barrier function and reduce the severity of lung injury. 3.6. Mitochondrial Transplantation Inhibits Pulmonary Cell Apoptosis and Necrosis and Alleviates Inflammatory Responses in Mice Following exogenous mitochondrial treatment, quantitative analyses of apoptosis and necrosis in lung tissue cells were performed via Western blotting and qPCR. Results showed that MT treatment significantly reduced the expression levels of pro-apoptotic protein BAX and apoptotic execution protein Caspase-3, while restoring the expression of anti-apoptotic protein BCL-2 (p < 0.05) (Fig. 6A). Additionally, immunohistochemistry and TUNEL staining of lung tissues further confirmed that MT treatment significantly reduced the number of apoptotic cells in lung tissues (Figs. 6B, C), consistent with the molecular-level findings. In terms of tissue inflammatory responses, MT treatment significantly decreased the expression levels of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 in lung tissues (p < 0.05) (Fig. 6D), indicating that it not only inhibited I/RI-induced apoptosis and necrosis of lung epithelial cells but also significantly alleviated inflammatory responses. Notably, the in vivo animal experiment results were highly consistent with the previous in vitro cell experiment results, further verifying that MT exerts significant anti-apoptotic and anti-inflammatory effects both in vitro and in vivo. To investigate the effect of MT treatment on oxidative stress, DHE fluorescence staining was used to detect ROS levels in lung tissues, and results showed that MT treatment significantly reduced ROS levels (Fig. 6E), suggesting that it improved the redox balance in lung tissues after I/RI by alleviating oxidative stress. Collectively, exogenous MT treatment significantly improved I/RI-induced lung tissue injury by inhibiting cell apoptosis, alleviating inflammatory responses, and reducing oxidative stress. 3.7. Mitochondrial Transplantation Upregulates Tight Junction Protein Expression and Improves Ultrastructural Damage of the Alveolar Epithelial Barrier Tight junction proteins ZO-1 and Occludin are key regulators of alveolar barrier permeability and play a central role in maintaining lung barrier function. The core pathological mechanism of lung ischemia/reperfusion injury (I/RI) involves damage to alveolar epithelial and endothelial cells, leading to altered vascular permeability, pulmonary edema, and disruption of the alveolar barrier function. Previous studies have shown that exogenous mitochondrial transplantation (MT) significantly alleviates inflammatory responses, reduces oxidative stress levels, and inhibits apoptosis and necrosis in both in vitro I/RI models (MLE-12 cells) and in vivo mouse models, suggesting that MT may protect the damaged alveolar barrier. To validate this hypothesis, in vitro and in vivo models were established for further investigation: Western blotting and qPCR results showed that ZO-1 and Occludin expression was significantly downregulated in MLE-12 cells and mouse lung tissues in the I/RI group, while their expression levels were significantly restored in the MT treatment group (Figs. 7A, B). Immunofluorescence staining of MLE-12 cells revealed that tight junctions in alveolar epithelial cells of the MT group exhibited a smooth linear distribution with a more intact structure (Fig. 7C). Immunohistochemistry of lung tissue sections confirmed that ZO-1 and Occludin expression in the I/RI group was significantly reduced with irregular and discontinuous staining, whereas in the MT group, the distribution of both proteins along the alveolar epithelium was significantly restored with enhanced staining continuity (Fig. 7D). Transmission electron microscopy (TEM) observations showed that the I/RI group exhibited pathological changes such as swollen and irregularly thickened epithelial cells, widened tight junctions, mitochondrial vacuolization, cristae lysis, and lamellar body vacuolization, while the MT group showed regular and flat epithelial cell morphology, reduced intracellular vacuoles, and significantly improved lamellar body exocytosis (Fig. 7E). Collectively, MT treatment effectively alleviated I/RI-induced disruption of the alveolar barrier function and improved lung epithelial ultrastructure by upregulating the expression of ZO-1 and Occludin. 4. Discussion In recent years, MT treatment for mitochondrial damage and dysfunction has garnered significant attention, with the protective effects of exogenous MT being intensively studied in various I/RI models, including cardiac, stroke, kidney, lung, and Parkinson’s disease [17–20] . Guariento et al. [21] demonstrated in a porcine myocardial infarction model that intracoronary infusion of autologous mitochondria significantly alleviated ischemia-reperfusion injury, improved cardiac function, and reduced infarct size. Similarly, studies have shown that transferring mitochondria from healthy muscle cells to damaged renal cells significantly reduces apoptosis rates by regulating cell replication repair and apoptotic protein expression [22] . In the field of lung I/RI (LI/RI), Moskowitzova et al. [23] found that MT treatment could serve as a potential therapy for acute lung injury in I/RI mice, with both pulmonary artery injection and airway nebulization routes significantly improving lung mechanics, enhancing lung viability and recovery, and reducing tissue damage. Beyond warm ischemia-induced acute lung injury, MT efficacy has recently been investigated in an extended ex vivo lung transplantation model: Cloer et al. [24] used intra-arterial mitochondrial transplantation in a porcine ex vivo lung perfusion model to assess whether exogenous MT could improve functional and molecular outcomes in large animal models and human lung equivalents, demonstrating that MT promoted cellular rescue and improved lung function during ex vivo lung perfusion after ischemia-reperfusion. In this study, we further validated the therapeutic potential of MT for LI/RI through in vitro and in vivo experiments, showing that MT significantly reduced oxidative stress levels in lung tissue cells, inhibited excessive release of inflammatory cytokines, regulated apoptosis-related pathways to reduce programmed cell death, mitigated disruption of alveolar epithelial tight junctions, alleviated alveolar barrier dysfunction, and ultimately reduced I/RI-induced lung injury. Studies have shown that mitochondria can be transferred from donor to recipient cells via gap junctions [25] , tunneling nanotubes [26] , or extracellular vesicles [27] . Additionally, free mitochondria can be incorporated into host cells from the extracellular space [28] . These findings provide a theoretical basis for exogenous MT treatment. In an in vitro cell model, our fluorescence labeling analysis confirmed that exogenous mitochondria can enter MLE-12 cells via simple internalization and persist within the cells. In in vivo experiments, we administered mitochondria via jugular vein injection and observed transplanted mitochondria in tissues and organs within 1 hour post-injection, with fluorescent exogenous mitochondria still detectable in various organs at 24 hours. These results confirm the feasibility of MT for treating LI/RI. However, our findings also indicate that intravenously injected exogenous mitochondria exhibit widespread distribution in vivo, which may severely impact their therapeutic effect on targeted organs. The broad uptake of mitochondria by multiple organs could reduce the therapeutic concentration of mitochondria in the target organ, potentially limiting MT efficacy. Therefore, achieving targeted delivery of MT will be a key focus of our future work. LI/RI triggers an inflammatory response, leading to infiltration of numerous inflammatory cells such as neutrophils and macrophages into lung tissues [29] . These inflammatory cells release various pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6 [30,31] , which can impair mitochondrial function through multiple pathways [29–31] . In both our in vitro and in vivo models, exogenous MT treatment reduced the expression of these inflammatory cytokines (TNF-α, IL-1β, IL-6) in LI/RI, thereby alleviating lung tissue damage. Oxidative stress is a critical factor in tissue injury during ischemia-reperfusion. During the ischemic phase, hypoxia impairs the mitochondrial electron transport chain, disrupting electron transfer and causing electron leakage that reacts with oxygen to form superoxide anions. Upon reperfusion, massive oxygen influx triggers a burst of reactive oxygen species (ROS) production [32] . ROS generation damages mitochondrial membrane structure and function, leading to reduced membrane fluidity, increased permeability, impaired selective material transport, and disrupted membrane potential maintenance, ultimately injuring tissue cells [33] . Studies have shown that MT may alleviate cellular/tissue oxidative stress by reducing ROS production [34,35] . As healthy mitochondria enter damaged cells, they can reduce ROS levels and restore balance between antioxidants and ROS generation, subsequently decreasing membrane phospholipid damage, lipid peroxidation, and cellular injury [36] . Similar results were observed in our study: MT treatment reduced ROS and MitoSOX levels in damaged MLE-12 cells and lung tissues, alleviating I/RI-induced oxidative stress. Research suggests that healthy mitochondria isolated from the liver, upon entering damaged cells, may replace or fuse with endogenous damaged mitochondria to prevent further cellular injury [34–37] . Consistent with this, our fluorescence labeling revealed that exogenous mitochondria entered cells and exerted functional effects. JC-1 assays also showed increased mitochondrial membrane potential after MT, likely due to exogenous mitochondria replacing damaged mitochondria within cells. Epithelial barrier dysfunction and high alveolar capillary permeability to proteins and fluids are hallmarks of lung injury [38 ]. Alveolar capillary permeability is determined by intercellular junctions, including tight junctions, gap junctions, and adherens junctions [39] . As a key component of the capillary-alveolar structure, tight junctions are essential for maintaining epithelial barrier integrity. Occludin, a primary transmembrane protein composed of three cytoplasmic domains and two extracellular loops, enables epithelial cells to adhere to each other. The carboxyl terminus of Occludin directly binds to the amino terminus of ZO-1, which in turn interacts with intracellular cytoskeletal proteins, with ZO-1 serving as a bridge between tight junctions and cytoskeletal proteins [40] . Current studies indicate that reduced Occludin and ZO-1 expression in lung tissues contributes to excessive epithelial barrier permeability and pulmonary edema [38–40] . Given the critical role of tight junction protein expression in alveolar permeability, we investigated the effects of MT treatment on tight junction protein expression in alveolar epithelial tissues and its protective role against alveolar epithelial barrier dysfunction after I/RI through in vitro and in vivo experiments. Our results showed that MT treatment reduced epithelial barrier permeability, alleviated pulmonary edema, and upregulated ZO-1 and Occludin expression, with cell immunofluorescence and lung tissue immunohistochemistry further corroborating these findings. Transmission electron microscopy (TEM) observations of the alveolar epithelial barrier ultrastructure revealed that MT treatment restored damaged alveolar ultrastructure and reduced intracellular mitochondrial vacuolar swelling, potentially due to exogenous mitochondria entering lung tissue cells and replacing dysfunctional endogenous mitochondria. Previous studies have shown that healthy mitochondria, upon entering damaged cells, may replace or fuse with native damaged mitochondria to prevent further injury to cardiomyocytes [12,37]. In Lin et al.’s [12] study, exogenous mitochondria were found to engage in mitochondrial fusion via fusion-related proteins after entering cardiomyocytes. This may also explain the TEM observations in our study, where MT treatment reduced mitochondrial swelling and increased the presence of healthy mitochondria in alveolar epithelial cells. 5. Research limitations It is important to emphasize that despite the encouraging results of our study, the specific mechanisms by which MT improves LI/RI remain unclear. The dosage and timing of MT administration in our research may not have been optimized. As measurements were performed only at specific time points, significant changes at other time points might have been overlooked. Additionally, mitochondrial transplantation exhibited widespread organ distribution in vivo, and the enrichment concentration in lung tissues may not have reached the optimal therapeutic level. 6. Conclusion This study systematically validated the potential of mitochondrial transplantation (MT) in treating lung ischemia/reperfusion injury (LI/RI) through in vitro and in vivo experiments. The results demonstrated that MT treatment significantly reduced oxidative stress levels in alveolar epithelial cells, inhibited the excessive release of inflammatory cytokines, regulated apoptotic pathways to reduce programmed cell death, and maintained alveolar epithelial barrier function by preserving the structural integrity of tight junction proteins such as ZO-1 and occludin, thereby ameliorating LI/RI. These findings provide a critical theoretical basis for the clinical application of MT in treating LI/RI. In future research, we will further optimize the treatment protocol, explore targeted pulmonary mitochondrial transplantation, and investigate its clinical translation prospects to provide new strategies for the prevention and treatment of LI/RI. Abbreviation MT Mitochondrial transplantation LI/RI Lung ischaemia-reperfusion injury I/RI Ischemia-reperfusion injury ATP Adenosine triphosphate ROS Reactive oxygen species IL-1β Interleukin-1β IL-6 Interleukin-6 TNF-α Tumor Necrosis Factor-α BCL-2 B-cell lymphoma 2 BAX BCL-2-associated X protein Caspase-3 Cysteine-aspartic acid protease 3 Z0-1 Zonula occludens-1 Occlundin Occlundin Declarations Announcement Description: The three authors, Guangdong Weng, Jie Zhao, and Xiedong Zhu, contributed equally to this study and are listed as co-first authors; Yao Chen is the second author; and Chengxin Zhang and Wenhui Gong are the co-corresponding authors and were responsible for the direction and coordination of the study. Author contributions: Guangdong Weng: research design, study implementation, and paper writing. Jie Zhao: study design, model construction, review, and editing. Xiedong Zhu : research design, model construction, review, and editing. Yao Chen: thesis review and revision. Chengxin Zhang: study design, paper review and revision, funding acquisition. Wenhui Gong: research design, paper review and revision, funding acquisition. Funding: We thank the Natural Science Foundation of Anhui Province (1908085MH241) for funding this project. Acknowledgements: We thank Center for Scientific Research of Anhui Medical University for supporting this study. Declaration of Interests: The authors declare that they have no known financial interests or personal relationships that might influence the work reported in this paper. Ethical Statement: All animal experimental procedures and steps were approved by the Laboratory Animal Ethics Committee of Anhui Medical University (LLSC20241781). Data availability：Data will be made available on request. References Diamond JM, Lee JC, Kawut SM, et al. Clinical risk factors for primary graft dysfunction after lung transplantation. Am J Respir Crit Care Med. 2013;187(5):527–34. Laubach VE, Sharma AK. 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Erratum in: Nat Nanotechnol. 2024 Aug;19(8):1236. Riou A, Broeglin A, Grimm A. Mitochondrial transplantation in brain disorders: Achievements, methods, and challenges. Neurosci Biobehav Rev. 2025 Feb;169:105971. doi: 10.1016/j.neubiorev.2024.105971. Ma H, Jiang T, Tang W, et al. Transplantation of platelet-derived mitochondria alleviates cognitive impairment and mitochondrial dysfunction in db/db mice. Clin Sci (Lond). 2020 Aug 28;134(16):2161-2175. Guariento A, Doulamis IP, Duignan T, et al. Mitochondrial transplantation for myocardial protection in ex-situ-perfused hearts donated after circulatory death. J Heart Lung Transplant. 2020;39(11):1279–1288. Huang H, Oo TT, Apaijai N, et al. An Updated Review of Mitochondrial Transplantation as a Potential Therapeutic Strategy Against Cerebral Ischemia and Cerebral Ischemia/Reperfusion Injury. Mol Neurobiol. 2023 Apr;60(4):1865-1883. Moskowitzova K, Orfany A, Liu K, et al. Mitochondrial transplantation enhances murine lung viability and recovery after ischemia-reperfusion injury. Am J Physiol Lung Cell Mol Physiol. 2020 Jan 1;318(1):L78-L88. Cloer CM, Givens CS, Buie LK, et al. Mitochondrial transplant after ischemia reperfusion promotes cellular salvage and improves lung function during ex-vivo lung perfusion. J Heart Lung Transplant. 2023 May;42(5):575-584. Qin Y, Jiang X, Yang Q,et al. The functions, methods, and mobility of mitochondrial transfer between cells. Front Oncol. 2021;11 . Drab M, Stopar D, Kralj-Iglič V, et al. Inception mechanisms of tunneling nanotubes. Cells. 2019;8:626. Gao J,Qin A, Liu D,, et al. Endoplasmic reticulum mediates mitochondrial transfer within the osteocyte dendritic network. Sci Adv. 2019;5:eaaw7215. Kubat GB, Ulger O, Akin S. Requirements for successful mitochondrial transplantation. J Biochem Mol Toxicol. 2021 Nov;35(11):e22898. Lin L, Xu H, Bishawi M,et al. Circulating mitochondria in organ donors promote allograft rejection. Am J Transplant. 2019 Jul;19(7):1917-1929. Long G, Gong R, Wang Q, Zhang D, Huang C. Role of released mitochondrial DNA in acute lung injury. Front Immunol. 2022 Aug 18;13:973089. Zhao M, Wang Y, Li L, et al. Mitochondrial ROS promote mitochondrial dysfunction and inflammation in ischemic acute kidney injury by disrupting TFAM-mediated mtDNA maintenance. Theranostics. 2021 Jan 1;11(4):1845-1863. Zhao J, Wang G, Han K, et al. Mitochondrial PKM2 deacetylation by procyanidin B2-induced SIRT3 upregulation alleviates lung ischemia/reperfusion injury. Cell Death Dis. 2022 Jul 11;13(7):594. Capuzzimati M, Hough O, Liu M. Cell death and ischemia-reperfusion injury in lung transplantation. J Heart Lung Transplant. 2022 Aug;41(8):1003-1013. Maleki F, Rabbani S, Shirkoohi R, Rezaei M. Allogeneic mitochondrial transplantation ameliorates cardiac dysfunction due to doxorubicin: An in vivo study. Biomed Pharmacother. 2023 Dec;168:115651. Bourebaba L, Bourebaba N, Galuppo L, Marycz K. Artificial mitochondrial transplantation (AMT) reverses aging of mesenchymal stromal cells and improves their immunomodulatory properties in LPS-induced synoviocytes inflammation. Biochim Biophys Acta Mol Cell Res. 2024 Oct;1871(7):119806. Ulger O, Eş I, Proctor CM, Algin O. Stroke studies in large animals: Prospects of mitochondrial transplantation and enhancing efficiency using hydrogels and nanoparticle-assisted delivery. Ageing Res Rev. 2024 Sep;100:102469. Yamada Y, Ito M, Arai M,et al. Challenges in Promoting Mitochondrial Transplantation Therapy. Int J Mol Sci. 2020 Sep 2;21(17):6365. Englert JA, Macias AA, Amador-Munoz D, et al. Isoflurane Ameliorates Acute Lung Injury by Preserving Epithelial Tight Junction Integrity. Anesthesiology. 2015 Aug;123(2):377-88. Yang J, Wang Y, Liu H, et al. C2-ceramide influences alveolar epithelial barrier function by downregulating Zo-1, occludin and claudin-4 expression. Toxicol Mech Methods. 2017 May;27(4):293-297. Li J, Qi Z, Li D, et al. Alveolar epithelial glycocalyx shedding aggravates the epithelial barrier and disrupts epithelial tight junctions in acute respiratory distress syndrome. Biomed Pharmacother. 2021 Jan;133:111026. Supplementary Files Preparesupportinginformation.docx Cite Share Download PDF Status: Published Journal Publication published 26 Nov, 2025 Read the published version in Journal of Translational Medicine → Version 1 posted Reviewers agreed at journal 04 Jun, 2025 Reviewers invited by journal 04 Jun, 2025 Editor assigned by journal 28 May, 2025 First submitted to journal 26 May, 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-6752547\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":false,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":466331662,\"identity\":\"99171a1f-d0d5-4544-bed1-f318c011f279\",\"order_by\":0,\"name\":\"Guangdong Weng\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABBklEQVRIiWNgGAWjYHCDBCCuqJFjY28/QIqWM8eM+XjOJJCghbGNOXGehIMBXnUGx3sPv/zaZpcn75587OEXNrb0Ngmg5h8V23BrOXMuzVq2LbnY8MyzdGMZHpncNunGA4w9Z27j1GJ2I8fMWBLono0zcsykJSTYcttkDiQwM7bh0XL/DUhLPVSLAXM6m0SCAX4tN3iMH35sO5w4XyLHTPJDAnMCQS32Z3LMmBnOHU/cwPMsTZrhwDHDNmAgH8TnF8n2M8Yff5RVJ85vTz4m+fNfjbx8e/vBBz8qcGsBAjZpHiBpcICBgZkHKnQAn3ogYP74A0jKNwBj8gcBpaNgFIyCUTAyAQAAwVwX3stApQAAAABJRU5ErkJggg==\",\"orcid\":\"https://orcid.org/0009-0000-8869-4796\",\"institution\":\"First Affiliated Hospital of Anhui Medical University\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Guangdong\",\"middleName\":\"\",\"lastName\":\"Weng\",\"suffix\":\"\"},{\"id\":466331663,\"identity\":\"3de180bc-89d4-4183-9287-ace3094cd5b3\",\"order_by\":1,\"name\":\"Jie Zhao\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"First Affiliated Hospital of Anhui Medical University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Jie\",\"middleName\":\"\",\"lastName\":\"Zhao\",\"suffix\":\"\"},{\"id\":466331664,\"identity\":\"1e6b96bb-ca22-4c3d-a189-90c19d058f67\",\"order_by\":2,\"name\":\"Xiedong Zhu\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"First Affiliated Hospital of Anhui Medical University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Xiedong\",\"middleName\":\"\",\"lastName\":\"Zhu\",\"suffix\":\"\"},{\"id\":466331665,\"identity\":\"21947f06-9394-43ed-b73a-143a242b1d3c\",\"order_by\":3,\"name\":\"Yao Chen\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"First Affiliated Hospital of Anhui Medical University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Yao\",\"middleName\":\"\",\"lastName\":\"Chen\",\"suffix\":\"\"},{\"id\":466331666,\"identity\":\"cf565829-5847-4f0e-8875-3f1d19fc57f3\",\"order_by\":4,\"name\":\"ChengXin Zhang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"First Affiliated Hospital of Anhui Medical University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"ChengXin\",\"middleName\":\"\",\"lastName\":\"Zhang\",\"suffix\":\"\"},{\"id\":466331667,\"identity\":\"bbdf9822-ac58-4929-a96b-7c43719d87f6\",\"order_by\":5,\"name\":\"WenHui Gong\",\"email\":\"\",\"orcid\":\"https://orcid.org/0009-0000-0848-0879\",\"institution\":\"First Affiliated Hospital of Anhui Medical University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"WenHui\",\"middleName\":\"\",\"lastName\":\"Gong\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2025-05-26 16:17:58\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-6752547/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-6752547/v1\",\"draftVersion\":[],\"editorialEvents\":[{\"content\":\"https://doi.org/10.1186/s12967-025-07360-y\",\"type\":\"published\",\"date\":\"2025-11-26T15:56:53+00:00\"}],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":84064896,\"identity\":\"cca9f7b5-80b8-46e3-8df7-f0cbb1a1c785\",\"added_by\":\"auto\",\"created_at\":\"2025-06-06 10:54:05\",\"extension\":\"jpg\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":1481430,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eCharacterization of isolated mitochondria from liver tissues.\\u003c/strong\\u003e (A) Morphology and purity of isolated mitochondria observed by electron microscopy (scale bar 500 nm, Carl Zeiss AG, Germany). (B) Assessment of ATP content in isolated mitochondria in the presence of different concentrations of ADP (n = 3; data are presented as mean ± SD). (C) Flow cytometry analysis of JC-1-stained mitochondria revealed normal membrane potential in the isolated mitochondria. (D) Characterization of isolated mitochondria labeled with MitoTracker Green FM (200 nM) and MitoTracker Red CMXRos (150 nM) observed by confocal microscopy (scale bar 20 μm).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig1.tif.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6752547/v1/937647732591d2426e8eeff7.jpg\"},{\"id\":84064898,\"identity\":\"9e68e2e0-392e-4478-a744-90779d9e74bc\",\"added_by\":\"auto\",\"created_at\":\"2025-06-06 10:54:05\",\"extension\":\"jpg\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":225422,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eLabeling of exogenous mitochondria transferred into MLE-12 cells.\\u003c/strong\\u003e (A) Exogenous mitochondria were labeled with MitoTracker Red CMXRos (red), and endogenous MLE-12 mitochondria were labeled with MitoTracker Green FM (green). (Scale bar 20 μm)\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig2.tiff.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6752547/v1/a7fbaedce2380b2d78760df2.jpg\"},{\"id\":84064902,\"identity\":\"044eeddb-a6cf-4262-af29-eafbe1c8250d\",\"added_by\":\"auto\",\"created_at\":\"2025-06-06 10:54:06\",\"extension\":\"jpg\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":14513444,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eMT treatment inhibits I/RI-induced apoptosis and necrosis in MLE-12 cells and alleviates inflammatory responses and oxidative stress levels.\\u003c/strong\\u003e (A) Expression levels of Caspase-3 and BAX were significantly decreased, while BCL-2 expression was partially restored; (B) flow cytometry results showed that MT treatment significantly inhibited cell apoptosis and necrosis; (C) EdU proliferation fluorescence assays further confirmed that MT treatment reduced apoptosis and necrosis in MLE-12 cells (scale bar 50 μm); (D) MT treatment significantly decreased the expression levels of TNF-α, IL-1β, and IL-6; (E, F) ROS, MitoSOX, and JC-1 staining results indicated that MT treatment alleviated oxidative stress and promoted the recovery of mitochondrial membrane potential (scale bar 50 μm). Experiments were repeated three times (*p \\u0026lt; 0.05, **p \\u0026lt; 0.01, ***p \\u0026lt; 0.001).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig3.tif.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6752547/v1/a81aceb8c01d795f6f7f4442.jpg\"},{\"id\":84064900,\"identity\":\"ec69c945-9ffc-4959-b323-2ca7dac6280d\",\"added_by\":\"auto\",\"created_at\":\"2025-06-06 10:54:06\",\"extension\":\"jpg\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":543242,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eIn vivo study of exogenous mitochondrial uptake.\\u003c/strong\\u003e Assessment of mitochondrial uptake in lung tissue cells at 1, 4, 8, and 24 hours post-treatment. Exogenous mitochondria were stained with MitoTracker Red (red), and cell nuclei were stained with DAPI (blue). Arrows indicate exogenous red-labeled mitochondria. (Scale bar: 50 μm)\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig4.tiff.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6752547/v1/e98b2e4ed6c9ac329e57b360.jpg\"},{\"id\":84064877,\"identity\":\"dea92742-bec5-4278-91ad-14a4725d3063\",\"added_by\":\"auto\",\"created_at\":\"2025-06-06 10:54:04\",\"extension\":\"jpg\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":2769371,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eMT treatment alleviates IRI-induced lung injury in mice. (A) H\\u0026amp;E staining of lung tissues revealed pathological changes, including inflammatory cell infiltration and alveolar/interstitial edema; (B) quantification of stained areas via lung injury scoring system, which comprehensively evaluated the degree of lung tissue damage, showed significantly lower scores in the MT treatment group compared to the IRI group; (C) lung wet/dry weight ratio in mice demonstrated a significant reduction in the MT treatment group versus the IRI group (P \\u0026lt; 0.01). n=3–5 per group. Experiments were repeated three times (*p \\u0026lt; 0.05, **p \\u0026lt; 0.01, ***p \\u0026lt; 0.001).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig5.tif.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6752547/v1/2030e780a70b98af97a766cf.jpg\"},{\"id\":84064903,\"identity\":\"227f04f7-c2d9-4e25-8409-083babb60d61\",\"added_by\":\"auto\",\"created_at\":\"2025-06-06 10:54:06\",\"extension\":\"png\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":66209163,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eEffects of MT treatment on lung tissue cells: (A) MT treatment inhibited apoptosis and alleviated pulmonary inflammatory responses in mouse lung tissues, as shown by the expression levels of apoptosis-related proteins and mRNAs; (B) tissue TUNEL staining showed TUNEL-positive cells with dark brown-stained apoptotic nuclei under light microscopy (scale bar 50 μm); (C) immunohistochemistry of lung tissues for apoptosis-related proteins BCL-2 and BAX; (D) expression of inflammation-related proteins and RNAs; (E) DHE fluorescence results in lung tissues (scale bar 50 μm). Experiments were repeated three times (*p \\u0026lt; 0.05, **p \\u0026lt; 0.01, ***p \\u0026lt; 0.001).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig6.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6752547/v1/3f3adf361d2f04864b36db67.png\"},{\"id\":84064901,\"identity\":\"51966984-49a6-43ad-b029-2f9099cc6350\",\"added_by\":\"auto\",\"created_at\":\"2025-06-06 10:54:06\",\"extension\":\"png\",\"order_by\":7,\"title\":\"Figure 7\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":70874343,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eEffects of exogenous MT on alveolar epithelial barrier and tight junction protein expression. (A, B) Western blotting and qPCR were used to detect the expression of ZO-1 and Occludin proteins in MLE-12 cells and lung tissues. (C) Merged images of cell immunofluorescence (IF) staining showed the expression of Occludin (green signal), ZO-1 (red signal), and cell nuclei (DAPI staining, blue) in MLE-12 cells. IF staining revealed that tight junction protein expression was reduced in the I/RI group, with discontinuous tight junction expression compared to the NC group, whereas MT treatment restored tight junction protein levels and the staining was qualitatively similar to the NC group (scale bar 50 μm). (D) Quantification of immunohistochemical staining of lung sections confirmed that Occludin and ZO-1 levels were significantly reduced in the IRI group, and MT treatment prevented this decline. (E) TEM images showed that the I/RI group exhibited swollen and irregularly thickened epithelial cells, widened tight junctions between epithelial cells, mitochondrial vacuolization, cristae lysis, and lamellar body emptying. MT treatment improved the ultrastructure of lung epithelial cells, manifested as regular flat epithelial cells, fewer vesicles, and reduced swelling.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Fig7.png\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6752547/v1/46637e31420b1d37d454fcc3.png\"},{\"id\":84064897,\"identity\":\"bdb0a787-e587-4bf9-ba2a-266e06c95fd7\",\"added_by\":\"auto\",\"created_at\":\"2025-06-06 10:54:05\",\"extension\":\"docx\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":14571,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"Preparesupportinginformation.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-6752547/v1/1acc96d7c0efbefc6b0a6107.docx\"}],\"financialInterests\":\"\",\"formattedTitle\":\"Mitochondrial transplantation attenuates alveolar epithelial cell dysfunction and reduces disruption of tight junction proteins to alleviate lung ischaemia-reperfusion injury\",\"fulltext\":[{\"header\":\"1. Introduction\",\"content\":\"\\u003cp\\u003eLung ischaemia-reperfusion injury (LI/RI) is a common and challenging pathological process in clinical practice, frequently occurring in scenarios such as lung transplantation, cardiopulmonary bypass surgery in cardiac surgery, and blood flow restoration after severe trauma, which seriously threatens patients\\u0026apos; life safety and prognosis \\u003csup\\u003e[1-3]\\u003c/sup\\u003e. Despite the continuous advancement of medical technology and the development of treatment methods for LI/RI, no breakthrough progress has been made yet. The pathophysiological mechanism of LI/RI is complex, in which mitochondrial damage plays a key role \\u003csup\\u003e[4]\\u003c/sup\\u003e. During the lung ischaemia phase, tissue cells suffer from hypoxia, leading to impairment of the mitochondrial electron transport chain function and obstruction of electron transport. This results in electron leakage, which reacts with oxygen to generate reactive oxygen species (ROS) such as superoxide anions \\u003csup\\u003e[5]\\u003c/sup\\u003e. During reperfusion, ROS are explosively produced, while the activity of antioxidant enzymes in mitochondria decreases, failing to promptly clear excessive ROS, thereby triggering lipid peroxidation and damaging the structure and function of the mitochondrial membrane \\u003csup\\u003e[6]\\u003c/sup\\u003e. At the same time, intracellular calcium homeostasis is disrupted, and mitochondria take up excessive calcium ions, activating the mitochondrial permeability transition pore, leading to the loss of mitochondrial membrane potential, inhibition of respiratory chain complex activity, and interference with the oxidative phosphorylation process \\u003csup\\u003e[7]\\u003c/sup\\u003e. In addition, the release of inflammatory factors also affects mitochondrial function through multiple pathways\\u003csup\\u003e\\u0026nbsp;[8]\\u003c/sup\\u003e. These factors collectively lead to changes in mitochondrial morphology, respiratory dysfunction, mitochondrial deoxynucleotide damage, and ultimately trigger cell apoptosis, exacerbation of inflammatory responses, and impairment of lung function.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eMitochondrial transplantation (MT), an innovative therapeutic approach for mitochondrial dysfunction and related disorders, has emerged as a focal area of research in recent years \\u003csup\\u003e[9]\\u003c/sup\\u003e. The core principle of MT lies in delivering exogenous healthy mitochondria into impaired cells to replace or supplement dysfunctional organelles, thereby restoring cellular bioenergetics and physiological homeostasis [10]. Accumulating evidence demonstrates that mitochondria isolated from diverse cellular sources can be internalized by any cell type in vitro and can migrate to target tissues via local or systemic administration in vivo. Notably, intravenously transplanted mitochondria exhibit tropism for tissues with mitochondrial damage, enabling preferential accumulation in affected sites\\u003csup\\u003e\\u0026nbsp;[10,11]\\u003c/sup\\u003e.As preclinical evidence for mitochondrial transfer expands, transplantation of intact, functional mitochondria has gained traction as a therapeutic strategy for multiple diseases. Using autologous, allogeneic, or xenogeneic mitochondrial sources, MT has demonstrated efficacy in preclinical models of ischemia-reperfusion injury, neurodegenerative diseases, and inflammatory conditions by enhancing ATP production, promoting cell survival, dampening inflammatory cascades, and mitigating oxidative stress \\u003csup\\u003e[12,13]\\u003c/sup\\u003e. Despite these advancements, the therapeutic potential of exogenous MT in lung ischemia-reperfusion injury (LI/RI) remains underexplored. Here, we postulated that MT could ameliorate alveolar epithelial dysfunction and preserve tight junction integrity to mitigate LI/RI. We tested this hypothesis using in vitro models with MLE-12 alveolar epithelial cells and in vivo experiments in C57BL/6 mice.\\u003c/p\\u003e\"},{\"header\":\"2. Materials and methods\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003e2.1 Isolation, Extraction, and Identification of Mitochondria\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eFresh liver tissues from 6-week-old male C57BL/6 mice were used as the source for mitochondrial extraction, and mitochondria were isolated using previously described methods \\u003csup\\u003e[14]\\u003c/sup\\u003e. The extracted mitochondria were resuspended in 0.3 mL of respiration buffer (250 mmol/L sucrose, 20 mmol/L K+-HEPES buffer, pH 7.2, 0.5 mmol/L K+-EGTA, pH 8.0), quantified by flow cytometry, stored on ice at 4\\u0026deg;C, and used within 2 hours. For ultrastructural imaging by transmission electron microscopy (TEM), isolated mitochondria were fixed in 2.5% glutaraldehyde followed by 1% osmium tetroxide, dehydrated in acetone, embedded in resin, and ultrathin sections (50 nm) were prepared, transferred to 200-mesh copper grids, stained with uranyl acetate and lead citrate, and imaged using a TEM (LEO906, Zeiss). ATP content was measured using a luciferin/luciferase-based luminometer system (Sigma-Aldrich) in the presence of adenosine diphosphate (ADP). Mitochondrial membrane potential was assessed using a JC-1 staining kit (Beyotime, Shanghai, China), and mitochondrial viability was evaluated by staining with MitoTracker Green FM (200 nM, Beyotime) and MitoTracker Red CMXRos (150 nM, Beyotime), followed by confocal microscopy (Carl Zeiss AG, Germany) for image analysis.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e2.2. Animal Models and Grouping\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAll animals were purchased from the Animal Experiment Center of Anhui Medical University, including 8\\u0026ndash;12-week-old male C57BL/6 mice used as experimental subjects and 6-week-old male C57BL/6 mice used for mitochondrial isolation. Animal studies were performed with approval from the Animal Research Ethics and Use Committee of Anhui Medical University (Approval No.: LLSC: 20241781).\\u003c/p\\u003e\\n\\u003cp\\u003eThirty-two male C57BL/6 mice (8\\u0026ndash;12 weeks old) were randomly divided into four groups: Control group (n=8), receiving only normal saline; I/RI group (n=8), receiving only normal saline; I/RI+Buf group (n=8), receiving blank respiratory buffer without mitochondria; and MT group (n=8), receiving mitochondrial treatment. A mouse model of lung ischemia/reperfusion injury (LI/RI) was established using the pulmonary hilum ligation method \\u003csup\\u003e[15]\\u003c/sup\\u003e. Mice were anesthetized by intraperitoneal injection of tribromoethanol (250\\u0026ndash;400 mg/kg), followed by tracheal intubation with a 20G tracheal catheter and connection to a small animal ventilator set to positive pressure ventilation mode at a frequency of 100\\u0026ndash;120 breaths/min and tidal volume of 0.8\\u0026ndash;1 mL. The ventilator inlet was connected to a small animal anesthesia machine, and 1%\\u0026ndash;1.5% isoflurane was administered via oxygen flow to maintain anesthesia. Mice were given heparin (20 U/kg) before ischemia. A left thoracotomy was performed by incising the fourth intercostal space to expose the left pulmonary hilum, and a 7-0 polypropylene suture was placed around the hilum. Ischemia was induced by ligating the hilum with a slipknot. After 1 hour of ischemia, the slipknot was released to initiate reperfusion. Control mice underwent thoracotomy without hilum ligation and received mechanical ventilation for 1 hour. At the start of reperfusion, the MT group was injected with 0.3 mL of respiratory buffer containing extracted mitochondria (1 \\u0026times; 10⁸ mitochondria suspended in 0.3 mL buffer) via the jugular vein, while other groups received corresponding treatments. Mice were sacrificed by over-anesthesia 4 hours after reperfusion, and lung tissues and other specimens were collected for subsequent analysis.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e2.3. Cell Culture and Model Construction\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eMouse alveolar epithelial MLE-12 cells were purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Cells were maintained in a 37\\u0026deg;C incubator with 5% CO₂, grown in Dulbecco\\u0026rsquo;s Modified Eagle Medium (11965, Gibco, USA) containing 10% fetal bovine serum (0500, Gibco, USA), penicillin (100 IU/mL), and streptomycin (100 \\u0026mu;g/mL). Cells were divided into four groups: NC group, I/RI group, IRI+Buf group, and MT group. For the hypoxia/reoxygenation model, when cells reached the logarithmic growth phase, they were washed with PBS and switched to serum-free and glucose-free medium, then cultured in a hypoxic incubator (37\\u0026deg;C, 5% CO₂, 1% O₂, 94% N₂) for 2 hours. Afterward, the medium was replaced with normal culture medium, and cells were reoxygenated in a standard incubator (37\\u0026deg;C, 5% CO₂, 95% O₂) for 1 hour\\u003csup\\u003e\\u0026nbsp;[16]\\u003c/sup\\u003e. Corresponding treatments were added during medium replacement: PBS for NC and I/RI groups, blank buffer for I/RI+Buf group, and mitochondrial-containing buffer (100 mitochondria per cell) for MT group. Cells were harvested 4 hours after treatment for subsequent analysis.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e2.4. Intracellular Localization of Mitochondrial Transplantation\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eBefore mitochondrial treatment, exogenous donor mitochondria were stained with MitoTracker Red CMXRos (150 nM, Beyotime, Shanghai, China), then co-cultured with MLE-12 cells after model establishment. Endogenous mitochondria were stained with MitoTracker Green FM (200 nM, Beyotime). After 2 hours of co-culture, cells were washed with PBS, and nuclei were stained with DAPI followed by PBS washing. Intracellular co-localization of mitochondria was observed under a confocal microscope (Carl Zeiss AG, Germany).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e2.5. Cellular MitoSOX, ROS, JC-1, and Edu Proliferation Staining\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eMitochondrial superoxide production was measured using MitoSOX Red Mitochondrial Superoxide Indicator (Beyotime, Shanghai, China). Total intracellular reactive oxygen species (ROS) were detected using dihydroethidium (DHE, Beyotime) and 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA, Beyotime). Mitochondrial membrane potential was assessed using a JC-1 staining kit (Beyotime). Cell proliferation was evaluated using a 5-ethynyl-2\\u0026apos;-deoxyuridine (EdU) assay kit. Four hours after treatment, cells were stained according to the manufacturers\\u0026apos; instructions. Fluorescence intensity was measured using a fluorescence microscope (Leica, Germany).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e2.6. Flow Cytometry Analysis\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eApoptosis of MLE-12 cells in each group was assessed using an Annexin V staining kit (Beyotime, Shanghai, China). Briefly, cells were washed with 1\\u0026times; binding buffer and stained with FITC-conjugated Annexin V and propidium iodide (PI) for 15 minutes according to the manufacturer\\u0026rsquo;s instructions. Samples were immediately analyzed by flow cytometry, and data were processed using FlowJo software.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e2.7. In Vivo Localization of Mitochondrial Transplantation in Mice\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eIsolated mitochondria were resuspended in prewarmed (37\\u0026deg;C) staining solution containing MitoTracker Red CMXRos (150 nM) probe in PBS and incubated in the dark for 30 minutes. After removing the staining solution, labeled mitochondria were washed twice with PBS, quantified by flow cytometry, and kept on ice until transplantation. The labeled mitochondria were resuspended in 0.3 mL of fresh prewarmed buffer (1\\u0026times;10⁸ mitochondria) and administered via jugular vein injection for MT treatment. Mice were euthanized at 1, 4, 8, and 24 hours post-administration, and lung tissues were excised, frozen in liquid nitrogen, and prepared into 10-\\u0026mu;m-thick cryosections. Tissue cell nuclei were stained with 4\\u0026apos;,6-diamidino-2-phenylindole (DAPI), and images were observed and analyzed using a fluorescence microscope (Leica, Germany).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e2.8. Lung Histopathology, Injury Scoring, and Wet/Dry Weight Ratio\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eLeft lung tissues were fixed in 4% paraformaldehyde, paraffin-embedded, sectioned, and stained with hematoxylin and eosin (H\\u0026amp;E). Slides were scanned and analyzed using a slide scanner. Lung injury was assessed using a five-point scoring system (0\\u0026ndash;4 points) evaluating parameters including alveolar and interstitial inflammation, edema, hemorrhage, necrosis, atelectasis, and hyaline membrane formation: 0 = no injury; 1 = injury in 25% of the area; 2 = 50% injury; 3 = 75% injury; 4 = total injury. The wet/dry (W/D) weight ratio was used to evaluate pulmonary edema. The left lung was weighed immediately after harvesting (wet weight), then dried in a vacuum oven at 80\\u0026deg;C for 24 hours to obtain the dry weight. The W/D ratio was calculated as follows:\\u003c/p\\u003e\\n\\u003cp\\u003eW/D ratio= Dry weight/Wet weight.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e2.9. TUNEL Staining and DHE Staining of Lung Tissues\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTerminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays were performed on paraffin-embedded lung tissue sections using a TUNEL in situ cell death detection kit according to the manufacturer\\u0026rsquo;s instructions. Negative and positive control sections were prepared with labeling solution only, and cell nuclei were counterstained with DAPI. Frozen lung tissue sections were stained with a dihydroethidium (DHE) staining kit to detect reactive oxygen species (ROS). Nuclei were stained with DAPI, and fluorescence intensity in different tissue samples was detected using a fluorescence microscope (Leica, Germany).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e2.10. Tissue Immunohistochemistry\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eLung tissue paraffin sections were baked overnight at 37\\u0026deg;C, then incubated with 0.2% Triton X-100 in PBS for 10 minutes and blocked with 5% bovine serum albumin at room temperature for 1 hour. The sections were incubated overnight with primary antibodies including rabbit anti-BAX antibody (1:200, Abcam, Cambridge), rabbit anti-BCL-2 antibody (1:200, Abcam, Cambridge), rabbit anti-Occludin-1 antibody (1:200, Abcam, Cambridge), and rabbit anti-ZO-1 antibody (1:200, Abcam, Cambridge). After washing, fluorescein isothiocyanate-conjugated anti-rabbit IgG (1:200, Beyotime, Shanghai, China) was used as the secondary antibody for counterstaining. The sections were re-stained with hematoxylin and eosin (H\\u0026amp;E), washed, covered with cover slips, and fixed. Expression of BAX, BCL-2, Occludin, and ZO-1 proteins in lung tissues was analyzed via a slide scanner to determine their levels.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e2.11. Transmission Electron Microscopy (TEM) of Lung Tissues\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eLung tissues were fixed and cut into 1 mm-thick slices, then placed in 2% paraformaldehyde/2% glutaraldehyde electron microscopy fixative and fixed overnight at 4\\u0026deg;C. After thorough washing, samples were post-fixed with 1% osmium tetroxide, dehydrated, embedded, and sectioned into ultrathin slices (70 nm) using an ultramicrotome. Sections were stained with lead citrate and uranyl acetate and observed under a transmission electron microscope (Leica, Germany).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e2.12. Western Blotting Analysis\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eProtein expression in lung tissues and MLE-12 cells was detected by Western blotting. After homogenization of lung tissues or MLE-12 cells, protein concentrations in supernatants were measured using a BCA protein assay kit (Beyotime, Shanghai, China). Equal amounts of protein were loaded onto sodium dodecyl sulfate-polyacrylamide gels, transferred to nylon membranes, and incubated with primary antibodies: rabbit monoclonal anti-IL-1\\u0026beta; (1:1000, Abcam, Cambridge), rabbit monoclonal anti-TNF-\\u0026alpha; (1:1000, CST, USA), rabbit monoclonal anti-IL-6 (1:1000, Abcam, Cambridge), rabbit monoclonal anti-BAX (1:1000, Abcam, Cambridge), rabbit monoclonal anti-BCL-2 (1:1000, Abcam, Cambridge), rabbit monoclonal anti-Caspase-3 (1:1000, Abcam, Cambridge), rabbit monoclonal anti-Occludin (1:1000, Abcam, Cambridge), and rabbit monoclonal anti-ZO-1 (1:1000, Abcam, Cambridge). Membranes were then incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibodies. Bands were detected by chemiluminescence, and band optical density was quantified by densitometry using ImageJ software. Experiments were repeated three times.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e2.13. RT-qPCR Analysis\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTotal RNA was extracted from lung tissues and MLE-12 cells using TRIzol reagent (Takara, Shiga, Japan) according to the manufacturer\\u0026rsquo;s instructions. cDNA synthesis was performed following the manufacturer\\u0026rsquo;s protocol. qPCR reactions were conducted in a 10-\\u0026mu;L system using SYBR-Green Supermix (Invitrogen Life Technologies). All reactions were performed in triplicate, with \\u0026beta;-actin serving as the internal reference. Primer sequences used in RT-qPCR are listed in Table 1.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eTable 1 Sequences of RT-PCR primers\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003ctable border=\\\"1\\\" cellspacing=\\\"0\\\" cellpadding=\\\"0\\\" class=\\\"fr-table-selection-hover\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eSpecies\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eGene\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eForward\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eReverse\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eMouse\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eIL-1\\u0026beta;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e5\\u0026apos;-TTCCTTGTGCAAGTGTCTGAAG-3\\u0026apos;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e5\\u0026apos;-ACTGTCAAAAGGTGGCATTT-3\\u0026apos;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eTNF-\\u0026alpha;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e5\\u0026apos;-TTCTCATTCCTGCTTGTGG-3\\u0026apos;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e5\\u0026apos;-ACTTGGTGGTTTGCTACG-3\\u0026apos;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eIL-6\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e5\\u0026apos;-CTGCAAGAGACTTCCATCCAG-3\\u0026apos;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e5\\u0026apos;-AGTGGTATAGACAGGTCTGTTGG-3\\u0026apos;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eBAX\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e5\\u0026apos;-TTTCATCCAGGATCGAGCAG-3\\u0026apos;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e5\\u0026apos;-AATCATCCTCTGCAGCTCCA-3\\u0026apos;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eBCL-2\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e5\\u0026apos;-GACTTTGCAGAGATGTCCAG-3\\u0026apos;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e5\\u0026apos;-TCAGGTACTCAGTCATCCAC-3\\u0026apos;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eCaspase-3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e5\\u0026apos;-ACTGGAATGTCAGCTCGCAA-3\\u0026apos;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e5\\u0026apos;-GCAGTAGTCGCCTCTGAAGA-3\\u0026apos;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eOccludin\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e5\\u0026apos;-CCTTCTGCTTCATCGCTTCCTTA-3\\u0026apos;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e5\\u0026apos;-CGTCGGGTTCACTCCCATTAT-3\\u0026apos;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\u003cbr\\u003e\\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003eZO-1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e5\\u0026apos;-GATAGTTTGGCAGCAAGAGATGGTA-3\\u0026apos;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd valign=\\\"top\\\"\\u003e\\n \\u003cp\\u003e5\\u0026apos;-AGGTCAGGGACGTTCAGTAAGGTAG-3\\u0026apos;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e2.14. Statistical analysis of data\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAll experiments were repeated at least three times independently; data are expressed as mean and standard error. All statistics were calculated using GraphPad Prism 8 (Graphpad, San Diego, CA, USA) using unpaired t-tests or one-way ANOVA with multiple comparisons tests. Flowjo (software version 7.6.1) was used to analyse flow cytometry data.TEM and fluorescence microscopy images were processed by Image J software.\\u003c/p\\u003e\"},{\"header\":\"3. Results\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003e3.1 Characterisation of isolated mitochondria\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTEM results showed that the isolated mitochondria exhibited intact morphology with well-preserved mitochondrial cristae and membrane structures, indicating high purity (Fig. 1A). Measurement of ATP content in mitochondria revealed that adding different concentrations of ADP to the isolated mitochondria increased ATP production in a dose-dependent manner, confirming their normal functional activity (Fig. 1B). Flow cytometry analysis using the JC-1 kit demonstrated that the isolated mitochondria maintained normal membrane potential levels (Fig. 1C). Confocal microscopy images of MitoTracker Green FM and MitoTracker Red CMXRos staining showed that the isolated mitochondria had high viability and purity, further verifying their normal mitochondrial function (Fig. 1D). Collectively, these results indicate that the isolated mitochondria possess high viability, intact functionality, and high purity, making them suitable for mitochondrial transplantation (MT) treatment.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e3.2. Uptake of Isolated Mitochondria into MLE-12 Cells\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTo investigate whether exogenous healthy mitochondria can enter damaged MLE-12 cells and exert protective effects, we initially investigated the feasibility of mitochondrial transplantation into MLE-12 cells. Mitochondria were isolated from liver tissues of 6-week-old healthy C57/BL6 male mice and stained with MitoTracker Red CMXRos, while endogenous mitochondria in MLE-12 cells were pre-stained with MitoTracker Green FM. After incubating the isolated mitochondria with I/RI-modeled MLE-12 cells for 2 hours, confocal live imaging was performed to observe mitochondrial uptake. Confocal images showed that fluorescently labeled exogenous mitochondria were successfully internalized into I/RI-damaged MLE-12 cells via simple co-incubation (Fig. 2).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e3.3. Mitochondrial Transplantation Reduces Apoptosis and Necrosis in MLE-12 Cells, Alleviates Inflammatory Responses and Oxidative Stress\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eInflammatory responses and oxidative stress are not only core mechanisms of cellular damage after I/RI but also key pathological processes driving cell apoptosis and necrosis. To investigate the effects of MT treatment on alveolar epithelial MLE-12 cells after I/RI injury, relevant assays were performed 4 hours post-treatment. Results showed that protein and mRNA expression levels of pro-apoptotic protein BAX and apoptotic execution protein Caspase-3 were significantly increased in MLE-12 cells of the I/RI group, while these apoptosis-related molecules were significantly decreased in the MT treatment group (P \\u0026lt; 0.05) (Fig. 3A). Flow cytometry and EdU proliferation fluorescence assays further confirmed that MT treatment significantly reduced cell apoptosis and necrosis (Figs. 3B, C). Additionally, MT treatment significantly inhibited the expression of I/RI-induced inflammatory cytokines (such as TNF-\\u0026alpha;, IL-1\\u0026beta;, and IL-6) (P \\u0026lt; 0.05) (Fig. 3D). Further studies revealed that MT treatment exerted protective effects by alleviating oxidative stress and restoring mitochondrial function. ROS and MitoSOX staining results showed that MT treatment significantly reduced intracellular ROS levels; meanwhile, JC-1 staining indicated that MT treatment promoted the recovery of mitochondrial membrane potential (Figs. 3E, F). Collectively, exogenous MT treatment significantly alleviated I/RI-induced apoptosis and necrosis in alveolar epithelial MLE-12 cells by inhibiting inflammatory responses, reducing oxidative stress, and restoring mitochondrial function.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e3.4. In Vivo Uptake and Localization of Exogenous Mitochondrial Transplantation in Mice\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTo investigate the biodistribution characteristics of exogenous mitochondria in mice, in vivo localization studies were performed using fluorescent labeling techniques. In a single-group animal experiment, fluorescent microscopy was used for dynamic tracking of transplanted exogenous mitochondria in key organs and tissues of LI/RI mice. Extracted mitochondria were fluorescently labeled with MitoTracker Red CMXRos (150 nM) and administered via jugular vein injection into mice. Lung, heart, liver, kidney, and spleen tissues were collected at 1, 4, 8, and 24 hours post-injection for frozen section analysis. Results showed that transplanted mitochondria were detected in the lung, heart, liver, kidney, and spleen as early as 1 hour after MT administration (Fig. 4), with the highest mitochondrial content observed in the lungs and heart. Additionally, transplanted mitochondria persisted in lung tissue within 24 hours, though tissue fluorescence signals gradually weakened over time.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e3.5. MT Treatment Alleviates Pathological Damage in Lung Tissues of Mice Caused by I/RI\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eIn H\\u0026amp;E-stained pathological sections of lung tissues, the I/RI group exhibited obvious pathological changes, microscopically showing extensive inflammatory cell infiltration and aggregation in various parts of the lung tissue, along with significant alveolar and interstitial edema (Fig. 5A). MT treatment alleviated I/RI-induced lung injury at the histopathological level: lung injury scoring showed that the IRI group had a higher score and more severe lung injury, while the mitochondrial treatment group had a significantly lower score than the I/RI group (P \\u0026lt; 0.01) (Fig. 5B); lung tissue wet/dry ratio results also supported this conclusion, as the IRI group showed a higher wet/dry ratio due to pulmonary edema, while the mitochondrial treatment group had a significantly lower wet/dry ratio (P \\u0026lt; 0.01), indicating that MT treatment effectively reduced lung tissue edema (Fig. 5C). These results indicate that MT treatment can improve alveolar barrier function and reduce the severity of lung injury.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e3.6. Mitochondrial Transplantation Inhibits Pulmonary Cell Apoptosis and Necrosis and Alleviates Inflammatory Responses in Mice\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eFollowing exogenous mitochondrial treatment, quantitative analyses of apoptosis and necrosis in lung tissue cells were performed via Western blotting and qPCR. Results showed that MT treatment significantly reduced the expression levels of pro-apoptotic protein BAX and apoptotic execution protein Caspase-3, while restoring the expression of anti-apoptotic protein BCL-2 (p \\u0026lt; 0.05) (Fig. 6A). Additionally, immunohistochemistry and TUNEL staining of lung tissues further confirmed that MT treatment significantly reduced the number of apoptotic cells in lung tissues (Figs. 6B, C), consistent with the molecular-level findings. In terms of tissue inflammatory responses, MT treatment significantly decreased the expression levels of pro-inflammatory cytokines such as TNF-\\u0026alpha;, IL-1\\u0026beta;, and IL-6 in lung tissues (p \\u0026lt; 0.05) (Fig. 6D), indicating that it not only inhibited I/RI-induced apoptosis and necrosis of lung epithelial cells but also significantly alleviated inflammatory responses. Notably, the in vivo animal experiment results were highly consistent with the previous in vitro cell experiment results, further verifying that MT exerts significant anti-apoptotic and anti-inflammatory effects both in vitro and in vivo. To investigate the effect of MT treatment on oxidative stress, DHE fluorescence staining was used to detect ROS levels in lung tissues, and results showed that MT treatment significantly reduced ROS levels (Fig. 6E), suggesting that it improved the redox balance in lung tissues after I/RI by alleviating oxidative stress. Collectively, exogenous MT treatment significantly improved I/RI-induced lung tissue injury by inhibiting cell apoptosis, alleviating inflammatory responses, and reducing oxidative stress.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e3.7. Mitochondrial Transplantation Upregulates Tight Junction Protein Expression and Improves Ultrastructural Damage of the Alveolar Epithelial Barrier\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTight junction proteins ZO-1 and Occludin are key regulators of alveolar barrier permeability and play a central role in maintaining lung barrier function. The core pathological mechanism of lung ischemia/reperfusion injury (I/RI) involves damage to alveolar epithelial and endothelial cells, leading to altered vascular permeability, pulmonary edema, and disruption of the alveolar barrier function. Previous studies have shown that exogenous mitochondrial transplantation (MT) significantly alleviates inflammatory responses, reduces oxidative stress levels, and inhibits apoptosis and necrosis in both in vitro I/RI models (MLE-12 cells) and in vivo mouse models, suggesting that MT may protect the damaged alveolar barrier. To validate this hypothesis, in vitro and in vivo models were established for further investigation: Western blotting and qPCR results showed that ZO-1 and Occludin expression was significantly downregulated in MLE-12 cells and mouse lung tissues in the I/RI group, while their expression levels were significantly restored in the MT treatment group (Figs. 7A, B). Immunofluorescence staining of MLE-12 cells revealed that tight junctions in alveolar epithelial cells of the MT group exhibited a smooth linear distribution with a more intact structure (Fig. 7C). Immunohistochemistry of lung tissue sections confirmed that ZO-1 and Occludin expression in the I/RI group was significantly reduced with irregular and discontinuous staining, whereas in the MT group, the distribution of both proteins along the alveolar epithelium was significantly restored with enhanced staining continuity (Fig. 7D). Transmission electron microscopy (TEM) observations showed that the I/RI group exhibited pathological changes such as swollen and irregularly thickened epithelial cells, widened tight junctions, mitochondrial vacuolization, cristae lysis, and lamellar body vacuolization, while the MT group showed regular and flat epithelial cell morphology, reduced intracellular vacuoles, and significantly improved lamellar body exocytosis (Fig. 7E). Collectively, MT treatment effectively alleviated I/RI-induced disruption of the alveolar barrier function and improved lung epithelial ultrastructure by upregulating the expression of ZO-1 and Occludin.\\u003c/p\\u003e\"},{\"header\":\"4. Discussion\",\"content\":\"\\u003cp\\u003eIn recent years, MT treatment for mitochondrial damage and dysfunction has garnered significant attention, with the protective effects of exogenous MT being intensively studied in various I/RI models, including cardiac, stroke, kidney, lung, and Parkinson’s disease\\u003csup\\u003e\\u0026nbsp;[17–20]\\u003c/sup\\u003e. Guariento et al.\\u003csup\\u003e\\u0026nbsp;[21]\\u0026nbsp;\\u003c/sup\\u003edemonstrated in a porcine myocardial infarction model that intracoronary infusion of autologous mitochondria significantly alleviated ischemia-reperfusion injury, improved cardiac function, and reduced infarct size. Similarly, studies have shown that transferring mitochondria from healthy muscle cells to damaged renal cells significantly reduces apoptosis rates by regulating cell replication repair and apoptotic protein expression\\u003csup\\u003e\\u0026nbsp;[22]\\u003c/sup\\u003e. In the field of lung I/RI (LI/RI), Moskowitzova et al.\\u003csup\\u003e[23]\\u003c/sup\\u003e found that MT treatment could serve as a potential therapy for acute lung injury in I/RI mice, with both pulmonary artery injection and airway nebulization routes significantly improving lung mechanics, enhancing lung viability and recovery, and reducing tissue damage. Beyond warm ischemia-induced acute lung injury, MT efficacy has recently been investigated in an extended ex vivo lung transplantation model: Cloer et al.\\u003csup\\u003e[24]\\u0026nbsp;\\u003c/sup\\u003eused intra-arterial mitochondrial transplantation in a porcine ex vivo lung perfusion model to assess whether exogenous MT could improve functional and molecular outcomes in large animal models and human lung equivalents, demonstrating that MT promoted cellular rescue and improved lung function during ex vivo lung perfusion after ischemia-reperfusion. In this study, we further validated the therapeutic potential of MT for LI/RI through in vitro and in vivo experiments, showing that MT significantly reduced oxidative stress levels in lung tissue cells, inhibited excessive release of inflammatory cytokines, regulated apoptosis-related pathways to reduce programmed cell death, mitigated disruption of alveolar epithelial tight junctions, alleviated alveolar barrier dysfunction, and ultimately reduced I/RI-induced lung injury.\\u003c/p\\u003e\\n\\u003cp\\u003eStudies have shown that mitochondria can be transferred from donor to recipient cells via gap junctions \\u003csup\\u003e[25]\\u003c/sup\\u003e, tunneling nanotubes \\u003csup\\u003e[26]\\u003c/sup\\u003e, or extracellular vesicles \\u003csup\\u003e[27]\\u003c/sup\\u003e. Additionally, free mitochondria can be incorporated into host cells from the extracellular space \\u003csup\\u003e[28]\\u003c/sup\\u003e. These findings provide a theoretical basis for exogenous MT treatment. In an in vitro cell model, our fluorescence labeling analysis confirmed that exogenous mitochondria can enter MLE-12 cells via simple internalization and persist within the cells. In in vivo experiments, we administered mitochondria via jugular vein injection and observed transplanted mitochondria in tissues and organs within 1 hour post-injection, with fluorescent exogenous mitochondria still detectable in various organs at 24 hours. These results confirm the feasibility of MT for treating LI/RI. However, our findings also indicate that intravenously injected exogenous mitochondria exhibit widespread distribution in vivo, which may severely impact their therapeutic effect on targeted organs. The broad uptake of mitochondria by multiple organs could reduce the therapeutic concentration of mitochondria in the target organ, potentially limiting MT efficacy. Therefore, achieving targeted delivery of MT will be a key focus of our future work.\\u003c/p\\u003e\\n\\u003cp\\u003eLI/RI triggers an inflammatory response, leading to infiltration of numerous inflammatory cells such as neutrophils and macrophages into lung tissues \\u003csup\\u003e[29]\\u003c/sup\\u003e. These inflammatory cells release various pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6 \\u003csup\\u003e[30,31]\\u003c/sup\\u003e, which can impair mitochondrial function through multiple pathways \\u003csup\\u003e[29–31]\\u003c/sup\\u003e. In both our in vitro and in vivo models, exogenous MT treatment reduced the expression of these inflammatory cytokines (TNF-α, IL-1β, IL-6) in LI/RI, thereby alleviating lung tissue damage. Oxidative stress is a critical factor in tissue injury during ischemia-reperfusion. During the ischemic phase, hypoxia impairs the mitochondrial electron transport chain, disrupting electron transfer and causing electron leakage that reacts with oxygen to form superoxide anions. Upon reperfusion, massive oxygen influx triggers a burst of reactive oxygen species (ROS) production \\u003csup\\u003e[32]\\u003c/sup\\u003e. ROS generation damages mitochondrial membrane structure and function, leading to reduced membrane fluidity, increased permeability, impaired selective material transport, and disrupted membrane potential maintenance, ultimately injuring tissue cells \\u003csup\\u003e[33]\\u003c/sup\\u003e. Studies have shown that MT may alleviate cellular/tissue oxidative stress by reducing ROS production \\u003csup\\u003e[34,35]\\u003c/sup\\u003e. As healthy mitochondria enter damaged cells, they can reduce ROS levels and restore balance between antioxidants and ROS generation, subsequently decreasing membrane phospholipid damage, lipid peroxidation, and cellular injury \\u003csup\\u003e[36]\\u003c/sup\\u003e. Similar results were observed in our study: MT treatment reduced ROS and MitoSOX levels in damaged MLE-12 cells and lung tissues, alleviating I/RI-induced oxidative stress. Research suggests that healthy mitochondria isolated from the liver, upon entering damaged cells, may replace or fuse with endogenous damaged mitochondria to prevent further cellular injury\\u003csup\\u003e\\u0026nbsp;[34–37]\\u003c/sup\\u003e. Consistent with this, our fluorescence labeling revealed that exogenous mitochondria entered cells and exerted functional effects. JC-1 assays also showed increased mitochondrial membrane potential after MT, likely due to exogenous mitochondria replacing damaged mitochondria within cells.\\u003c/p\\u003e\\n\\u003cp\\u003eEpithelial barrier dysfunction and high alveolar capillary permeability to proteins and fluids are hallmarks of lung injury \\u003csup\\u003e[38\\u003c/sup\\u003e]. Alveolar capillary permeability is determined by intercellular junctions, including tight junctions, gap junctions, and adherens junctions\\u003csup\\u003e\\u0026nbsp;[39]\\u003c/sup\\u003e. As a key component of the capillary-alveolar structure, tight junctions are essential for maintaining epithelial barrier integrity. Occludin, a primary transmembrane protein composed of three cytoplasmic domains and two extracellular loops, enables epithelial cells to adhere to each other. The carboxyl terminus of Occludin directly binds to the amino terminus of ZO-1, which in turn interacts with intracellular cytoskeletal proteins, with ZO-1 serving as a bridge between tight junctions and cytoskeletal proteins\\u003csup\\u003e\\u0026nbsp;[40]\\u003c/sup\\u003e. Current studies indicate that reduced Occludin and ZO-1 expression in lung tissues contributes to excessive epithelial barrier permeability and pulmonary edema \\u003csup\\u003e[38–40]\\u003c/sup\\u003e. Given the critical role of tight junction protein expression in alveolar permeability, we investigated the effects of MT treatment on tight junction protein expression in alveolar epithelial tissues and its protective role against alveolar epithelial barrier dysfunction after I/RI through in vitro and in vivo experiments. Our results showed that MT treatment reduced epithelial barrier permeability, alleviated pulmonary edema, and upregulated ZO-1 and Occludin expression, with cell immunofluorescence and lung tissue immunohistochemistry further corroborating these findings. Transmission electron microscopy (TEM) observations of the alveolar epithelial barrier ultrastructure revealed that MT treatment restored damaged alveolar ultrastructure and reduced intracellular mitochondrial vacuolar swelling, potentially due to exogenous mitochondria entering lung tissue cells and replacing dysfunctional endogenous mitochondria. Previous studies have shown that healthy mitochondria, upon entering damaged cells, may replace or fuse with native damaged mitochondria to prevent further injury to cardiomyocytes [12,37]. In Lin et al.’s [12] study, exogenous mitochondria were found to engage in mitochondrial fusion via fusion-related proteins after entering cardiomyocytes. This may also explain the TEM observations in our study, where MT treatment reduced mitochondrial swelling and increased the presence of healthy mitochondria in alveolar epithelial cells.\\u003c/p\\u003e\"},{\"header\":\"5. Research limitations\",\"content\":\"\\u003cp\\u003eIt is important to emphasize that despite the encouraging results of our study, the specific mechanisms by which MT improves LI/RI remain unclear. The dosage and timing of MT administration in our research may not have been optimized. As measurements were performed only at specific time points, significant changes at other time points might have been overlooked. Additionally, mitochondrial transplantation exhibited widespread organ distribution in vivo, and the enrichment concentration in lung tissues may not have reached the optimal therapeutic level.\\u003c/p\\u003e\"},{\"header\":\"6. Conclusion\",\"content\":\"\\u003cp\\u003eThis study systematically validated the potential of mitochondrial transplantation (MT) in treating lung ischemia/reperfusion injury (LI/RI) through in vitro and in vivo experiments. The results demonstrated that MT treatment significantly reduced oxidative stress levels in alveolar epithelial cells, inhibited the excessive release of inflammatory cytokines, regulated apoptotic pathways to reduce programmed cell death, and maintained alveolar epithelial barrier function by preserving the structural integrity of tight junction proteins such as ZO-1 and occludin, thereby ameliorating LI/RI. These findings provide a critical theoretical basis for the clinical application of MT in treating LI/RI. In future research, we will further optimize the treatment protocol, explore targeted pulmonary mitochondrial transplantation, and investigate its clinical translation prospects to provide new strategies for the prevention and treatment of LI/RI.\\u003c/p\\u003e\"},{\"header\":\"Abbreviation\",\"content\":\"\\u003ctable border=\\\"1\\\" cellspacing=\\\"0\\\" cellpadding=\\\"0\\\" width=\\\"573\\\"\\u003e\\n \\u003ctbody\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 119px;\\\"\\u003e\\n \\u003cp\\u003eMT\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 454px;\\\"\\u003e\\n \\u003cp\\u003eMitochondrial transplantation\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 119px;\\\"\\u003e\\n \\u003cp\\u003eLI/RI\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 454px;\\\"\\u003e\\n \\u003cp\\u003eLung ischaemia-reperfusion injury\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 119px;\\\"\\u003e\\n \\u003cp\\u003eI/RI\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 454px;\\\"\\u003e\\n \\u003cp\\u003eIschemia-reperfusion injury\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 119px;\\\"\\u003e\\n \\u003cp\\u003eATP\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 454px;\\\"\\u003e\\n \\u003cp\\u003eAdenosine triphosphate\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 119px;\\\"\\u003e\\n \\u003cp\\u003eROS\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 454px;\\\"\\u003e\\n \\u003cp\\u003eReactive oxygen species\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 119px;\\\"\\u003e\\n \\u003cp\\u003eIL-1\\u0026beta;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 454px;\\\"\\u003e\\n \\u003cp\\u003eInterleukin-1\\u0026beta;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003eIL-6\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd\\u003e\\n \\u003cp\\u003eInterleukin-6\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 119px;\\\"\\u003e\\n \\u003cp\\u003eTNF-\\u0026alpha;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 454px;\\\"\\u003e\\n \\u003cp\\u003eTumor Necrosis Factor-\\u0026alpha;\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 119px;\\\"\\u003e\\n \\u003cp\\u003eBCL-2\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 454px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eB-cell lymphoma 2\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 119px;\\\"\\u003e\\n \\u003cp\\u003eBAX\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 454px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eBCL-2-associated X protein\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 119px;\\\"\\u003e\\n \\u003cp\\u003eCaspase-3\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 454px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eCysteine-aspartic acid protease 3\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 119px;\\\"\\u003e\\n \\u003cp\\u003eZ0-1\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 454px;\\\"\\u003e\\n \\u003cp\\u003e\\u003cstrong\\u003eZonula occludens-1\\u003c/strong\\u003e\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003ctr\\u003e\\n \\u003ctd style=\\\"width: 119px;\\\"\\u003e\\n \\u003cp\\u003eOcclundin\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003ctd style=\\\"width: 454px;\\\"\\u003e\\n \\u003cp\\u003eOcclundin\\u003c/p\\u003e\\n \\u003c/td\\u003e\\n \\u003c/tr\\u003e\\n \\u003c/tbody\\u003e\\n\\u003c/table\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAnnouncement Description:\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe three authors, Guangdong Weng, Jie Zhao, and Xiedong Zhu, contributed equally to this study and are listed as co-first authors; Yao Chen is the second author; and Chengxin Zhang and Wenhui Gong are the co-corresponding authors and were responsible for the direction and coordination of the study.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor contributions:\\u0026nbsp;\\u003c/strong\\u003eGuangdong Weng: research design, study implementation, and paper writing. Jie Zhao: study design, model construction, review, and editing. Xiedong Zhu : research design, model construction, review, and editing. Yao Chen: thesis review and revision. Chengxin Zhang: study design, paper review and revision, funding acquisition. Wenhui Gong: research design, paper review and revision, funding acquisition.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding:\\u0026nbsp;\\u003c/strong\\u003eWe thank the Natural Science Foundation of Anhui Province (1908085MH241) for funding this project.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgements:\\u0026nbsp;\\u003c/strong\\u003eWe thank Center for Scientific Research of Anhui Medical University for supporting this study.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eDeclaration of Interests:\\u003c/strong\\u003e The authors declare that they have no known financial interests or personal relationships that might influence the work reported in this paper.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthical Statement:\\u0026nbsp;\\u003c/strong\\u003eAll animal experimental procedures and steps were approved by the Laboratory Animal Ethics Committee of Anhui Medical University (LLSC20241781).\\u003c/p\\u003e\\n\\u003cp\\u003eData availability：Data will be made available on request.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eDiamond JM, Lee JC, Kawut SM, et al. Clinical risk factors for primary graft dysfunction after lung transplantation. Am J Respir Crit Care Med. 2013;187(5):527\\u0026ndash;34.\\u003c/li\\u003e\\n\\u003cli\\u003eLaubach VE, Sharma AK. Mechanisms of lung ischemia-reperfusion injury. Curr Opin Organ Transplant. 2016;21:246\\u0026ndash;252. \\u003c/li\\u003e\\n\\u003cli\\u003eHu Q, Luo W, Huang L, et. al Multiorgan protection of remote ischemic perconditioning in valve replacement surgery. J Surg Res. 2016 Jan;200(1):13-20.\\u003c/li\\u003e\\n\\u003cli\\u003eVaseva AV, Marchenko ND, Ji K, et al. p53 opens the mitochondrial permeability transition pore to trigger necrosis. Cell. 2012 Jun 22;149(7):1536-48. \\u003c/li\\u003e\\n\\u003cli\\u003eVlastos D, Zeinah M, Ninkovic-Hall G, et al. The effects of ischaemic conditioning on lung ischaemia-reperfusion injury. Respir Res. 2022 Dec 16;23(1):351. \\u003c/li\\u003e\\n\\u003cli\\u003ePak O, Sydykov A, Kosanovic D, et al. Lung Ischaemia-Reperfusion Injury: The Role of Reactive Oxygen Species. Adv Exp Med Biol. 2017;967:195-225. \\u003c/li\\u003e\\n\\u003cli\\u003eKalogeris T, Bao Y, Korthuis RJ. Mitochondrial reactive oxygen species: a double edged sword in ischemia/reperfusion vs preconditioning. Redox Biol. 2014 Jun 2;2:702-14. \\u003c/li\\u003e\\n\\u003cli\\u003eMortaz E, Alipoor SD, Adcock IM, Mumby S, Koenderman L. Update on Neutrophil Function in Severe Inflammation. Front Immunol. 2018 Oct 2;9:2171. \\u003c/li\\u003e\\n\\u003cli\\u003eJiao Q, Xiang L, Chen Y. Mitochondrial transplantation: A promising therapy for mitochondrial disorders. Int J Pharm. 2024 Jun 10;658:124194. doi: 10.1016/j.ijpharm.2024.124194. Epub 2024 May 3. \\u003c/li\\u003e\\n\\u003cli\\u003eHayashida K, Takegawa R, Shoaib M, et al. Mitochondrial transplantation therapy for ischemia reperfusion injury: a systematic review of animal and human studies. J Transl Med. 2021 May 17;19(1):214. \\u003c/li\\u003e\\n\\u003cli\\u003eGorick C, Debski A. Mitochondrial transplantation for ischemic heart disease. Nat Nanotechnol. 2024 Sep;19(9):1247-1248. \\u003c/li\\u003e\\n\\u003cli\\u003eLin RZ, Im GB, Luo AC, et al. Mitochondrial transfer mediates endothelial cell engraftment through mitophagy. Nature. 2024 May;629(8012):660-668. \\u003c/li\\u003e\\n\\u003cli\\u003eHu C, Shi Z, Liu X, Sun C. The Research Progress of Mitochondrial Transplantation in the Treatment of Mitochondrial Defective Diseases. Int J Mol Sci. 2024 Jan 18;25(2):1175. \\u003c/li\\u003e\\n\\u003cli\\u003eZhang A, Liu Y, Pan J, Pontanari F, Chia-Hao Chang A, Wang H, Gao S, Wang C, Chang AC. Delivery of mitochondria confers cardioprotection through mitochondria replenishment and metabolic compliance. Mol Ther. 2023 May 3;31(5):1468-1479. \\u003c/li\\u003e\\n\\u003cli\\u003eBai YZ, Yokoyama Y, Li W, et al. 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Biomed Pharmacother. 2021 Jan;133:111026.\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":true,\"isPdf\":false,\"isPdfUpToDate\":false,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"journal-of-translational-medicine\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"jtrm\",\"sideBox\":\"Learn more about [Journal of Translational Medicine](http://translational-medicine.biomedcentral.com)\",\"snPcode\":\"\",\"submissionUrl\":\"https://www.editorialmanager.com/jtrm/default.aspx\",\"title\":\"Journal of Translational Medicine\",\"twitterHandle\":\"@BioMedCentral\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"BMC/SO AJ\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Mitochondrial transplantation, Lung ischaemia-reperfusion injury, Tight junction proteins, Repair of lung damage\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-6752547/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-6752547/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"Objective\\n\\nThe aim of this study was to investigate the therapeutic effect of exogenous mitochondrial transplantation (MT) on lung ischemia-reperfusion injury (LI/RI) and to investigate the effect of mitochondrial transplantation on alveolar epithelial cell function as well as on the ultrastructural changes of the alveolar epithelial barrier ultrastructural changes.\\n\\nMethods\\n\\nThe therapeutic effect of mouse liver-derived mitochondrial transplantation on LI/RI was assessed by constructing a hypoxia-reoxygenation model of mouse alveolar epithelial cells (MLE-12 cells) and a lung ischaemia-reperfusion injury model in C57BL/6 male mice, which simulated the pathological process of LI/RI.\\n\\nResults\\n\\nThe study results showed that MT exhibited significant therapeutic potential in LI/RI. In vitro and in vivo experiments revealed that MT significantly improved lung tissue injury by reducing oxidative stress, inflammatory response, apoptosis, and necrosis. Meanwhile, MT could alleviate alveolar epithelial cell dysfunction, reduce the disruption of tight junction proteins, and protect the alveolar epithelial barrier, thereby mitigating LI/RI.\\n\\nConclusion\\n\\nThis study confirmed in a lung ischemia-reperfusion injury model that MT treatment can repair the structural damage of the alveolar barrier caused by ischemia-reperfusion by targeting and regulating the expression of tight junction proteins in alveolar epithelial cells, providing a new perspective for elucidating the functional targets of MT treatment in protecting the alveolar barrier.\",\"manuscriptTitle\":\"Mitochondrial transplantation attenuates alveolar epithelial cell dysfunction and reduces disruption of tight junction proteins to alleviate lung ischaemia-reperfusion injury\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-06-06 10:53:55\",\"doi\":\"10.21203/rs.3.rs-6752547/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"reviewerAgreed\",\"content\":\"\",\"date\":\"2025-06-04T11:52:32+00:00\",\"index\":0,\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2025-06-04T09:19:39+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2025-05-28T15:33:07+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"Journal of Translational Medicine\",\"date\":\"2025-05-26T12:15:43+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"journal-of-translational-medicine\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"jtrm\",\"sideBox\":\"Learn more about [Journal of Translational Medicine](http://translational-medicine.biomedcentral.com)\",\"snPcode\":\"\",\"submissionUrl\":\"https://www.editorialmanager.com/jtrm/default.aspx\",\"title\":\"Journal of Translational Medicine\",\"twitterHandle\":\"@BioMedCentral\",\"acdcEnabled\":true,\"dfaEnabled\":true,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"BMC/SO AJ\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"b8ebb257-43e8-4c36-b75e-16c5bf00c23d\",\"owner\":[],\"postedDate\":\"June 6th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2025-12-01T16:00:30+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-6752547\",\"link\":\"https://doi.org/10.1186/s12967-025-07360-y\",\"journal\":{\"identity\":\"journal-of-translational-medicine\",\"isVorOnly\":false,\"title\":\"Journal of Translational Medicine\"},\"publishedOn\":\"2025-11-26 15:56:53\",\"publishedOnDateReadable\":\"November 26th, 2025\"},\"versionCreatedAt\":\"2025-06-06 10:53:55\",\"video\":\"\",\"vorDoi\":\"10.1186/s12967-025-07360-y\",\"vorDoiUrl\":\"https://doi.org/10.1186/s12967-025-07360-y\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-6752547\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-6752547\",\"identity\":\"rs-6752547\",\"version\":[\"v1\"]},\"buildId\":\"8U1c8b4HqxoKbykW_rLl7\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}