Mitochondrial transplantation attenuates alveolar epithelial cell dysfunction and reduces disruption of tight junction proteins to alleviate lung ischaemia-reperfusion injury

Abstract

Objective

This study aims to investigate the therapeutic effect of exogenous mitochondrial transplantation (MT) on lung ischemia-reperfusion injury (LI/RI), and analyze the impact of MT on alveolar epithelial cell function and the ultrastructure of the alveolar epithelial barrier.

Methods

To simulate the pathological process of LI/RI, we established a hypoxia-reoxygenation model using mouse alveolar epithelial cells (MLE-12 cells) and a LI/RI model in male C57BL/6 mice. Based on these models, we further evaluated the therapeutic effect of mouse liver-derived mitochondrial transplantation on LI/RI.

Results

The results of this study showed that MT exhibits significant therapeutic potential in LI/RI. Both in vitro and in vivo experiments confirmed that MT can significantly ameliorate lung tissue injury by reducing oxidative stress levels, alleviating inflammatory responses, and decreasing cell apoptosis and necrosis. Meanwhile, MT is capable of alleviating alveolar epithelial cell dysfunction, reducing the disruption of tight junction proteins, and preserving the integrity of the alveolar epithelial barrier, thereby mitigating LI/RI.

Conclusion

Our study confirmed in the LI/RI model that MT therapy can repair the alveolar barrier structural damage caused by ischemia-reperfusion by targeting the regulation of the expression levels of tight junction proteins in alveolar epithelial cells. This finding provides a new perspective for clarifying the target of action through which MT therapy protects alveolar barrier function.

Introduction

Lung ischaemia-reperfusion injury (LI/RI) is a common and intractable clinical pathological condition, which is frequently observed in scenarios such as lung transplantation, cardiopulmonary bypass surgery in cardiac surgery, and blood flow restoration after severe trauma. It seriously impairs patient prognosis and endangers life safety [1–3]. Despite continuous advances in current medical technology and the development of therapeutic approaches for LI/RI, no breakthrough progress has been achieved. The pathophysiological mechanism of LI/RI is complex, and mitochondrial damage plays a central role in its pathological process [4].During the lung ischaemia phase, tissue cells suffer from hypoxia-induced impairment of mitochondrial electron transport chain function. This leads to blocked electron transfer, with some electrons leaking out and reacting with oxygen to generate reactive oxygen species (ROS) such as superoxide anions [5]. During the reperfusion phase, ROS are produced in a burst, while the activity of mitochondrial antioxidant enzymes decreases significantly, making it difficult to scavenge excessive ROS in a timely manner. This further induces lipid peroxidation, disrupting the structure and function of mitochondrial membranes [6].Meanwhile, intracellular calcium homeostasis is disturbed, and mitochondria take up excessive calcium ions, which activates the mitochondrial permeability transition pore (mPTP). This activation results in the loss of mitochondrial membrane potential, which not only inhibits the activity of respiratory chain complexes but also interferes with the process of oxidative phosphorylation [7]. In addition, the release of inflammatory factors can further affect mitochondrial function through multiple pathways [8].These factors collectively contribute to abnormal mitochondrial morphology, impaired respiratory function, and damage to mitochondrial deoxyribonucleic acid (mtDNA), ultimately triggering cell apoptosis, exacerbated inflammatory responses, and impaired lung function.

Mitochondrial transplantation (MT), as an emerging strategy for treating mitochondrial functional damage and dysfunctional diseases, has attracted considerable attention in recent years [9]. Its core lies in delivering exogenous healthy mitochondria into damaged cells; by supplementing or replacing dysfunctional mitochondria, it restores cellular energy metabolism and normal physiological functions [10].Studies have confirmed that mitochondria isolated from different cell sources can not only enter various cell types in vitro but also be internalized by tissues through local or systemic injection in vivo. Furthermore, intravenously injected mitochondria can preferentially migrate to target cells and tissues with mitochondrial damage [10, 11]. With the continuous accumulation of evidence related to mitochondrial transfer, the strategy of transplanting intact functional mitochondria for treating various mitochondrial diseases has been extensively studied.When MT therapy is conducted using autologous, allogeneic, or xenogeneic mitochondria, the transplanted mitochondria can demonstrate potential therapeutic value in ischemia-reperfusion injury, neurodegenerative diseases, tissue damage, and inflammatory diseases. This is achieved by supplementing ATP, promoting cell proliferation, alleviating inflammatory responses, and inhibiting excessive oxidative damage [12, 13].To our knowledge, research on exogenous MT in LI/RI treatment remains to be further explored. Therefore, this study proposes the hypothesis that MT can alleviate LI/RI by reducing alveolar epithelial cell dysfunction and the disruption of tight junction proteins. We verified this hypothesis using MLE-12 cells (in vitro) and C57BL/6 mice (in vivo) to establish experimental models.

Materials and methods

Isolation, extraction, and identification of mitochondria

Fresh livers from 6-week-old male C57BL/6 mice were selected as the source for mitochondrial extraction. Since the liver is a mitochondrion-rich organ, the extracted mitochondria possess high quantity and purity, which can meet the experimental requirements. The isolation and extraction method of liver-derived mitochondria was as previously described [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, then stored on ice at 4℃and used within 2 h.Calibration of the flow cytometer (BD FACSCanto II) was required prior to detection: ①The forward scatter (FSC)/side scatter (SSC) parameters were adjusted using 4.2 μm and 10 μm standard fluorescent microspheres, and a threshold was set to exclude debris < 0.5 μm and aggregates > 2 μm; (For whole-cell detection, cell populations were gated based on a size range of 10–20 μm, while for mitochondrial detection, only particles within the 0.5–2 μm size range were analyzed.) ②MitoTracker Red fluorescence was used to validate the gating specificity, ensuring that over 90% of the gated particles were mitochondria.Direct counting with a Coulter counter was used for mitochondrial quantification: A Beckman Coulter Z2 particle counter was employed to determine the mitochondrial concentration (unit: particles/mL), with measurements repeated three times and the average value taken. Mitochondrial purity was verified using the mitochondrial-specific protein COX-IV and the cytoplasmic component α-tubulin (α-TUB). 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 Sect. (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.

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 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 h of ischemia, the slipknot was released to initiate reperfusion. Control mice underwent thoracotomy without hilum ligation and received mechanical ventilation for 1 h. 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 h after reperfusion, and lung tissues and other specimens were collected for subsequent analysis.

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. To establish the glucose-oxygen deprivation model, when the cells grew to the logarithmic growth phase, they were washed with PBS and then replaced with glucose-free and serum-free medium. The cells were cultured in a hypoxic incubator (37 °C) containing 5% CO₂, 1% O₂, and 94% N₂ for 2 h. After that, the glucose-free medium was replaced with normal medium, and the cell plates were reoxygenated in a regular incubator (37 °C, 5% CO₂ + 95% air) for 1 h [16]. Corresponding drugs were added during the replacement with normal medium: the NC group and I/RI group were given PBS, the I/RI + Buf group was given blank buffer, and the MT group was given buffer containing mitochondria (100 mitochondria per cell). Cells were collected for subsequent analysis 4 h after drug administration.

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 h 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).

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 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).

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 min according to the manufacturer’s instructions. Samples were immediately analyzed by flow cytometry, and data were processed using FlowJo software.

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 min. 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 h 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).

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 h to obtain the dry weight. The W/D ratio was calculated as follows:

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 ROS. Nuclei were stained with DAPI, and fluorescence intensity in different tissue samples was detected using a fluorescence microscope (Leica, Germany).

Tissue immunohistochemistry

Lung tissue paraffin sections were baked overnight at 37 °C, then incubated with 0.2% Triton X-100 in PBS for 10 min and blocked with 5% bovine serum albumin at room temperature for 1 h. 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.

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).

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.

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’
	Cleaved Caspase-3	5’-CTCGCTCTGGTACGGATGTG-3’	5’-TCCCATAAATGACCCCTTCATCA-3’
	Occludin	5’-CCTTCTGCTTCATCGCTTCCTTA-3’	5’-CGTCGGGTTCACTCCCATTAT-3’
	ZO-1	5’-GATAGTTTGGCAGCAAGAGATGGTA-3’	5’-AGGTCAGGGACGTTCAGTAAGGTAG-3’

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.

Results

Characterisation of isolated mitochondria

To evaluate the purity of the isolated mitochondria, we detected the mitochondrial inner membrane marker protein COX-IV and the cytoplasmic marker protein α-tubulin (α-TUB) via Western blot; the results showed that COX-IV protein was detectable in the isolated mitochondria, while α-TUB was not detected, confirming their high purity (Fig. 1A). Meanwhile, observation under transmission electron microscopy (TEM) revealed that the isolated mitochondria had intact morphology, with well-preserved mitochondrial membranes and cristae structures, and no obvious impurity contamination, further verifying their purity (Fig. 1B). Furthermore, the ATP production capacity assay indicated that after adding different concentrations of ADP to the isolated mitochondria, the ATP yield increased with the elevation of ADP concentration, demonstrating that the mitochondria possessed normal energy synthesis function (Fig. 1C). JC-1 staining flow cytometry analysis showed that the freshly isolated mitochondria maintained a normal membrane potential level, whereas the membrane potential decreased significantly after CCCP treatment (Fig. 1D). In addition, confocal microscopy observation and co-localization analysis of double staining with MitoTracker Green FM (which labels total mitochondria) and MitoTracker Red CMXRos (which labels active mitochondria) revealed that the isolated mitochondria had high purity, good activity, and normal function (Figs. 1E, F). Collectively, the mitochondria isolated and extracted in this study exhibited high purity, strong activity, and intact function, which meet the experimental requirements for MT therapy.

Fig. 1.

Characterization of isolated mitochondria from liver tissues. (A) Western blot analysis of COX-IV and α-tubulin (α-TUB) for the purity of the extracted mitochondria. (B) Morphology and purity of isolated mitochondria observed by electron microscopy (scale bar: 500 nm) (Carl Zeiss AG, Germany).(C) Assessment of ATP content in post-isolation mitochondria in the presence of different concentrations of ADP (n = 3) (data are presented as mean ± SD). (D) Flow cytometric analysis of JC-1-stained mitochondria showing that the isolated mitochondria had normal membrane potential.(E) Characterization of isolated mitochondria observed by confocal microscopy after labeling with MitoTracker Green FM (200 nM) and MitoTracker Red CMXRos (150 nM) (scale bar: 20 μm). (F) Co-localization analysis of MitoTracker Green FM (200 nM) and MitoTracker Red CMXRos (150 nM) staining

Uptake of isolated mitochondria into MLE-12 cells

To investigate whether exogenous healthy mitochondria can enter damaged MLE-12 cells and exert potential protective effects, this study first verified the feasibility of transplanted mitochondria entering these cells: after isolating mitochondria from the liver tissue of 6-week-old healthy male C57BL/6 mice, we stained them with MitoTracker Red CMXRos, while pre-staining the endogenous mitochondria of MLE-12 cells with MitoTracker Green FM; we then co-incubated the isolated mitochondria with MLE-12 cells subjected to I/RI modeling for 2 h, and observed the cellular uptake of mitochondria via confocal live-cell imaging. Confocal images showed that fluorescently labeled exogenous mitochondria could be successfully internalized into damaged MLE-12 cells after I/RI modeling (Fig. 2A). We observed that the rod-shaped and filamentous mitochondria in cells gradually shortened and became rounded after I/RI modeling, undergoing significant morphological changes (Fig. 2B). Subsequently, we compared the mitochondrial uptake between normal MLE-12 cells and MLE-12 cells after I/RI modeling, and found that MLE-12 cells damaged by I/RI took up significantly more mitochondria (Fig. 2C).

Fig. 2.

Labeling of exogenous mitochondrial transfer into MLE-12 cells. (A) Exogenous mitochondria were labeled with MitoTracker Red CMXRos (red), and endogenous mitochondria in MLE-12 cells were labeled with MitoTracker Green FM (green). (B) Mitochondria in normal MLE-12 cells and damaged mitochondria in MLE-12 cells after I/RI modeling. (C) Comparison of mitochondrial uptake amount between MLE-12 cells after I/RI modeling and normal MLE-12 cells

Mitochondrial transplantation reduces apoptosis and necrosis in MLE-12 Cells, alleviates inflammatory responses and oxidative stress

Inflammatory response and oxidative stress are not only the core mechanisms of cell damage after I/RI, but also the key pathological processes driving cell apoptosis and necrosis. To clarify the effect of MT therapy on alveolar epithelial MLE-12 cells after I/RI injury, we performed relevant cell assays 4 h after treatment administration. The results showed that the protein and mRNA expression levels of the pro-apoptotic protein BAX and the executioner apoptotic protein Cleaved Caspase-3 were significantly increased in MLE-12 cells of the I/RI group, whereas the expression levels of these apoptosis-related molecules were significantly decreased in the MT treatment group (p < 0.05) (Fig. 3A). Further verification by flow cytometry confirmed that MT therapy significantly reduced cell apoptosis and necrosis (Figs. 3B, C). EdU proliferation fluorescence assay indicated that MT therapy restored cell proliferation ability to a certain extent; this observation is consistent with the findings of Wang et al., who reported that mitochondrial transplantation into bone marrow stromal cells promotes angiogenesis during bone repair and enhances endothelial cell growth [17].In addition, MT therapy significantly inhibited the expression of inflammatory factors (e.g., TNF-α, IL-1β, IL-6) induced by I/RI (p < 0.05) (Fig. 3D). Further studies revealed that its protective effect is associated with alleviating oxidative stress and restoring mitochondrial function: ROS and MitoSOX staining results showed that MT therapy significantly reduced intracellular ROS levels, while JC-1 staining results demonstrated that MT therapy promoted the recovery of mitochondrial membrane potential (Figs. 3E, F). Collectively, exogenous MT therapy can significantly alleviate I/RI-induced apoptosis and necrosis of alveolar epithelial MLE-12 cells by inhibiting inflammatory responses, reducing oxidative stress, and restoring mitochondrial function.

Fig. 3.

MT treatment inhibits I/RI-induced apoptosis and necrosis in MLE-12 cells and alleviates inflammatory responses and oxidative stress levels. (A) Expression levels of Cleaved 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 < 0.05, **p < 0.01, ***p < 0.001,****p < 0.0001)

In vivo uptake and localization of exogenous mitochondrial transplantation in mice

To investigate the biodistribution characteristics of exogenous mitochondria in mice, this study conducted in vivo localization experiments using fluorescence labeling technology: in single-group animal experiments, fluorescence microscopy was used to dynamically track the transplanted exogenous mitochondria in key organs and tissues of LI/RI mice. First, the extracted mitochondria were fluorescently labeled with 150 nM MitoTracker Red CMXRos, then injected into normal mice and model group mice via the jugular vein. At 1 h, 4 h, 8 h, and 24 h after injection, tissues including the lung, heart, liver, kidney, and spleen were collected respectively to prepare frozen sections for analysis (Fig. 4A).The results showed that 1 h after administration, the transplanted mitochondria could be detected in the lung, heart, liver, kidney, and spleen of both normal mice and model group mice (Fig. 4B), with the highest mitochondrial content observed in the lung and heart. Meanwhile, the transplanted mitochondria persisted in lung tissue within 24 h, but the tissue fluorescence signal gradually weakened over time. In addition, a comparison of the uptake of exogenous mitochondria by lung tissue between normal control group mice and LI/RI model mice revealed that although normal lung tissue could take up exogenous mitochondria, its uptake amount was significantly lower than that of lung tissue after LI/RI (Fig. 4C).

Fig. 4.

In vivo study of exogenous mitochondrial uptake. (A) Schematic diagram of in vivo mitochondrial transplantation in mice; (B) Distribution of transplanted mitochondria in the heart, liver, kidney, and spleen of normal mice and model group mice 1 h after administration (Scale bar: 50 μm); (C) Evaluation of mitochondrial uptake in lung tissue cells at 1, 4, 8, and 24 h; exogenous mitochondria were stained with MitoTracker Red (red), cell nuclei were stained with DAPI (blue), and arrows indicate exogenous red mitochondria (Scale bar: 50 μm)

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MT treatment alleviates pathological damage in lung tissues of mice caused by I/RI

Gross observation of lung tissue specimens showed that the degree of pulmonary edema in mice was significantly reduced after MT therapy (Fig. 5A). Lung injury score results revealed that the score of the I/RI group was significantly increased, indicating more severe lung injury, while the score of the MT treatment group was significantly lower than that of the I/RI group (p < 0.01) (Fig. 5B). This conclusion was further verified by the lung tissue wet/dry weight ratio results: the I/RI group exhibited a higher wet/dry weight ratio due to pulmonary edema, whereas the MT treatment group showed a significant decrease in the wet/dry weight ratio (p < 0.001) (Fig. 5C).Observation of HE-stained pathological sections of lung tissue showed obvious pathological changes in the I/RI group: massive inflammatory cell infiltration and accumulation in various parts of lung tissue were observed under the microscope, along with significant edema in alveoli and interstitium (Fig. 5D). In contrast, MT therapy alleviated I/RI-induced lung injury to a certain extent at the histopathological level, suggesting that it could effectively relieve lung tissue edema. Collectively, these results indicate that MT therapy can improve alveolar barrier function and reduce the degree of lung injury.

Fig. 5.

MT treatment alleviates IRI-induced lung injury in mice. (A) Gross specimens of lung tissue; (B) Quantification of stained areas via lung injury score, a scoring system used to comprehensively assess the degree of lung tissue injury; (C) Mouse lung wet/dry weight ratio (p < 0.01); (D) HE staining of lung tissue. n = 3 ~ 5 per group, with experiments repeated 3 times (*p < 0.05, **p < 0.01, ***p < 0.001,****p < 0.0001)

Mitochondrial transplantation inhibits pulmonary cell apoptosis and necrosis and alleviates inflammatory responses in mice

To evaluate the effect of exogenous mitochondrial therapy on the levels of apoptosis and necrosis in lung tissue cells, we performed quantitative analysis using Western blotting and qPCR techniques. The results showed that MT therapy significantly decreased the expression levels of the pro-apoptotic protein BAX and the executioner apoptotic protein Cleaved Caspase-3, while the expression level of the anti-apoptotic protein BCL-2 was restored (p < 0.01) (Fig. 6A). In addition, the results of lung tissue immunohistochemistry and TUNEL staining further confirmed that MT therapy significantly reduced the number of apoptotic cells in lung tissue (Figs. 6B, C), which was consistent with the aforementioned molecular-level detection results.In terms of regulating tissue inflammatory responses, MT therapy significantly decreased the expression levels of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 in lung tissue (p < 0.05) (Fig. 6D), suggesting that it can not only inhibit I/RI-induced apoptosis and necrosis of lung epithelial cells but also significantly alleviate inflammatory responses. Notably, the results of in vivo animal experiments were highly consistent with the previous in vitro cellular experiment results, further verifying that MT exerts significant anti-apoptotic and anti-inflammatory effects both in vitro and in vivo.To clarify the regulatory effect of MT therapy on oxidative stress, DHE fluorescent staining was used to detect the ROS level in lung tissue. The results showed that MT therapy significantly reduced the ROS level (Fig. 6E), indicating that it can improve the redox balance of lung tissue after I/RI by alleviating oxidative stress. Collectively, exogenous MT therapy significantly ameliorates I/RI-induced lung tissue injury by inhibiting cell apoptosis, reducing inflammatory responses, and alleviating oxidative stress.

Fig. 6.

Effects of MT treatment on lung tissue cells: (A) MT treatment inhibited the apoptosis of lung tissue cells in mice and alleviated the pulmonary inflammatory response to a certain extent. (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 < 0.05, **p < 0.01, ***p < 0.001,****p < 0.0001)

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 proteins that maintain alveolar barrier permeability and play a central role in regulating lung barrier function, whereas the core pathophysiological mechanism of LI/RI involves damage to alveolar epithelial and endothelial cells, which in turn leads to altered vascular permeability, lung tissue edema, and impairment of alveolar barrier function. Previous studies have confirmed that exogenous mitochondrial therapy can significantly alleviate inflammatory responses, reduce oxidative stress levels, and inhibit cell apoptosis and necrosis in MLE-12 cells and lung tissue in both in vitro I/RI models and in vivo experiments, suggesting that MT may exert a protective effect on the damaged alveolar barrier.To verify this hypothesis, this study established in vitro and in vivo models for in-depth investigation: Results from Western blotting and qPCR assays showed that the expression levels of ZO-1 and Occludin were significantly downregulated in MLE-12 cells and mouse lung tissue of the I/RI group, whereas the expression levels of these two proteins were significantly restored in the MT treatment group (Figs. 7A, B). Immunofluorescence staining of MLE-12 cells revealed that the tight junctions of alveolar epithelial cells in the MT group showed a smooth linear distribution with a more intact structure (Fig. 7C). Immunohistochemistry (IHC) results of lung tissue sections confirmed that in the I/RI group, the expression of ZO-1 and Occludin was significantly reduced, with irregular and discontinuous staining; in the MT group, the distribution of these two proteins along the alveolar epithelium was significantly restored, and the continuity of staining was significantly enhanced (Fig. 7D).Observation under transmission electron microscopy (TEM) showed that in the I/RI group, epithelial cells were swollen with irregular thickening, the tight junctions between cells were widened, and pathological changes such as mitochondrial vacuolization, cristae dissolution, and lamellar body vacuolation were observed. In the MT group, epithelial cells showed a regular and flat morphology, the number of intracellular vacuoles was reduced, and the lamellar body emptying phenomenon was also significantly improved (Fig. 7E). Collectively, MT therapy can effectively alleviate LI/RI-induced impairment of alveolar barrier function and improve the ultrastructure of lung epithelium by upregulating the expression of ZO-1 and Occludin.

Fig. 7.

Effects 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 in the I/RI group, epithelial cells exhibited swelling and irregular thickening, with widened tight junctions between epithelial cells (as indicated by the arrow), along with mitochondrial vacuolization, cristae dissolution, and lamellar body depletion. MT treatment improved the ultrastructure of lung epithelial cells, characterized by regular flattened epithelial cells, fewer vesicles, and reduced swellin.Experiments were repeated three times (*p < 0.05, **p < 0.01, ***p < 0.001,****p < 0.0001)

Discussion

Recently, MT has attracted considerable attention in the field of treating mitochondrial damage and dysfunction, and its exogenous protective effects have been extensively studied in various ischaemia-reperfusion injury (I/RI) models, including those of heart disease, stroke, kidney disease, lung disease, and Parkinson’s disease [18–21].Guariento et al. [22] confirmed in a porcine myocardial infarction model that intracoronary infusion of autologous mitochondria significantly alleviated ischaemia-reperfusion injury, improved cardiac function, and reduced infarct size. Similarly, another study showed that transferring mitochondria derived from healthy muscle cells into damaged renal cells significantly reduced the apoptosis rate by regulating cell replication and repair processes and the expression of apoptosis-related proteins [23]. In the field of LI/RI, Moskowitzova et al. [24] found that MT could serve as a potential therapeutic approach for acute lung injury in I/RI mice; both pulmonary artery injection and airway nebulization (two routes of administration) significantly improved pulmonary mechanical parameters, promoted lung function recovery, and alleviated tissue damage. Beyond acute lung injury caused by warm ischaemia, Cloer et al. [25] also conducted a study in a porcine ex vivo lung perfusion model, investigating whether exogenous MT (administered via intra-arterial route) could improve the functional and molecular outcomes of donor lungs in large animal models and humans. The results showed that after ischaemia-reperfusion, MT promoted cell survival and improved lung function during ex vivo lung perfusion.Our study further verified the therapeutic potential of MT for LI/RI through in vitro and in vivo experiments: MT therapy could significantly reduce the level of oxidative stress in lung tissue cells, inhibit the excessive release of inflammatory factors, reduce programmed cell death by regulating apoptosis-related pathways, and simultaneously reduce the disruption of tight junctions in alveolar epithelial cells and alleviate alveolar barrier function injury, ultimately mitigating lung injury caused by I/RI.

Studies have shown that mitochondria can be transferred from donor cells to recipient cells via gap junctions [26], tunneling nanotubes [27], or extracellular vesicles [28]; free mitochondria can also be incorporated into host cells from the extracellular space. These findings provide a theoretical basis for exogenous MT therapy. In the in vitro cell model, we confirmed through cell fluorescence labeling analysis that exogenous mitochondria can enter MLE-12 cells and exert therapeutic effects; however, the specific pathway and molecular mechanism by which exogenous mitochondria enter these cells still need further verification.In in vivo experiments, we administered mitochondria via jugular vein injection for treatment and found that the transplanted mitochondria could be detected in tissues and organs within 1 h after injection, and fluorescent exogenous mitochondria could still be continuously observed in lung tissue within 24 h. The retention of exogenously transplanted mitochondria may achieve acute therapeutic effects by improving cellular energy metabolism and inhibiting early inflammatory responses; nevertheless, their long-term persistence and sustained effects require verification by extending observation time points. These results all confirm the feasibility of MT for treating LI/RI.A study by Bechet et al. [29] achieved targeted mitochondrial transplantation by delivering mitochondria via the pulmonary artery to treat ischaemia-reperfusion injury during ex vivo lung perfusion. However, to better align with clinical practice, our study did not adopt the pulmonary artery-targeted delivery method. The results showed that exogenously transplanted mitochondria administered via intravenous injection were widely distributed in the body, which may affect the therapeutic effect on the targeted organ. Moreover, the extensive uptake of mitochondria by various organs in the body may lead to a decrease in the therapeutic concentration of mitochondria in the target organ, thereby influencing the efficacy of MT.Therefore, in future studies, we will further compare the therapeutic effects of pulmonary artery-targeted transplantation; meanwhile, exploring other methods to achieve targeted MT therapy will also be the focus of our next work. Subsequently, we will also investigate the combination of nanocarrier modification to achieve targeted delivery of intravenous MT, aiming to solve the problem of uneven distribution of mitochondria in vivo organs.

LI/RI triggers inflammatory responses, with massive infiltration of neutrophils, macrophages, and other inflammatory cells into lung tissue [30], followed by the release of inflammatory factors such as TNF-α, IL-1β, and IL-6 [31, 32]; these factors can affect mitochondrial function through multiple pathways [30–32]. Our in vitro and in vivo studies showed that exogenous MT can reduce the expression of these inflammatory factors in LI/RI, thereby alleviating lung tissue injury.Furthermore, oxidative stress is another crucial factor in ischaemia-reperfusion-induced tissue damage: during the ischaemic phase, cellular hypoxia impairs the mitochondrial electron transport chain, leading to electron leakage and the generation of superoxide anions; during reperfusion, a large amount of O₂ influx causes explosive production of ROS [33]. ROS disrupt the structure and function of mitochondrial membranes, reducing membrane fluidity, increasing permeability, impairing substance transport and mitochondrial membrane potential maintenance, and ultimately damaging cells [34]. Studies have demonstrated that MT can alleviate oxidative stress by reducing ROS production [35, 36]: after healthy mitochondria enter damaged cells, they reduce the total amount of ROS, balance antioxidants and ROS production, decrease membrane phospholipid damage and lipid peroxidation, and thereby mitigate cell injury [37]. Consistent with these findings, our study also showed decreased levels of ROS and MitoSOX in damaged MLE-12 cells and lung tissue after MT, indicating alleviation of LI/RI-induced oxidative stress.In addition, other studies have found that healthy mitochondria derived from the liver can replace or fuse with endogenous damaged mitochondria after entering impaired cells, preventing further cell damage [35–38]. Our study observed a similar phenomenon: fluorescence labeling experiments confirmed that exogenous mitochondria could successfully enter alveolar epithelial cells, and JC-1 staining also showed a significant increase in mitochondrial membrane potential after MT. This may be related to the functional replacement of damaged mitochondria by exogenous mitochondria after entering cells. However, the functional effects of MT may not be limited to intracellular actions, and its specific sites of action still need further verification through mitochondrial endocytosis blocking experiments.

Epithelial barrier dysfunction and high alveolar capillary permeability to proteins and fluids are hallmarks of lung injury [39]. Alveolar capillary permeability is determined by intercellular junctions, including tight junctions, gap junctions, and adherens junctions [40]. 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 [41]. Current studies indicate that reduced Occludin and ZO-1 expression in lung tissues contributes to excessive epithelial barrier permeability and pulmonary edema [39–41]. 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, 38]. 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.

This study confirmed that MT downregulated the expression of pro-inflammatory cytokines such as TNF-α, IL-1β, and IL-6 in the LI/RI model (Figs. 3D and 6D). Besides repairing tight junctions to block the “barrier disruption-inflammatory positive feedback loop”, combined with the current experimental results and literature, there are other anti-inflammatory pathways: First, MT significantly reduced ROS levels in MLE-12 cells and lung tissue (Figs. 3E and 6E), while restoring mitochondrial membrane potential (Fig. 3F). This not only directly scavenges ROS by supplementing antioxidant enzymes such as SOD, but also reduces endogenous mitochondrial electron leakage to inhibit ROS production, thereby blocking the activation of the TLR4-MyD88-NF-κB inflammatory pathway [6].Second, MT enhanced mitochondrial ATP production capacity. The increase in local ATP content can neutralize excessive ROS to reduce inflammatory responses, and can inhibit AMPK-NF-κB-mediated inflammatory responses by restoring cellular energy homeostasis [34, 42]. This is consistent with the anti-inflammatory mechanism of MT observed by Guariento et al. [22] in a myocardial I/RI model.Regarding the role of immune cells, although our experiment did not directly detect immune cell phenotypes, HE staining of lung tissue showed that MT significantly reduced neutrophil infiltration (Fig. 5D). Combined with the report by Moskowitzova et al. [24] that “MT can reduce the secretion of pro-inflammatory cytokines by macrophages in lung I/RI”, it is speculated that MT may induce the polarization of macrophages to the M2 anti-inflammatory phenotype by releasing mitochondria-derived substances (e.g., humanin) [36, 43], thereby participating in anti-inflammation indirectly. However, this mechanism still needs to be further verified by subsequent experiments.

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.

Conclusion

Our study confirmed the application potential of MT in treating LI/RI through systematic in vitro and in vivo experiments. The results showed that MT therapy can significantly reduce the oxidative stress level in alveolar epithelial cells, inhibit the excessive release of inflammatory factors, and reduce programmed cell death by regulating the apoptotic pathway. Meanwhile, it can protect the structural integrity of tight junction proteins (e.g., ZO-1 and Occludin), maintain the function of the alveolar epithelial barrier, and thereby alleviate LI/RI.These findings provide an important theoretical basis for the clinical application of MT in treating LI/RI. In future studies, we will further optimize the treatment regimen, deeply explore the strategy of mitochondrial-targeted lung transplantation therapy, investigate its clinical transformation prospects, and provide a new direction for the prevention and treatment of LI/RI.

Abbreviations

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

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.

Data availability

Data will be made available on request.

Declarations

Ethical statement

All animal experimental procedures and steps were approved by the Laboratory Animal Ethics Committee of Anhui Medical University (LLSC20241781).

Conflict of interest

The authors declare that they have no known financial interests or personal relationships that might influence the work reported in this paper.