Investigation of the Protective Effects of Mesenchymal Stem Cell-Derived Exosomes on Hyperoxia-Induced Type II Alveolar Epithelial Cell Injury Based on Ferroptosis and Autophagy

Investigation of the Protective Effects of Mesenchymal Stem Cell-Derived Exosomes on Hyperoxia-Induced Type II Alveolar Epithelial Cell Injury Based on Ferroptosis and Autophagy | 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 Investigation of the Protective Effects of Mesenchymal Stem Cell-Derived Exosomes on Hyperoxia-Induced Type II Alveolar Epithelial Cell Injury Based on Ferroptosis and Autophagy Guoyue Liu, Guiyang Jia, yingcong Ren, qianxia Huang, Cunzhi Yin, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4538714/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective To investigate the protective effects of mesenchymal stem cell-derived exosomes on ferroptosis and autophagy in hyperoxia-induced type II alveolar epithelial cell injury. Methods Cells were treated with exosomes under hyperoxic conditions and divided into the following groups: control group (oxygen volume fraction of 0.21), hyperoxia group (oxygen volume fraction of 0.95), hyperoxia+exosome group, hyperoxia+exosome+Fer-1 (10 μmol/L) ferroptosis inhibitor group, and hyperoxia+exosome+3-MA (25 μM) autophagy inhibitor group. High-throughput analysis was performed to analyze the transcriptomic changes in type II alveolar epithelial cells treated with exosomes under hyperoxic exposure. GO analysis and KEGG enrichment analysis were conducted to investigate the regulatory effects of differentially expressed genes in cells. Quantitative PCR was used to verify the high-throughput sequencing results. Cell proliferation was detected by EdU assay. ROS levels were measured by DCFH-DA probe. The expression of ferroptosis factors (GPX4, SLC7A11) and autophagy-related factors (Wnt1, β-catenin, p62, ATG5, Beclin1) was detected by Western blotting. LC3B staining in cells was examined by immunofluorescence. Results Sequencing results showed that exosome treatment caused significant transcriptomic changes in cells compared to the hyperoxia group. Quantitative PCR results confirmed the expression changes of genes such as HSPA1A and NR4A1, consistent with the sequencing results. EdU assay showed that the hyperoxia group significantly decreased EdU positivity compared to the control group, which was alleviated by exosome treatment. Compared to the control group, the hyperoxia group promoted ROS accumulation, while exosome treatment alleviated ROS accumulation. Western blotting results showed that, compared to the control group, the hyperoxia group significantly decreased GPX4 and SLC7A11 expression, while exosome treatment significantly increased GPX4 and SLC7A11 expression. In the hyperoxia+exosome+ferroptosis inhibitor group, GPX4 and SLC7A11 expression were significantly decreased. Immunofluorescence results showed that hyperoxia significantly increased LC3B positivity, while exosome treatment significantly decreased LC3B positivity. In the hyperoxia+exosome+3-MA autophagy inhibitor group, LC3B positivity was significantly increased. Western blotting results showed that the hyperoxia group significantly decreased the expression of Wnt1, β-catenin, and p62, and significantly increased the expression of ATG5 and Beclin1, while the exosome group significantly increased the expression of Wnt1, β-catenin, and p62, and significantly decreased the expression of ATG5 and Beclin1. In the hyperoxia+exosome+3-MA autophagy inhibitor group, the expression of Wnt1, β-catenin, and p62 was significantly decreased, and the expression of ATG5 and Beclin1 was significantly increased. Conclusion Mesenchymal stem cell-derived exosomes alleviate hyperoxia-induced damage to alveolar epithelial cells by inducing cell proliferation, alleviating ROS accumulation, inhibiting ferroptosis, and inhibiting autophagy. Mesenchymal stem cell-derived exosomes Type II alveolar epithelial cells Hyperoxia Oxidative stress Ferroptosis Autophagy Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Oxygen therapy is widely used in the treatment of patients with respiratory failure and hypoxemia. It plays a crucial role in saving lives, preventing multi-organ failure caused by hypoxia, and buying time for emergency treatment. However, hyperoxia can also trigger oxygen toxicity, including acute lung injury (ALI) [1] . Prolonged exposure to hyperoxia can cause severe lung damage in premature infants, leading to ALI and bronchopulmonary dysplasia (BPD) [2] , and exacerbate acute respiratory distress syndrome (ARDS) in adults, which is one of the causes of death. Studies have shown that long-term hyperoxic exposure can lead to the destruction of the alveolar-capillary barrier and structural changes in the alveoli, as well as a large amount of apoptosis in alveolar epithelial cells[3, 4]. Therefore, elucidating the pathogenesis of this disease and implementing appropriate lung protective measures during oxygen therapy is crucial. Research has shown that type II alveolar epithelial cells (AEC II) are the stem cells of the lung tissue and can differentiate into other lung tissue cells, but hyperoxia has been proven to promote apoptosis [5] and excessive differentiation of AEC II cells into AEC I[6]. After AEC II cell injury and apoptosis, the self-repair capacity is limited, which can easily lead to damage to the alveolar structure and pulmonary fibrosis [7] . Therefore, exogenous cell supplementation plays an important role in the repair of damaged alveolar epithelial cells. Mesenchymal stem cells (MSCs) are a group of adult stem cells derived from tissues such as bone marrow, adipose tissue, and umbilical cord, possessing self-renewal, multi-lineage differentiation, and tissue repair-promoting capabilities [8] . In recent years, there have been increasing reports on the therapeutic effects of MSCs in lung injury diseases, showing antimicrobial, anti-inflammatory, tissue regeneration-promoting, anti-fibrotic, antioxidant stress, and anti-apoptotic properties, which have opened up new avenues for the treatment of lung injury [9] . Although MSCs have certain advantages in terms of immunogenicity, in vitro expansion capability, genetic characteristics, and acquisition methods, they also have defects such as potential tumorigenicity, embolism, and teratogenicity after transplantation, and there are still many controversies in clinical treatment [10] . In recent years, research has found that MSCs exert their therapeutic effects primarily through paracrine secretion of exosomes, which have similar biological functions as the parent cells, such as anti-inflammatory, tissue regeneration-promoting, anti-apoptotic, and anti-fibrotic effects. However, compared to MSCs, exosomes have a smaller size, lower risk of embolism, and lower immunogenicity. Their specific surface receptors allow them to deliver drugs or specific components to target cells. Due to these advantages, exosomes have promising applications in lung injury and are currently a research hotspot in the stem cell field. MSC-derived exosomes have been shown to have protective effects similar to those of MSCs themselves in cardiovascular diseases, osteoarthritis, wound healing, various acute kidney injury animal models, and rat models of stroke [11] In recent studies, ferroptosis and autophagy have been implicated in ALI. The present study aims to investigate the protective effects of MSC-derived exosomes in a hyperoxia-induced alveolar epithelial cell injury model by evaluating their biological effects on cell proliferation, oxidative stress response, ferroptosis, and autophagy, and exploring the underlying mechanisms. This study may provide new insights into the treatment of alveolar epithelial cell injury. Experimental materials and methods 1.1 Experimental materials Lysate, SDS-PAGE gel configuration kit, 5× protein uploading buffer were purchased from Beyotime Biotech. Inc. Trypsin, collagenase, deoxyribonuclease I, fetal bovine serum, DMEM were purchased from Gibco (USA). CD9, CD81, Nrf2, GPX4, SLC7A11, Wnt1, β-catenin, p62, ATG5, Beclin1 were purchased from Abcam (UK). Trizol and M-MLV reverse transcriptase were purchased from Promega (USA). 3-MA was purchased from Selleck (USA). The ferroptosis inhibitor ferrostatin-1 (Fer-1) was purchased from MedChe- mExpress. 4% paraformaldehyde was purchased from Beijing Ruier Xinde Technology Co., Ltd. The fluorescent probe 2,7-dichloro dihydro fluorescein diacetate (DCFH-DA) was purchased from Sigma-Aldrich. 1.2 Extraction and Identification of Exosomes MSCs were isolated using the tissue cell adherence method, cultured and expanded to the third generation, and exosomes from the MSCs supernatant were obtained by ultracentrifugation. In most studies, transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blotting are often considered the preferred methods for identifying exosomes. In this study, the extracted MSC-derived exosomes were identified using TEM, NTA, and Western blotting. Transmission Electron Microscopy: 10 μL of the extracted exosomes were diluted with an equal volume of phosphate-buffered saline (PBS), dropped onto a 2 mm copper grid, and left at room temperature for 1 min. Excess liquid was gently removed with filter paper, and the grid was stained with 3% uranyl acetate solution for 5 min at room temperature. After gentle rinsing with double-distilled water, the grid was air-dried at room temperature and observed under a transmission electron microscope, and images were captured. Nanoparticle Tracking Analysis: 10 μL of exosomes were diluted to 30 μL. First, the instrument performance was tested using a standard sample. After passing the test, the exosome sample was loaded, and the particle size information was obtained upon completion of the analysis. Western Blotting: Western blotting was used to detect exosomal surface markers. 1.3 Uptake Experiment for MSC-derived Exosomes The extracted MSC-derived exosomes were first mixed with diluent C, and then PKH-67 dye solution was quickly added and mixed with diluent C, followed by light-protected incubation for 15 min. The mixture was then placed on ice, and 0.5% BSA was added to terminate the reaction. After centrifugation and extraction according to the instructions, PKH-67-labeled MSC-derived exosomes were obtained. ACEII were digested, resuspended, and counted. 100 μL of the cell suspension was mixed with an equal volume of PKH-67-labeled exosome suspension and added to the bottom wells of a confocal culture dish. The total cell number was approximately 1×10 5 . The culture dish was transferred to an incubator and cultured for 1 h, after which 2 mL of complete culture medium was added using a pipette. After 12 h of culture, the cells were prepared for imaging. The culture medium was removed, and the cells were washed three times with PBS, fixed with 4% paraformaldehyde (2 mL) at room temperature for 10 min, and washed three times with PBS. The cells were then stained with DAPI (10 min) to label the cell nuclei. After removing the DAPI, the confocal culture dish was placed on a horizontal shaker and washed 3 times with PBS for 5 min each. The PBS was removed, and the dish was allowed to dry slightly before carefully removing the glass bottom and mounting it onto a slide. The cells were observed and imaged under a laser confocal microscope. 1.4 RNA-seq High-throughput Sequencing Cells were treated with MSC-derived exosomes under hyperoxic conditions, with the cells divided into hyperoxia and hyperoxia + MSC-derived exosomes groups. After modeling, the cells were collected, and high-throughput sequencing was performed to identify significantly upregulated or downregulated genes compared to the hyperoxia group. GO analysis and KEGG pathway analysis of the differentially expressed genes were conducted to determine the relevant genes, and quantitative PCR was used to validate the high-throughput sequencing results. 1.5 Real-time PCR Detection of Relative mRNA Expression of Differentially Expressed Genes Total RNA was extracted from cells using the Trizol one-step method. Cells from different treatment groups were collected, and trichloromethane was added. After reverting for 15 s and standing at room temperature for 2 min, the mixture was centrifuged. The supernatant was transferred to a new PE tube, and isopropanol was added. After standing for 10 min, the mixture was centrifuged. The supernatant was discarded, and pre-cooled 75% ethanol was added for washing. After drying for 20 min, RNase-free water was added to fully dissolve the precipitate. The concentration and purity were determined. The cDNA was synthesized by reverse transcription of RT-PCR. The reaction system was then prepared according to the real-time PCR kit instructions and detected on a real-time fluorescence quantitative PCR instrument. The ΔΔCt values and relative quantities (RQ, where RQ = 2-ΔΔCt) were calculated using the internal reference as a control to determine the relative expression levels of the target genes. 1.6 EdU Detection of Cell Proliferation Changes in Different Groups Cells induced by hyperoxia were treated with MSC-derived exosomes and the cells were divided into control, hyperoxia, and hyperoxia + MSC-derived exosomes groups. The complete culture medium was discarded, and a medium containing 50 μmol/L EdU was added for co-incubation for 2 h. Cells were fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.5% Triton X-100 for 10 min, and washed with PBS. DAPI staining solution was added, and cells were incubated in the dark for 10 min at room temperature. After washing with PBS, the cells were mounted with glycerol. An inverted fluorescence microscope was used to observe the stained cells, and 5 random fields were selected to calculate the EdU-positive cell rate. Blue fluorescence represents DAPI-stained cell nuclei (all cells), while red fluorescence represents EdU-labeled proliferating cells. The cell proliferation rate was calculated as (number of red fluorescent cells / number of blue fluorescent cells) × 100%. 1.7 Western Blotting Detection of Exosomal Marker Proteins (CD9, CD81), Ferroptosis-related Factors (GPX4, SLC7A11), and Autophagy-related Factors (Wnt1, β-catenin, p62, ATG5, Beclin1) After treatment, cells were washed 3 times with pre-cooled PBS and lysed with RIPA cell lysis buffer for 30 min. The mixture was centrifuged at 12,000 rpm for 10 min at 4°C. After BCA protein quantification, protein loading buffer was added in proportion, and the proteins were separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane. The membrane was blocked with 5% non-fat milk for 1 h at room temperature and incubated with primary antibodies against CD9, CD81, GPX4, SLC7A11, Wnt1, β-catenin, p62, ATG5, Beclin1, and GAPDH overnight at 4°C. The next day, the membrane was washed 3 times and incubated with the corresponding rabbit anti-secondary antibody for 1 h at room temperature. ECL reagent was used for exposure and development in an automatic gel imaging system. 1.8 Determination of ROS Levels The DCFH-DA probe (10 μmol/L) method was used to determine cellular ROS levels. ACEII cells were seeded in 6-well plates at a density of 1×10 5 cells per well. After treatment, cells were incubated with the probe molecule (10 μmol/L) at 37°C for 30 min, stained with nuclear staining solution B for 2 min, and washed three times with PBS. A fluorescence microscope was used to collect fluorescence images of the cells, and Image J software was used to analyze all the images. 1.9 Immunofluorescence Detection of LC3B Puncta in Cells Cells from different groups were seeded onto slides, fixed with 4% paraformaldehyde for 15 min, washed with PBS, and permeabilized with 0.3% Triton X-100 for 20 min. Cells were blocked with 5% BSA for 1 h at room temperature and incubated with anti-LC3B primary antibody (1:1000) overnight at 4°C. After washing, cells were incubated with fluorescent-labeled secondary antibody (1:1000) for 1 h at room temperature. Cells were stained with DAPI and observed under a laser scanning confocal microscope to scan images of LC3B puncta. Image J software was used to analyze the optical density values of the images and calculate the fluorescence intensity. Fluorescence intensity = optical density value / fluorescence area. 2.0 Data Processing All data were processed using SPSS 29.0 statistical software. Continuous data are presented as "mean ± standard deviation". For multiple group comparisons, one-way ANOVA analysis of variance was used. For pairwise comparisons between groups, the LSD test was used for equal variances, and the Dunnett's test was used for unequal variances. P < 0.05 was considered statistically significant. Images were processed using Image J 8.0. Experimental Results 2.1 Morphological and Phenotypic Identification of MSC-derived exosomes Transmission electron microscopy results showed that the exosomes had a cup-shaped morphology with relatively uniform diameters, appearing round or oval, with a membrane-bound vesicular structure containing dense cloud-like substances, which are the contents of exosomes. Nanoparticle tracking analysis revealed that the exosome diameters ranged from 80 to 100 nm (Figure 1A-B). Western blotting results showed the expression of the exosomal marker proteins CD9 and CD81 in the extracted exosomes (Figure 1C). 2.2 ACEII Cells Can Uptake MSC-derived exosomes To investigate whether ACEII cells can uptake MSC-derived exosomes, this study used PKH-67 to label MSC-derived exosomes. PKH-67 is a green fluorescent biomembrane tracking dye widely used in various in vivo or in vitro experiments. After co-culturing the labeled MSC-derived exosomes with ACEII cells, we observed distinct green fluorescence surrounding the nuclei of most cells (Figure 2). 2.3 MSC-derived Exosomes Induce Transcriptional Changes in Hyperoxia-induced ACEII High-throughput sequencing results showed that, compared to the hyperoxia group, 1,254 genes were differentially expressed in the MSC-derived exosomes treated group, with 684 upregulated genes and 570 downregulated genes (Figure 3A-B). This suggests that MSC-derived exosomes treatment caused significant changes in the cellular transcriptome. Through GO analysis and KEGG pathway analysis, it was found that the differentially expressed genes are involved in the regulation of MAPK signaling, NF-κB signaling, p53 signaling, and other pathways, as well as biological processes such as RNA degradation, DNA repair, and cell cycle regulation (Figure 3C-E). Quantitative PCR results showed that, except for RIK, GM28118, MMP3, ATP2A3, and IGF2, which did not show differential expression, the expression levels of other factors were generally consistent with the sequencing results. Among them, HSPA1A and NR4A1 showed more significant changes in expression (Figure 3F-G), suggesting that these factors play important roles in the protective effects of MSC-derived exosomes against ACEII injury. 2.4 MSC-Derived Exosomes Promote ACEII Cell Proliferation ACEII cells were subjected to hyperoxic treatment, followed by treatment with MSC-derived exosomes. Compared to the control group, the number of EdU-positive cells was significantly reduced in the hyperoxia group, while treatment with MSC-derived exosomes alleviated this reduction (Figure 4). This suggests that hyperoxia inhibits cell proliferation, and exosome treatment can improve this inhibitory effect. 2.5 MSC-Derived Exosomes Alleviate Hyperoxic Injury to ACEII by Reducing ROS Accumulation and Inhibiting Ferroptosis ACE Ⅱ was subjected to hyperoxia treatment, followed by exosome treatment. The results showed that hyperoxia promoted ROS accumulation in cells, while exosome treatment alleviated ROS accumulation Compared to the control group(Figure 5A). The expression of GPX4 and SLC7A11 expression were significantly decreased in the hyperoxia group. In the hyperoxia + exosome group, GPX4 and SLC7A11 expression were significantly increased. In the hyperoxia + exosome + ferroptosis inhibitor group, GPX4 and SLC7A11 expression was significantly decreased (Figure 5B-C). 2.6 MSC-Derived Exosomes Regulate ACEII Cell Injury Through Autophagy ACE Ⅱ was subjected to hyperoxia treatment, followed by exosome treatment. Immunofluorescence results showed that hyperoxia induced a significant increase in LC3B-positive cells, while exosome treatment led to a significant decrease in cytoplasmic LC3B-positive cells. Treatment with the autophagy inhibitor 3-MA significantly increased LC3B-positive cells(Figure 6A). Western blot analysis showed that hyperoxia treatment significantly decreased the expression of Wnt1, β-catenin, and p62, while significantly increasing the expression of ATG5 and Beclin1. After exosome treatment, the expression of Wnt1, β-catenin, and p62 was significantly increased, while the expression of ATG5 and Beclin1 was significantly decreased. Treatment with the autophagy inhibitor 3-MA significantly decreased the expression of Wnt1, β-catenin, and p62, while significantly increasing the expression of ATG5 and Beclin1(Figure 6B-C). These results suggest that MSC-derived exosomes can affect cellular autophagy. Discussion The main features of hyperoxia-induced lung injury are damage to alveolar epithelial and endothelial cells, pulmonary edema, and excessive inflammatory responses [ 1 ] . Alveolar epithelial cells play a regulatory role in maintaining the integrity of alveolar structure and function. AEC I is responsible for gas exchange, while AEC II has multiple functions, such as regulating pulmonary immune function, defending against harmful substances invading the lungs, synthesizing and secreting surfactants to maintain surface tension, proliferating and differentiating into AEC I to repair alveoli, and regulating water and salt balance to maintain osmotic pressure [ 12 ] . Therefore, maintaining AEC II homeostasis is crucial for preserving normal lung tissue function [ 13 ] . Studies have shown that prolonged exposure to hyperoxia can lead to damage to the alveolar-capillary barrier and alveolar structure, with AEC II being the primary affected component[4]. Hyperoxia therapy inhibits AEC II proliferation and promotes AEC II apoptosis[5]. Our results show that hyperoxic injury clearly reduces the proliferation of AEC II on the alveolar wall, promotes ROS accumulation, exhibits ferroptotic features, and increases autophagy. Increased AEC II apoptosis can affect alveolar surface tension, leading to impaired alveolar ventilation and gas exchange. Therefore, during oxygen therapy, reasonable interventions should be taken to prevent and treat hyperoxia-induced lung injury, including targeted regulation and protection of AEC II. Alveolar epithelial cell injury is the primary pathological change in ALI and ARDS, so promoting the repair of damaged alveolar epithelium is key to treating lung injury. MSCs are multipotent progenitor cells with self-renewal and multi-lineage differentiation abilities. MSCs residing in the lungs can interact with epithelial cells, promoting alveolar cell growth, differentiation, and self-renewal [ 10 ] . There are increasing studies on the use of MSCs in the treatment of lung injury diseases, and experimental data show that MSCs can significantly reduce pulmonary inflammation and tissue damage [ 14 ] . Studies have shown that MSCs can repair lung injury caused by various factors, including LPS, bleomycin, colchicine, and hypoxia [ 15 ] . However, direct transplantation of MSCs carries risks such as tumorigenicity, embolism, abnormal differentiation, and uncontrolled immune regulation, which limits their application. Increasing evidence[14]suggests that MSCs exert therapeutic effects through paracrine mechanisms, with exosomes being a major paracrine component and key to their biological effects. Due to their advantages such as selective assembly, targeted delivery, efficient repair of damaged tissues, high safety, and chemical stability for easy storage, exosomes have become a hot topic in research. Therefore, in this study, we observed the effects of MSC-derived exosomes on hyperoxia-induced ACEII injury. The results showed that MSC-derived exosomes significantly improved cell proliferation, alleviated ROS accumulation, inhibited ferroptosis and autophagy under hyperoxic conditions. Studies have found that hyperoxia can promote ROS generation, upregulate LC3B expression, and stimulate autophagosome formation. Activated LC3B interacts with the Fas-mediated death-inducing signaling complex, inhibiting the Fas-associated apoptotic pathway and protecting lung epithelial cells [ 16 ] . Hyperoxia can also promote autophagosome formation and reduce lung epithelial cell death in a Trp53-dependent manner[17]. However, autophagy itself is a highly regulated dynamic mechanism, and excessive induction of autophagy may damage cells, while inhibition of autophagy can also protect lung tissue [ 18 ] . The Wnt/β-catenin pathway is a classic signaling pathway involved in regulating various cellular processes, including proliferation, differentiation, and apoptosis. The Wnt signaling pathway is an important signal transduction pathway for cellular autophagy. Inhibition of Wnt signaling can increase the induction of autophagy, and when autophagy is enhanced, β-catenin in the Wnt signaling pathway can be reduced through autophagic degradation [ 19 ] . JIA et al. suggested that the Wnt/β-catenin signaling pathway may be involved in the process of hyperoxia-induced acute lung injury (HALI) in neonatal rats by negatively regulating autophagy. ZHANG et al. showed that autophagy was activated and the Wnt/β-catenin signaling pathway was inhibited in H9c2 cells after H/R treatment. Our results showed that after hyperoxia treatment of ACEII cells, ROS accumulation increased, autophagy was activated, and the Wnt/β-catenin signaling pathway was inhibited. Elevated ROS levels not only lead to oxidative cell damage but also play an important role in ferroptosis [ 20 ] . Ferroptosis is a newly identified form of regulated, programmed cell death that occurs due to excessive accumulation of intracellular iron-dependent ROS[21]. Ferroptosis lacks the morphological features or phenomena of apoptosis, and is mainly characterized by the accumulation of ROS and iron, significant mitochondrial shrinkage, and increased membrane density [ 22 ] . Glutathione peroxidase 4 (GPX4) is a central molecule in the body's antioxidant system and a key regulator of ferroptosis. Ferroptosis-inducing factors can directly or indirectly affect GPX4 through various pathways, leading to a decrease in cellular antioxidant capacity, ROS accumulation, and ultimately oxidative cell death[23]. GSH is a necessary cofactor for GPX4 synthesis and is required for GPX4 to remove cellular lipid peroxides. GSH deficiency will impair GPX4 function, promoting ferroptosis[24]. Additionally, studies have shown that GSH deficiency leads to increased expression of the autophagy marker protein LC3 and an increase in the number of autophagic vesicles, significantly activating autophagy. In yeast, GSH deficiency specifically activates mitochondrial autophagy [ 25 ] . Therefore, various signaling molecules involved in iron metabolism and lipid peroxidation participate in the regulation of iron metabolism and lipid peroxidation. Studies have shown that the occurrence of ferroptosis requires the involvement of autophagy, and certain regulators of autophagy (such as Beclin-1, Nrf2, STAT3, p53, p62) as well as selective autophagy (such as ferritinophagy, lipophagy, and circadian autophagy) play important roles in the ferroptosis process[26]. Beclin-1 is a key regulator of autophagy and can participate in the regulation of ferroptosis as an inhibitor of system Xc-[27]. AMPK phosphorylates Beclin-1, and phosphorylated Beclin-1 interacts with the solute carrier family 7 member 11 (SLC7A11), a component of the glutamate-cystine reverse transport system Xc-, inhibiting the transport activity of Xc- and promoting ferroptosis. Inhibition of AMPK activity disrupts the interaction between Beclin-1 and SLC7A11, or mutation of the interaction site between Beclin-1 and SLC7A11, can inhibit ferroptosis[28]. Additionally, the ELAV-like RNA-binding protein 1 (ELAVL1) can bind to the 3' untranslated region of Beclin-1 mRNA, maintaining its stability and thereby activating autophagy, leading to enhanced ferritinophagy and promoting ferroptosis [ 29 ] . These studies suggest that Beclin-1 regulates ferroptosis by modulating autophagy activity. The nuclear factor erythroid 2-related factor 2 (Nrf2) is a core protein in the antioxidant stress response of the cellular protective pathway. Under normal physiological conditions, Keap1 (Kelch-like ECH-associated protein 1) interacts with Nrf2, mediating its ubiquitination and proteasomal degradation[30]. Under oxidative stress conditions, Nrf2 trans-activates the SQSTM1 gene encoding the p62 protein, and the accumulation of p62 promotes the p62-dependent autophagic degradation of Keap1, inhibiting Keap1-mediated Nrf2 degradation [ 31 ] . p62 can competitively bind to Keap1, mediating the autophagic degradation of Keap1 and activating Nrf2, while simultaneously upregulating iron- and ROS-related genes to exert an anti-ferroptotic effect. Furthermore, impaired cellular autophagy leads to the accumulation of p62, oxidized proteins, or damaged organelles, which can also activate Nrf2, thereby inducing the expression of autophagy-related genes such as p62, ATG5, and ULK1, enhancing autophagic activity to remove damaged proteins and organelles [ 32 ] . As a transcription factor, Nrf2 can also directly regulate the expression of important genes involved in the ferroptosis process, including HO-1, SLC7A11, and GPX4, thereby regulating cellular iron metabolism, GSH levels, GPX4 synthesis, and lipid oxidation. Therefore, Nrf2 plays a crucial role in the regulation of ferroptosis[33]. In summary, hyperoxia induces ROS generation, upregulates LC3B expression, and stimulates autophagosome formation. Additionally, ROS not only causes oxidative cell damage but also plays an important role in ferroptosis. Studies have shown that the occurrence of ferroptosis requires the involvement of autophagy, and certain regulators of autophagy (such as GPX4, Beclin-1, p62, etc.) play important roles in ferroptosis. Both ferroptosis and autophagy play crucial regulatory roles in the cell proliferation process. Excessive ferroptosis and autophagy promote cell apoptosis; therefore, ferroptosis and autophagy are important for cell proliferation. MSC-derived exosomes have similar functions to MSCs. In this study, we treated hyperoxia-induced ACE II with MSC-derived exosomes. The results showed that MSC-derived exosomes could improve the inhibitory effect of hyperoxia on ACE II proliferation, reduce ROS accumulation and downregulate LC3B expression. Ferroptosis and autophagy were significantly improved, while the addition of ferroptosis inhibitors and autophagy inhibitors increased ferroptosis and autophagy. Therefore, when hyperoxia damages lung epithelial cells, MSC-derived exosomes may alleviate the damaging effects of hyperoxia on ACE II by improving the inhibitory effect of hyperoxia on ACE II, promoting ACE II proliferation, and reducing ROS accumulation to inhibit ferroptosis and autophagy, thereby decreasing cell apoptosis. Thus, MSC-derived exosomes have potential application value in the treatment of hyperoxia-induced acute lung injury. However, research on MSC-derived exosomes is still in its early stages, and many issues need to be addressed, such as the administration route and purification techniques of MSC-derived exosomes. Moreover, most studies focus on cells and genes, and research on the treatment of ALI with MSC-derived exosomes is very limited, with many uncertainties. Nevertheless, numerous related studies have shown the great prospects of using MSC-derived exosomes for the treatment of acute lung injury, and they may become an effective therapeutic approach for acute lung injury. However, there are still many difficulties before their actual clinical application, and more research is needed for further exploration. List Of Abbreviations ALI acute lung injury BPD bronchopulmonary dysplasia ARDS acute respiratory distress syndrome ACE II type II alveolar epithelial cells MSCs Mesenchymal stem cells DCFH-DA 2,7-dichloro dihydro fluorescein diacetate TEM transmission electron microscopy NTA nanoparticle tracking analysis PBS phosphate-buffered saline HALI hyperoxia-induced acute lung injury SLC7A11 solute carrier family 7 member 11 ELAVL1 ELAV-like RNA-binding protein 1 Nrf2 nuclear factor erythroid 2-related factor 2 Keap1 Kelch-like ECH-associated protein 1. Declarations Ethics approval and consent to participate: Not applicable. Consent for publication: Not applicable. Availability of data and materials: All data generated or analysed during this study are included in this published article [and its supplementary information files]. Competing interests: The authors declare that they have no competing interests. Funding: The research was supported by: Science and Technology Plan Project of Guizhou Provincial (Qiankehe Fundamentals ZK [2024] General 345); National Natural Science Foundation of China (No. 82160369); Zunyi Medical University［2022］(No.21). Authors' contributions: GY. L designed the experimental protocol and establish cell models, and was a major contributor in writing the manuscript. GY. J used GO analysis and KEGG enrichment analysis to study the regulatory role of differentially expressed genes in cells, and conducted Western blotting experiments. YC. R performed cell proliferation detection. QX. H and CZ. Y validated the high-throughput sequencing results using quantitative PCR. X. X conducted ROS level detection experiment. H. W conducted immunofluorescence tests. M. C supervised the experiment and revised the manuscript of the paper. All authors read and approved the final manuscript. Acknowledgements: Not applicable. References Wan M, Tajuddin WNB et al. 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Inhibition of Regulatory-Associated Protein of Mechanistic Target of Rapamycin Prevents Hyperoxia-Induced Lung Injury by Enhancing Autophagy and Reducing Apoptosis in Neonatal Mice. Am J Respir Cell Mol Biol. 2016;55(5):722–35. Wu XT, et al. Visfatin Plays a Significant Role in Alleviating Lipopolysaccharide-Induced Apoptosis and Autophagy Through PI3K/AKT Signaling Pathway During Acute Lung Injury in Mice. Arch Immunol Ther Exp (Warsz). 2019;67(4):249–61. Nàger M, et al. Inhibition of WNT-CTNNB1 signaling upregulates SQSTM1 and sensitizes glioblastoma cells to autophagy blockers. Autophagy. 2018;14(4):619–36. Su LJ et al. Reactive Oxygen Species-Induced Lipid Peroxidation in Apoptosis, Autophagy, and Ferroptosis. Oxid Med Cell Longev, 2019. 2019: p. 5080843. Santana-Codina N, Gikandi A, Mancias JD. The Role of NCOA4-Mediated Ferritinophagy in Ferroptosis. Adv Exp Med Biol. 2021;1301:41–57. Liu X, et al. 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Activation of ferritinophagy is required for the RNA-binding protein ELAVL1/HuR to regulate ferroptosis in hepatic stellate cells. Autophagy. 2018;14(12):2083–103. Ichimura Y, Komatsu M. Activation of p62/SQSTM1-Keap1-Nuclear Factor Erythroid 2-Related Factor 2 Pathway in Cancer. Front Oncol. 2018;8:210. Sun X, et al. Activation of the p62-Keap1-NRF2 pathway protects against ferroptosis in hepatocellular carcinoma cells. Hepatology. 2016;63(1):173–84. Pajares M, et al. Transcription factor NFE2L2/NRF2 is a regulator of macroautophagy genes. Autophagy. 2016;12(10):1902–16. Dodson M, Castro-Portuguez R, Zhang DD. NRF2 plays a critical role in mitigating lipid peroxidation and ferroptosis. Redox Biol. 2019;23:101107. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4538714","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":322348449,"identity":"4db31333-5df3-40b6-aa7c-aa22b8f7ceb1","order_by":0,"name":"Guoyue Liu","email":"","orcid":"","institution":"The Second Affiliated Hospital of Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Guoyue","middleName":"","lastName":"Liu","suffix":""},{"id":322348450,"identity":"c3e4af0c-a714-4035-b424-07b44ff6a812","order_by":1,"name":"Guiyang Jia","email":"","orcid":"","institution":"The Second Affiliated Hospital of Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Guiyang","middleName":"","lastName":"Jia","suffix":""},{"id":322348451,"identity":"28774ce4-ec84-4eb4-8076-0dfb7f6dcb03","order_by":2,"name":"yingcong Ren","email":"","orcid":"","institution":"Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"yingcong","middleName":"","lastName":"Ren","suffix":""},{"id":322348452,"identity":"ddbfb470-d74e-420b-9906-f60be8004c37","order_by":3,"name":"qianxia Huang","email":"","orcid":"","institution":"Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"qianxia","middleName":"","lastName":"Huang","suffix":""},{"id":322348453,"identity":"3dbf3cf9-0202-4fcd-81fd-bfa8dd9d05f7","order_by":4,"name":"Cunzhi Yin","email":"","orcid":"","institution":"The Second Affiliated Hospital of Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Cunzhi","middleName":"","lastName":"Yin","suffix":""},{"id":322348454,"identity":"9dd88815-25f8-4332-bcc1-cc38d7d7da97","order_by":5,"name":"Xuan Xiao","email":"","orcid":"","institution":"The Second Affiliated Hospital of Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Xuan","middleName":"","lastName":"Xiao","suffix":""},{"id":322348455,"identity":"3e5fcaa7-caa1-4b60-baf1-60da8ba0bb5b","order_by":6,"name":"Hang Wu","email":"","orcid":"","institution":"The Second Affiliated Hospital of Zunyi Medical University","correspondingAuthor":false,"prefix":"","firstName":"Hang","middleName":"","lastName":"Wu","suffix":""},{"id":322348456,"identity":"1886922e-bee2-4bb6-a996-7d1b48273c38","order_by":7,"name":"Miao Chen","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAzElEQVRIiWNgGAWjYDACCRBhwMDAz3D4wIEPP0jRItl4LPHgzB6itYB0NZ8xPszBRoQO/tnNxx7dKLCTM2A78+EwAw+DPL/YAQKW3DmWbpxjkGxsznN2w+ECCwbDmbMT8GsxkMgxk84xYE7cOQOoZQYPQ4LBbYJa8r8BtdQnbrj/5sFhHjaitOSwAbUcTtxw4AwDcVokbqSBHHbcWLLhmAEwkCUI+4V/RvIz6Zw/1XLAqHz84cMPG3l+aQJaMGwlTfkoGAWjYBSMAuwAAGFuRptjeio9AAAAAElFTkSuQmCC","orcid":"","institution":"Affiliated Hospital of Zunyi Medical University","correspondingAuthor":true,"prefix":"","firstName":"Miao","middleName":"","lastName":"Chen","suffix":""}],"badges":[],"createdAt":"2024-06-06 08:56:10","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4538714/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4538714/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":59658005,"identity":"59b19ca6-7996-4727-b1f9-4ade700e55bb","added_by":"auto","created_at":"2024-07-04 11:14:11","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":977554,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphological, Size Distribution, and Phenotypic Identification of MSC-derived exosomes. \u003c/strong\u003eA-B: shows the morphology of exosomes under transmission electron microscopy (left), appearing as round or oval cup-shaped membrane-bound small vesicles, and the nanoparticle tracking analysis results (right) showing exosome diameters ranging from 80 to 100 nm. C: shows the Western blotting detection of protein expression related to MSC-derived exosomes.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-4538714/v1/c5117cc30437777619c341b0.png"},{"id":59657030,"identity":"7949d868-ad18-4b62-8411-e00ae5ef6a2b","added_by":"auto","created_at":"2024-07-04 10:58:11","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1200196,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eUptake of MSC-derived exosomes by ACEII. \u003c/strong\u003eDAPI stains the ACEII cell nuclei in blue, PKH-67 labels the exosomes in green, and the merged image shows that the exosomes are present around the ACEII cell nuclei with a high nesting rate.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-4538714/v1/575fcdf62dee1d2c56aff8e0.png"},{"id":59657478,"identity":"a95fce5d-21d7-4adc-be5f-597c93a10fb1","added_by":"auto","created_at":"2024-07-04 11:06:11","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":602902,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eSignificant transcriptional changes in ACEII Induced by MSC-derived exosomes Treatment. \u003c/strong\u003eA-B: shows the significant changes in the cellular transcriptome induced by MSC-derived exosomes treatment. C-E: shows the GO analysis and KEGG pathway analysis results. F-G: shows the quantitative PCR validation results.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-4538714/v1/bb57e9d0f90231b690bb99c7.png"},{"id":59657034,"identity":"ac5a313d-15ad-44ab-9a31-34cf429a686b","added_by":"auto","created_at":"2024-07-04 10:58:11","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":701254,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMSC-Derived Exosomes Alleviate the effect of Hyperoxic on ACEII cell proliferation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e2.5 MSC-Derived Exosomes Alleviate Hyperoxic Injury to ACEII by Reducing ROS Accumulation and Inhibiting Ferroptosis\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-4538714/v1/fb9d3614c70ae2b0103a5f46.png"},{"id":59657031,"identity":"9a4f00b7-c842-4952-942a-0a75fce52ef8","added_by":"auto","created_at":"2024-07-04 10:58:11","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":910794,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMSC-Derived Exosomes Alleviate Hyperoxic Injury to ACEII Cells. \u003c/strong\u003eA: Changes in ROS levels in ACEII cells in different groups. B-C: Expression of ferroptosis-related factors GPX4, and SLC7A11 in different groups.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-4538714/v1/9470c30f516c903a97c331a1.png"},{"id":59657036,"identity":"86704bb3-05a4-4753-bf88-fa4f09770033","added_by":"auto","created_at":"2024-07-04 10:58:11","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":782322,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eEffect of MSC-Derived Exosomes on ACEII Cell Autophagosomes and Autophagy-Related Proteins. \u003c/strong\u003eA: Immunofluorescence staining to observe the number of LC3B puncta in cells from different groups. B-C: Expression of autophagy-related proteins.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-4538714/v1/aafcba2aeede90e85d7f14c5.png"},{"id":61325912,"identity":"7eca8edc-e25c-4b1e-8f7c-f46264f5a22b","added_by":"auto","created_at":"2024-07-29 14:00:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6441887,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4538714/v1/3209f505-e1a0-4d7c-b779-c88ff35deb59.pdf"},{"id":59657035,"identity":"89c7576f-0599-4a49-b8c7-56ce9109d8e9","added_by":"auto","created_at":"2024-07-04 10:58:11","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":778498,"visible":true,"origin":"","legend":"","description":"","filename":"supplementaryfile.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4538714/v1/175f8a440b324575913c8c46.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Investigation of the Protective Effects of Mesenchymal Stem Cell-Derived Exosomes on Hyperoxia-Induced Type II Alveolar Epithelial Cell Injury Based on Ferroptosis and Autophagy","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOxygen therapy is widely used in the treatment of patients with respiratory failure and hypoxemia. It plays a crucial role in saving lives, preventing multi-organ failure caused by hypoxia, and buying time for emergency treatment. However, hyperoxia can also trigger oxygen toxicity, including acute lung injury (ALI)\u003csup\u003e[1]\u003c/sup\u003e. Prolonged exposure to hyperoxia can cause severe lung damage in premature infants, leading to ALI and bronchopulmonary dysplasia (BPD)\u003csup\u003e[2]\u003c/sup\u003e, and exacerbate acute respiratory distress syndrome (ARDS) in adults, which is one of the causes of death. Studies have shown that long-term hyperoxic exposure can lead to the destruction of the alveolar-capillary barrier and structural changes in the alveoli, as well as a large amount of apoptosis in alveolar epithelial cells[3, 4]. Therefore, elucidating the pathogenesis of this disease and implementing appropriate lung protective measures during oxygen therapy is crucial. Research has shown that type II alveolar epithelial cells (AEC II) are the stem cells of the lung tissue and can differentiate into other lung tissue cells, but hyperoxia has been proven to promote apoptosis\u003csup\u003e[5]\u003c/sup\u003e\u003csup\u003e\u0026nbsp;\u003c/sup\u003eand excessive differentiation of AEC II cells into AEC I[6]. After AEC II cell injury and apoptosis, the self-repair capacity is limited, which can easily lead to damage to the alveolar structure and pulmonary fibrosis\u003csup\u003e[7]\u003c/sup\u003e. Therefore, exogenous cell supplementation plays an important role in the repair of damaged alveolar epithelial cells.\u003c/p\u003e\n\u003cp\u003eMesenchymal stem cells (MSCs) are a group of adult stem cells derived from tissues such as bone marrow, adipose tissue, and umbilical cord, possessing self-renewal, multi-lineage differentiation, and tissue repair-promoting capabilities\u003csup\u003e[8]\u003c/sup\u003e.\u0026nbsp;In recent years, there have been increasing reports on the therapeutic effects of MSCs in lung injury diseases, showing antimicrobial, anti-inflammatory, tissue regeneration-promoting, anti-fibrotic, antioxidant stress, and anti-apoptotic properties, which have opened up new avenues for the treatment of lung injury\u003csup\u003e[9]\u003c/sup\u003e. Although MSCs have certain advantages in terms of immunogenicity, in vitro expansion capability, genetic characteristics, and acquisition methods, they also have defects such as potential tumorigenicity, embolism, and teratogenicity after transplantation, and there are still many controversies in clinical treatment\u003csup\u003e[10]\u003c/sup\u003e. In recent years, research has found that MSCs exert their therapeutic effects primarily through paracrine secretion of exosomes, which have similar biological functions as the parent cells, such as anti-inflammatory, tissue regeneration-promoting, anti-apoptotic, and anti-fibrotic effects. However, compared to MSCs, exosomes have a smaller size, lower risk of embolism, and lower immunogenicity. Their specific surface receptors allow them to deliver drugs or specific components to target cells. Due to these advantages, exosomes have promising applications in lung injury and are currently a research hotspot in the stem cell field.\u0026nbsp;MSC-derived exosomes have been shown to have protective effects similar to those of MSCs themselves in cardiovascular diseases, osteoarthritis, wound healing, various acute kidney injury animal models, and rat models of stroke\u003csup\u003e[11]\u003c/sup\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn recent studies, ferroptosis and autophagy have been implicated in ALI. The present study aims to investigate the protective effects of MSC-derived exosomes in a hyperoxia-induced alveolar epithelial cell injury model by evaluating their biological effects on cell proliferation, oxidative stress response, ferroptosis, and autophagy, and exploring the underlying mechanisms. This study may provide new insights into the treatment of alveolar epithelial cell injury.\u003c/p\u003e"},{"header":"Experimental materials and methods","content":"\u003cp\u003e1.1\u0026nbsp;Experimental materials\u003c/p\u003e\n\u003cp\u003eLysate, SDS-PAGE gel configuration kit, 5\u0026times; protein uploading buffer were purchased from Beyotime Biotech. Inc. Trypsin, collagenase, deoxyribonuclease I, fetal bovine serum, DMEM were purchased from Gibco (USA). CD9, CD81, Nrf2, GPX4, SLC7A11, Wnt1, \u0026beta;-catenin, p62, ATG5, Beclin1 were purchased from Abcam (UK). Trizol and M-MLV reverse transcriptase were purchased from Promega (USA). 3-MA was purchased from Selleck (USA). The ferroptosis inhibitor ferrostatin-1 (Fer-1) was purchased from MedChe- mExpress. 4% paraformaldehyde was purchased from Beijing Ruier Xinde Technology Co., Ltd. The fluorescent probe 2,7-dichloro dihydro fluorescein diacetate (DCFH-DA) was purchased from Sigma-Aldrich.\u003c/p\u003e\n\u003cp\u003e1.2\u0026nbsp;Extraction and Identification of Exosomes\u003c/p\u003e\n\u003cp\u003eMSCs were isolated using the tissue cell adherence method, cultured and expanded to the third generation, and exosomes from the MSCs supernatant were obtained by ultracentrifugation. In most studies, transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and Western blotting are often considered the preferred methods for identifying exosomes. In this study, the extracted MSC-derived exosomes were identified using TEM, NTA, and Western blotting.\u003c/p\u003e\n\u003cp\u003eTransmission Electron Microscopy: 10 \u0026mu;L of the extracted exosomes were diluted with an equal volume of phosphate-buffered saline (PBS), dropped onto a 2 mm copper grid, and left at room temperature for 1 min. Excess liquid was gently removed with filter paper, and the grid was stained with 3% uranyl acetate solution for 5 min at room temperature. After gentle rinsing with double-distilled water, the grid was air-dried at room temperature and observed under a transmission electron microscope, and images were captured.\u003c/p\u003e\n\u003cp\u003eNanoparticle Tracking Analysis: 10 \u0026mu;L of exosomes were diluted to 30 \u0026mu;L. First, the instrument performance was tested using a standard sample. After passing the test, the exosome sample was loaded, and the particle size information was obtained upon completion of the analysis.\u003c/p\u003e\n\u003cp\u003eWestern Blotting: Western blotting was used to detect exosomal surface markers.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e1.3\u0026nbsp;Uptake Experiment for MSC-derived Exosomes\u003c/p\u003e\n\u003cp\u003eThe extracted MSC-derived exosomes were first mixed with diluent C, and then PKH-67 dye solution was quickly added and mixed with diluent C, followed by light-protected incubation for 15 min. The mixture was then placed on ice, and 0.5% BSA was added to terminate the reaction. After centrifugation and extraction according to the instructions, PKH-67-labeled MSC-derived exosomes were obtained. ACEII were digested, resuspended, and counted. 100 \u0026mu;L of the cell suspension was mixed with an equal volume of PKH-67-labeled exosome suspension and added to the bottom wells of a confocal culture dish. The total cell number was approximately 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e. The culture dish was transferred to an incubator and cultured for 1 h, after which 2 mL of complete culture medium was added using a pipette. After 12 h of culture, the cells were prepared for imaging. The culture medium was removed, and the cells were washed three times with PBS, fixed with 4% paraformaldehyde (2 mL) at room temperature for 10 min, and washed three times with PBS. The cells were then stained with DAPI (10 min) to label the cell nuclei. After removing the DAPI, the confocal culture dish was placed on a horizontal shaker and washed 3 times with PBS for 5 min each. The PBS was removed, and the dish was allowed to dry slightly before carefully removing the glass bottom and mounting it onto a slide. The cells were observed and imaged under a laser confocal microscope.\u003c/p\u003e\n\u003cp\u003e1.4\u0026nbsp;RNA-seq High-throughput Sequencing\u003c/p\u003e\n\u003cp\u003eCells were treated with MSC-derived exosomes under hyperoxic conditions, with the cells divided into hyperoxia and hyperoxia + MSC-derived exosomes groups. After modeling, the cells were collected, and high-throughput sequencing was performed to identify significantly upregulated or downregulated genes compared to the hyperoxia group. GO analysis and KEGG pathway analysis of the differentially expressed genes were conducted to determine the relevant genes, and quantitative PCR was used to validate the high-throughput sequencing results.\u003c/p\u003e\n\u003cp\u003e1.5 \u0026nbsp;Real-time PCR Detection of Relative mRNA Expression of Differentially Expressed Genes\u003c/p\u003e\n\u003cp\u003eTotal RNA was extracted from cells using the Trizol one-step method. Cells from different treatment groups were collected, and trichloromethane was added. After reverting for 15 s and standing at room temperature for 2 min, the mixture was centrifuged. The supernatant was transferred to a new PE tube, and isopropanol was added. After standing for 10 min, the mixture was centrifuged. The supernatant was discarded, and pre-cooled 75% ethanol was added for washing. After drying for 20 min, RNase-free water was added to fully dissolve the precipitate. The concentration and purity were determined. The cDNA was synthesized by reverse transcription of RT-PCR. The reaction system was then prepared according to the real-time PCR kit instructions and detected on a real-time fluorescence quantitative PCR instrument. The \u0026Delta;\u0026Delta;Ct values and relative quantities (RQ, where RQ = 2-\u0026Delta;\u0026Delta;Ct) were calculated using the internal reference as a control to determine the relative expression levels of the target genes.\u003c/p\u003e\n\u003cp\u003e1.6\u0026nbsp;EdU Detection of Cell Proliferation Changes in Different Groups\u003c/p\u003e\n\u003cp\u003eCells induced by hyperoxia were treated with MSC-derived exosomes and the cells were divided into control, hyperoxia, and hyperoxia + MSC-derived exosomes groups. The complete culture medium was discarded, and a medium containing 50 \u0026mu;mol/L EdU was added for co-incubation for 2 h. Cells were fixed with 4% paraformaldehyde for 30 min, permeabilized with 0.5% Triton X-100 for 10 min, and washed with PBS. DAPI staining solution was added, and cells were incubated in the dark for 10 min at room temperature. After washing with PBS, the cells were mounted with glycerol. An inverted fluorescence microscope was used to observe the stained cells, and 5 random fields were selected to calculate the EdU-positive cell rate. Blue fluorescence represents DAPI-stained cell nuclei (all cells), while red fluorescence represents EdU-labeled proliferating cells. The cell proliferation rate was calculated as (number of red fluorescent cells / number of blue fluorescent cells) \u0026times; 100%.\u003c/p\u003e\n\u003cp\u003e1.7\u0026nbsp;Western Blotting Detection of Exosomal Marker Proteins (CD9, CD81), Ferroptosis-related Factors (GPX4, SLC7A11), and Autophagy-related Factors (Wnt1, \u0026beta;-catenin, p62, ATG5, Beclin1)\u003c/p\u003e\n\u003cp\u003eAfter treatment, cells were washed 3 times with pre-cooled PBS and lysed with RIPA cell lysis buffer for 30 min. The mixture was centrifuged at 12,000 rpm for 10 min at 4\u0026deg;C. After BCA protein quantification, protein loading buffer was added in proportion, and the proteins were separated by SDS-PAGE electrophoresis and transferred to a PVDF membrane. The membrane was blocked with 5% non-fat milk for 1 h at room temperature and incubated with primary antibodies against CD9, CD81, GPX4, SLC7A11, Wnt1, \u0026beta;-catenin, p62, ATG5, Beclin1, and GAPDH overnight at 4\u0026deg;C. The next day, the membrane was washed 3 times and incubated with the corresponding rabbit anti-secondary antibody for 1 h at room temperature. ECL reagent was used for exposure and development in an automatic gel imaging system.\u003c/p\u003e\n\u003cp\u003e1.8\u0026nbsp;Determination of ROS Levels\u003c/p\u003e\n\u003cp\u003eThe DCFH-DA probe (10 \u0026mu;mol/L) method was used to determine cellular ROS levels. ACEII cells were seeded in 6-well plates at a density of 1\u0026times;10\u003csup\u003e5\u003c/sup\u003e cells per well. After treatment, cells were incubated with the probe molecule (10 \u0026mu;mol/L) at 37\u0026deg;C for 30 min, stained with nuclear staining solution B for 2 min, and washed three times with PBS. A fluorescence microscope was used to collect fluorescence images of the cells, and Image J software was used to analyze all the images.\u003c/p\u003e\n\u003cp\u003e1.9 Immunofluorescence Detection of LC3B Puncta in Cells\u003c/p\u003e\n\u003cp\u003eCells from different groups were seeded onto slides, fixed with 4% paraformaldehyde for 15 min, washed with PBS, and permeabilized with 0.3% Triton X-100 for 20 min. Cells were blocked with 5% BSA for 1 h at room temperature and incubated with anti-LC3B primary antibody (1:1000) overnight at 4\u0026deg;C. After washing, cells were incubated with fluorescent-labeled secondary antibody (1:1000) for 1 h at room temperature. Cells were stained with DAPI and observed under a laser scanning confocal microscope to scan images of LC3B puncta. Image J software was used to analyze the optical density values of the images and calculate the fluorescence intensity. Fluorescence intensity = optical density value / fluorescence area.\u003c/p\u003e\n\u003cp\u003e2.0 Data Processing\u003c/p\u003e\n\u003cp\u003eAll data were processed using SPSS 29.0 statistical software. Continuous data are presented as \u0026quot;mean \u0026plusmn; standard deviation\u0026quot;. For multiple group comparisons, one-way ANOVA analysis of variance was used. For pairwise comparisons between groups, the LSD test was used for equal variances, and the Dunnett\u0026apos;s test was used for unequal variances. P \u0026lt; 0.05 was considered statistically significant. Images were processed using Image J 8.0.\u003c/p\u003e"},{"header":"Experimental Results","content":"\u003cp\u003e2.1 Morphological and Phenotypic Identification of MSC-derived exosomes\u003c/p\u003e\n\u003cp\u003eTransmission electron microscopy results showed that the exosomes had a cup-shaped morphology with relatively uniform diameters, appearing round or oval, with a membrane-bound vesicular structure containing dense cloud-like substances, which are the contents of exosomes. Nanoparticle tracking analysis revealed that the exosome diameters ranged from 80 to 100 nm (Figure 1A-B). Western blotting results showed the expression of the exosomal marker proteins CD9 and CD81 in the extracted exosomes (Figure 1C).\u003c/p\u003e\n\u003cp\u003e2.2 ACEII Cells Can Uptake MSC-derived exosomes\u003c/p\u003e\n\u003cp\u003eTo investigate whether ACEII cells can uptake MSC-derived exosomes, this study used PKH-67 to label MSC-derived exosomes. PKH-67 is a green fluorescent biomembrane tracking dye widely used in various in vivo or in vitro experiments. After co-culturing the labeled MSC-derived exosomes with ACEII cells, we observed distinct green fluorescence surrounding the nuclei of most cells (Figure 2).\u003c/p\u003e\n\u003cp\u003e2.3 MSC-derived Exosomes Induce Transcriptional Changes in Hyperoxia-induced ACEII\u003c/p\u003e\n\u003cp\u003eHigh-throughput sequencing results showed that, compared to the hyperoxia group, 1,254 genes were differentially expressed in the MSC-derived exosomes treated group, with 684 upregulated genes and 570 downregulated genes (Figure 3A-B). This suggests that MSC-derived exosomes treatment caused significant changes in the cellular transcriptome. Through GO analysis and KEGG pathway analysis, it was found that the differentially expressed genes are involved in the regulation of MAPK signaling, NF-\u0026kappa;B signaling, p53 signaling, and other pathways, as well as biological processes such as RNA degradation, DNA repair, and cell cycle regulation (Figure 3C-E). Quantitative PCR results showed that, except for RIK, GM28118, MMP3, ATP2A3, and IGF2, which did not show differential expression, the expression levels of other factors were generally consistent with the sequencing results. Among them, HSPA1A and NR4A1 showed more significant changes in expression (Figure 3F-G), suggesting that these factors play important roles in the protective effects of MSC-derived exosomes against ACEII injury.\u003c/p\u003e\n\u003cp\u003e2.4 MSC-Derived Exosomes Promote ACEII Cell Proliferation\u003c/p\u003e\n\u003cp\u003eACEII cells were subjected to hyperoxic treatment, followed by treatment with MSC-derived exosomes. Compared to the control group, the number of EdU-positive cells was significantly reduced in the hyperoxia group, while treatment with MSC-derived exosomes alleviated this reduction (Figure 4). This suggests that hyperoxia inhibits cell proliferation, and exosome treatment can improve this inhibitory effect.\u003c/p\u003e\n\u003cp\u003e2.5 MSC-Derived Exosomes Alleviate Hyperoxic Injury to ACEII by Reducing ROS Accumulation and Inhibiting Ferroptosis\u003c/p\u003e\n\u003cp\u003eACE Ⅱ was subjected to hyperoxia treatment, followed by exosome treatment. The results showed that hyperoxia promoted ROS accumulation in cells, while exosome treatment alleviated ROS accumulation Compared to the control group(Figure 5A). The expression of GPX4 and SLC7A11 expression were significantly decreased in the hyperoxia group. In the hyperoxia + exosome group, GPX4 and SLC7A11 expression were significantly increased. In the hyperoxia + exosome + ferroptosis inhibitor group, GPX4 and SLC7A11 expression was significantly decreased (Figure 5B-C).\u003c/p\u003e\n\u003cp\u003e2.6 MSC-Derived Exosomes Regulate ACEII Cell Injury Through Autophagy\u003c/p\u003e\n\u003cp\u003eACE Ⅱ was subjected to hyperoxia treatment, followed by exosome treatment. Immunofluorescence results showed that hyperoxia induced a significant increase in LC3B-positive cells, while exosome treatment led to a significant decrease in cytoplasmic LC3B-positive cells. Treatment with the autophagy inhibitor 3-MA significantly increased LC3B-positive cells(Figure 6A). Western blot analysis showed that hyperoxia treatment significantly decreased the expression of Wnt1, \u0026beta;-catenin, and p62, while significantly increasing the expression of ATG5 and Beclin1. After exosome treatment, the expression of Wnt1, \u0026beta;-catenin, and p62 was significantly increased, while the expression of ATG5 and Beclin1 was significantly decreased. Treatment with the autophagy inhibitor 3-MA significantly decreased the expression of Wnt1, \u0026beta;-catenin, and p62, while significantly increasing the expression of ATG5 and Beclin1(Figure 6B-C). These results suggest that MSC-derived exosomes can affect cellular autophagy.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe main features of hyperoxia-induced lung injury are damage to alveolar epithelial and endothelial cells, pulmonary edema, and excessive inflammatory responses\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. Alveolar epithelial cells play a regulatory role in maintaining the integrity of alveolar structure and function. AEC I is responsible for gas exchange, while AEC II has multiple functions, such as regulating pulmonary immune function, defending against harmful substances invading the lungs, synthesizing and secreting surfactants to maintain surface tension, proliferating and differentiating into AEC I to repair alveoli, and regulating water and salt balance to maintain osmotic pressure\u003csup\u003e[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]\u003c/sup\u003e. Therefore, maintaining AEC II homeostasis is crucial for preserving normal lung tissue function\u003csup\u003e[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]\u003c/sup\u003e. Studies have shown that prolonged exposure to hyperoxia can lead to damage to the alveolar-capillary barrier and alveolar structure, with AEC II being the primary affected component[4]. Hyperoxia therapy inhibits AEC II proliferation and promotes AEC II apoptosis[5]. Our results show that hyperoxic injury clearly reduces the proliferation of AEC II on the alveolar wall, promotes ROS accumulation, exhibits ferroptotic features, and increases autophagy. Increased AEC II apoptosis can affect alveolar surface tension, leading to impaired alveolar ventilation and gas exchange. Therefore, during oxygen therapy, reasonable interventions should be taken to prevent and treat hyperoxia-induced lung injury, including targeted regulation and protection of AEC II.\u003c/p\u003e \u003cp\u003eAlveolar epithelial cell injury is the primary pathological change in ALI and ARDS, so promoting the repair of damaged alveolar epithelium is key to treating lung injury. MSCs are multipotent progenitor cells with self-renewal and multi-lineage differentiation abilities. MSCs residing in the lungs can interact with epithelial cells, promoting alveolar cell growth, differentiation, and self-renewal\u003csup\u003e[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]\u003c/sup\u003e. There are increasing studies on the use of MSCs in the treatment of lung injury diseases, and experimental data show that MSCs can significantly reduce pulmonary inflammation and tissue damage\u003csup\u003e[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]\u003c/sup\u003e. Studies have shown that MSCs can repair lung injury caused by various factors, including LPS, bleomycin, colchicine, and hypoxia\u003csup\u003e[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]\u003c/sup\u003e. However, direct transplantation of MSCs carries risks such as tumorigenicity, embolism, abnormal differentiation, and uncontrolled immune regulation, which limits their application. Increasing evidence[14]suggests that MSCs exert therapeutic effects through paracrine mechanisms, with exosomes being a major paracrine component and key to their biological effects. Due to their advantages such as selective assembly, targeted delivery, efficient repair of damaged tissues, high safety, and chemical stability for easy storage, exosomes have become a hot topic in research. Therefore, in this study, we observed the effects of MSC-derived exosomes on hyperoxia-induced ACEII injury. The results showed that MSC-derived exosomes significantly improved cell proliferation, alleviated ROS accumulation, inhibited ferroptosis and autophagy under hyperoxic conditions.\u003c/p\u003e \u003cp\u003eStudies have found that hyperoxia can promote ROS generation, upregulate LC3B expression, and stimulate autophagosome formation. Activated LC3B interacts with the Fas-mediated death-inducing signaling complex, inhibiting the Fas-associated apoptotic pathway and protecting lung epithelial cells\u003csup\u003e[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]\u003c/sup\u003e. Hyperoxia can also promote autophagosome formation and reduce lung epithelial cell death in a Trp53-dependent manner[17]. However, autophagy itself is a highly regulated dynamic mechanism, and excessive induction of autophagy may damage cells, while inhibition of autophagy can also protect lung tissue\u003csup\u003e[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. The Wnt/β-catenin pathway is a classic signaling pathway involved in regulating various cellular processes, including proliferation, differentiation, and apoptosis. The Wnt signaling pathway is an important signal transduction pathway for cellular autophagy. Inhibition of Wnt signaling can increase the induction of autophagy, and when autophagy is enhanced, β-catenin in the Wnt signaling pathway can be reduced through autophagic degradation\u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. JIA et al. suggested that the Wnt/β-catenin signaling pathway may be involved in the process of hyperoxia-induced acute lung injury (HALI) in neonatal rats by negatively regulating autophagy. ZHANG et al. showed that autophagy was activated and the Wnt/β-catenin signaling pathway was inhibited in H9c2 cells after H/R treatment. Our results showed that after hyperoxia treatment of ACEII cells, ROS accumulation increased, autophagy was activated, and the Wnt/β-catenin signaling pathway was inhibited.\u003c/p\u003e \u003cp\u003eElevated ROS levels not only lead to oxidative cell damage but also play an important role in ferroptosis\u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Ferroptosis is a newly identified form of regulated, programmed cell death that occurs due to excessive accumulation of intracellular iron-dependent ROS[21]. Ferroptosis lacks the morphological features or phenomena of apoptosis, and is mainly characterized by the accumulation of ROS and iron, significant mitochondrial shrinkage, and increased membrane density\u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Glutathione peroxidase 4 (GPX4) is a central molecule in the body's antioxidant system and a key regulator of ferroptosis. Ferroptosis-inducing factors can directly or indirectly affect GPX4 through various pathways, leading to a decrease in cellular antioxidant capacity, ROS accumulation, and ultimately oxidative cell death[23]. GSH is a necessary cofactor for GPX4 synthesis and is required for GPX4 to remove cellular lipid peroxides. GSH deficiency will impair GPX4 function, promoting ferroptosis[24]. Additionally, studies have shown that GSH deficiency leads to increased expression of the autophagy marker protein LC3 and an increase in the number of autophagic vesicles, significantly activating autophagy. In yeast, GSH deficiency specifically activates mitochondrial autophagy\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e. Therefore, various signaling molecules involved in iron metabolism and lipid peroxidation participate in the regulation of iron metabolism and lipid peroxidation. Studies have shown that the occurrence of ferroptosis requires the involvement of autophagy, and certain regulators of autophagy (such as Beclin-1, Nrf2, STAT3, p53, p62) as well as selective autophagy (such as ferritinophagy, lipophagy, and circadian autophagy) play important roles in the ferroptosis process[26].\u003c/p\u003e \u003cp\u003eBeclin-1 is a key regulator of autophagy and can participate in the regulation of ferroptosis as an inhibitor of system Xc-[27]. AMPK phosphorylates Beclin-1, and phosphorylated Beclin-1 interacts with the solute carrier family 7 member 11 (SLC7A11), a component of the glutamate-cystine reverse transport system Xc-, inhibiting the transport activity of Xc- and promoting ferroptosis. Inhibition of AMPK activity disrupts the interaction between Beclin-1 and SLC7A11, or mutation of the interaction site between Beclin-1 and SLC7A11, can inhibit ferroptosis[28]. Additionally, the ELAV-like RNA-binding protein 1 (ELAVL1) can bind to the 3' untranslated region of Beclin-1 mRNA, maintaining its stability and thereby activating autophagy, leading to enhanced ferritinophagy and promoting ferroptosis\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. These studies suggest that Beclin-1 regulates ferroptosis by modulating autophagy activity.\u003c/p\u003e \u003cp\u003eThe nuclear factor erythroid 2-related factor 2 (Nrf2) is a core protein in the antioxidant stress response of the cellular protective pathway. Under normal physiological conditions, Keap1 (Kelch-like ECH-associated protein 1) interacts with Nrf2, mediating its ubiquitination and proteasomal degradation[30]. Under oxidative stress conditions, Nrf2 trans-activates the SQSTM1 gene encoding the p62 protein, and the accumulation of p62 promotes the p62-dependent autophagic degradation of Keap1, inhibiting Keap1-mediated Nrf2 degradation\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. p62 can competitively bind to Keap1, mediating the autophagic degradation of Keap1 and activating Nrf2, while simultaneously upregulating iron- and ROS-related genes to exert an anti-ferroptotic effect. Furthermore, impaired cellular autophagy leads to the accumulation of p62, oxidized proteins, or damaged organelles, which can also activate Nrf2, thereby inducing the expression of autophagy-related genes such as p62, ATG5, and ULK1, enhancing autophagic activity to remove damaged proteins and organelles\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. As a transcription factor, Nrf2 can also directly regulate the expression of important genes involved in the ferroptosis process, including HO-1, SLC7A11, and GPX4, thereby regulating cellular iron metabolism, GSH levels, GPX4 synthesis, and lipid oxidation. Therefore, Nrf2 plays a crucial role in the regulation of ferroptosis[33].\u003c/p\u003e \u003cp\u003eIn summary, hyperoxia induces ROS generation, upregulates LC3B expression, and stimulates autophagosome formation. Additionally, ROS not only causes oxidative cell damage but also plays an important role in ferroptosis. Studies have shown that the occurrence of ferroptosis requires the involvement of autophagy, and certain regulators of autophagy (such as GPX4, Beclin-1, p62, etc.) play important roles in ferroptosis. Both ferroptosis and autophagy play crucial regulatory roles in the cell proliferation process. Excessive ferroptosis and autophagy promote cell apoptosis; therefore, ferroptosis and autophagy are important for cell proliferation. MSC-derived exosomes have similar functions to MSCs. In this study, we treated hyperoxia-induced ACE II with MSC-derived exosomes. The results showed that MSC-derived exosomes could improve the inhibitory effect of hyperoxia on ACE II proliferation, reduce ROS accumulation and downregulate LC3B expression. Ferroptosis and autophagy were significantly improved, while the addition of ferroptosis inhibitors and autophagy inhibitors increased ferroptosis and autophagy. Therefore, when hyperoxia damages lung epithelial cells, MSC-derived exosomes may alleviate the damaging effects of hyperoxia on ACE II by improving the inhibitory effect of hyperoxia on ACE II, promoting ACE II proliferation, and reducing ROS accumulation to inhibit ferroptosis and autophagy, thereby decreasing cell apoptosis. Thus, MSC-derived exosomes have potential application value in the treatment of hyperoxia-induced acute lung injury. However, research on MSC-derived exosomes is still in its early stages, and many issues need to be addressed, such as the administration route and purification techniques of MSC-derived exosomes. Moreover, most studies focus on cells and genes, and research on the treatment of ALI with MSC-derived exosomes is very limited, with many uncertainties. Nevertheless, numerous related studies have shown the great prospects of using MSC-derived exosomes for the treatment of acute lung injury, and they may become an effective therapeutic approach for acute lung injury. However, there are still many difficulties before their actual clinical application, and more research is needed for further exploration.\u003c/p\u003e"},{"header":"List Of Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eALI\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eacute lung injury\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eBPD\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ebronchopulmonary dysplasia\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eARDS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eacute respiratory distress syndrome\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eACE II\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etype II alveolar epithelial cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eMSCs\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMesenchymal stem cells\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eDCFH-DA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e2,7-dichloro dihydro fluorescein diacetate\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eTEM\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003etransmission electron microscopy\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eNTA\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enanoparticle tracking analysis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003ePBS\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ephosphate-buffered saline\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eHALI\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ehyperoxia-induced acute lung injury\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eSLC7A11\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003esolute carrier family 7 member 11\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eELAVL1\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eELAV-like RNA-binding protein 1\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eNrf2\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003enuclear factor erythroid 2-related factor 2\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e\u003cb\u003eKeap1\u003c/b\u003e\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eKelch-like ECH-associated protein 1.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eEthics approval and consent to participate: Not applicable.\u003c/p\u003e\n\u003cp\u003eConsent for publication: Not applicable.\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials: All data generated or analysed during this study are included in this published article [and its supplementary information files].\u003c/p\u003e\n\u003cp\u003eCompeting interests: The authors declare that they have no competing interests.\u003c/p\u003e\n\u003cp\u003eFunding: The research was supported by: Science and Technology Plan Project of Guizhou Provincial (Qiankehe Fundamentals ZK [2024] General 345); National Natural Science Foundation of China (No. 82160369); Zunyi Medical University［2022］(No.21).\u003c/p\u003e\n\u003cp\u003eAuthors\u0026apos; contributions:\u0026nbsp;GY. L designed the experimental protocol and establish cell models, and was a major contributor in writing the manuscript. GY. J used GO analysis and KEGG enrichment analysis to study the regulatory role of differentially expressed genes in cells, and conducted Western blotting experiments. YC. R performed cell proliferation detection. QX. H and CZ. Y validated the high-throughput sequencing results using quantitative PCR. X. X conducted ROS level detection experiment. H. W conducted immunofluorescence tests. M. C supervised the experiment and revised the manuscript of the paper. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003eAcknowledgements: Not applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWan M, Tajuddin WNB et al. Mechanistic Understanding of Curcumin's Therapeutic Effects in Lung Cancer. Nutrients, 2019. 11(12).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMaturu P, et al. 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Redox Biol. 2019;23:101107.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Mesenchymal stem cell-derived exosomes, Type II alveolar epithelial cells, Hyperoxia, Oxidative stress, Ferroptosis, Autophagy","lastPublishedDoi":"10.21203/rs.3.rs-4538714/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4538714/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eObjective\u003c/strong\u003e To investigate the protective effects of mesenchymal stem cell-derived exosomes on ferroptosis and autophagy in hyperoxia-induced type II alveolar epithelial cell injury.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMethods\u003c/strong\u003e Cells were treated with exosomes under hyperoxic conditions and divided into the following groups: control group (oxygen volume fraction of 0.21), hyperoxia group (oxygen volume fraction of 0.95), hyperoxia+exosome group, hyperoxia+exosome+Fer-1 (10 μmol/L) ferroptosis inhibitor group, and hyperoxia+exosome+3-MA (25 μM) autophagy inhibitor group. High-throughput analysis was performed to analyze the transcriptomic changes in type II alveolar epithelial cells treated with exosomes under hyperoxic exposure. GO analysis and KEGG enrichment analysis were conducted to investigate the regulatory effects of differentially expressed genes in cells. Quantitative PCR was used to verify the high-throughput sequencing results. Cell proliferation was detected by EdU assay. ROS levels were measured by DCFH-DA probe. The expression of ferroptosis factors (GPX4, SLC7A11) and autophagy-related factors (Wnt1, β-catenin, p62, ATG5, Beclin1) was detected by Western blotting. LC3B staining in cells was examined by immunofluorescence.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e Sequencing results showed that exosome treatment caused significant transcriptomic changes in cells compared to the hyperoxia group. Quantitative PCR results confirmed the expression changes of genes such as HSPA1A and NR4A1, consistent with the sequencing results. EdU assay showed that the hyperoxia group significantly decreased EdU positivity compared to the control group, which was alleviated by exosome treatment. Compared to the control group, the hyperoxia group promoted ROS accumulation, while exosome treatment alleviated ROS accumulation. Western blotting results showed that, compared to the control group, the hyperoxia group significantly decreased GPX4 and SLC7A11 expression, while exosome treatment significantly increased GPX4 and SLC7A11 expression. In the hyperoxia+exosome+ferroptosis inhibitor group, GPX4 and SLC7A11 expression were significantly decreased. Immunofluorescence results showed that hyperoxia significantly increased LC3B positivity, while exosome treatment significantly decreased LC3B positivity. In the hyperoxia+exosome+3-MA autophagy inhibitor group, LC3B positivity was significantly increased. Western blotting results showed that the hyperoxia group significantly decreased the expression of Wnt1, β-catenin, and p62, and significantly increased the expression of ATG5 and Beclin1, while the exosome group significantly increased the expression of Wnt1, β-catenin, and p62, and significantly decreased the expression of ATG5 and Beclin1. In the hyperoxia+exosome+3-MA autophagy inhibitor group, the expression of Wnt1, β-catenin, and p62 was significantly decreased, and the expression of ATG5 and Beclin1 was significantly increased.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion\u003c/strong\u003e Mesenchymal stem cell-derived exosomes alleviate hyperoxia-induced damage to alveolar epithelial cells by inducing cell proliferation, alleviating ROS accumulation, inhibiting ferroptosis, and inhibiting autophagy.\u003c/p\u003e","manuscriptTitle":"Investigation of the Protective Effects of Mesenchymal Stem Cell-Derived Exosomes on Hyperoxia-Induced Type II Alveolar Epithelial Cell Injury Based on Ferroptosis and Autophagy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-04 10:58:06","doi":"10.21203/rs.3.rs-4538714/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"46976a66-5672-4c53-bd96-f3e34c56f4de","owner":[],"postedDate":"July 4th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-08-27T06:36:32+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-04 10:58:06","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4538714","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4538714","identity":"rs-4538714","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}