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
Authors: Liu Guoyue , Jia Guiyang , Ren yingcong , Huang qianxia , Yin Cunzhi , Xiao Xuan , Wu Hang , Chen Miao 
Published: July 2024

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.