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. 2023 Apr 14;13(1):6126.
doi: 10.1038/s41598-023-33151-w.

EPC-exosomal miR-26a-5p improves airway remodeling in COPD by inhibiting ferroptosis of bronchial epithelial cells via PTGS2/PGE2 signaling pathway

Caihong Liu #  1 , Junjuan Lu #  1 , Ting Yuan  2 , Lihua Xie  1 , Li Zhang  3
Affiliations

EPC-exosomal miR-26a-5p improves airway remodeling in COPD by inhibiting ferroptosis of bronchial epithelial cells via PTGS2/PGE2 signaling pathway

Caihong Liu et al. Sci Rep. .

Abstract

We aimed to investigate whether exosomes (Exo) affected chronic obstructive pulmonary disease (COPD) by influencing ferroptosis of bronchial epithelial cells (BECs) and the mechanisms involved. Here we took the peripheral blood samples of normal subjects and COPD patients, extracted and identified endothelial progenitor cells (EPCs) and EPC-Exo. An animal model of COPD was established. Then human BECs were taken and treated with cigarette smoke extract (CSE) for 24 h to construct a COPD cell model. Next, we screened differentially expressed ferroptosis-related genes in COPD patients by bioinformatics. Bioinformatics predicted the miRNA targeting PTGS2. Then, the mechanism of action of miR-26a-5p and Exo-miR-26a-5p was investigated in vitro. We successfully isolated and identified EPC and Exo. In vitro, EPC alleviated CSE-induced ferroptosis in BECs by transporting Exo. In vivo, Exo alleviated cigarette smoke-induced ferroptosis and airway remodeling in mice. Through further validation, we found that CSE-induced ferroptosis promoted the epithelial-mesenchymal transition (EMT) of BECs. Bioinformatics analysis and validation showed that PTGS2/PGE2 pathway affected CSE-induced ferroptosis in BECs. Meanwhile, miR-26a-5p targeting PTGS2 affected CSE-induced ferroptosis in BECs. Additionally, we found that miR-26a-5p affected CSE-induced BECs EMT. Exo-miR-26a-5p alleviated CSE-induced ferroptosis and EMT. In conclusion, EPC-exosomal miR-26a-5p improved airway remodeling in COPD by inhibiting ferroptosis of BECs via the PTGS2/PGE2 pathway.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
EPC alleviated CSE-induced ferroptosis in BECs by transporting Exo. (A) The concentration of Exo in the cell supernatant was evaluated by ELISA. *P < 0.05 vs. EPC + DMSO. (B) MDA and SOD levels in the cell supernatant were determined by ELISA. (C) The relative content of catalytic Fe (II) in BECs was detected by FCM. (D) Western blot detection of ferroptosis markers TfR, FtL, and GPX4 expressions in BECs. *P < 0.05 vs. Control, #P < 0.05 vs. CSE, &P < 0.05 vs. EPC + DMSO. The data were presented in the form of mean ± standard deviation. n = 3.
Figure 2
Figure 2
Exo alleviated ferroptosis induced by CS in vivo. (A) FCM detection of ROS content in lung tissue. (B) ELISA detection of MDA and SOD in lung tissue. (C) The relative content of catalytic Fe (II) in lung tissue was detected by FCM. (D) The relative expression of ferroptosis markers TfR, FtL, and GPX4 in lung tissue was evaluated by western blot. *P < 0.05 vs. Control, #P < 0.05 vs. Model. The data were presented in the form of mean ± standard deviation. n = 5 mice/group.
Figure 3
Figure 3
Exo alleviated CS-induced airway remodeling in mice. (A) Masson staining of the bronchus. (B) Expression of TNF-α, IL-1β, IL-6, HMGB1 and TGF-β in BALF were determined by ELISA. (C) Western blot detection of Vimentin, E-cadherin, and ZO-1 expressions. *P < 0.05 vs. Control, #P < 0.05 vs. Model. The data were presented in the form of mean ± standard deviation. n = 5 mice/group.
Figure 4
Figure 4
CSE-induced Ferroptosis promoted oxidative stress and EMT in BECs. (A) FCM detection of ROS content. (B) MDA and SOD levels. (C) The content of catalytic Fe (II) was detected by FCM. (D) The expression of ferroptosis markers TfR, FtL, and GPX4 was utilized by western blot. (E) The protein expression of EMT markers Vimentin, E-cadherin, and ZO-1. (F) IF staining of EMT markers Vimentin and E-cadherin. *P < 0.05 vs. Control, #P < 0.05 vs. CSE. The data were presented in the form of mean ± standard deviation. n = 3.
Figure 5
Figure 5
PTGS2/PGE2 signaling pathway affected CSE-induced ferroptosis in BECs. (A) Screening of DEGs. Red dots are differentially expressed mRNAs, and blue dots are stable genes. (B) Venn diagrams of DEGs and ferroptosis-related genes. (C) The cluster heatmap visualization of ferroptosis-related differential genes (drawn with R language). (D) qRT-PCR verification of PTGS2, HMOX1, and MT1G expression. (E) PTGS2 expression was determined by qRT-PCR and western blot. (F) ELISA detection of PGE2, and lipid peroxidation MDA and SOD. (G) The content of catalytic Fe (II) was detected by FCM. (H) The protein expression of ferroptosis markers TfR, FtL, and GPX4. *P < 0.05 vs. Control, #P < 0.05 vs. CSE, &P < 0.05 vs. CSE + NC. The data were presented in the form of mean ± standard deviation. n = 3.
Figure 5
Figure 5
PTGS2/PGE2 signaling pathway affected CSE-induced ferroptosis in BECs. (A) Screening of DEGs. Red dots are differentially expressed mRNAs, and blue dots are stable genes. (B) Venn diagrams of DEGs and ferroptosis-related genes. (C) The cluster heatmap visualization of ferroptosis-related differential genes (drawn with R language). (D) qRT-PCR verification of PTGS2, HMOX1, and MT1G expression. (E) PTGS2 expression was determined by qRT-PCR and western blot. (F) ELISA detection of PGE2, and lipid peroxidation MDA and SOD. (G) The content of catalytic Fe (II) was detected by FCM. (H) The protein expression of ferroptosis markers TfR, FtL, and GPX4. *P < 0.05 vs. Control, #P < 0.05 vs. CSE, &P < 0.05 vs. CSE + NC. The data were presented in the form of mean ± standard deviation. n = 3.
Figure 6
Figure 6
miR-26a-5p targeting PTGS2 affected CSE-induced ferroptosis in BECs. (A) Targeting sequences of miR-26a-5p and PTGS2. (B) Dual-luciferase verification of miR-26a-5p and PTGS2 relationship. (C) miR-26a-5p and PTGS2 mRNA expression, and PTGS2 protein expression. (D) ELISA detection of PGE2, lipid peroxidation MDA and SOD. (E) The content of catalytic Fe (II) was evaluated by FCM. (F) The protein expression of ferroptosis markers TfR, FtL, and GPX4. *P < 0.05 vs. NC, #P < 0.05 vs. miR-26a-5p mimics, &P < 0.05 vs. miR-26a-5p mimics + vector. The data were presented in the form of mean ± standard deviation. n = 3.
Figure 7
Figure 7
miR-26a-5p affected CSE-induced BECs EMT. (A) miR-26a-5p mRNA expression. (B) Western blot detection of EMT markers Vimentin, E-cadherin, and ZO-1. (C) IF staining of EMT markers Vimentin and E-cadherin. *P < 0.05 vs. Control, #P < 0.05 vs. CSE, &P < 0.05 vs. CSE + NC. The data were presented in the form of mean ± standard deviation. n = 3.
Figure 8
Figure 8
Exo-miR-26a-5p alleviated CSE-induced ferroptosis and EMT. (A) qRT-PCR verification of miR-26a-5p expression. *P < 0.05 vs. EPC. (B) Exo uptake assay. (C) miR-26a-5p mRNA expression. *P < 0.05 vs. Exo, #P < 0.05 vs. Exo-NC inhibitor. (D) miR-26a-5p mRNA expression. (E) ELISA detection of PGE2 expression. (F) The expressions of PTGS2 and ferroptosis markers TfR, FtL, and GPX4 were detected by western blot. G. EMT markers Vimentin, E-cadherin, and ZO-1 protein expression of BECs. *P < 0.05 vs. CSE, #P < 0.05 vs. CSE + Exo, &P < 0.05 vs. CSE + Exo-NC inhibitor. The data were presented in the form of mean ± standard deviation. n = 3.
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