Extracellular Vesicles Derived from Adipose Mesenchymal Stem Cells Alleviate PM2.5-Induced Lung Injury and Pulmonary Fibrosis

Yongheng Gao, Jinbo Sun, Chuan Dong, Mingxuan Zhao, Ying Hu, Faguang Jin, Yongheng Gao, Jinbo Sun, Chuan Dong, Mingxuan Zhao, Ying Hu, Faguang Jin

Abstract

BACKGROUND Exposure to PM2.5 (fine particulate matter ≤2.5 μm in aerodynamic diameter) in air increases the risk of lung injury and pulmonary fibrosis (PF). Extracellular vesicles (EVs) derived from adipose mesenchymal stem cells (ADSCs) have been identified as a potential treatment based on the proteins or RNAs delivery and immunomodulatory properties. Here, we assessed the protective effects and mechanisms of ADSCs-EVs on PM2.5-induced lung injury or PF. MATERIAL AND METHODS Rats (male, 6 weeks old) were exposed to PBS or PM2.5 (1.5 mg/kg/day) for 3 days a week for 4 weeks. ADSCs-EVs were extracted by ultracentrifugation. PBS and ADSCs-EVs were administrated through intratracheal instillation. After the end of exposure, the rats were anesthetized and killed. Lung tissues with different treatments were collected for Western blot analysis and HE, IHC, and IF staining analysis. Cells exposed to PM2.5 or "PM2.5+ADSCs-EVs" in vitro were also collected for further Western blotting, qRT-PCR, and IF staining evaluation. RESULTS The results indicated that the initial response of lungs exposed to PM2.5 was lung injury with oxidative stress and inflammation. Long-term PM2.5 exposure resulted in obvious PF in rats. Treatment with ADSCs-EVs decreased PM2.5-induced apoptosis and necrosis in type II alveolar epithelial cells and alleviated lung injury and PF in rats. ADSCs-EVs suppressed reactive oxygen species (ROS) levels and inflammation induced by PM2.5. Furthermore, ADSCs-EVs inhibited TGF-ßRI by transferring let-7d-5p and further mitigated PF. CONCLUSIONS Our results suggest that EVs derived from ADSCs can alleviate PM2.5-induced lung injury and PF.

Conflict of interest statement

Conflict of interest

None.

Figures

Figure 1
Figure 1
PM2.5 exposure induced lung injury and PF. (A) SEM image of PM2.5. (B) Particles size distribution of PM2.5. (C) HE staining of lungs at 6 h after single exposure to PBS (None) or PM2.5. (D) Relative ROS of cells, TGF-β1 and TNF-α concentrations in BALF at different time points (3, 6, 9, 12, and 24 h after PM2.5 single exposure) were detected. (E) TEM of type II alveolar epithelial cells 6 hours after rats were treated with PBS (None) or PM2.5. (F) Masson staining of lung tissues with treatments of PBS or PM2.5 for 4 weeks (Yellow arrows represented PF). (G) IHC staining of TGF-β1 in lungs after treatments of PBS or PM2.5 for 4 weeks. (* p<0.05; ** p<0.01; *** p<0.001).
Figure 2
Figure 2
The identification of ADSCs and AT II cells. (A) Multilineage differentiation of ADSCs (upper left: osteoblast differentiation, upper middle: adipocyte differentiation, upper right: cartilage differentiation) and typical image of ADSCs (lower). (B) SP-C immunofluorescence staining for AT II cells.
Figure 3
Figure 3
ADSCs-EVs alleviated lung injury induced by PM2.5. (A) TEM identification of ADSCs-EVs. (B) NTA analysis of ADSCs-EVs. (C) GM130, Alix, TSG101, and CD63 expressions in ADSCs-EVs and cell lysates were detected by Western blotting. (D) The uptake of ADSCs-EVs in ATII cells was detected by a confocal microscopy. (E) Apoptosis or necrosis of ATII cells with treatments of PBS (None), ADSCs-EVs, PM2.5, or PM2.5+ADSCs-EVs was detected by flow cytometry assay. (F) Six hours after the rats treated with PBS (None), ADSCs-EVs, PM2.5 or PM2.5+ADSCs-EVs, relative ROS level in BALF was measured. (G) Six hours after the rats treated with PBS (None), ADSCs-EVs, PM2.5, or PM2.5+ADSCs-EVs, TNF-α concentration in BALF was evaluated. (H) HE staining of lungs exposed to PBS (None), ADSCs-EVs, PM2.5 or PM2.5+ADSCs-EVs. (* p<0.05; ** p<0.01; *** p<0.001).
Figure 4
Figure 4
ADSCs-EVs alleviated PF induced by PM2.5. Masson staining, IHC staining for TGF-β1, IF staining for TGF-βRI and TGF-βRII in lungs exposed to PBS (None), ADSCs-EVs, PM2.5, or PM2.5+ADSCs-EVs for 4 weeks. (* p

Figure 5

ADSCs-EVs repressed TGF-βRI expression. (…

Figure 5

ADSCs-EVs repressed TGF-βRI expression. ( A ) Western blotting analysis of TGF-β1, TGF-βRI,…

Figure 5
ADSCs-EVs repressed TGF-βRI expression. (A) Western blotting analysis of TGF-β1, TGF-βRI, and TGF-βRII levels in ATII cells under different treatments. (B) The miRNA levels in ATII cells with different treatments were measured by qRT-PCR. (* p<0.05; ** p<0.01; *** p<0.001).

Figure 6

let-7d-5p directly inhibited the expression…

Figure 6

let-7d-5p directly inhibited the expression of TGF-βRI. ( A ) The expression of…

Figure 6
let-7d-5p directly inhibited the expression of TGF-βRI. (A) The expression of TGF-βRI under different treatments was tested by Western blotting. (B) The expression of TGF-βRI mRNA under different treatments was tested by qRT-PCR. (C) Schematic representation of matching sequence between TGF-βRI 3′UTR mRNA and let-7d-5p. (D) Luciferase activity of ATII cells co-transfected with reporter plasmid (“TGF-βRI wt” or “TGF-βRI mt”) and let-7d-5p or miRNA-NC. (* p<0.05; ** p<0.01; *** p<0.001).
Figure 5
Figure 5
ADSCs-EVs repressed TGF-βRI expression. (A) Western blotting analysis of TGF-β1, TGF-βRI, and TGF-βRII levels in ATII cells under different treatments. (B) The miRNA levels in ATII cells with different treatments were measured by qRT-PCR. (* p<0.05; ** p<0.01; *** p<0.001).
Figure 6
Figure 6
let-7d-5p directly inhibited the expression of TGF-βRI. (A) The expression of TGF-βRI under different treatments was tested by Western blotting. (B) The expression of TGF-βRI mRNA under different treatments was tested by qRT-PCR. (C) Schematic representation of matching sequence between TGF-βRI 3′UTR mRNA and let-7d-5p. (D) Luciferase activity of ATII cells co-transfected with reporter plasmid (“TGF-βRI wt” or “TGF-βRI mt”) and let-7d-5p or miRNA-NC. (* p<0.05; ** p<0.01; *** p<0.001).

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