Glucagon-like peptide-1 receptor signaling activation in alveolar type II cells enhances lung development in neonatal rats exposed to hyperoxia

Figures

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  Fig. 1. Hyperoxia exposure leads to the change of GLP-1R level in lung tissues and lung histological changes. (A) Venn diagram of five differentially expressed genes at 1, 3, 7, 10, and 14 days after birth. (B) Log2FC of five differentially expressed genes at 1, 3, 7, 10, and 14 days after birth. (C–D) Western blot and analysis of GLP-1R in the lung tissues in the control group and hyperoxia group at 3, 7, 10, and 14 days after birth. (E) mRNA level and analysis of GLP-1R in the lung tissues in the control group and hyperoxia group at 3, 7, 10, and 14 days after birth. (F) HE staining of the lung tissues of neonatal rats was observed at 3, 7, 10, and 14 days after birth using microscopy (400 magnification; scale bar, 50 μm). Lung morphology changes were evaluated by (G) RAC value and (H) alveolar wall thickness. The data are expressed as mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 compared with control group.

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  Fig. 2. The expression of GLP-1R in lung development after birth. (A–B) Western blot and analysis of GLP-1R in the lung tissues after birth in the control group. (C) HE staining of the control and KD-GLP-1R groups of lung tissues at 14 days after birth using microscopy (400 magnification; scale bar, 50 μm). Lung morphology changes were evaluated by (D) alveolar wall thickness and (E) RAC value. (F) Secretion of TGF-β1, VEGF-A, and BMP4 in lung tissues between the control and KD-GLP-1R groups tested by ELISA. (G–H) Western blot analysis of GLP-1R levels in fetal lung tissues under normoxia. The data are expressed as mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 compared with the control group.

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  Fig. 3. The role of GLP-1R in ATII cells measured by single-cell sequencing. (A) tSNE of all cells from Control and GLP-1R lungs. (B) Pie charts of Control and GLP-1R lung different cell type proportions. (C) tSNE of all cells from Control and GLP-1R lung epithelial cells. (D) Pie charts of Control and GLP-1R lung different epithelial cell type proportions. (E) GLP-1R gene expression level in ATI and ATII cells. (F–J) Lung development associated genes level in ATII cells of Control and GLP-1R knocked-down groups. (K–L) Cell apoptosis ratio of control, and KD-GLP-1R groups tested by flow cytometer. (M) Proliferation capacity of control, and KD-GLP-1R groups tested by CCK8. The data are expressed as mean ± SD. ∗∗P < 0.01, ∗∗∗∗P < 0.0001 vs. control.

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  Fig. 4. The expression of GLP-1R in primary ATII cells of rats with BPD. (A–B) Western blot analysis of GLP-1R in primary ATII cells of rats with BPD in the control and hyperoxia groups at 3, 7, 10, and 14 days after birth. (C) mRNA level and analysis of GLP-1R in primary ATII cells of rats with BPD in the control and hyperoxia groups at 3, 7, 10, and 14 days after birth. (D–E) Representative immunostaining of lung sections and ATII cell slides from the control and hyperoxia groups using anti-GLP-1R and anti-SPC. Yellow puncta denote co-localization pointed by white arrows ( × 400 magnification; scale bar, 50 μm). The data are expressed as mean ± SD. ∗P < 0.05, ∗∗P < 0.01 vs. control.

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  Fig. 5. Activation of GLP-1R signaling in ATII cells promotes lung development. (A) HE staining of the control, hyperoxia, and OE-GLP-1R groups of lung tissues at 14 days after birth using microscopy (400 magnification; scale bar, 50 μm). Lung morphology changes were evaluated by (B) alveolar wall thickness and (C) RAC value. The data are expressed as mean ± SD. ∗P < 0.05, ∗∗P < 0.01 compared with control group. (D–F) Secretion of TGF-β1, VEGF-A, and BMP4 in lung tissues between control, hyperoxia and OE-GLP-1R groups tested by ELISA. (G–H) Cell apoptosis ratio of control, hyperoxia, and OE-GLP-1R groups tested by flow cytometer. (I) Proliferation capacity of control, hyperoxia, and OE-GLP-1R groups tested by CCK8. The data are expressed as mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 vs. control.

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  Fig. 6. Mitochondria metabolic reprogramming under hyperoxia and regulated by GLP-1R. (A) ATP concentration in the primary ATII cells at 14 days after birth in the hyperoxia group was decreased and alleviated in the GLP-1R overexpression group. (B–C) ECAR value was elevated in hyperoxia group compared with control group, and decrease in the GLP-1R overexpression group. The OCR was decreased in hyperoxia group in contrast with control group, and elevated in the GLP-1R overexpression group. (D) MitoTracker Red CMXRos staining exhibited that the fragmentation of mitochondria was improved in GLP-1R overexpression group (white arrows, 1000 magnification; scale bar, 10 μm). (E–G) Western blot analyses of DRP1, PFKM, HK2, and LDHA in primary ATII cells at 14 days after birth. The data are expressed as mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 vs. control.

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  Fig. 7. m6A methylated modification of GLP-1R mRNA in RLE-6TN cells. (A) mRNA level and analysis of GLP-1R in RLE-6TN cells in the control group and hyperoxia group at 24, 48, and 72 h after exposed to hyperoxia. (B) m6A modification site of GLP-1R confirmed by MeRIP-qPCR. (C) Heat map of m6A modification associated proteins. (D–G) mRNA level and analysis of METTL3, METTL14, WTAP, and VIRMA in RLE-6TN cells in the control and hyperoxia group at 48 h after knockdown by siRNA. (H) mRNA level and analysis of GLP-1R in RLE-6TN cells in the hyperoxia group at 48 h after knockdown of METTL3, METTL14, WTAP, and VIRMA. (I) Relative luciferase activity of psiCHECK2 with either wild-type or mutant m6A sites after co-transfection with pCDNA3.1/pCDNA3.1-METTL14 into HEK-293T cells. Renilla luciferase activity was measured and normalized to that of Firefly luciferase. The data are expressed as mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001 vs. control.