Human breast milk-derived exosomes through inhibiting AT II cell apoptosis to prevent bronchopulmonary dysplasia in rat lung

Abstract

Human breast milk (HBM) effectively prevents and cures neonatal bronchopulmonary dysplasia (BPD). Exosomes are abundant in breast milk, but the function of HBM-derived exosomes (HBM-Exo) in BPD is still unclear. This study was to investigate the role and mechanism of HBM-Exo in BPD. Overall lung tissue photography and H&E staining showed that HBM-Exo improved the lung tissue structure collapse, alveolar structure disorder, alveolar septum width, alveolar number reduction and other injuries caused by high oxygen exposure. Immunohistochemical results showed that HBM-Exo improved the inhibition of cell proliferation and increased apoptosis caused by hyperoxia. qPCR and Western blot results also showed that HBM-Exo improved the expression of Type II alveolar epithelium (AT II) surface marker SPC. In vivo study, CCK8 and flow cytometry showed that HBM-Exo improved the proliferation inhibition and apoptosis of AT II cells induced by hyperoxia, qPCR and immunofluorescence also showed that HBM-Exo improved the down-regulation of SPC. Further RNA-Seq results in AT II cells showed that a total of 88 genes were significantly different between the hyperoxia and HBM-Exo with hyperoxia groups, including 24 up-regulated genes and 64 down-regulated genes. KEGG pathway analysis showed the enrichment of IL-17 signalling pathway was the most significant. Further rescue experiments showed that HBM-Exo improved AT II cell damage induced by hyperoxia through inhibiting downstream of IL-17 signalling pathway (FADD), which may be an important mechanism of HBM-Exo in the prevention and treatment of BPD. This study may provide new approach in the treatment of BPD.

Keywords: AT II; FADD; HBM-Exo; IL-17; apoptosis; bronchopulmonary dysplasia.

FIGURE 1

Characterization of human breast milk (HBM)‐Exo. (A) Nano‐Sight analysis was used to determine the average size and strength of exosomes. (B) Representative microscope images of HBM‐Exos. The sample was fixed and analysed using electron microscopy. The scale is 100 nm; (C) expression of CD63, HSP70 and CD9 markers in HBM‐Exos by Western blot. Unpaired t‐test, *p < 0.05, **p < 0.01, ***p < 0.001. Exo: HBM‐Exo, Sup: Supernatant. *VS Sup

FIGURE 2

Human breast milk (HBM)‐Exo Function in Animal Model. (A) Fluorescence imaging of a mouse model at 12 h post‐administration of DiR‐labelled HBM‐Exo. (B) The body weight of control, hyperoxia and hyperoxia with HBM‐Exo treatment group. (C) The morphology of lung tissue in the control, hyperoxia and hyperoxia with HBM‐Exo treatment group. (D) Histopathological changes in the lung by H&E, quantification of mean linear intercept (MLI) represents a surrogate of average air space diameter, quantification of mean alveolar number (MAN) represents the average number of alveoli, data represent results from 6 individual studies. Micrographs are representative and were obtained at the same magnification. (E) Representative images of Ki67 immunostaining showing the proliferation and the C‐caspase3 immunostaining showing the apoptosis (brown staining) in the control, hyperoxia and hyperoxia with HBM‐Exo treatment group. Micrographs are representative and were obtained at the same magnification. (F) SPC mRNA relative expression in the control, hyperoxia and hyperoxia with HBM‐Exo treatment group, data represent results from 3 individual studies. (G) SPC protein relative expression in the control, hyperoxia and hyperoxia with HBM‐Exo treatment group, data represent results from 6 individual studies. The pictures shown are representative. Unpaired t‐test, */#p < 0.05,**/##p < 0.01, ***/###p < 0.001. Con: control, Exo: HBM‐Exo, Hyp: Hyperoxia, C‐Caspas: cleaved‐caspase. * VS Con, # VS Hyp. The concentration of HBM‐Exo: 200 μg/ml protein

FIGURE 3

Human breast milk (HBM)‐Exo Function in the AT II cells. (A) Cellular internalization of HBM‐Exo in MLE‐12 cells. The PKH26‐labelled HBM‐Exo were incubated with MLE‐12 cells for 6 h (red) and stained with DAPI (blue). Original magnification: 200x. (B) Proliferation curves of MLE‐12 cells co‐cultured with HBM‐Exos, data represent results from 3 individual studies. (C) The apoptosis rates of MLE‐12 cells following co‐culture with HBM‐Exo, data represent results from 3 individual studies. (D) qPCR evaluate the relative expression of SPC mRNA, data represent results from 3 individual studies. (E) Immunofluorescence evaluate the expression of SPC protein. Original magnification: 200x. The pictures shown are representative. Unpaired t‐test, */#p < 0.05,**/##p < 0.01, ***/###p < 0.001. Con: control, Exo: HBM‐Exo, Hyp: Hyperoxia, * VS Con, # VS Hyp. The concentration of HBM‐Exo: 15.4 μg/ml protein

FIGURE 4

General Properties of the differentially expressed genes. (A) The number of DEGs. (B) Heatmap analysis of the genes. (C) Scatter plot showing the significantly changed genes identified in two groups. (D) Volcano plot showing the significantly changed genes identified in two groups. (Red dots indicate significantly up‐regulated genes; blue dots indicate significantly down‐regulated genes)

FIGURE 5

GO and KEGG Pathways Analysis. (A) Biological process. (B) Cellular component (C) Molecular function (D) KEGG Pathway analysis

FIGURE 6

Human breast milk (HBM)‐Exo Decreased the FADD and Apoptosis Expression. (A) FADD protein expression in clinical lung tissue samples by Western blot; (B) FADD protein expression in rats by Western blot; (C) FADD protein expression in cells by Western blot; (D) Protein expression of apoptosis‐related C‐Caspase3 and C‐Caspase9 in cells by Western blot. Data represent results from 3 individual studies. The pictures shown are representative. Unpaired t‐test, */#p < 0.05,**/##p <.01, ***/###p < 0.001. Con: control, Exo: HBM‐Exo, Hyp: Hyperoxia, C‐Caspas: cleaved‐caspase. * VS Con, # VS Hyp. The concentration of HBM‐Exo: 15.4μg/ml protein

FIGURE 7

Activation of IL‐17 signalling pathway inhibits the mechanism improvement of human breast milk (HBM)‐Exo. (A) IL‐17 and FADD protein expression in control, hyperoxia and hyperoxia with HBM‐Exo treatment group, as well as hyperoxia with HBM‐Exo treatment plus IL‐17 cytokines group by Western blot. (B) Protein expression of the apoptotic‐related C‐Caspase3 and C‐Caspase9 in control, hyperoxia, hyperoxia with HBM‐Exo treatment group, as well as hyperoxia with HBM‐Exo treatment plus IL‐17 cytokines group by western blot. Data represent results from 3 individual studies. The pictures shown are representative. Unpaired t‐test, */#p < 0.05,**/##p < 0.01, ***/###p < 0.001. Con: control, Exo: HBM‐Exo, Hyp: Hyperoxia, C‐Caspas: cleaved‐caspase. * VS Con, # VS BPD. The concentration of HBM‐Exo: 15.4μg/ml protein

FIGURE 8

Activation of IL‐17 signalling pathway inhibits the function improvement of human breast milk (HBM)‐Exo. (A) Cell proliferation in the control, hyperoxia, hyperoxia with HBM‐Exo treatment group, as well as hyperoxiawith HBM‐Exo treatment plus IL‐17 cytokines group by CCK8 kit; (B) SPC protein expression in the control, hyperoxia and hyperoxia +HBM‐Exo treatment group, as well as hyperoxia with HBM‐Exo treatment plus IL‐17 cytokines group by immunoflurescenc. (C) Cell apoptosis in the control, hyperoxia, and hyperoxia with HBM‐Exo treatment group, as well as hyperoxia with HBM‐Exo treatment plus IL‐17 cytokines group by flow cytometry. Data represent results from 3 individual studies. The pictures shown are representative. Unpaired t‐test, */#p < 0.05, **/##p < 0.01, ***/###p < 0.001. Con: control, Exo: HBM‐Exo, Hyp: Hyperoxia, C‐Caspas: cleaved‐caspase. * VS Con, # VS BPD. The concentration of HBM‐Exo: 15.4 μg/ml protein