FGF10 ameliorates lipopolysaccharide-induced acute lung injury in mice via the BMP4-autophagy pathway

Abstract

Introduction: Damage to alveolar epithelial cells caused by uncontrolled inflammation is considered to be the main pathophysiological change in acute lung injury. FGF10 plays an important role as a fibroblast growth factor in lung development and lung diseases, but its protective effect against acute lung injury is unclear. Therefore, this study aimed to investigate protective effect and mechanism of FGF10 on acute lung injury in mice. Methods: ALI was induced by intratracheal injection of LPS into 57BL/6J mice. Six hours later, lung bronchoalveolar lavage fluid (BALF) was acquired to analyse cells, protein and the determination of pro-inflammatory factor levels, and lung issues were collected for histologic examination and wet/dry (W/D) weight ratio analysis and blot analysis of protein expression. Results: We found that FGF10 can prevent the release of IL-6, TNF-α, and IL-1β, increase the expression of BMP4 and autophagy pathway, promote the regeneration of alveolar epithelial type Ⅱ cells, and improve acute lung injury. BMP4 gene knockdown decreased the protective effect of FGF10 on the lung tissue of mice. However, the activation of autophagy was reduced after BMP4 inhibition by Noggin. Additionally, the inhibition of autophagy by 3-MA also lowered the protective effect of FGF10 on alveolar epithelial cells induced by LPS. Conclusions: These data suggest that the protective effect of FGF10 is related to the activation of autophagy and regeneration of alveolar epithelial cells in an LPS-induced ALI model, and that the activation of autophagy may depend on the increase in BMP4 expression.

Keywords: AECs; BMP4; FGF10; acute lung injury; autophagy.

FIGURE 1

LPS treatment results in alveolar injury and increases FGF10 expression in mice. (A) Pathological change in lung tissue was detected by H&E (200×). (B) Quantification analysis of wet/dry weight ratio. (C–E) The expression of IL-6, TNF-α, and IL-1β in BALF were determined by ELISA. (F) The expression of FGF10 and FGFR2B were detected by Western blotting. (G) Quantitative analysis of FGF10 and FGFR2B protein. The data were presented as the mean ± SEM, n = 5. *p < 0.05, * *p < 0.01.

FIGURE 2

FGF10 attenuates LPS-induced structural damage and inflammatory mediator production in mice. (A) Pathological change in lung tissue was detected by H&E (200×). (B) Quantitative analysis of wet/dry weight ratio. (C) BAL fluid was obtained to differentially count total cells.(D–F) The expression of IL-6, TNF-α, and IL-1β in BALF were determined by ELISA. The data were presented as the mean ± SEM, n = 5. *p < 0.05, * *p < 0.01.

FIGURE 3

FGF10 promotes autophagy and the regeneration of alveolar epithelial cells in LPS-induced lung injury. (A) The expression of LC3, P62, and SPC in mice was detected by Western blotting. (B–D) Quantitative analyses of LC3, P62, and SPC protein. (E) The viability of AECs with different concentrations of LPS administration was detected by CCK-8. (F) After exposure to LPS and FGF10, the viability of AECs were detected by CCK-8. (G) The expression of LC3, P62, and SPC in AECs was detected by Western blotting. (H–J) Quantitative analysis of LC3, P62, and SPC protein. (K) The expression of LC3 (green) of AECs was analyzed by immunostaining (magnification ×400). The data were presented as the mean ± SEM, n = 3–5. *p < 0.05, * *p < 0.01.

FIGURE 4

FGF10 promotes the expression of BMP4. (A) The expression of BMP4 in mice was detected by Western blotting. (B) Quantitative analysis of BMP4 protein. (C) The expression of BMP4 in AECs was detected by Western blotting. (D) Quantitative analysis of BMP4 protein. (E) BMP4 expression (green) of AECs was analyzed by immunostaining (magnification ×400). The data were presented as the mean ± SEM, n = 3–5. *p < 0.05, * *p < 0.01.

FIGURE 5

BMP4 knockdown weakens the protective effect of FGF10 on acute lung injury by autophagy. (A) The expression of BMP4 in mice was detected by Western blotting. (B) Quantitative analysis of BMP4 protein. (C) Pathological change in lung tissue was detected by H&E (200×). (D) Quantitative analysis of wet/dry weight ratio. (E) BAL fluid was obtained to differentially count total cells. (F–H) The expression of IL-6, TNF-α, and IL-1β in BALF fluid was analyzed by ELISA. (I,J) The expression of LC3, P62, and SPC in mice was detected by Western blotting. (K–M) Quantitative analysis of LC3, P62, and SPC protein. The data were presented as the mean ± SEM, n = 5. *p < 0.05, * *p < 0.01.

FIGURE 6

Inhibition of BMP4 decreases the protective role of FGF10 on AECs via autophagy. (A) After exposure to 200 ng/ml of Noggin for 0.5 h and 10 ng/ml of FGF10 for 1 h, the viability of AECs with 10 μg/ml LPS administration was detected by CCK-8. (B) The protein expression of LC3, P62, BMP4, and SPC was examined by Western blotting. (C–F) Quantitative analyses of LC3, P62, BMP4, and SPC protein. (G,H) The expression of LC3 (green) and BMP4 (green) of AECs were analyzed by immunostaining (magnification ×400). The data were presented as the mean ± SEM, n = 3. *p < 0.05, * *p < 0.01.

FIGURE 7

Inhibition of autophagy exacerbates cytotoxicity induced by LPS in AECs. (A) After exposure to 2.5 mM of 3-MA for 2 h and 10 ng/ml of FGF10 for 1 h, the viability of AECs with the administration of 10 μg/ml of LPS was detected by CCK-8. (B) The protein expression of LC3, P62, BMP4, and SPC was examined by Western blotting. (C–F) Quantitative analyses of LC3, P62, BMP4, and SPC protein. (G,H) The expression of LC3 (green) and BMP4 (green) of AECs was analyzed by immunostaining (magnification ×400). The data are presented as the mean ± SEM, n = 3. *p < 0.05, * *p < 0.01.