FGF1 alleviates LPS-induced acute lung injury via suppression of inflammation and oxidative stress

Abstract

Background: Acute lung injury (ALI) and its severe form, acute respiratory distress syndrome (ARDS), are devastating clinical disorders with high mortality, and for which more effective therapies are urgently needed. FGF1, the prototype member of the FGF family, is shown to exert protective effects against injurious stimuli in multiple disease models. Here we aimed to evaluate whether FGF1 pretreatment is protective against LPS-induced ALI and elucidate the potential underlying mechanisms.

Methods: For drug-treated groups, C57B/6 mice received a single i.p. injection of FGF1 (1 mg/kg) 1 h before the LPS challenge or not. To induce the ALI model, the mice were treated by intratracheal instillation of LPS (5 mg/kg). Then, histopathological changes in lung tissues were assessed by hematoxylin and eosin staining and transmission electron microscopy. ELISA and qPCR assays were used to detect pro-inflammatory cytokine levels in BALF and lung tissues, respectively. The total number of inflammatory cells (neutrophils and macrophages) in BALF were counted using the Wright-Giemsa method. The expressions of reactive oxygen species (ROS) and malondialdehyde (MDA) were measured using their respective kits. Western blot and immunostaining were used to evaluate the expressions of antioxidants (Nrf-2, HO-1, SOD2, GPX4, and Catalase), as well as the inflammatory and/or apoptosis-related factors (TLR4, NF-κB, and Cleaved- caspase 3).

Results: FGF1 pretreatment significantly ameliorated the LPS-induced histopathological changes, reduced lung wet/dry ratios, ROS and MDA levels, total BALF protein, inflammatory cell infiltration, proinflammatory cytokine levels, and significantly increased the expression of antioxidant proteins (Nrf-2, HO-1, Catalase, and SOD2). In addition, FGF1 pretreatment significantly reduced the expression of TLR4 and cleaved- caspase 3, inhibited NF-κB activation, and reduced LPS-induced cell apoptosis.

Conclusions: Altogether, our results suggest that FGF1 pretreatment is protective against LPS-induced ALI through mediating anti-inflammatory and antioxidant effects, which may be attributed to the downregulation of TLR4 expression and inhibition of NF-κB activation, as well as promotion of antioxidant defenses. Therefore, FGF1 administration may prove beneficial in preventative strategies for ALI/ARDS.

Keywords: Acute lung injury; FGF1; Inflammation; Lipopolysaccharide; NF-κB; Nrf2; Oxidative stress; TLR4.

Fig. 1

FGF1 pretreatment protects against LPS-induced ALI. A H&E staining of lung sections at 12 and 24 h after LPS stimulation, magnification (10 ×). B Histological mean lung injury scores from 28 lung sections (n = 4 per group). C, D Total BAL protein and lung wet/dry weight ratios were evaluated at 12 h post LPS treatment. E, F The mRNA expression of TNF- α, IL-6 and IL-1β were determined at 12- and 24 h time points. Data are presented as mean ± SD (n = 4 per group). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001; One-way or Two-way ANOVA with Tukey post hoc-analysis were applied where appropriate

Fig. 2

FGF1 pretreatment ameliorates LPS-induced oxidative stress via promoting antioxidant defenses. Effects of FGF1 pretreatment on ROS (A), MDA (B), in RAW264.7 cells and mice lung tissues, respectively; scale bar = 64 μm. C, D Immunohistochemistry and WB analysis of SOD2 in lung tissues; scale bar = 64 μm. E, F WB analysis of Nrf2, HO-1 in mice, and cells incubated with varying concentrations of FGF1, respectively. All data were analyzed at 12 h after LPS treatment. Data are presented as mean ± SD (n = 5 per group). *p < 0.05, **p < 0.01; One-way ANOVA with Tukey’s post-hoc test

Fig. 3

FGF1 pretreatment dampens LPS-induced inflammatory cell infiltration and cytokine production. A Inflammatory cell infiltration was quantified by counting the total number of inflammatory cells, neutrophils, and macrophages in BALF using the Wright-Giemsa method (red arrows indicate the neutrophils and black arrows indicate the macrophages). Scale bar: 64 μm, magnification: 40×. B Representative immunofluorescence analysis of A549 cells for TNF-α expression, (scale bar = 64 μm, magnification: 10×). C WB analysis and quantification of the relative protein expression of murine IL-6, IL-1β, and TNF-α; β-actin was used for normalization. Data are presented as mean ± SD (n = 3 to 5 per group). *p < 0.05, **p < 0.01; One-way ANOVA with Tukey’s post-hoc test

Fig. 4

FGF1 pretreatment inhibits NF-κB nuclear translocation. A Immunofluorescence analysis and quantification of NF-κB p65 nuclear expression in A459 cells across different groups (scale bar = 64 μm). The fluorescence levels were quantified using ImageJ software. Corrected Total cellular Fluorescence (CTCF) = integrated density (area of selected region × mean fluorescence of background readings). B The relative protein expression levels of nuclear NF-κB p65, Lamin B, IκBα, p-IκBα were analyzed by western blot. Bar graphs show the quantification of protein expression levels normalized to GAPDH. All data were analyzed at 12 h after LPS treatment. Data are presented as mean ± SD (n = 3 to 5 per group). **p < 0.01, ***p < 0.001; One-way ANOVA with Tukey’s post-hoc test

Fig. 5

The effects of FGF1 pretreatment of LPS-induced cell death. A Representative images of TUNEL assay staining to detect apoptotic cells in the lung sections of mice at 12- and 24 h. Scale bar = 100 μm. Apoptotic cells were observed under fluorescence microscopy and the percentage of TUNEL positive cells per 40x magnification field was calculated. B WB analysis and quantification of TLR4 and the downstream apoptosis-related protein cleaved-caspase 3 in murine lungs. WB assays were performed at 12 h post LPS treatment. Data are presented as mean ± SD (n = 3 per group). **p < 0.01, ***p < 0.001, ****p < 0.0001; One-way ANOVA with Tukey’s post-hoc test