Nintedanib Mitigates Radiation-Induced Pulmonary Fibrosis by Suppressing Epithelial Cell Inflammatory Response and Inhibiting Fibroblast-to-Myofibroblast Transition

Abstract

Radiation-induced pulmonary fibrosis (RIPF) represents a serious complication observed in individuals undergoing thoracic radiation therapy. Currently, effective interventions for RIPF are unavailable. Prior research has demonstrated that nintedanib, a Food and Drug Administration (FDA)-approved anti-fibrotic agent for idiopathic pulmonary fibrosis, exerts therapeutic effects on chronic fibrosing interstitial lung disease. This research aimed to investigate the anti-fibrotic influences of nintedanib on RIPF and reveal the fundamental mechanisms. To assess its therapeutic impact, a mouse model of RIPF was established. The process involved nintedanib administration at various time points, both prior to and following thoracic radiation. In the RIPF mouse model, an assessment was conducted on survival rates, body weight, computed tomography features, histological parameters, and changes in gene expression. In vitro experiments were performed to discover the mechanism underlying the therapeutic impact of nintedanib on RIPF. Treatment with nintedanib, administered either two days prior or four weeks after thoracic radiation, significantly alleviated lung pathological changes, suppressed collagen deposition, and improved the overall health status of the mice. Additionally, nintedanib demonstrated significant mitigation of radiation-induced inflammatory responses in epithelial cells by inhibiting the PI3K/AKT and MAPK signaling pathways. Furthermore, nintedanib substantially inhibited fibroblast-to-myofibroblast transition by suppressing the TGF-β/Smad and PI3K/AKT/mTOR signaling pathways. These findings suggest that nintedanib exerts preventive and therapeutic effects on RIPF by modulating multiple targets instead of a single anti-fibrotic pathway and encourage the further clinical trials to determine the efficacy of nintedanib in patients with RIPF.

Keywords: Epithelial cells; Fibroblast-to-myofibroblast transition.; Inflammatory response; Nintedanib; Radiation-induced pulmonary fibrosis.

Figure 1

(A) Schematic representation of the study design illustrating the treatment regimen, timing of sacrifice, and parameters that were investigated. (B) Photograph of representative mice at week 22 post-radiation vs. control. (C) Kaplan-Meier analysis of the survival of mice subjected to thoracic radiation. (D) Body weight, a general health status indicator of mice. (E, F, and G) Kaplan-Meier survival curves based on nintedanib treatment. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ns: not significant.

Figure 2

Effect of nintedanib on radiation-induced radiographic, morphological, and histopathological changes in the lungs of mice. (A) Representative micro-computed tomography (CT) images of different treatment groups at weeks 4, 12, and 22 post-radiation. (B) Assessment of lung density in micro-CT images. (C) Representative gross views of the lungs at weeks 4, 12, and 22 post-radiation. (D) Assessment of relative lung weight. (E) The results of hematoxylin and eosin (H&E) staining of lung samples obtained at weeks 4, 12, and 22 post-radiation (scale bar = 200 μm). (F) Masson's trichrome staining of lung samples from various groups at weeks 4, 12, and 22 post-radiation (scale bar = 200 μm). (G) Ashcroft score was used to determine the pulmonary fibrosis degree. (H) The hydroxyproline content in lung tissues of different groups was analyzed and quantified at week 22 post-radiation (n = 6 per group). The data are expressed as mean ± standard error of the mean. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ns: not significant.

Figure 3

Effects of nintedanib on radiation-induced fibrotic response in vivo. The protein expressions of (A) α-SMA, (B) COL1A and (C) CTGF in mice lung tissue at 4, 12 and 22 weeks post-radiation were measured using IHC staining and semi-quantification with comparison to the control group. The levels of TGF-β1 and IL-6 in mice lung tissues (D, E) and serum (F, G) at 22 weeks post-radiation were measured by ELISA. Quantitative analysis of α-SMA, COL1A and CTGF were expressed as the means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant. Scale bar = 200 μm.

Figure 4

RNA sequencing (RNA-seq) analysis of pulmonary changes in different treatment groups. (A) Heatmap of significant differentially expressed genes (DEGs) (P < 0.05). (B) Venn diagram of total DEGs. (C) Venn diagram of DEGs enriched in the top 100 inflammatory-related Gene Ontology (GO) terms. (D) The expression levels of representative inflammation-related and fibrosis-related genes. (E) The top 10 substantially enriched fibrosis-related GO terms (radiation treatment (RT) group vs. control group).

Figure 5

Analysis of the mechanisms responsible for the anti-fibrotic effects of nintedanib. (A) Representative western blotting analysis results of COL1A1, α-SMA, CTGF, COX-2, TGF-β1 and IL-6 in lung tissue of mice. Quantitative statistical results of (B) fibrosis-related and (C) inflammatory-related protein expression levels. (D) Dot plot of Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of differentially expressed genes (DEGs) between the radiation treatment (RT) and control groups. (E) Inflammation-related Gene Ontology (GO) terms in which the DEGs between the RT and control groups are enriched. (F and G) GSEA plots for RNA-seq data in the RT group compared to the control group. Normalized enrichment score (NES), nominal p-value and FDR q-value are stated on the plots. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant.

Figure 6

Nintedanib inhibited the radiation-induced activation of the PI3K/AKT and MAPK signaling pathways and inflammatory response in epithelial cells. (A-D) The time and dose of radiation were optimized by analyzing the protein levels using western blotting. Representative results of western blotting analysis of p-PI3K, PI3K, p-AKT, AKT, p-p38, p38, p-ERK1/2, ERK1/2, p-p65 and p65 in MEL-12 cells (E) and BEAS-2B cells (F) in different treatment groups, as well their corresponding quantitative statistical results in MEL-12 cells (G) and BEAS-2B cells (H). Representative results of western blotting analysis of COX-2, TGF-β1, IL-1β and IL-6 in MEL-12 cells (I) and BEAS-2B cells (J) in different treatment groups, as well their corresponding quantitative statistical results in MEL-12 cells (K) and BEAS-2B cells (L). ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001.

Figure 7

Nintedanib inhibited the radiation-induced activation of signaling pathways, including TGF-β/Smad and PI3K-AKT-mTOR, and the upregulation of fibrosis-related proteins in pulmonary fibroblasts. (A-D) The time and dose of radiation were optimized by evaluating protein levels using western blotting. Representative western blotting analysis results of p-Smad2/3, Smad2/3, p-PI3K, PI3K, p-AKT, AKT, p-mTOR and mTOR, in L929 cells (E) and MRC-5 cells (G) in different treatment groups, as well as their corresponding quantitative statistical results of protein levels in L929 cells (F) and MRC-5 cells (H). Representative western blotting analysis results of COL1A1, α-SMA and CTGF in L929 cells (I) and MRC-5 cells (K) in different treatment groups, as well as their corresponding quantitative statistical results of protein levels in L929 cells (J) and MRC-5 cells (L). ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001.

Figure 8

Nintedanib effects on radiation-mediated cross-talk between epithelial cells and fibroblasts. (A) Schematic representation of the co-culture setup: MLE-12 cells were placed in the upper chamber and subjected to overnight culture. L929 cells were seeded in the lower chamber. Subsequently, the MLE-12 cells were exposed to 8 Gy X-ray irradiation and immediately co-cultured with non-radiation-treated L929 cells for 48 hours with or without nintedanib (1 μM) treatment. (B) Representative protein bands of COL1A1, α-SMA and CTGF in L929 cells after co-culture for 48 hours. (C, D) Transwell assay and quantitative analysis showed the migration of L929 cells that irradiated with 8 Gy X-ray, with or without treatment of 1 μM nintedanib (scale bar = 100 μm). (E and F) Transwell assay, along with quantitative analysis, exhibited the migration of L929 cells after incubation in CM for 24 hours with or without treatment of 1 μM nintedanib (scale bar = 100 μm). (G and H) Wound closure assay for the assessment migratory capacities of L929 cells after incubation in CM for 24 hours with or without treatment of 1 μM nintedanib (scale bar = 200 μm). ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001.

Figure 9

Schematic illustration of nintedanib effects in the RIPF.