Fluorofenidone alleviates cigarette smoke exposure-induced chronic lung injury by targeting ferroptosis

Abstract

Chronic obstructive pulmonary disease (COPD) is a common condition that poses significant health risks to humans. Pulmonary interstitial fibrosis (PIF) often manifests in advanced stages of COPD. Fluorofenidone (AKF) has a wide range of pharmacological effects, including anti-fibrotic, antioxidant, and anti-inflammatory effects. Therefore, this study aimed to assess the role of AKF in lung injury and its underlying mechanisms. The COPD mice model was constructed by cigarette smoke (CS) combined with lipopolysaccharide (LPS) treatment. The effect of AKF on COPD mice was evaluated by lung injury, lipid peroxidation, inflammatory factors, and the expression of ferroptosis markers. Furthermore, the normal human bronchial epithelial cell line, Beas-2B, was used to verify the mechanism underlying the association between ferroptosis and inflammation. AKF attenuated the cigarette smoke (CS)/LPS-induced inflammatory response in the mouse lungs. Additionally, AKF attenuated the CS/LPS-induced fibrosis response in the mouse lungs. AKF inhibits ferroptosis in lung tissues of CS/LPS-exposed mice. Furthermore, AKF suppressed the inflammatory response and ferroptosis in CSE-treated BEAS-2B cells via NF-κB signaling pathway. AKF can function as a novel ferroptosis inhibitor by inhibiting NF-κB to inhibit airway inflammation and fibrosis, providing a scientific basis for the use of AKF to prevent the progression of COPD and pulmonary fibrosis.

Keywords: Ferroptosis; Fluorofenidone; Lung fibrosis; Lung injury; NF-κB.

Fig. 1

AKF attenuated the CS/LPS-induced inflammatory response in the lungs of mice. (A) Schematic diagram of animal treatment. (B) HE staining of the lung tissue. (C) Lung injury scores of the three groups. (D–F) The contents of IL-1β, IL-6 and TNF-α in the BALF. (G–I) The contents of IL-1β, IL-6 and TNF-α in serum. * means compared to the control group, P < 0.05. # means compared to the CS/LPS group, P < 0.05.

Fig. 2

AKF attenuated CS/LPS-induced fibrosis in the lungs of mice. (A) Masson’s trichrome staining of lung tissues. (B) Lung fibrosis scores of the three groups. (C) The level of hydroxyproline. (D–F) The mRNA level of α-SMA, FAP and COL3A1. (G) The protein level of α-SMA, FAP and Collagen III. * means compared to the control group, P < 0.05. # means compared to the CS/LPS group, P < 0.05.

Fig. 3

AKF inhibited ferroptosis in lung tissues of CS /LPS-exposed mice. (A) IHC staining of ACSL4 and GPX4 in lung tissues. (B) Western blotting of ACSL4 and GPX4 in lung tissues. (C) The level of MDA in lung tissues. (D) The level of GSH in lung tissues. (E) The level of MDA in serum. (F) The level of GSH in serum. * means compared to the control group, P < 0.05. # means compared to the CS/LPS group, P < 0.05.

Fig. 4

AKF suppressed the inflammatory response and ferroptosis in BEAS-2B cells treated with CSE. (A) The cell viability was determined using CCK8 assay. (B–D) The contents of IL-1β, IL-6 and TNF-α in cell supernatant. (E–F) The level of MDA and GSH in cell supernatant. (G) Western blotting of ACSL4 and GPX4 in cells. (H) Morphological characteristics of ferroptosis was observed by transmission electron microscopy. # means compared to the CS/LPS group, P < 0.05.

Fig. 5

AKF suppressed ferroptosis-mediated inflammation in CSE-treated BEAS-2B cells via NF-κB signaling pathway. (A) The cell viability was determined using CCK8 assay. (B) The contents of IL-1β, IL-6 and TNF-α in cell supernatant. (E–F) The level of MDA and GSH in cell supernatant. (G) Western blotting of ACSL4 and GPX4 in cells. (H) Western blotting of P- NF-κB/ NF-κB in cells. Note: The control group, CSE group and AKF group were the same as in the Fig. 4.