Ferroptosis inhibitor alleviates Radiation-induced lung fibrosis (RILF) via down-regulation of TGF-β1

Abstract

Background: Radiation-induced lung fibrosis (RILF) is a severe and life-threatening complication of thoracic radiotherapy. Cell death is the key issue in RILF. Ferroptosis is a form programmed cell death implicated in the pathologies of inflammation. This study aimed to investigate the role of ferroptosis in RILF, and the effectiveness and the potential underlying mechanism of ferroptosis inhibitor on RILF.

Methods: Immunofluorescence, western blot and RT-PCR assays were performed to examine the ferroptosis maker glutathione peroxidase 4 (GPX4) in a mice RILF model. The lung tissue sections were stained with hematoxylin and eosin (H&E), Masson trichrome staining and Sirius-Red staining to evaluate the histopathological changes in RILF mice. Reactive oxygen species (ROS) and hydroxyproline (HYP) in lungs were measured by the relevant kits. The serum levels of inflammatory cytokines (TNF-α, IL-6, IL-10, and TGF-β1) were measured with Elisa. The protein and mRNA levels of GPX4, nuclear factor (erythroid-derived 2)-like 2 (Nrf2), hemeoxygenase-1 (HO1) and quinone oxidoreductase 1 (NQO1) in lungs were examined by western blot and RT-PCR.

Results: GPX4 levels of the irradiated lungs were significantly down-regulated than the groups with no irradiation, and the ferroptosis inhibitor, liproxstatin-1, increased GPX4 levels significantly in RILF mice. Treatment with liproxstatin-1 lowered the Szapiel and Ashcroft scores significantly, down-regulated the levels of ROS and HYP in lungs and reduced the serum inflammatory cytokines levels in RILF mice. The protein and the mRNA levels of Nrf2, HO1 and NQO1 were up-regulated by liproxsratin-1 in RILF.

Conclusions: Our data suggested that ferroptosis played a critical role in RILF, ferroptosis inhibitor liproxstatin-1 alleviated RILF via down-regulation of TGF-β1 by the activation of Nrf2 pathway. The effectiveness of ferroptosis inhibition on RILF provides a novel therapeutic target for RILF.

Keywords: Ferroptosis; Ferroptosis inhibitor; Nrf2; ROS; Radiation-induced lung fibrosis; TGF-β1.

Fig. 1

The effect of lipoxstatin-1 on GPX4 expressions following RILF. a Representative fluorescence micrographs of GPX4 staining in lungs. Scale bar is 20 μm. b Quantification of GPX4 expressions. c The protein levels of GPX4 in lungs were evaluated by western blotting. d Quantification of the protein levels of GPX4 in lungs. e Quantification of the mRNA levels of GPX4 in lungs were evaluated by Real time PCR. (Data shown as mean ± SD, one-way ANOVA followed by a Bonferroni correction, * P < 0.05, ** P < 0.01, *** P <0.001, n = 6)

Fig. 2

The effect of liproxstatin-1 on histology outcome following RILF. A H&E staining, Masson trichrome staining and Sirius-Red staining were used to evaluate IR-induced fibrosis of the lung tissues. Scale bar: 100 μm. The histopathological slides were evaluated using a semiquantitative scoring method. b The Szapiel score was used to evaluate the pulmonary fibrosis stained with H&E. c The pulmonary fibrosis stained by Masson trichrome and Sirius-Red staining were evaluated according to the Ashcroft score. (Data shown as mean ± SD, one-way ANOVA followed by a Bonferroni correction, * P < 0.05, ** P < 0.01, *** P <0.001, n = 6)

Fig. 3

The effect of lipoxstatin-1 on HYP content in RILF. (Data shown as mean ± SD, one-way ANOVA followed by a Bonferroni correction, *** P < 0.001, n = 6)

Fig. 4

Effects of liproxstatin-1 on serum cytokines following RILF. a, b, c, d: The levels of TNF-α, IL-6, IL-10 and TGF-β1 were evaluated by Elisa. (Data shown as mean ± SD, one-way ANOVA followed by a Bonferroni correction, * P < 0.05, ** P < 0.01, *** P < 0.001, n = 6)

Fig. 5

The effect of lipoxstatin-1 on ROS levels in irradiated lungs. (Data shown as mean ± SD, one-way ANOVA followed by a Bonferroni correction, *** P < 0.001, n = 6)

Fig. 6

Effects of liproxstain-1 on the proteins levels of Nrf2 pathway following RILF. a The expressions of Nrf2, HO1 and NQO1 in lungs were evaluated by western blotting. b, c, d Quantification of Nrf2, HO1 and NQO1 expressions. (Data shown as mean ± SD, one-way ANOVA followed by a Bonferroni correction, ^ns Represented no statistical difference. * P < 0.05, ** P < 0.01, *** P < 0.001, n = 6)

Fig. 7

Effects of liproxstain-1 on the mRNA levels of Nrf2 pathway following RILF. a, b, c Quantification of Nrf2, HO1 and NQO1expressions. The mRNA levels of Nrf2, HO1 and NQO1 in lungs were evaluated by real time PCR. d The schematic diagram of Nrf2 pathway following RILF. (Data shown as mean ± SD, one-way ANOVA followed by a Bonferroni correction, ^ns Represented no statistical difference. * P < 0.05, *** P < 0.001, n = 6)