NVP-AUY922 alleviates radiation-induced lung injury via inhibition of autophagy-dependent ferroptosis

Abstract

Radiation-induced lung injury (RILI) is a common complication of radiotherapy for which no effective interventions are available. NVP-AUY922, a resorcinylic isoxazole amide drug, exhibits anti-inflammatory, immunomodulatory, and therapeutic effects against various types of cancers. In this study, we explore the role and underlying mechanisms of NVP-AUY922 in the treatment of RILI. We established a model of BEAS-2B cell injury and a mouse model of RILI. Cell proliferation, death, gross weight, and survival rates of mice, and histological parameters were assessed. Additionally, inflammation-related indices and indicators related to ferroptosis were evaluated. Furthermore, immunofluorescence and co-immunoprecipitation were used to determine the interaction between GPX4, LAMP-2A, and HSC70. NVP-AUY922 significantly ameliorated radiation-induced lung tissue damage, inflammatory cell infiltration, proinflammatory cytokine release, and lung epithelial BEAS-2B cell damage. NVP-AUY922 markedly limited the activation of ferroptosis, which is involved in RILI. Mechanistically, NVP-AUY922 prevented chaperone-mediated autophagy of the GPX4 pathway in vitro and in vivo, and the autophagy inhibitor Baf-A1 significantly increased the level of GPX4 and alleviated lung inflammation. NVP-AUY922 can alleviate RILI by inhibiting chaperone-mediated lysosomal degradation of GPX4, demonstrating its potential as a novel protective agent against RILI.

Fig. 1. NVP-AUY922 alleviates radiation-induced BEAS-2B cell injury.

A, B Cell viability and LDH release assay was detected at different time points (24, 48, or 72 h) following radiation (0, 5, 10, or 15 Gy). C The chemical structure of NVP-AUY922. D, E Cell viability and LDH release assay in BEAS-2B. F–H Concentrations of IL-1β (F), IL-6 (G), and TNF-α (H) in cell culture supernatant. I, K Representative flow cytometric images. J, L Representative EDU assay fluorescence images. All data are expressed as the mean ± SD. (n = 6). ^#/*P < 0.05, ^##/**P < 0.01, ^###/***P < 0.001, ^# vs. IR group; * vs. control group.

Fig. 2. NVP-AUY922 improved survival and lung injury induced by IR in mice.

A Survival curves. B Body Weight. C W/D ratio of the lungs. D Depilation of mice after radiation. E Representative images of lung tissues. F Representative HE staining images of the lung tissues. G–I Concentrations of IL-1β (G), IL-6 (H), and TNF-α (I) in serum. J TUNEL staining of lung sections. Mice (n = 12) were pre-treated with NVP-AUY922 (5 mg/kg, 10 mg/kg, 15 mg/kg, i.p.) or vehicle (i.p.) 2 h prior to a single radiation dose of 10 Gy (dose rate of 2.0 Gy/min), then continuously administered from day 2 to day 5. Animals were sacrificed on day 16. ^#/*P < 0.05, ^##/**P < 0.01, ^###/***P < 0.001, ^# vs. IR group; * vs. control group.

Fig. 3. Ferroptosis is involved in radiation-induced lung injury.

The mice were treated with ferrostatin-1 (5 mg/kg) or DMSO. A H&E staining of lung. B–D Concentrations of IL-1β (B), IL-6 (C), and TNF-α (D) in the Serum. E Representative IHC images of GPX4 and 4HNE in the lung sections. F Representative DHE fluorescence images of the mouse lung tissues. G–I Activities of iron content, MDA, and GSH in the mouse lung tissues. ^#/*P < 0.05, ^##/**P < 0.01,^###/***P < 0.001, ^# vs. IR group; * vs. control group.

Fig. 4. NVP-AUY922 attenuates radiation-induced ferroptosis in BEAS-2B cells.

A BEAS-2B cell morphology and death were observed. B, C CCK-8 assays and LDH release assays in BEAS-2B cell. D The levels of GPX4 and 4HNE in BEAS-2B cells. E, F Representative DHE fluorescence images of BEAS-2B cells. G–I Activities of iron content, MDA, and GSH in cell culture supernatant. J Mitochondria with obvious characteristics of ferroptosis were detected by transmission electron microscopy. K–L Concentrations of TNF-α (K), IL-1β (I), and IL-6 (L) in cell culture supernatant. ^#/*P < 0.05, ^##/**P < 0.01, ^###/***P < 0.001, ^# vs. IR group; * vs. control group.

Fig. 5. NVP-AUY922 attenuates ferroptosis and inflammation induced by IR.

A Representative IHC images of GPX4 and 4HNE in the lung sections. B Representative DHE fluorescence images of the mouse lung tissues. C The levels of 4HNE and GPX4 in lung tissues. D–F Activities of iron content, MDA, and GSH in the mouse lung tissues. G–I Concentrations of IL-6 (G), TNF-α (H), and IL-1β (I) in serum. ^#/*P < 0.05, ^##/**P < 0.01, ^###/***P < 0.001, ^# vs. IR group; * vs. control group.

Fig. 6. NVP-AUY922 inhibited the interaction between GPX4, HSC70, and LAMP-2A induced by IR.

A Western blot assay for GPX4. B Quantification of GPX4 mRNA levels by qPCR. C Western blot using an antibody against GPX4. BEAS-2B cells were pre-treated with MG132 (10 µM), CQ (25 µM), NVP-AUY922 (10 nM) and Baf-A1 (200 nM) 2 h prior to the 15 Gy radiation for 10 min. D–E Microscopy was performed to detect the changes in GPX4, HSP90, and LAMP-2A. F Co-IP assay was performed using an antibody against GPX4 or control IgG and western blotting for GPX4, LAMP-2A, HSC70, and HSP90 was performed. G Co-IP assays were performed using an antibody against GPX4 or control IgG followed by western blotting for GPX4, LAMP-2A, HSC70, and HSP90. ^#/*P < 0.05, ^##/**P < 0.01, ^###/***P < 0.001, ^# vs. IR group; * vs. control group.

Fig. 7. Lysosomal inhibitor of Baf-A1 alleviates radiation-induced ferroptosis in mice.

The mice were treated with Baf-A1 (10 µM) or DMSO. A H&E staining of lung. B Representative IHC images of GPX4 and 4HNE in the lung sections. C TUNEL assay of lung sections. D The levels of 4HNE and GPX4 in the lung tissues. E–G Activities of MDA, GSH, and iron content in the mouse lung tissues. H–J Concentrations of TNF-α (H), IL-1β (I), and IL-6 (J) in serum. ^#/*P < 0.05, ^##/**P < 0.01, ^###/***P < 0.001, ^# vs. IR group; * vs. control group.