Melatonin alleviates septic ARDS by inhibiting NCOA4-mediated ferritinophagy in alveolar macrophages

Abstract

Ferroptosis is a novel form of programmed cell death which can exacerbate lung injury in septic acute respiratory distress syndrome (ARDS). Alveolar macrophages, crucial innate immune cells, play a pivotal role in the pathogenesis of ARDS. Ferritinophagy is a process of ferritin degradation mediated by nuclear receptor coactivator 4 (NCOA4) which releases large amounts of iron ions thus promoting ferroptosis. Recent evidence revealed that inhibiting macrophage ferroptosis can effectively attenuate pulmonary inflammatory injury. Melatonin (MT), an endogenous neurohormone, has antioxidant and anti-inflammatory effects and can reduce septic ARDS. However, it is not clear whether MT's pulmonary protective effect is related to the inhibition of macrophage ferritinophagy. Our in vitro experiments demonstrated that MT decreased intracellular malondialdehyde (MDA), Fe²⁺, and lipid peroxidation levels, increased glutathione (GSH) levels and cell proliferation, and upregulated glutathione peroxidase 4 (GPX4) and ferritin heavy chain 1 (FTH1) protein levels in LPS-treated macrophages. Mechanistically, the antiferroptotic effect of MT on LPS-treated macrophages was significantly compromised by the overexpression of NCOA4. Our in vivo experiments revealed that MT alleviated the protein expression of NCOA4 and FTH1 in the alveolar macrophages of septic mice. Furthermore, MT improved lipid peroxidation and mitigated damage in alveolar macrophages and lung tissue, ultimately increasing the survival rates of septic mice. These findings indicate that MT can inhibit ferroptosis in an NCOA4-mediated ferritinophagy manner, thereby ameliorating septic ARDS.

Fig. 1. Ferroptosis in RAW264.7 cells is activated with LPS (1 μg/ml) at different time points.

A–C The FTH1 and GPX4 protein levels were detected by western blot. Intracellular GSH (D), MDA (E), and Fe²⁺ (F) contents were detected by corresponding assay kits. Cell proliferation detected by the EdU assay kit was observed under a fluorescence microscope (I) and statistics as shown in (G). Lipid peroxidation levels detected by the C11 BODIPY assay kit were observed under a fluorescence microscope (J) and statistics as shown in (H). Data are expressed as mean ± SD (n = 6), *p < 0.05, **p < 0.01 indicate significant differences from each group, NS indicates no significance.

Fig. 2. Different doses of MT inhibit LPS-induced ferroptosis in RAW264.7 cells.

Intracellular GSH (A), MDA (B), and Fe²⁺ (C) contents were detected by corresponding assay kits. Lipid peroxidation levels detected by the C11 BODIPY assay kit were observed under a fluorescence microscope (E) and statistics as shown in (D). Data are expressed as mean ± SD (n = 6), *p < 0.05, **p < 0.01 indicate significant differences from each group, NS indicates no significance.

Fig. 3. MT markedly alleviates ferroptosis in macrophages after LPS stimulation.

A–C The FTH1 and GPX4 protein levels were detected by western blot. Cell proliferation detected by the EdU assay kit was observed under a fluorescence microscope (D) and statistics as shown in (E). Lipid peroxidation levels detected by the C11 BODIPY assay kit were observed under a fluorescence microscope (F) and statistics as shown in (G). Intracellular GSH (H), MDA (I), and Fe²⁺ (J) contents were detected by corresponding assay kits. Data are expressed as mean ± SD (n = 6), * p < 0.05, **p < 0.01 indicate significant differences from each group, NS indicates no significance.

Fig. 4. MT inhibits RAW264.7 cell ferroptosis via MT2.

Intracellular GSH (A), MDA (B), and Fe²⁺ (C) contents were detected by corresponding assay kits. Cell proliferation detected by the EdU assay kit was observed under a fluorescence microscope (H) and statistics as shown in (D). E–G The FTH1 and GPX4 protein levels were detected by western blot. Data are expressed as mean ± SD (n = 6), *p < 0.05, **p < 0.01 indicate significant differences from each group, NS indicates no significance.

Fig. 5. MT suppresses LPS-induced ferroptosis in RAW264.7 cells via MT1.

A The mRNA expression levels of MT1 after knockdown of MT1 receptor in RAW264.7 cells with siRNA. Intracellular GSH (B), MDA (C), and Fe²⁺ (D) contents were detected by corresponding assay kits. E–G The FTH1 and GPX4 protein levels were detected by western blot. Lipid peroxidation levels detected by the C11 BODIPY assay kit were observed under a fluorescence microscope (H) and statistics as shown in (I). Cell proliferation detected by the EdU assay kit was observed under a fluorescence microscope (J) and statistics as shown in (K). Data are expressed as mean ± SD (n = 6), *p < 0.05, **p < 0.01 indicate significant differences from each group, NS indicates no significance.

Fig. 6. MT alleviates LPS-induced ferroptosis in RAW264.7 cells by inhibiting ferritinophagy.

Intracellular MDA (A), and Fe²⁺ (B) contents were detected by corresponding assay kits. Lipid peroxidation levels detected by the C11 BODIPY assay kit were observed under a fluorescence microscope (I) and statistics as shown in (C). Cell proliferation detected by the EdU assay kit was observed under a fluorescence microscope (J) and statistics as shown in (D). E–H The FTH1, GPX4 and LC3 protein levels were detected by western blotting. Data are expressed as mean ± SD (n = 6), *p < 0.05, **p < 0.01 indicate significant differences from each group, NS indicates no significance.

Fig. 7. MT protects RAW264.7 cells from LPS-induced ferroptosis by inhibiting NCOA4-mediated ferritinophagy.

A The transfection efficiency of lentivirus was detected by qPCR. Intracellular GSH (B), MDA (C), and Fe²⁺ (D) contents were detected by corresponding assay kits. E–H The FTH1, GPX4 and NCOA4 protein levels were detected by western blotting. Lipid peroxidation levels detected by the C11 BODIPY assay kit were observed under a fluorescence microscope (I) and statistics as shown in (J). Cell proliferation detected by the EdU assay kit was observed under a fluorescence microscope (K) and statistics as shown in (L). Mitochondrial membrane potential changes detected by JC-1 mitochondrial assay kit was observed (M) under fluorescence microscope and statistics as shown in (N). O Mitochondrial morphological changes under electron microscopy (The arrows mark mitochondria). Data are expressed as mean ± SD (n = 6), *p < 0.05, **p < 0.01 indicate significant differences from each group, NS indicates no significance.

Fig. 8. MT alleviates septic ARDS by inhibiting ferritinophagy of alveolar macrophages in mice.

A The representative HE staining of lung tissue sections. B 4-HNE immunohistochemistry of mouse lung tissues. C The lung injury score analysis. D The protein concentration in BALF. E Alveolar macrophages were detected by flow cytometry with F4/80 and CD11c. F–H The NCOA4 and FTH1 protein levels were detected by western blot. The contents of GSH (I), MDA (J) and Fe²⁺ (K) in alveolar macrophages of mice. L The survival rate of mice. Data are expressed as mean ± SD (n = 6), *p < 0.05, **p < 0.01 indicate significant differences from each group, NS indicates no significance.