Obacunone alleviates ferroptosis during lipopolysaccharide-induced acute lung injury by upregulating Nrf2-dependent antioxidant responses

Abstract

Background: Acute lung injury (ALI) has received considerable attention in the field of intensive care as it is associated with a high mortality rate. Obacunone (OB), widely found in citrus fruits, is a natural bioactive compound with anti-inflammatory and antioxidant activities. However, it is not clear whether OB protects against lipopolysaccharide (LPS)-induced ALI. Therefore, in this study, we aimed to evaluate the protective effects of OB and the potential mechanisms against LPS-induced ALI and BEAS-2B cell injury.

Methods: We established a model of BEAS-2B cell injury and a mouse model of ALI by treating with LPS. Samples of in vitro model were subjected to cell death, Cell Counting Kit-8, and lactate dehydrogenase (LDH) release assays. The total number of cells and neutrophils, protein content, and levels of IL-6, TNF-α, and IL-1β were determined in bronchoalveolar lavage fluid (BALF). Glutathione, reactive oxygen species, and malondialdehyde levels were determined in lung tissue. Additionally, immunohistochemical analysis, immunofluorescence, western blot, quantitative real-time PCR, and enzyme-linked immunosorbent assay were conducted to examine the effects of OB. Furthermore, mice were treated with an Nrf2 inhibitor (ML385) to verify its role in ferroptosis. Data were analyzed using one-way analysis of variance or paired t-tests.

Results: Compared with the LPS group, OB effectively alleviated LPS-induced ALI by decreasing lung wet/dry weight ratio, reactive oxygen species and malondialdehyde production, and superoxide dismutase and glutathione consumption in vivo. In addition, OB significantly alleviated lung histopathological injury, reduced inflammatory cytokine secretion and Fe²⁺ and 4-HNE levels, and upregulated GPX4, SLC7A11, and Nrf2 expression. Mechanistically, OB activated Nrf2 by inhibiting Nrf2 ubiquitinated proteasome degradation. ML385 reversed the protective effects of OB against LPS-induced ALI.

Conclusion: Overall, OB alleviates LPS-induced ALI, making it a potential novel protective agent against LPS-induced ALI.

Keywords: Acute lung injury; Ferroptosis; Lipopolysaccharide; Nrf2; Obacunone.

Fig. 1

Protective effects of obacunone against lipopolysaccharide (LPS)-induced BEAS-2B damage. A, B LPS treatment decreased cell viability and increased lactate dehydrogenase (LDH) release as determined using Cell Counting Kit-8 (CCK-8) (A) and LDH (B) assays. C–G Obacunone (OB) increased cell viability (C), and inhibited LDH (D), IL-1β (E), IL-6 (F), and TNF-α (G) release in a dose-dependent manner (0, 10, 20, or 40 μM) following LPS treatment (10 μg/mL). H Representative EDU assay fluorescence images. I Representative flow cytometric images. Similar results were obtained from three independent experiments. All data are presented as mean ± standard error of the mean (SEM) (n = 6 for each group). ^#/*P < 0.05, ^##/**P < 0.01, ^###/***P < 0.001, ^# versus control group; * versus LPS group

Fig. 2

Protective effects of obacunone (OB) against lipopolysaccharide (LPS)-induced acute lung injury (ALI) in mice. A Representative images of mouse lung tissues. Lung tissues (n = 6) from each experimental group were processed for histological evaluation following the LPS challenge. B Representative hematoxylin and eosin (HE) staining images of the lung tissues. C Representative terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) fluorescence images of the lung tissues. D Lung wet weight/dry (W/D) weight ratio. E–G Total cell and neutrophil count and protein concentration in bronchoalveolar lavage fluid (BALF). H–J IL-1β, IL-6, and TNF-α concentrations in the BALF. K, L KL-6 and CRP in plasma. M, N LYM (%) and NEU (%) in EDTA anticoagulation blood. Similar results were obtained from three independent experiments. All data are presented as mean ± SEM (n = 6 for each group). ^#/*P < 0.05, ^##/**P < 0.01, ^###/***P < 0.001, ^# versus control group; * versus LPS group

Fig. 3

Effects of obacunone against lipopolysaccharide (LPS)-triggered oxidative stress in induced acute lung injury (ALI). A Representative dihydroethidium (DHE) fluorescence images of BEAS-2B cells. B Representative DHE fluorescence images of the mouse lung tissues. C–F Activities of malondialdehyde (MDA), catalase (CAT), glutathione (GSH), and superoxide dismutase (SOD) in the mouse lung tissues. Similar results were obtained from three independent experiments. All data are presented as mean ± SEM (n = 6 for each group). ^#/*P < 0.05, ^##/**P < 0.01, ^###/***P < 0.001, ^# versus control group; * versus LPS group; ns, no significant difference between control and OB group

Fig. 4

Ferroptosis was upregulated during lipopolysaccharide (LPS)-induced injury in vivo and in vitro. A Representative immunofluorescence images of GPX4 and 4-HNE in BEAS-2B cells. B, C Western blotting of GPX4 and SLC7A11 in mice (B) and BEAS-2B cells (C). D Expression levels of GPX4 in BEAS-2B cells as determined by RT-qPCR. E Expression level of Fe²⁺ in BEAS-2B cells. F Immunohistochemistry-based images of GPX4 and 4-HNE in the lung tissues. G Transmission electron microscopy (TEM) of ferroptosis in BEAS-2B cells. Similar results were obtained from three independent experiments. All data are presented as mean ± SEM (n = 6 for each group). ^##/**P < 0.01, ^###/***P < 0.001, ^# versus control group; * versus LPS group; ns, no significant difference between control and OB group

Fig. 5

Effects of the ferroptosis inhibitor on lipopolysaccharide (LPS)-induced acute lung injury (ALI). A Representative images of the mouse lung tissues. B Representative terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) fluorescence images of the lung tissues. C Representative immunohistochemistry-based images of GPX4 and 4-HNE in the lung tissues. D Lung wet weight/dry (W/D) weight ratio. E–G Concentrations of IL-1β, IL-6, and TNF-α in bronchoalveolar lavage fluid (BALF). Similar results were obtained from three independent experiments. All data are presented as mean ± SEM (n = 6 for each group). ^#/*P < 0.05, ^##/**P < 0.01, ^###/***P < 0.001, ^# versus control group; * versus LPS group

Fig. 6

Obacunone (OB) upregulated Nrf2 level in lipopolysaccharide (LPS)-induced acute lung injury (ALI) models. A Representative immunofluorescence images of Nrf2 in BEAS-2B cells. B Representative immunohistochemistry-based images of Nrf2 in the mouse lung tissues. C, D Nrf2 and HO-1 level in BEAS-2B cells (C) and the mouse lung tissues (D) as determined by western blotting. Similar results were obtained from three independent experiments. All data are presented as mean ± SEM (n = 6 for each group). ^##/**P < 0.01, ^###/***P < 0.001, # versus control group; * versus LPS group

Fig. 7

Obacunone (OB) inhibited ferroptosis by activating nuclear factor-2 associated factor 2 (Nrf2). A Representative dihydroethidium (DHE) fluorescence images of the mouse lung tissues. B Representative hematoxylin and eosin (HE) staining images of the lung tissues. C Representative immunohistochemistry-based images of GPX4 and 4-HNE in the lung tissues. D Representative transmission electron microscopy (TEM) images of ferroptosis in BEAS-2B cells. E–G IL-1β, IL-6, and TNF-α concentrations in bronchoalveolar lavage fluid (BALF). H Fe²⁺ level in BEAS-2B cells. Similar results were obtained from three independent experiments. All data are presented as mean ± SEM (n = 6 for each group). ^##/**P < 0.01, ^###/***P < 0.001, ^# versus control group; * versus LPS + OB group

Fig. 8

Obacunone (OB) reduced nuclear factor-2 associated factor 2 (Nrf2) ubiquitinated proteasome degradation. A Expression of Nrf2 and Keap1 in BEAS-2B cells as determined by western blotting. B Expression levels of Nrf2 and HO-1 in BEAS-2B cells as determined by RT-qPCR. C BEAS-2B cells were treated with 50 μg/mL cycloheximide (CHX) and incubated for a specified period. Nrf2 level in BEAS-2B cells as determined by western blotting. D BEAS-2B cells were either left untreated or treated with 10 μM MG132 for 4 h to inhibit the degradation of ubiquitinated proteins. Nrf2 level was determined by western blotting. E BEAS-2B cells were treated with 10 μM MG132 for 4 h, and endogenous ubiquitin-bound Nrf2 and Keap1 were detected. Cell lysates were immunoprecipitated using anti-NRF2 and anti-Keap1 antibodies and imprinted with anti-ubiquitin antibodies. F Diagram showing the mechanism of action of OB. Similar results were obtained from three independent experiments. All data are presented as mean ± SEM (n = 6 for each group). ^#/*P < 0.05, ^##/**P < 0.01, ^# versus control group; * versus LPS group; ns, no significant difference between control and LPS group