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Protective effects of lipid mediators, obtained from docosahexaenoic acid via soybean lipoxygenase, on lipopolysaccharide‑induced acute lung injury through the NF‑κB and Nrf2/HO‑1 signaling pathways

  • Authors:
    • Yan Su
    • Hack Sun Choi
    • Soon Kyu Kwon
    • Yunjon Han
    • Soon-Chang Cho
    • Jin Hyuk Shin
    • Yong-Suk Jang
    • Jong Hyun Choi
    • Jeong-Woo Seo
  • View Affiliations / Copyright

    Affiliations: Microbial Biotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology, Jeongeup, Jeonbuk 56212, Republic of Korea, Department of Biochemistry and Molecular Biology, Yonsei University College of Medicine, Seoul 03722, Republic of Korea, Biocorp Co., Ltd., Goheung‑gun, Jeollanam‑do 59551, Republic of Korea, Department of Bioactive Material Sciences, Jeonbuk National University, Jeonju, Jeonbuk 54896, Republic of Korea
    Copyright: © Su et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Article Number: 233
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    Published online on: June 17, 2025
       https://doi.org/10.3892/mmr.2025.13598
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Abstract

Acute lung injury (ALI), marked by acute and chronic inflammation, causes damage to alveolar epithelial and capillary endothelial cells. The present study investigated lipid mediators (LM) effects on lipopolysaccharide (LPS)‑induced RAW264.7 cells and ALI mice. LM, comprising 17S‑monohydroxy docosahexaenoic acid (DHA), resolvin D5 and protectin DX (in a 3:47:50 ratio), were derived from DHA via soybean lipoxygenase and demonstrated anti‑inflammatory properties. In vitro experiments revealed that LM decreased nitric oxide (NO) and prostaglandin E2 (PGE2) levels caused by LPS via downregulating inducible nitric oxide synthase and cyclooxygenase‑2. Additionally, LM inhibited the inflammation by suppressing NF‑κB signaling. The results also indicated that LM reduced oxidative stress by lowering reactive oxygen species and malondialdehyde (MDA) levels while enhancing glutathione (GSH) content and superoxide dismutase (SOD) activities, probably through activation of nuclear factor erythroid 2‑related factor 2 (Nrf2)/heme oxygenase‑1 (HO‑1) signaling pathway. Moreover, the benefits of LM on inflammation and oxidative stress were reversed when pretreated with ML385, an Nrf2 inhibitor. In vivo studies revealed that LM reduced the lung wet/dry ratio, increased GSH, catalase and SOD activities, along with lowered myeloperoxidase and MDA levels. In addition, LM reduced inflammatory cytokine levels in serum and bronchoalveolar lavage fluid. Mechanistically, LM inhibited NF‑κB signaling and activated Nrf2/HO‑1 signaling pathways.
View Figures

Figure 1

LM mediates the production of NO and
PGE2 in vitro. (A) RAW264.7 cells were treated with LM for 3
h, followed by stimulation with LPS at 1 µg/ml for 24 h. MTT assay
was performed to detect the cell viability. The production of (B)
NO and (C) PGE2 were quantified. (D-F) Western blot analysis was
conducted to assess the expression of iNOS and COX-2. Data are
presented as mean ± SD and was determined using Tukey's test.
**P<0.01, ***P<0.001, ****P<0.0001. LM, lipid mediators;
NO, nitric oxide; PGE2, prostaglandin E2; LPS, lipopolysaccharide;
iNOS, inducible nitric oxide synthase; COX-2, cyclooxygenase-2.

Figure 2

LM inhibits inflammatory cytokines
via NF-κB signaling pathway in vitro. RAW264.7 cells were
pre-incubated with LM for a period of 3 h, followed by LPS at 1
µg/ml for 24 h. (A) IL-6, (B) TNF-α and (C) IL-1β were measured via
ELISA. (D and E) Western blot analysis was conducted to evaluate
the expression levels of pp65. Data are presented as mean ± SD and
was determined using Tukey's test. **P<0.01, ****P<0.0001.
LM, lipid mediators; IL, interleukin; TNF-α, tumor necrosis
factor-α; ELISA, enzyme-linked immunosorbent assay; NC, normal
control.

Figure 3

LM suppresses the oxidative stress
in vitro. (A) RAW264.7 cells were pretreated with LM for 3
h, followed by a 24-h exposure to LPS. Cells were then stained with
DCFH-DA to detect ROS. Fluorescence intensity was visualized using
a fluorescence microscope, scale bar, 100 µm. Levels of (B) MDA,
(C) GSH and (D) SOD in the cells were measured. (E) Western blot
analysis was performed to evaluate the expression of Nrf2 and HO-1.
Relative expression of (F) Nrf2 and (G) HO-1 was quantified using
ImageJ software. Data are presented as mean ± SD and was determined
using Tukey's test. **P<0.01, ***P<0.001, ****P<0.0001.
LM, lipid mediators; LPS, lipopolysaccharide; ROS, reactive oxygen
species; MDA, malondialdehyde; GSH, glutathione; SOD, superoxide
dismutase; Nrf2, nuclear factor erythroid 2-related factor 2; HO-1,
heme oxygenase-1; NC, normal control.

Figure 4

LM suppresses the inflammation via
Nrf2 signaling pathway in vitro. RAW264.7 cells were
pretreated with ML385 at 5 µM for 2 h and then LM (2 µg/ml) for
further 3 h, followed by stimulated with LPS for 24 h. (A) and (B)
Nrf2 expression levels were assessed by western blotting. The
levels of (C) IL-6 and (D) TNF-α were measured using ELISA kits.
Data are presented as mean ± SD and determined using Tukey's test.
*P<0.05, **P<0.01, ****P<0.0001. LM, lipid mediators;
Nrf2, nuclear factor erythroid 2-related factor 2; LPS,
lipopolysaccharide; IL, interleukin; TNF-α, tumor necrosis
factor-α; ELISA, enzyme-linked immunosorbent assay.

Figure 5

LM inhibits the pathological
alterations in LPS-induced ALI mice. (A) H&E-stained images of
lung tissue injury. Scale bar, 100 µm. (B) Histological scoring of
lung injury; black arrows indicate hemorrhage and destruction of
alveolar structure. Data are presented as mean ± SD and was
determined using Kruskal-Wallis followed by Dunn's post hoc test.
*P<0.05, ***P<0.001. (C) The W/D ratio was measured in the
LPS-induced ALI model. (D) MPO from lung was determined. Data are
presented as mean ± SD and was assessed using Tukey's test.
**P<0.01, ****P<0.0001. LM, lipid mediators; LPS,
lipopolysaccharide; ALI, acute lung injury; H&E, hematoxylin
and eosin; W/D, wet-to-dry; MPO, myeloperoxidase.

Figure 6

LM reduces the inflammatory cytokines
in BALF and serum in LPS-induced ALI mice. (A) TNF-α, (B) IL-6 and
(C) IL-1β in BALF obtained from LPS-induced ALI mice, was measured
by ELISA kits. (D) TNF-α, (E) IL-6 and (F) IL-1β levels in serum of
LPS-induced ALI mice, was detected by ELISA kits. Data are
presented as mean ± SD and determined using Tukey's test.
***P<0.001, ****P<0.0001. LM, lipid mediators; BALF,
bronchoalveolar lavage fluid; LPS, lipopolysaccharide; ALI, acute
lung injury; TNF-α, tumor necrosis factor-α; IL, interleukin;
ELISA, enzyme-linked immunosorbent assay.

Figure 7

LM alters oxidative stress in
LPS-induced ALI mice. The level of (A) MDA, (B) GSH, (C) SOD and
(D) CAT were determined in lung homogenates from LPS-induced ALI
mice. Data are presented as mean ± SD and determined using Tukey's
test. **P<0.01, ***P<0.001, ****P<0.0001. LM, lipid
mediators; LPS, lipopolysaccharide; ALI, acute lung injury; MDA,
malondialdehyde; GSH, glutathione; SOD, superoxide dismutase; CAT,
catalase; NC, normal control.

Figure 8

LM modulates NF-κB and Nrf2/HO-1
signaling in LPS-induced ALI mice. (A) Western blotting was
conducted to determine p65 and pp65. (B) Quantification of these
proteins via densitometric analysis with GAPDH as the internal
control. (C) The expression of Nrf2 and HO-1 were analyzed by
western blotting. (D and E) Quantification of Nrf2 and HO-1
expression via densitometric analysis. Data are presented as mean ±
SD and determined using Tukey's test. ***P<0.001,
****P<0.0001. LM, lipid mediators; HO-1, heme oxygenase-1; LPS,
lipopolysaccharide; ALI, acute lung injury; NC, normal control.
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Copy and paste a formatted citation
Spandidos Publications style
Su Y, Choi HS, Kwon SK, Han Y, Cho S, Shin J, Jang Y, Choi JH and Seo J: Protective effects of lipid mediators, obtained from docosahexaenoic acid via soybean lipoxygenase, on lipopolysaccharide‑induced acute lung injury through the NF‑&kappa;B and Nrf2/HO‑1 signaling pathways. Mol Med Rep 32: 233, 2025.
APA
Su, Y., Choi, H.S., Kwon, S.K., Han, Y., Cho, S., Shin, J. ... Seo, J. (2025). Protective effects of lipid mediators, obtained from docosahexaenoic acid via soybean lipoxygenase, on lipopolysaccharide‑induced acute lung injury through the NF‑&kappa;B and Nrf2/HO‑1 signaling pathways. Molecular Medicine Reports, 32, 233. https://doi.org/10.3892/mmr.2025.13598
MLA
Su, Y., Choi, H. S., Kwon, S. K., Han, Y., Cho, S., Shin, J., Jang, Y., Choi, J. H., Seo, J."Protective effects of lipid mediators, obtained from docosahexaenoic acid via soybean lipoxygenase, on lipopolysaccharide‑induced acute lung injury through the NF‑&kappa;B and Nrf2/HO‑1 signaling pathways". Molecular Medicine Reports 32.3 (2025): 233.
Chicago
Su, Y., Choi, H. S., Kwon, S. K., Han, Y., Cho, S., Shin, J., Jang, Y., Choi, J. H., Seo, J."Protective effects of lipid mediators, obtained from docosahexaenoic acid via soybean lipoxygenase, on lipopolysaccharide‑induced acute lung injury through the NF‑&kappa;B and Nrf2/HO‑1 signaling pathways". Molecular Medicine Reports 32, no. 3 (2025): 233. https://doi.org/10.3892/mmr.2025.13598
Copy and paste a formatted citation
x
Spandidos Publications style
Su Y, Choi HS, Kwon SK, Han Y, Cho S, Shin J, Jang Y, Choi JH and Seo J: Protective effects of lipid mediators, obtained from docosahexaenoic acid via soybean lipoxygenase, on lipopolysaccharide‑induced acute lung injury through the NF‑&kappa;B and Nrf2/HO‑1 signaling pathways. Mol Med Rep 32: 233, 2025.
APA
Su, Y., Choi, H.S., Kwon, S.K., Han, Y., Cho, S., Shin, J. ... Seo, J. (2025). Protective effects of lipid mediators, obtained from docosahexaenoic acid via soybean lipoxygenase, on lipopolysaccharide‑induced acute lung injury through the NF‑&kappa;B and Nrf2/HO‑1 signaling pathways. Molecular Medicine Reports, 32, 233. https://doi.org/10.3892/mmr.2025.13598
MLA
Su, Y., Choi, H. S., Kwon, S. K., Han, Y., Cho, S., Shin, J., Jang, Y., Choi, J. H., Seo, J."Protective effects of lipid mediators, obtained from docosahexaenoic acid via soybean lipoxygenase, on lipopolysaccharide‑induced acute lung injury through the NF‑&kappa;B and Nrf2/HO‑1 signaling pathways". Molecular Medicine Reports 32.3 (2025): 233.
Chicago
Su, Y., Choi, H. S., Kwon, S. K., Han, Y., Cho, S., Shin, J., Jang, Y., Choi, J. H., Seo, J."Protective effects of lipid mediators, obtained from docosahexaenoic acid via soybean lipoxygenase, on lipopolysaccharide‑induced acute lung injury through the NF‑&kappa;B and Nrf2/HO‑1 signaling pathways". Molecular Medicine Reports 32, no. 3 (2025): 233. https://doi.org/10.3892/mmr.2025.13598
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