Fish Oil Containing Pro-Resolving Mediators Enhances the Antioxidant System and Ameliorates LPS-Induced Inflammation in Human Bronchial Epithelial Cells

Abstract

Fish oil, renowned for its high content of omega-3 fatty acids, particularly eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), has gained considerable attention for its potential health benefits. EPA and DHA exhibit anti-inflammatory effects by promoting the production of specialized pro-resolving mediators (SPMs), such as resolvins and protectins. Fish oil has been studied for its potential to reduce bronchial inflammation, a key feature of respiratory conditions like asthma and COPD. This study investigates the cellular mechanisms of fish oil in an in vitro model of lung inflammation using lipopolysaccharide (LPS) on a healthy human bronchial epithelium cell line. LPS exposure for 24 h reduced cell viability, elevated reactive oxygen species (ROS), depleted glutathione (GSH), and induced mitochondrial depolarization, indicating oxidative stress and inflammation. Fish oil administration significantly mitigated ROS production, prevented GSH depletion, and reduced mitochondrial depolarization. This was associated with the upregulation of the endogenous antioxidant system, evidenced by restored GSH levels and the increased gene expression of glutathione peroxidase (GPX), catalase (CAT), superoxide dismutase 1 (SOD1), and superoxide dismutase 2 (SOD2). Fish oil also suppressed IL-6 and IL-1β expression and increased anti-inflammatory cytokine IL-10 expression. Furthermore, fish oil upregulated the expression of pro-resolving mediator receptors, suggesting a role in inflammation resolution. These findings highlight the potential of fish oil supplementation as a preventive measure against pulmonary diseases characterized by unresolved inflammation such as lung inflammation.

Keywords: PUFAs; lung disease; omega-3; pro-resolving mediators; resolvins.

Figure 1

Biosynthetic pathways of specialized pro-resolving mediators (SPMs). Lipoxins (LxA4 and LxB4) are produced from AA, E-series of resolvins (RvE1, RvE2, and RvE3) are produced from EPA, and D-series of resolvins (RvD1, RvD2, RvD3, RvD4, RvD5, and RvD6), protectins (PD1 and PDX), and maresins (MaR1 and MaR2) are produced from DHA. HpEPE: hydroperoxyeicosapentaenoic acid; HpDHA: hydroperoxydocosahexaenoic acid.

Figure 2

FO and LPS impact on cell viability. (A) FO supplementation at 100 mg/mL decreases BEAS viability. MTT assay quantification detecting cell viability following FO treatment at 10, 50, and 100 μg/mL. BSA condition was included given its role as FO vehicle. (B) LPS supplementation hampers BEAS viability following 24 h treatment. Histograms are representative of MTT assay performed on BEAS treated with 1 and 10 μg/mL LPS at different timepoints. (C) FO supplementation recovers LPS-induced cell death. Quantification of MTT test assessing cell viability following FO 10 μg/mL, LPS 10 μg/mL single treatments, and their combination. Histograms are representative of 4 biological replicates. (* p ≤ 0.05; *** p ≤ 0.001; **** p ≤ 0.0001 compared to CTRL. #### p ≤ 0.0001 compared to LPS).

Figure 3

FO supplementation recovers cells’ LPS-induced mitochondrial potential loss and oxidative stress. (A) FO supplementation prevents LPS-induced JC-1 signal decrease. Representative images showing JC-1 assay in BEAS, supplemented with FO, LPS, and their combination. (B) FO recovers LPS-induced mitochondrial depolarization. Quantification of JC-1 signal as in (A). Histogram showing JC-1 quantification following BEAS treatment with FO, LPS, and their combination. (C) FO restores LPS-induced oxidative stress. Histogram showing ROS quantification by fluoresceine fluorescence measurement following BEAS treatment with FO, LPS, and their combination. Histograms are representative of 4 biological replicates. (** p ≤ 0.01; **** p ≤ 0.0001 compared to CTRL; ## p ≤ 0.01; #### p ≤ 0.0001 compared to LPS).

Figure 4

FO recovers cellular ROS scavenging ability. (A) FO supplementation implement GSH production upon LPS treatment. Histogram showing GSH quantification in BEAS following treatment with FO, LPS, and their combination. RT-PCR evaluating the expression GPX (B), CAT (C), SOD2 (D), IL-6 (E), IL-10 (F), and TNF- α (G) following treatment with FO, LPS, and their combination. Histograms are representative of 4 biological replicates. (* p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001 compared to CTRL. # p ≤ 0.05; ## p ≤ 0.01; ### p ≤ 0.001; #### p ≤ 0.0001 compared to LPS).

Figure 5

FO effect relies on resolvin-mediated inflammation resolution. RT- PCR analysis showing PTGS1 (A), ALOX15 (B), FPR2 (C), and GPR32 (D) expression following BEAS supplementation with FO, LPS, and their combination. Histograms are representative of 4 biological replicates. (* p ≤ 0.05; *** p ≤ 0.001; **** p ≤ 0.0001 compared to CTRL. ### p ≤ 0.001; #### p ≤ 0.0001 compared to LPS).