Coconut Oil Alleviates the Oxidative Stress-Mediated Inflammatory Response via Regulating the MAPK Pathway in Particulate Matter-Stimulated Alveolar Macrophages

Abstract

Exposure to particulate matter (PM) is related to various respiratory diseases, and this affects the respiratory immune system. Alveolar macrophages (AMs), which are defenders against pathogens, play a key role in respiratory inflammation through cytokine production and cellular interactions. Coconut oil demonstrates antioxidant and anti-inflammatory properties, and it is consumed worldwide for improved health. However, reports on the protective effects of coconut oil on the PM-induced respiratory immune system, especially in AMs, are limited. In this study, we generated artificial PM (APM) with a diameter approximately of 30 nm by controlling the temperature, and compared its cytotoxicity with diesel exhaust particles (DEP). We also investigated the antioxidant and anti-inflammatory effects of coconut oil in APM− and DEP−stimulated AMs, and the underlying molecular mechanisms. Our results showed that APM and DEP had high cytotoxicity in a dose-dependent manner in AMs. In particular, APM or DEP at 100 μg/mL significantly decreased cell viability (p < 0.05) and significantly increased oxidative stress markers such as reactive oxygen species (p < 0.01); the GSSH/GSH ratio (p < 0.01); and cytokine production, such as tumor necrosis factor-α (p < 0.001), interleukin (IL)-1β (p < 0.001), and IL-6 (p < 0.001). The expression of the genes for chemokine (C-X-C motif) ligand-1 (p < 0.05) and monocyte chemoattractant protein-1 (p < 0.001); and the proteins toll-like receptor (TLR) 4 (p < 0.01), mitogen-activated protein kinase (MAPK), and c-Jun N-terminal kinase (p < 0.001), p38 (p < 0.001); and extracellular receptor-activated kinase (p < 0.001), were also upregulated by PM. These parameters were reversed upon treatment with coconut oil in APM− or DEP−stimulated AMs. In conclusion, coconut oil can reduce APM− or DEP−induced inflammation by regulating the TLR4/MAPK pathway in AMs, and it may protect against adverse respiratory effects caused by PM exposure.

Keywords: alveolar macrophage; artificial particulate matter; coconut oil; diesel exhaust particulates; inflammation; mitogen-activated protein kinase; oxidative stress.

Figure 1

Cytotoxicity of artificial particulate matter (APM) and diesel exhaust particles (DEP) in alveolar macrophages (MH-S). Negative controls (NC) are untreated samples. MH-S was treated with APM and DEP of different concentrations for 6 h. (A) Cell viability was measured using the 3–(4,5–dimethylthiazol–2–yl)–2,5–diphenyltetrazolium bromide (MTT) assay in the APM– and DEP–stimulated MH-S cells. (B) Morphological changes in MH-S by exposure to APM/DEP. Data are presented as the mean ± SD (n = 6 per group). * p < 0.05, ** p < 0.01, or *** p < 0.001 vs. NC.

Figure 2

Artificial particulate matter (APM) and diesel exhaust particles (DEP) induce oxidative stress in alveolar macrophages (MH−S). (A) Cytotoxicity of N−Acetyl Cysteine (NAC). (B) Reactive oxygen species level was measured using 2′,7′−dichlorofluorescein diacetate (DCF−DA) staining. Negative controls (NC) are untreated samples. MH−S was pretreated with NAC for 3 h and was stimulated with 100 μg/mL APM or DEP for 3 h. Data are presented as the means ± SD (n = 4 per group). ** p < 0.01, or *** p < 0.001 vs. NC; ^### p < 0.001 vs. APM; ^§§§ p < 0.001 vs. DEP group.

Figure 3

Effects of coconut oil on oxidative stress in artificial particulate matter (APM) and diesel exhaust particles (DEP)−stimulated alveolar macrophages (MH−S). (A,B) Cytotoxicity assessment of distilled water (DW) and coconut oil the 3–(4,5–dimethylthiazol–2–yl)–2,5–diphenyltetrazolium bromide (MTT) assay. (C) Cytotoxicity assessment of coconut oil in APM and DEP−stimulated MH−S. (D) Reactive oxygen species (ROS) production was measured using 2′,7′−dichlorofluorescein diacetate (DCF−DA) staining. Data represent the mean ± SD (n = 8 per group). (E) NADPH dehydrogenase (quinone) (NQO1) gene level was measured using real−time PCR. (F) Glutathione (GSH), (G) glutathione disulfide (GSSG), (H) GSSG/GSH ratio, and (I) Nicotinamide adenine dinucleotide phosphate (NADPH) levels in MH−S. Negative controls (NC) are untreated samples. Vehicle control (VC) was treated with cell medium including 10% DW. Data are presented as the means ± SD of three independent experiments. * p < 0.05, ** p < 0.01 or *** p < 0.001, **** p < 0.0001 vs. NC or VC; ^## p < 0.01 or ^### p < 0.001 vs. APM group; ^§§ p < 0.01 or ^§§§ p < 0.001 vs. DEP group.

Figure 4

Effects of coconut oil on artificial particulate matter (APM) and diesel exhaust particles (DEP)−induced inflammation in alveolar macrophages (MH−S). Protein levels of (A) tumor necrosis factor−α (TNF−α), (B) interleukin (IL)−1β, and (C) IL−6 in MH−S were determined using enzyme−linked immunosorbent assay (ELISA). (D,E) Relative gene expression of inflammatory factors in APM/DEP–stimulated MH−S cells. Vehicle control (VC) was treated with cell medium including 10% distilled water. All experiments were independently performed in triplicate. * p < 0.5 or *** p < 0.001 vs. VC; ^# p < 0.05 or ^### p < 0.001 vs. APM; ^§ p < 0.05 or ^§§ p < 0.01, ^§§§ p < 0.001 vs. DEP.

Figure 5

Effect of coconut oil on mitogen−activated protein kinase (MAPK) pathway in artificial particulate matter (APM) and diesel exhaust particles (DEP)−stimulated alveolar macrophages (MH−S). (A–C) Phosphorylation of extracellular receptor-activated kinase (ERK), C−Jun N−terminal kinase (JNK), and p38 MAPK was detected via Western blotting. (D–F) Relative densities of p−JNK, p−p38 MAPK, and p−ERK proteins. Vehicle control (VC) was treated with cell medium including 10% distilled water. Data are presented as the means ± SD (n = 3 per group). The normalized value of target protein expression level in the control group was set to 1. *** p < 0.001 vs. VC; ^### p < 0.001 vs. APM; ^§§§ p < 0.001 vs. DEP groups.

Figure 6

(A) Representative Western blots and (B) relative density of toll−like receptor (TLR) 4 expression in artificial particulate matter (APM) and diesel exhaust particles (DEP)−stimulated alveolar macrophages (MH−S). Vehicle control (VC) was treated with cell medium including 10% distilled water. Data are presented as the means ± SD (n = 3 per group). ** p < 0.01 or *** p < 0.001 vs. VC; ^## p < 0.01 vs. APM group; ^§§§ p < 0.001 vs. DEP group.