The medium-chain fatty acid decanoic acid reduces oxidative stress levels in neuroblastoma cells

Abstract

Enhanced oxidative stress is a contributing factor in the pathogenesis of several neurodegenerative disorders such as Alzheimer´s disease. Beneficial effects have been demonstrated for medium-chain fatty acids (MCFAs) nutritionally administered as medium-chain triglycerides (MCTs) or coconut oil (CO). The observed effects on cognitive impairment are generally attributed to the hepatic metabolism of MCFAs, where resulting ketone bodies serve as an alternate energy source to compensate for the impaired glucose utilisation in the human brain. Here we show that the saturated MCFA decanoic acid (10:0) reduces the oxidative stress level in two different neuroblastoma cell lines. Phosphatidylcholine (PC) containing decanoic acid (10:0) (PC10:0/10:0) reduced the cellular H₂O₂ release in comparison to solvent, L-α-Glycerophosphorylcholine and PC containing the long-chain fatty acid (LCFA) arachidic acid (20:0). This effect seems to be at least partially based on an upregulation of catalase activity, independent of alterations in catalase gene expression. Further, PC10:0/10:0 decreased the intracellular oxidative stress level and attenuated the H₂O₂-induced cell death. It did not affect the level of the ketone body β-hydroxybutyrate (βHB). These results indicate that decanoic acid (10:0) and possibly MCFAs in general directly reduce oxidative stress levels independent of ketone levels and thus may promote neuronal health.

Figure 1

Impact of PC10:0/10:0, PC20:0/20:0 or solvent on the extracellular H₂O₂ level and H₂O₂ release. Cells were incubated with the solvent EtOH (0.2%) (set as 100%), PC10:0/10:0, PC20:0/20:0 (10 µM except for e) for 18 h (except for g) before H₂O₂ accumulating in the medium during the treatment and H₂O₂ release was measured by using Amplex Red (5 µM) and HRP (0.01 U/ml). 10 µM L-α-Glycerophosphorylcholine was used as an additional reference (f, 10 µM). H₂O₂ level in the conditioned medium of (a) SH-SY5Y cells (n = 12) and (b) Neuro2a cells (n = 8). H₂O₂ released by (c) SH-SY5Y cells (n = 28), (d) Neuro2a cells (n = 8), (e) SH-SY5Y cells treated with the solvent EtOH (0.2%) or different concentrations (1, 3 and 10 µM) of PC10:0/10:0 or PC20:0/20:0 (n ≥ 6), (f) SH-SY5Y cells additionally treated with 10 µM l-α-Glycerophosphorylcholine as reference (n = 6) and (g) SH-SY5Y cells after short-term treatment for 30 min (n = 14). Error bars represent SEM. Asterisks show the statistical significance calculated by one-way ANOVA followed by post hoc testing using Tukey's test (* p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001). Figure was created using Origin Pro 2020b and CorelDRAW Graphics Suite 2020.

Figure 2

Impact of PC10:0/10:0, PC20:0/20:0 or solvent on the intracellular ROS level and H₂O₂-induced cytotoxicity. Cells were incubated with the solvent EtOH (0.2%) (set as 100%), PC10:0/10:0 or PC20:0/20:0 (10 µM) in absence (a,b) or presence (c) of 500 µM H₂O₂ for 18 h before intracellular ROS level and H₂O₂-induced changes in cell viability were examined. Intracellular ROS level measured by using CellROX Green in (a) SH-SY5Y cells (n = 13) and (b) Neuro2a cells (n = 12). (c) H₂O₂-induced changes in the viability of SH-SY5Y cells determined by labeling dead cells with propidiumiodide (n = 4). Error bars represent SEM. Asterisks show the statistical significance calculated by one-way ANOVA followed by post hoc testing using Tukey's test (* p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001). Figure was created using Origin Pro 2020b and CorelDRAW Graphics Suite 2020.

Figure 3

Impact of PC10:0/10:0, PC20:0/20:0 or solvent on the intra- and extracellular βHB level. Cells were incubated with the solvent EtOH (0.2%) (set as 100%), PC10:0/10:0 or PC20:0/20:0 (10 µM) for 18 h before the level of βHB was measured by using the β-Hydroxybutyrate (Ketone Body) Colorimetric Assay Kit. βHB level in the homogenates of (a) SH-SY5Y cells (n = 8) and (b) Neuro2a cells (n ≥ 5). βHB level in the conditioned medium of (c) SH-SY5Y cells (n = 8) and (d) Neuro2a cells (n ≥ 5). Error bars represent SD. The statistical significance was calculated by one-way ANOVA followed by post hoc testing using Tukey's test, no significant differences were observed. Figure was created using Origin Pro 2020b and CorelDRAW Graphics Suite 2020.

Figure 4

Impact of PC10:0/10:0, PC20:0/20:0 or solvent on the activity of antioxidative enzymes and on catalase gene expression. Cells were incubated with the solvent EtOH (0.2%) (set as 100%), PC10:0/10:0, PC20:0/20:0 (10 µM) for 18 h in absence (a,b,e) or presence of PPARγ antagonists (c) and inhibitors of protein biosynthesis (d). Catalase, GPx and SOD activity in the homogenates of (a) SH-SY5Y cells (catalase: n ≥ 5, GPx: n ≥ 5, SOD: n ≥ 6) and (b) Neuro2a cells (catalase: n ≥ 6, GPx: n ≥ 5, SOD: n ≥ 6) determined by using the corresponding enzyme activity assay kits. The activity level of the respective enzyme in untreated cells is indicated by a dotted line. H₂O₂ released by SH-SY5Y cells measured by using Amplex Red (5 µM) and HRP (0.01 U/ml) after treatment with phospholipids or solvent along with (c) 5 µM BADGE / 5 µM GW9662/ DMSO (n ≥ 4) and (d) 20 µM cycloheximide/ 2 µM puromycin/ DMSO (n ≥ 5). (e) Catalase gene (CAT) expression in SH-SY5Y cells measured by RT-PCR using two different CAT primer pairs and two different house keeping genes (ACTB: β-actin, TBP: TATA-binding protein) for normalization (n = 7). Error bars represent SD (a,b,e) or SEM (c,d). Asterisks show the statistical significance calculated by one-way ANOVA followed by post hoc testing using Tukey's test (** p ≤ 0.01 and *** p ≤ 0.001). Figure was created using Origin Pro 2020b and CorelDRAW Graphics Suite 2020.

Figure 5

Schematic overview of the cellular generation and detoxification of O₂^•− and H₂O₂ including the observed effects of decanoic acid (10:0). The mitochondrial electron transport chain and NOX activity are the major cellular sources of O₂^•−, which is rapidly converted to H₂O₂ by distinct SODs (mitochondrial SOD2, cytosolic SOD1, extracellular SOD3). In contrast to other NOX isoenzymes generating O₂^•−, NOX4 predominatly releases H₂O_2. H₂O₂ diffuses across cellular membranes and is intracellularly detoxified by antioxidant enzymes such as GPx and catalase. Decanoic acid (10:0) administered in the form of PC10:0/10:0 reduces the intracellullar ROS level with O₂^•− probably representing the major detected ROS species. Additionally, the steady state level of extracellular H₂O₂ as well as H₂O₂ release is decreased in cells treated with PC10:0/10:0. The latter effect might be at least partially based on an upregulation of catalase activity independent of alterations in CAT gene expression. Figure was created using CorelDRAW Graphics Suite 2020.