Linoleic Acid Induces Metabolic Reprogramming and Inhibits Oxidative and Inflammatory Effects in Keratinocytes Exposed to UVB Radiation

Abstract

Linoleic acid (LA), the primary ω-6 polyunsaturated fatty acid (PUFA) found in the epidermis, plays a crucial role in preserving the integrity of the skin's water permeability barrier. Additionally, vegetable oils rich in LA have been shown to notably mitigate ultraviolet (UV) radiation-induced effects, including the production of reactive oxygen species (ROS), cellular damage, and skin photoaging. These beneficial effects are primarily ascribed to the LA in these oils. Nonetheless, the precise mechanisms through which LA confers protection against damage induced by exposure to UVB radiation remain unclear. This study aimed to examine whether LA can restore redox and metabolic equilibria and to assess its influence on the inflammatory response triggered by UVB radiation in keratinocytes. Flow cytometry analysis unveiled the capacity of LA to diminish UVB-induced ROS levels in HaCaT cells. GC/MS-based metabolomics highlighted significant metabolic changes, especially in carbohydrate, amino acid, and glutathione (GSH) metabolism, with LA restoring depleted GSH levels post-UVB exposure. LA also upregulated PI3K/Akt-dependent GCLC and GSS expression while downregulating COX-2 expression. These results suggest that LA induces metabolic reprogramming, protecting against UVB-induced oxidative damage by enhancing GSH biosynthesis via PI3K/Akt signaling. Moreover, it suppresses UVB-induced COX-2 expression in HaCaT cells, making LA treatment a promising strategy against UVB-induced oxidative and inflammatory damage.

Keywords: keratinocyte; linoleic acid; photodamage.

Figure 1

Determination of cell viability according to the CCK-8 (Cell Counting Kit-8) assay. The bar graphs represent the percentages of cell viability of HaCaT cells (A) irradiated with different doses of UVB, (B) treated with different concentrations of LA, and (C) irradiated with UVB and treated with 25 µM or 50 µM of LA. The evaluation of cell viability was determined at 6 h after treatment in all cases. Significance was calculated using the one-way ANOVA test and was corrected for multiple comparisons using Dunnett’s test. Black bars: non-irradiated cells; red bars: untreated UVB-irradiated cells; white bars: LA-treated cells.

Figure 2

Effect of LA on UVB-induced ROS production in keratinocytes. (A) A representative histogram of four different experiments. A shift to the left of the histogram corresponding to the HaCaT cells irradiated and treated with 50 µM of LA can be observed, similar to that obtained from the non-irradiated control. (B) The average fluorescence intensity was analyzed using FACS. The average dichlorofluorescein (DCF) levels in the UVB-irradiated HaCaT cells were higher than in the non-irradiated control, while the average DCF values in the irradiated cells treated with 50 μM LA were lower than in the irradiated cells. Bar graphs represent the mean ± the standard error of the mean (SEM) from four independent experiments. * p < 0.05; ** p < 0.01 compared to cells exposed to UVB radiation without treatment. Black bar: non-irradiated cells; red bar: untreated UVB-irradiated cells; blue bars: UVB and LA-treated cells.

Figure 3

Partial least squares-discriminant analysis (PLS-DA) score plots based on metabolomic analysis. (A) HaCaT cells irradiated with 45 mJ/cm² (green) and non-irradiated control cells (red) at 6 h post-irradiation. (B) Cells irradiated with 45 mJ/cm² of UVB (green) and cells irradiated with UVB and treated with 50 µM LA (blue), at 6 h post-irradiation. The explained variances of the selected components are shown in brackets. n = 5, independent experiments.

Figure 4

Heatmap of changes in metabolite profiles induced by (A) UVB radiation and (B) LA treatment in UVB-irradiated cells. Rows represent specific intracellular metabolites and columns represent conditions. n = 5, independent experiments.

Figure 5

Overview of the main metabolic pathways affected by (A) radiation with 45 mJ/cm² UVB and (B) HaCaT cells irradiated with UVB and treated with 50 µM LA, after 6 h. n = 5, independent experiments.

Figure 6

Effects of LA treatment on the synthesis of glutathione and its precursors in UVB-irradiated HaCaT cells. (A–E) Relative abundances of glutathione and typical metabolites related to their synthesis (axis Y = relative abundance with respect to ribitol). (F) Quantification of GSH with the method based on Ellman’s reagent (DTNB, 5,5′-dithio-bis-2-nitrobenzoic acid). Data are presented as mean ± SEM. ** p < 0.01, compared to control cells. n = 5, independent experiments. Black bar: non-irradiated cells; red bar: untreated UVB-irradiated cells; blue bars: UVB and LA-treated cells.

Figure 7

Involvement of the PI3K/Akt pathway in LA-induced GSH levels in UVB-irradiated HaCaT cells. (A) Levels of the phosphorylated form of Akt were analyzed by Western blot and total Akt was used as a loading control. A representative image from an experiment is shown (below). The densitometric ratios of p-Akt/Akt are shown in the bar graph as mean ± SEM (top). (B) Quantification of GSH in the presence or absence of the PI3K inhibitor, LY204002. * p < 0.05, ** p < 0.01, compared to the UVB control. n = 5, independent experiments. ns: not significant. Black bar: non-irradiated cells; red bar: untreated UVB-irradiated cells; blue bars: UVB and LA-treated cells; grey bars: cells treated with inhibitor LY294002.

Figure 8

Linoleic acid increases GCLC and GSS levels in UVB-irradiated HaCaT cells. Bar graphs show the relative mRNA expression levels of (A) GCLC and (B) GSS. RPS9 was used as a housekeeping gene. The levels of the proteins (C) GCLC and (D) GSS were determined through Western blot and GAPDH was used as a loading control. Each bar represents the mean ± SEM. * p < 0.05; *** p < 0.005 and **** p < 0.001, compared to the UVB control. n = 5, independent experiments. Black bar: non-irradiated cells; red bar: untreated UVB-irradiated cells; blue bars: UVB and LA-treated cells.

Figure 9

LA-induced GCLC and GSS expression is mediated through the PI3K/Akt pathway. Total proteins were analyzed through immunoblotting using specific antibodies against (A) GCLC and (B) GSS in the presence or absence of the PI3K inhibitor. GAPDH was used as a loading control. (Bottom) Images from a representative experiment are shown. (Top) Densitometric ratios of GCLC/GAPDH or GSS/GAPDH are shown in the graphs as mean ± SEM. * p < 0.05; ** p < 0.01 compared to the UVB control. n = 5, independent experiments. Black bar: non-irradiated cells; red bar: untreated UVB-irradiated cells; blue bar: UVB and LA-treated cells; grey bar: cells treated with inhibitor LY294002.

Figure 10

Linoleic acid reduces COX-2 expression and PGE2 synthesis in UVB-irradiated HaCaT cells. (A) Bar graphs show the relative mRNA expression levels of COX-2. RPS9 was used as a housekeeping gene. (B) The levels of proteins of COX-2 were determined through Western blot and GAPDH was used as a loading control. (C) The graph shows the concentration of PGE2 determined through ELISA. Each bar represents the mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.005, compared to the UVB control. n = 5, independent experiments. ns: not significant. Black bar: non-irradiated cells; red bar: untreated UVB-irradiated cells; blue bars: UVB and LA-treated cells.