Influence of Omega 3 and 6 Fatty Acids on Human Meibomian Gland Epithelial Cells

Abstract

Purpose: Oral supplementation with omega 3 (ω-3) and/or 6 (ω-6) fatty acids (FAs) has been reported to alleviate the signs and symptoms of dry eye disease and to improve the expressibility and quality of meibum, in patients with meibomian gland dysfunction. We tested our hypothesis that these FA effects may reflect a direct FA action on human meibomian gland epithelial cells.

Methods: Immortalized human meibomian gland epithelial cells (IHMGECs) were cultured with ω-3, ω-6, or both FAs together for up to 7 days in the presence or absence of serum. After FA exposure, cells were analyzed for lipid expression, lysosome content, and proliferative ability.

Results: Our research shows that ω-3 and ω-6 stimulate the accumulation of small neutral lipid-containing vesicles, but not lysosomes, in IHMGECs. This vesicular effect was associated with a 2.4- to 3.7-fold increase in the cellular content of triglycerides after ω-3 and ω-6 treatment, respectively. The combination of both FAs together also enhanced triglyceride levels. Of interest, culture of IHMGECs with ω-3 and azithromycin, a known inducer of IHMGEC differentiation, led to a significantly greater amount of total neutral lipids, relative to that found with azithromycin alone. Cellular exposure to the FAs did not alter the expression of free or esterified cholesterol, or phospholipids. Further, these FAs, alone or together, prevented the proliferation of IHMGECs in serum-free, but not serum-containing, media.

Conclusions: Our findings support our hypothesis and demonstrate that ω-3 and ω-6 can act directly on IHMGECs to influence the quality and quantity of intracellular lipids.

Figure 1

Effects of ω-3 and/or ω-6 on the quantity and quality of lipids in IHMGECs. Cells were treated with vehicle, 10⁻⁵M ω-3, 10⁻⁵M ω-6, ω-3+ω-6 (each 0.5 × 10⁻⁵), or FAs combined with 10 μg/ml AZM for 7 days. a. Cells were stained with LipidTOX, and the green color indicates neutral lipids. The images were obtained with a fluorescent microscope. b. The fluorescence intensity of Lipid TOX staining was measured by using ImageJ. ***p<0.001 and ****p < 0.0001 versus control. **†<0.01 and ***†<0.001 versus AZM. The experiments were repeated 3 times, and the results from a single experiment are shown. c. The lipid extractions were analyzed by HPTLC, and the band intensities were measured by using ImageJ; control band instensity was set to 1, and data (mean ± SE) are reported as fold-change compared to control values. The results displayed are from 5 separate experiments.

Figure 2

Influence of ω-3 and/or ω-6 on the accumulation of acidic organelles in IHMGECs. Cells were cultured with vehicle, 10⁻⁵M ω-3, 10⁻⁵M ω-6, ω-3+ω-6 (each 0.5 × 10⁻⁵), or FAs combined with 10 μg/ml AZM for 7 days in a and b, and 5 days for c, d and e. a. Cell samples in duplicate were exposed to LysoTracker, which stains acidic organelles a red color. Images were obtained with a fluorescent microscope. b. The fluorescence intensities were measured by using ImageJ. ****p < 0.0001 versus control. *†<0.05, **†<0.01 versus AZM. The staining was repeated 3 times, and the results shown are from a single experiment. c. Cell lysates were evaluated on Western blots for Lamp-1 and LC3 in triplicate. d. The samples containing AZM showed a significant increase of Lamp-1 level comparing to control. *p<0.05. e. There was no significant difference between the effects of ω-3 or ω-6 combined with AZM or AZM alone. The remaining samples displayed weak signals for LC3 expression.

Figure 3

Impact of ω-3 and/or ω-6 on the proliferation of IHMGECs. Cells were seeded at a 10,000 cells/well in serum-free media (a), and 50,000 cells/well in serum-containing media (b), in12-well plates (n=3 wells/treatment). IHMGECs were treated with vehicle, 10⁻⁵M ω-3, 10⁻⁵M ω-6, ω-3+ω-6 (each 0.5 × 10⁻⁵), or AZM (10 μg/ml) for 5 days before cell counting. Results were reported as mean ± SE. Data from one experiment are shown as a representative of three studies performed under the same conditions. ***p<0.001 and ****p<0.0001.