Abstract
Purpose: :
Recent studies showed that systemic polyunsaturated fatty acids (PUFAs) may improve the symptoms of dry eye syndrome due to their anti-inflammatory effects. We have evaluated the anti-inflammatory effects of PUFAs and mechanism of action on human corneal epithelial (HCE) cells in-vitro.
Methods: :
HCE cells were incubated for 3 hours with different concentrations of three PUFAs: Alpha-linolenic acid (ALA), Gamma-linolenic acid (GLA) and Linolenic acid (LA). Oleic acid (OA) and Dexamethasone (DM) served as negative and positive controls, respectively. Cells were stimulated with either polyinosinic : polycytidylic acid (poly I:C) or lipopolysaccharide (LPS) complex (LPS combined with CD14 and LPS binding protein). After exposure to PUFAs, cells and supernatants were collected and tested for the expression of several pro-inflammatory cytokines. The protein contents and mRNA expression levels of Interleukin (IL)-6, IL-8, IL-1β, Tumor necrosis factor-α (TNF-α) and inhibitory factor-ΚBα (I-ΚBα) were tested with multiplex fluorescent bead immunoassay and real time-PCR, respectively. Viability of cells was tested with FITC-Annexin V/PI at the end of each treatment.
Results: :
The protein contents and mRNA expression levels of IL-6, IL-8, IL-1β and TNF-α were significantly increased after stimulation with LPS or poly I:C. These levels were dramatically decreased following treatment with ALA as compared to OA or bovine serum albumin (p < 0.05). HCE cells incubation with LPS complex stimulation elicited up to 10-fold higher levels of IL-6 (P<0.001), 4-fold IL-1β (P<0.001), 20-fold TNFα (P<0.001) and 2.5-fold IL-8 (P<0.001) compared to cells incubated in medium alone. Following treatment with ALA, a significant decrease was demonstrated in the protein content of TNF-α to 23.81% (P<0.001), IL-6 to 46.71% (P<0.001), IL-1β to 20.86% (P<0.05) and IL-8 to 52.21% (P<0.001). Similar results were demonstrated at the mRNA level, as measured by real time PCR. The anti-inflammatory effects of ALA were similar to those of DM for all of the pro-inflammatory cytokines. A significant dose-dependent relation was demonstrated for the effects of ALA on the suppression of all of the cytokines tested. The ALA inhibition of the LPS-induced pro-inflammatory cytokines was associated with a significant reduction of the mRNA expression level of I-ΚBα.
Conclusions: :
ALA may serve as a potent anti-inflammatory agent in ocular surface inflammation, as evaluated in cultured HCE. The anti-inflammatory effects of ALA are comparable to those of the corticosteroid DM, and are mediated through I-ΚBα signal transduction.
Keywords: inflammation • cytokines/chemokines • cornea: epithelium