Role of Phospholipase A2 (PLA2) Inhibitors in Prevention of Apotheosis of the Corneal Epithelial Cells and Mitigation of Acanthamoeba keratitis

Abstract

Purpose: : We have identified the mannose-induced protein (MIP-133) from A. castellanii trophozoites. MIP-133 plays an important role in the pathogenesis of infection and induced apoptosis of human and Chinese hamster corneal epithelial cells. The aim of this study is to determine if MIP-133 induces apoptosis of corneal epithelial cells through phospholipase A₂ (PLA₂) mediated pathways.The efficacy of PLA₂ inhibitors to provide protection against Acanthamoeba keratitis was examine in vivo.

Methods: : Human corneal epithelial cells (HCE) were incubated in the presence or absence of MIP-133 for 24 hr and extracellular arachidonic acid release assay were performed. Percent apoptosis was determined by the caspase-3 assay. The effect of various PLA₂ inhibitors on apoptosis and arachidonic acid release was tested in vitro. Inhibition experiments involved pre-incubating the corneal epithelial cells for 1 hr with either 10 uM of MAFP (Methyl-arachidonyl fluorophosphonate) or 20 uM AACOCF3 (Arachidonyl trifluoromethyl ketone) selective inhibitor of human cytosolic PLA₂(cPLA ₂). Animals were infected with A. castellanii-laden contact lenses. MAFP (50ug/5ul) was injected under the contact lens of an infected cornea three times a day for 7 days after infection. Control animals were treated with 5ul of PBS. Keratitis was observed 7 days after infection

Results: : MIP-133 induced significant arachidonic acid release from HCE cells and PLA₂ inhibitors significantly reduced arachidonic acid release from HCE cells (P<0.05). MIP-133-induced apoptosis in corneal cells and PLA₂ inhibitors significantly inhibited MIP-133-induced apoptosis in the HCE cells (P<0.05). Treatment with the PLA₂ inhibitor had a profound effect on the severity and chronicity of keratitis. In addition, hamsters treated with PLA₂ inhibitor had significantly less severe keratitis as compared with control groups.

Conclusions: : These results indicate that PLA₂ is involved in apoptosis and arachidonic acid release from corneal epithelial cells that induced by MIP-133. Moreover, PLA₂ inhibitors can be used as a therapeutic target in Acanthamoeba keratitis.

Keywords: Acanthamoeba • cornea: basic science • keratitis