CYTOSOLIC PHOSPHOLIPASE A2 ALPHA INHIBITORS ATTENUATE APOPTOSIS OF THE CORNEAL EPITHELIAL CELLS AND MITIGATE ACANTHAMOEBA KERATITIS

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2013-04-12

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Tripathi, Trivendra

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Purpose: The aim of this study was to explore if MIP-133 from Acanthamoeba castellanii trophozoites induces apoptosis of Chinese hamster corneal epithelial cells (HCORN) in vitro via cPLA2ɑ pathway, and to determine the efficacy of cPLA2ɑ inhibitors to alleviate AK in vivo. Methods: HCORN were incubated with or without MIP-133 at doses of 7.5, 15, and 50µg/ml for 6, 12, and 24hrs. Inhibition of cPLA2ɑ was carried out by pre-incubating HCORN for 1hr with cPLA2ɑ inhibitors (10µM MAFP and 20µM AACOCF3) with or without 15µg/ml MIP-133 for 24hrs. Chinese hamsters were injected subconjunctivally with MIP-133 at dosage 40µg/40µl and eyes were removed 3 days after infection. Chinese hamsters were infected with parasite-laden contact lens and treated with cPLA2ɑ inhibitors (AACOCF3 and CAY10650) topically 50µg/5µl three times a day for 14 days post-infection. Expression of cPLA2ɑ at mRNA and enzyme levels was examined by RT-PCR and cPLA2ɑ enzyme assay. Apoptosis was determined by DNA fragmentation assay. CXCL2 expression was examined by RT-PCR and ELISA. In vivo infections were examined by clinical severity of disease scored on a scale of 0 to 5 based on corneal infiltration, corneal neovascularization, and corneal ulceration. Clinical pathology of cornea was examined by histopathology. Results: MIP-133 induces significant increase in cPLA2ɑ and CXCL2 levels from corneal cells. Moreover, cPLA2ɑ inhibitors, MAFP and AACOCF3, significantly reduce cPLA2ɑ and CXCL2 from these cells (P<0.05). Additionally, cPLA2ɑ inhibitors significantly inhibit MIP-133-induced apoptosis in HCORN cells (P<0.05). Subconjunctival injection of purified MIP-133 produced cytopathic properties characteristic of MIP-133. Treatments of cPLA2ɑ inhibitors showed significantly less severe keratitis and decreased clinical pathology as compared with control animals. Conclusions: Results suggest that cPLA2ɑ inhibitors therapeutically attenuate apoptosis of the corneal epithelial cells and mitigate AK.

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