Docosahexaenoic acid (DHA, 22:6 n-3) from fish oil, and butyrate, a fiber fermentation product, work coordinately to protect against colon tumorigenesis in part by inducing apoptosis. We have recently demonstrated that dietary DHA is incorporated into mitochondrial membrane phospholipids, thereby enhancing oxidative stress induced by butyrate metabolism. In order to elucidate the subcellular origin of oxidation induced by DHA and butyrate, immortalized young adult mouse colonocytes were treated with 0-200 microM DHA or linoleic acid (LA, 18:2 n-6; control) for 72 h with or without 5 mM butyrate for the final 24 h. Cytosolic reactive oxygen species, membrane lipid oxidation, and mitochondrial membrane potential (MP), were measured by live-cell fluorescence microscopy. After 24 h of butyrate treatment, DHA primed cells exhibited a 151% increase in lipid oxidation (P < 0.01), compared with no butyrate treatment, which could be blocked by a mitochondria-specific antioxidant, 10-(6'-ubiquinoyl) decyltriphenylphosphonium bromide (MitoQ) (P < 0.05). Butyrate treatment of LA pretreated cells did not show any significant effect. In the absence of butyrate, DHA treatment, compared with LA, increased resting MP by 120% (P < 0.01). In addition, butyrate-induced mitochondrial membrane potential (MP), dissipation was 21% greater in DHA primed cells as compared with LA at 6 h. This effect was reversed by preincubation with inhibitors of the mitochondrial permeability transition pore, cyclosporin A or bongkrekic acid (1 microM). The functional importance of these events is supported by the demonstration that DHA and butyrate-induced apoptosis is blocked by MitoQ. These data indicate that DHA and butyrate potentiate mitochondrial lipid oxidation and the dissipation of MP which contribute to the induction of apoptosis.

Lupton JR, Chapkin RS. Synergy between docosahexaenoic acid and butyrate elicits p53-independent apoptosis via mitochondrial Ca 2ϩ accumulation in colonocytes.

We have previously shown that butyrate, a short-chain fatty acid fiber fermentation product, induces colonocyte apoptosis via a nonmitochondrial, Fas-mediated, extrinsic pathway. Interestingly, fermentable fiber when combined with fish oil containing docosahexaenoic acid (DHA, 22:6n-3) exhibits an enhanced ability to induce apoptosis and protect against colon tumorigenesis. To determine the molecular mechanism of action, the effect of DHA and butyrate cotreatment on intracellular Ca 2+ homeostasis was examined. Mouse colonocytes were treated with 50 Mmol/L DHA or linoleic acid (LA) for 72 h F butyrate (0-10 mmol/L) for the final 24 h. Cytosolic and mitochondrial Ca 2+ levels were measured using Fluo-4 and Rhod-2. DHA did not alter basal Ca 2+ or the intracellular inositol trisphosphate (IP 3 ) pool after 6 h butyrate cotreatment. In contrast, at 12 and 24 h, DHA-and butyrate-treated cultures exhibited a 25% and 38% decrease in cytosolic Ca 2+ compared with LA and butyrate. Chelation of extracellular Ca 2+ abolished the effect of thapsigargin on the IP 3 -releasable Ca 2+ pool. DHA and butyrate cotreatment compared with untreated cells increased the mitochondrial-to-cytosolic Ca 2+ ratio at 6, 12, and 24 h by 73%, 18%, and 37%, respectively. The accumulation of mitochondrial Ca 2+ preceded the onset of apoptosis. RU-360, a mitochondrial-uniporter inhibitor, abrogated mitochondrial Ca 2+ accumulation and also partially blocked apoptosis in DHA and butyrate cotreated cells. Collectively, these data show that the combination of DHA and butyrate, compared with butyrate alone, further enhances apoptosis by additionally recruiting a Ca 2+

Pseudomonas aeruginosa is the primary bacterial pathogen causing contact lens related keratitis. Available ophthalmic agents have reduced efficacy and antimicrobial peptides (AMPs) hold promise as future antibiotics. Here we investigated the in vitro and in vivo anti-Pseudomonal activity of esculentin-1a(1-21)-NH2, derived from a frog skin AMP. The data revealed a minimum inhibitory concentration between 2 and 16 μM against reference strains or drug-resistant clinical isolates of P. aeruginosa without showing toxicity to human corneal epithelial cells up to 50 μM. At 1 μM the peptide rapidly killed bacterial cells and this activity was fully retained in 150 mM sodium chloride and 70% (v/v) human basal tears, particularly against the virulent ATCC 19660 strain. Furthermore, its dropwise administration at 40 μM to the ocular surface in a murine model of P. aeruginosa keratitis (three times daily, for 5 days post-infection) resulted in a significant reduction of infection. The mean clinical score was 2.89 ± 0.26 compared to 3.92 ± 0.08 for the vehicle control. In addition, the corneal level of viable bacteria in the peptide treated animals was significantly lower with a difference of 4 log10 colony counts, compared to 7.7 log10 cells recovered in the control. In parallel, recruitment of inflammatory cells was reduced by half compared to that found in the untreated eyes. Similar results were obtained when esculentin-1a(1-21)NH2 was applied prior to induction of keratitis. Overall, our findings highlight esculentin-1a(1-21)NH2 as an attractive candidate for the development of novel topical pharmaceuticals against Pseudomonas keratitis.

The eye and its associated tissues including the lacrimal system and lids have evolved several defence mechanisms to prevent microbial invasion. Included among this armory are several host-defence peptides. These multifunctional molecules are being studied not only for their endogenous antimicrobial properties but also for their potential therapeutic effects. Here the current knowledge of host-defence peptide expression in the eye will be summarized. The role of these peptides in eye disease will be discussed with the primary focus being on infectious keratitis, inflammatory conditions including dry eye and wound healing. Finally the potential of using host-defence peptides and their mimetics/derivatives for the treatment and prevention of eye diseases is addressed.