Peroxynitrite-induced expression of inducible nitric oxide synthase and activated apoptosis via nuclear factor-kappa B pathway in retinal pigment epithelial cells and antagonism of cholecystokinin octapeptide-8 in vitro

AIM: To explore that if peroxynitrite induced the expression of inducible nitric oxide synthase (iNOS)via nuclear factor-kappa B (NF-kappa B)pathway in retinal pigment epithelial (RPE) cells and the antagonism of cholecystokinin octapeptide-8 (Melatonin, CCK-8) in vitro. METHODS: RPE cells were obtained from eyes of C57BL/6 mouse and divided into control, peroxynitrite and CCK-8 groups. Control group was treated with saline, peroxynitrite group was treated with peroxynitrite, and CCK-8 group was treated with CCK-8 after added with peroxynitrite. All changes were observered at 6, 12 and 24 hours after treatment. Gene array analysis, Reverse Transcription Polymerase Chain Reaction (RT-PCR) were used to determine the expression of inducible nitric oxide synthase ( iNOS)mRNA in RPE cells. Western blotting was used to test the apoptosis of RPE cells. Immunofluorescent staining was used to determine the NF-kappa B pathway signal transduction. RESULTS: Compared to the control group, the expression of iNOS mRNA was up-regulated in peroxynitrite group and down-regulated in CCK-8 group with gene array analysis. Apoptosis was increased in peroxynitrite group and decreased in CCK-8 group with western blotting. The NF-kappa B pathway signal transduction was more and more stronger in the peroxynitrite group. But in CCK-8 group, little stronger could be observed at 12 hours, then weak at 24 hours with immunofluorescent staining (P<0.001). CONCLUSION: This study suggested that apoptosis of RPE cells was partly induced by peroxynitrite, which may be the new way of oxidative damage to the RPE cells. The NF-kappa B signal transduction may affect and reinforce apoptosis mediated by peroxynitrite. CCK-8 decreased apoptosis of RPE cells induced by peroxynitrite and is a potential agent for therapy of retinopathy. The mechanism of CCK-8 dealing with RPE cells may be related to its direct inhibition of the formation of iNOS to produce peroxynitrite and antagnism of damage of peroxynitrite to the RPE cells.