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 chol...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.展开更多
AIM: To explore if peroxyntrite (ONOO(-)) induced iNOS vi;7 Fas/ Fas/L pathway in diabetic rats and the effection of cholecystokinin octapeptide-8 (CCK-8) as therapeutic agent for decrease diabetic retinopathy. METHOD...AIM: To explore if peroxyntrite (ONOO(-)) induced iNOS vi;7 Fas/ Fas/L pathway in diabetic rats and the effection of cholecystokinin octapeptide-8 (CCK-8) as therapeutic agent for decrease diabetic retinopathy. METHODS: Thirty-six rats were taken as control group, seventy two were given (streptozotocin) STZ (45mg/kg) and then divided into ONOO(-) group and CCK-8 group (peritoneal injection CCK-8). STZ-induced diabetic rats were treated with CCK-8 for 60 days. Western blotting analysis, DNA ladder, RT-PCR, immunohistochemistry and flow cytometry were used for determining the expression of nitrotyrosine (NT, the foot print of ONOO(-)); apoptosis and inducible nitric oxide synthase (iNOS) mRNA as well as Fas/Fasl signal transduction in RPE cells. RESULTS: Both RPE cells in ONOO(-) and CCK-8 group developed apoptosis and expressed NT, iNOS mRNA and Fas/Fasl. But latter delayed the all changes in a time-dependent manner compared with control and ONOO(-) group (P<0.001). iNOS and Fas/Fasl were up-regulated and associated with an increase of expression of ONOO(-) in vivo. CONCLUSION: The study suggested that apoptosis of RPE was partly induced by ONOO(-) may be the new way of oxidative damage to the RPE cells. CCK-8 decreased RPE cells apoptosis partly induced by ONOO(-) and is a potential drug for therapy of diabetic retinopathy. The mechanism of CCK-8 dealing with RPE cells may be related to its direct inhibition of the formation of iNOS to produce ONOO(-) and antagnism of damage of ONOO(-) to RPE cells.展开更多
基金Supported by Hebei Province Science foundation,China(No.07276101D-3)Clinical Science Project Fund of the Ministry of Health in Hebei Province,China(No. 03078)Foreign Studying Project Fund in Hebei Province,China
文摘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.
文摘AIM: To explore if peroxyntrite (ONOO(-)) induced iNOS vi;7 Fas/ Fas/L pathway in diabetic rats and the effection of cholecystokinin octapeptide-8 (CCK-8) as therapeutic agent for decrease diabetic retinopathy. METHODS: Thirty-six rats were taken as control group, seventy two were given (streptozotocin) STZ (45mg/kg) and then divided into ONOO(-) group and CCK-8 group (peritoneal injection CCK-8). STZ-induced diabetic rats were treated with CCK-8 for 60 days. Western blotting analysis, DNA ladder, RT-PCR, immunohistochemistry and flow cytometry were used for determining the expression of nitrotyrosine (NT, the foot print of ONOO(-)); apoptosis and inducible nitric oxide synthase (iNOS) mRNA as well as Fas/Fasl signal transduction in RPE cells. RESULTS: Both RPE cells in ONOO(-) and CCK-8 group developed apoptosis and expressed NT, iNOS mRNA and Fas/Fasl. But latter delayed the all changes in a time-dependent manner compared with control and ONOO(-) group (P<0.001). iNOS and Fas/Fasl were up-regulated and associated with an increase of expression of ONOO(-) in vivo. CONCLUSION: The study suggested that apoptosis of RPE was partly induced by ONOO(-) may be the new way of oxidative damage to the RPE cells. CCK-8 decreased RPE cells apoptosis partly induced by ONOO(-) and is a potential drug for therapy of diabetic retinopathy. The mechanism of CCK-8 dealing with RPE cells may be related to its direct inhibition of the formation of iNOS to produce ONOO(-) and antagnism of damage of ONOO(-) to RPE cells.
