Background Retinal pigment epithelial （RPE） cell is a monolayer of multifunctional cells between the retina and the choroid. Peroxynitrite （ONOO-） is known to induce toxicity on RPE cells. This study aimed to eval...Background Retinal pigment epithelial （RPE） cell is a monolayer of multifunctional cells between the retina and the choroid. Peroxynitrite （ONOO-） is known to induce toxicity on RPE cells. This study aimed to evaluate ONOO- induced expression of inducible nitric oxide synthase （iNOS） and complement 3 （C3） via Fas/FasL pathway in RPE cells and the values of puerarin as a therapeutic target for inhibiting the apoptosis of RPE cells. Methods RPE cells were obtained from eyes of C57BL/6 mice. RPE cells were divided into control, ONOO-and puerarin groups. Control group was treated with saline, ONOO-group was treated with ONOO-, and puerarin group was treated with puerarin after added with ONOO-. All changes were observered at 6, 12 and 24 hours after treatment. Western blotting analysis was used to determine the expression of nitrotyrosine （NT, the foot print of ONOO-） and C3; flow cytometry was used to determine the apoptosis of RPE cells. Immunohistochemistry and Western blotting were used to determine Fas/FasL signal transduction. Gene array analysis, reverse transcription polymerase chain reaction （RT-PCR） and Western blotting were used to determine the expression of iNOS mRNA and iNOS protein in RPE cells. Results There were minor expression of NT, C3, Fas/FasL and iNOS mRNA in control group, and strong expression of NT and C3 in ONOO- group, while in puerarin group weak expressions of NT and C3 were detected as time passed by （P 〈0.001）. Apoptosis of RPE cells occured and reached a higher level at 6 and 24 hours after addition of ONOO- respectively in ONOO- group, but delayed apoptosis in puerarin group （P 〈0.05）. Compared to control group, the expression of Fas/FasL was up-regulated in ONOO- group, but was down-regulated in puerarin group （P 〈0.001）. Similarly, the expressions of iNOS mRNA and iNOS protein in ONOO-group were up-regulated in ONOO- group, but down-regulated in puerarin group （P 〈0.001）. Conclusions ONOO- expresseion in RPE cells may constitute the n展开更多
植物干细胞与动物干细胞的比较研究可以深入认识干细胞生物学的核心问题。植物干细胞与动物干细胞在细胞特征、功能、信号传导、研究方法等方面均有所不同。但是近年来对argonaute(AGO)与polycomb group proteins(PcG)蛋白研究表明动、...植物干细胞与动物干细胞的比较研究可以深入认识干细胞生物学的核心问题。植物干细胞与动物干细胞在细胞特征、功能、信号传导、研究方法等方面均有所不同。但是近年来对argonaute(AGO)与polycomb group proteins(PcG)蛋白研究表明动、植物干细胞行为之间有着某种分子相似性。展开更多
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
To clarify the possible mechanisms of α anordin and probimane on calmodulin Ca ++ Mg ++ APTase system, enzyme dynamic study was carried out by determining three dynamic parameters [the substrate concentra...To clarify the possible mechanisms of α anordin and probimane on calmodulin Ca ++ Mg ++ APTase system, enzyme dynamic study was carried out by determining three dynamic parameters [the substrate concentration(ATP) response curve, dose(inhibitors) response curve and time response curve]. Our data have shown that the inhibitory rates of α anordrin and probimane are unrelated to substrate(APT) concentrations, but related to calmodulin concentrations. The inhibition of α anordrin and probimane is very quick that is completed within 1 to 5 min and can maintain more than 1 hr in the same inhibitory rates. So it is possible that α anordrin and probimane are calmodulin competitors with calmodulin like binding whose actions can occur by affecting the reaction balance of substrate and product on target enzymes of calmodulin( Ca ++ Mg ++ ATPase).展开更多
基金supported by the Science and Technology Foundation of Hebei Province,China(No.07276101D-3)Clinical Science Program of the Department of Health,Hebei Province,China(No.03078)the Project of Study Abroad of Hebei Province,China(No.06016)
基金This study was supported by grants from Hebei Province Science Foundation （No. 07276101D-3） and Hebei Province Abroad Studying Foundation （2007）.
文摘Background Retinal pigment epithelial （RPE） cell is a monolayer of multifunctional cells between the retina and the choroid. Peroxynitrite （ONOO-） is known to induce toxicity on RPE cells. This study aimed to evaluate ONOO- induced expression of inducible nitric oxide synthase （iNOS） and complement 3 （C3） via Fas/FasL pathway in RPE cells and the values of puerarin as a therapeutic target for inhibiting the apoptosis of RPE cells. Methods RPE cells were obtained from eyes of C57BL/6 mice. RPE cells were divided into control, ONOO-and puerarin groups. Control group was treated with saline, ONOO-group was treated with ONOO-, and puerarin group was treated with puerarin after added with ONOO-. All changes were observered at 6, 12 and 24 hours after treatment. Western blotting analysis was used to determine the expression of nitrotyrosine （NT, the foot print of ONOO-） and C3; flow cytometry was used to determine the apoptosis of RPE cells. Immunohistochemistry and Western blotting were used to determine Fas/FasL signal transduction. Gene array analysis, reverse transcription polymerase chain reaction （RT-PCR） and Western blotting were used to determine the expression of iNOS mRNA and iNOS protein in RPE cells. Results There were minor expression of NT, C3, Fas/FasL and iNOS mRNA in control group, and strong expression of NT and C3 in ONOO- group, while in puerarin group weak expressions of NT and C3 were detected as time passed by （P 〈0.001）. Apoptosis of RPE cells occured and reached a higher level at 6 and 24 hours after addition of ONOO- respectively in ONOO- group, but delayed apoptosis in puerarin group （P 〈0.05）. Compared to control group, the expression of Fas/FasL was up-regulated in ONOO- group, but was down-regulated in puerarin group （P 〈0.001）. Similarly, the expressions of iNOS mRNA and iNOS protein in ONOO-group were up-regulated in ONOO- group, but down-regulated in puerarin group （P 〈0.001）. Conclusions ONOO- expresseion in RPE cells may constitute the n
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.
文摘To clarify the possible mechanisms of α anordin and probimane on calmodulin Ca ++ Mg ++ APTase system, enzyme dynamic study was carried out by determining three dynamic parameters [the substrate concentration(ATP) response curve, dose(inhibitors) response curve and time response curve]. Our data have shown that the inhibitory rates of α anordrin and probimane are unrelated to substrate(APT) concentrations, but related to calmodulin concentrations. The inhibition of α anordrin and probimane is very quick that is completed within 1 to 5 min and can maintain more than 1 hr in the same inhibitory rates. So it is possible that α anordrin and probimane are calmodulin competitors with calmodulin like binding whose actions can occur by affecting the reaction balance of substrate and product on target enzymes of calmodulin( Ca ++ Mg ++ ATPase).
