摘要
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 new way of oxidant stress. Fas/FasL signal transduction pathway and C3 may affect and reinforce apoptosis mediated by ONOO-. Puerarin could reverse ONOO- damage on RPE cells. The antagonizing mechanism of puerarin may be related to its inhibitory to the expression of iNOS mRNA, and therefore decrease ONOO- formation as well as directly antagonize the effect of ONOO-. Furthermore, puerarin may be an useful therapeutic agent against apoptosis of RPE cells.
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 new way of oxidant stress. Fas/FasL signal transduction pathway and C3 may affect and reinforce apoptosis mediated by ONOO-. Puerarin could reverse ONOO- damage on RPE cells. The antagonizing mechanism of puerarin may be related to its inhibitory to the expression of iNOS mRNA, and therefore decrease ONOO- formation as well as directly antagonize the effect of ONOO-. Furthermore, puerarin may be an useful therapeutic agent against apoptosis of RPE cells.
基金
This study was supported by grants from Hebei Province Science Foundation （No. 07276101D-3） and Hebei Province Abroad Studying Foundation （2007）.
