title: Inducible nitric oxide synthase and Fas/FasL with C3 expression of mouse retinal pigment epithelial cells in response to stimulation by peroxynitrite and antagonism of puerarin.
abstract: 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.
authors.affiliation: Department of Ophthalmology, Hebei Province People's Hospital, Shijiazhuang, Hebei 050051, China. jhaolina@yahoo.com.cn
authors.forename: Li-Na
authors.initials: LN
authors.lastname: Hao
authors.forename: Yan-Qing
authors.initials: YQ
authors.lastname: Zhang
authors.forename: Yu-Hua
authors.initials: YH
authors.lastname: Shen
authors.forename: Zhi-Yun
authors.initials: ZY
authors.lastname: Wang
authors.forename: Yan-Hua
authors.initials: YH
authors.lastname: Wang
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D003176:Complement C3 / Q000235:genetics / Q000378:metabolism*; D004847:Epithelial Cells / Q000187:drug effects / Q000378:metabolism*; D053222:Fas Ligand Protein / Q000235:genetics / Q000378:metabolism*; D005434:Flow Cytometry; D007150:Immunohistochemistry; D007529:Isoflavones / Q000494:pharmacology*; D051379:Mice; D008810:Mice, Inbred C57BL; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism*; D030421:Peroxynitrous Acid / Q000494:pharmacology*; D010857:Pigment Epithelium of Eye / Q000166:cytology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D019014:fas Receptor / Q000235:genetics / Q000378:metabolism*
chemical_list: D003176:Complement C3; D053222:Fas Ligand Protein; C491899:Fas protein, mouse; D007529:Isoflavones; D019014:fas Receptor; D030421:Peroxynitrous Acid; D052247:Nitric Oxide Synthase Type II; C033607:puerarin