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.
- Author:
Li-Na HAO
1
;
Yan-Qing ZHANG
;
Yu-Hua SHEN
;
Zhi-Yun WANG
;
Yan-Hua WANG
Author Information
- Publication Type:Journal Article
- MeSH: Animals; Blotting, Western; Cells, Cultured; Complement C3; genetics; metabolism; Epithelial Cells; drug effects; metabolism; Fas Ligand Protein; genetics; metabolism; Flow Cytometry; Immunohistochemistry; Isoflavones; pharmacology; Mice; Mice, Inbred C57BL; Nitric Oxide Synthase Type II; genetics; metabolism; Peroxynitrous Acid; pharmacology; Pigment Epithelium of Eye; cytology; Reverse Transcriptase Polymerase Chain Reaction; fas Receptor; genetics; metabolism
- From: Chinese Medical Journal 2011;124(16):2522-2529
- CountryChina
- Language:English
-
Abstract:
BACKGROUNDRetinal 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.
METHODSRPE 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.
RESULTSThere 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).
CONCLUSIONSONOO(-) 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.
