Prevention of Oxidative Stress-Induced Retinal Pigment Epithelial Cell Death by the PPAR
Agonists, 15-Deoxy-Delta 12, 14-Prostaglandin
Figure 2
Prevention of H2O2-induced
mitochondrial membrane depolarization by 15d-PGJ2. Binding of the JC-1
dyes to mitochondria leads to the appearance of two peaks. The green peak (at 545 nm) represents JC-1
monomers of this dye. The red peak (at 595 nm) represents JC-1 aggregates, which is caused by the negative charge of
mitochondrial membrane. Depolarization
of mitochondrial membrane causes a shift in the emission spectrum from red to
green color, which can be quantified by a fluorescence plate reader. The
relative intensity of these two peaks is a measurement of relative mitochondrial
potential such that a higher ratio represents more mitochondrial membrane
depolarization. (a)–(d): The JC-1 emission spectra between 520 nm to 620 nm were
determined for cells under various conditions [53]; (a): untreated cells; (B): cells
treated with 1 M 15d-PGJ2 overnight; (c): cells treated with 1.5 mM
H2O2 for 2 hours; (d): Cells treated with 1 M 15d-PGJ2 overnight, then with 1.5 mM H2O2 (without 15d-PGJ2)
for 2 hours. Note H2O2 caused a shift of the relative intensity of the peaks, and 15d-PGJ2 pretreatment restored membrane potential to a condition closer to untreated
cells. (e)-(f): Cells were pretreated with 1 M 15d-PGJ2 overnight, then with 1.5 mM H2O2 (without 15d-PGJ2)
for 2 hours (e) or 4 hours (f); then the 545/595 emission intensity ratios
were determined. Note in either 2-hour
or 4-hour treatment, H2O2 caused an increase of the
545/595 emission intensity ratio, indicating mitochondrial depolarization. 15d-PGJ2 pretreatment restored the
ratio to that similar to control value (P < .001 between H2O2-treated
and 15d-PGJ2+H2O2-treated cells in (e) and (f)).
