Cite as Chin J Exp Ophthalmol, 2015,33(2): 109-114.
Abstract [Download PDF] [Read Full Text]
Recent researches show that oxidative stress is involved in the progress of keratoconus.Nuclear factor-E2-related factor 2-antioxidant response element (Nrf2-ARE) pathway plays a critical role in the defense against oxidative stress,but its function in keratoconus is unclear.
To investigate the differences of Nrf2-ARE signaling activation and matrix degenerating enzymes between keratoconus and normal corneal stromal cells.
Corneal stromal cells were isolated from keratoconus and normal cornea by using dispase and collagenase digestion.The cells were treated with hydrogen peroxide (H2O2) to mimic in vivo oxidative stress condition.Reactive oxygen species (ROS) production was measured by fluorescence substrate DCHF-DA incubation.Nrf2 level and the expression of Nrf2-ARE downstream antioxidant genes were analyzed by Western blot and real-time quantitative-PCR(RT-qPCR).The activity of matrix degenerating enzymes,including urokinase-type plasminogen activator (uPA)-uPA receptor (uPAR) system and matrix metalloproteinase-2 (MMP-2) were assessed by Western blot and gelatin zymography respectively.
In normal culture,keratoconus corneal stromal cells assumed increased basal ROS and Nrf2 level when compared with normal cells(t=18.155,P<0.01).However,after H2O2 treatment,the keratoconus corneal stromal cells showed increased ROS production,while decreased Nrf2 translocation and no significant difference in expression levels of Nrf2-ARE downstream antioxidant genes(Nrf2:t=62.123,P<0.01;(nicotinamide adenine dinucleotide phosphate quinine oxidoreductase-1[NQO-1]:t=2.209,P=0.092;hemo oxygenase-1[HO-1]:t=0.293,P=0.784;superoxide dismutase[SOD2]:t=0.749,P=0.495).The contents of uPA-uPAR and the activity of MMP-2 also showed a higher level in keratoconus corneal stromal cells than normal cells,with significant differences between them (t=19.164,15.458,4.818,all at P<0.01).
The defect of Nrf2-ARE signaling activation exists in the keratoconus corneal stromal cells, and correlats with the abnormal expression level of stromal degeneration enzymes, which suggests that the defect of Nrf2-ARE signaling activation may be involved in the progression of keratoconus.