The Defect Of Nrf2-ARE Signaling Activation In Corneal Stromal Cells Of Keratoconus

Objective:To investigate the differences of Nrf2-ARE signaling activation and matrix degenerating enzymes between Keratoconus and normal corneal stromal cells.Methods: 1. Corneal stromal cells were isolated from Keratoconus and normal cornea by using Dispase and collagenase digestion. Human corneal fibroblasts(HCF) were cultured in Dulbecco modified Eagle medium: Nutrient mixture F12(DMEM/F-12, 1:1) supplemented with 10% fetal bovine serum(FBS), 100 U/ml penicillin and 100 μg/ml streptomycin. The cells were treated with hydrogen peroxide(H2O2) to mimic in vivo oxidative stress condition. 2. The groups were divided as follows,(1) HCF-CON group: normal corneal fibroblasts were incubated in normal medium,(2) HCF-H2O2 group: normal corneal fibroblasts were incubated in DMEM/F-12 with 200 μM H2O2,(3) KCF-CON group: Keratoconus corneal fibroblasts were incubated in normal medium,(4) KCF-H2O2 group: Keratoconus corneal fibroblasts were incubated in DMEM/F-12 with 200 μM H2O2. 3.Reactive oxygen species(ROS) production was measured by fluorescence substrate DCHF-DA incubation. 4.Nuclear Nrf2 level and the expression of Nrf2-ARE downstream antioxidant genes including SOD2、HO-1、NQO-1 were analyzed by western blot and real time quantitative-polymerase chain reaction(RT-q PCR). 5、The activity of matrix degenerating enzymes, including urokinase-type plasminogen activator(u PA)-u PA receptor(u PAR) system and matrix metalloproteinase-2(MMP-2) were assessed by western blot and gelatin zymography respectively.Result:1. In the normal incubated condition, KC fibroblasts showed a higher expression of ROS compared with normal samples. The addition of H2O2 extended the differences between KCF and HCF more significant, although both of the fluorescence intensity of two samples were strengthen.2. In normal culture, Keratoconus corneal stromal cells assumed increased basal nuclear Nrf2 level compared with normal cells(t=18.155, P<0.01). However, after H2O2 treatment, the Keratoconus corneal stromal cells showed decreased nuclear Nrf2 translocation, while no significant difference in expression levels of Nrf2-ARE downstream antioxidant genes(Nrf2:t=62.123, P<0.01;NQO-1:t=2.209, P=0.092;HO-1:t=0.293, P=0.784;SOD2:t=0.749, P=0.495). 3. The contents of u PA-u PAR and the activity of MMP-2 also showed a higher level in Keratoconus corneal stromal cells than normal cells(u PA:t=19.164,P<0.01;u PAR:t=15.458,P<0.01; MMP-2:t=4.818,P<0.01).Conclusion:The defect of Nrf2-ARE signaling activation existed in the Keratoconus corneal stromal cells, and correlated with the abnormal expression level of stromal degeneration enzymes, which suggests the defect of Nrf2-ARE signaling activation may be involved in the progression of Keratoconus.