Effects Of Cyclic Mechanical Strain-induced Oxidative Stress On The Expression Of Inflammatory Mediators And MMPs In Corneal Fibroblasts

Keratoconus is a progressive corneal ectatic disease characterized by its focal thinning and protrusion,which eventually may lead to a conical shape.Early lesions often cause high myopia and irregular astigmatism,in the later stages,corneal turbidity and scarring may occur in the patients of keratoconus,resulting in severe visual impairment and even blindness.It is widely believed that the following factor are involved with the development of keratoconus,such as genetic,environmental,biological mechanics,matrix degradation,oxidative stress and other factors.However,the pathogenic mechanism of this disease is still unclear.The mechanical stimulation and oxidative stress may play a role in the development of the occurrence of keratoconus,In this study,Flexercell4000 tension system was used for applying cyclic mechanical strain on normal and keratoconus corneal fibroblasts in vitro(magnitude 15%;frequency 0.1 Hz;duration 12 h),to simulate a mechanical environment of corneal tissue.Our aim is to explore the effects ofcyclic mechanical strain on oxidative stress level,antioxidant capacity,the expression of inflammatory cytokines and matrix-degrading enzymes in human corneal fibroblasts.The main work and results are as follows:(1)To investigate the effects of cyclic mechanical stretch on the oxidative stress level of human normal and keratoconus corneal fibroblasts.The reactive oxygen species(ROS)production in human corneal fibroblasts was measured using the fluorescent dye 2',7'-Dichlorofluorescin diacetate(DCFH-DA),the fluorescence intensity of ROS can indirectly reflect cellular oxidative stress state.The results showed that the basal ROS level of keratoconus corneal fibroblasts was significantly higher than that of normal ones.The ROS levels of normal and keratoconus corneal fibroblasts increased significantly after cyclic mechanical stretch.(2)To investigate the effects of cyclic mechanical strain on the expression of antioxidant enzymes in human normal and keratoconus corneal fibroblasts.The expression levels of superoxide dismutase-2(SOD-2),heme oxygenase-1(HO-1)and NADPH quinine oxidoreductase-1(NQO-1)in human corneal fibroblasts were detected by real-time fluorescence quantitative PCR.The results have shown that part of the antioxidant enzymes of normal corneal fibroblasts increased significantly after cyclic mechanical stretch.In one case,the expressions of antioxidant enzymes of keratoconus corneal fibroblasts were significantly lower thannormal ones,and significantly increased after stretching.In the other case,keratoconus corneal fibroblasts had significantly higher antioxidase gene levels than normal ones,which significantly reduced or did not change after cyclic stretch.(3)To investigate the effects of cyclic mechanical strain on the expression of inflammatory cytokines in human normal and keratoconus corneal fibroblasts.The gene or protein expression levels of interleukin 6(IL-6),interleukin 1?(IL-1?)and tumor necrosis factor ?(TNF-?)in human corneal fibroblasts were detected by real-time fluorescent quantitative PCR or enzyme-linked immunosorbent assay(ELISA).The results showed that the gene and protein expression levels of inflammatory cytokines in keratoconus corneal fibroblasts were higher than those of normal ones.After cyclic stretching,the gene and protein expression levels of inflammatory cytokines had a significant increase in both normal and keratoconus corneal fibroblasts.(4)To investigate the effects of cyclic mechanical strain on the expression of matrix metalloproteinase in human normal and keratoconus corneal fibroblasts.Real-time fluorescence quantitative PCR and enzyme-linked immunosorbent assay(ELISA)were used to detect the gene or protein expression levels of MMP-1,MMP-9 in human corneal fibroblasts.The results showed that the gene expression level of MMP-1 and the protein expression level of MMP-9 in keratoconus corneal fibroblasts weresignificantly higher than that of normal ones.After cyclic stretching,the gene or protein expression level of MMP-1 and MMP-9 were significantly increased in normal and keratoconus corneal fibroblasts.In summary,keratoconus corneal fibroblasts maintained higher oxidative stress state,the oxidative stress level significantly increased after cells exposured to the mechanical stimulation,but due to the defects of antioxidant system in keratoconus,corneal fibroblasts cannot effectively cope with oxidative damage.In addition,the keratoconus fibroblasts themselves may release some of the inflammatory mediators and the matrix degradation enzyme,which are over-released after mechanical stimulation.This may result in corneal matrix degradation,and promote the development of keratoconus.