Effects Of Mechanical Stretch And PGE₂ On LOXs Expression In Keratoconus Corneal Fibroblasts

Keratoconus is a corneal disorder characterized by progressive corneal thinning, protrusion and scarring, which could result in decreased vision in bad situation. Both environmental and genetic factors were considered to result in this disease, however the cause and underlying pathological mechanism haven’t been figured out yet. Corneal stroma is the main part of the cornea, determining the majority of refractive power and desired transmittance of cornea. The synthesis and degradation of corneal stroma is related to the production and development of keratoconus.Several studies had investigated that Inflammatory cytokines and mechanical stretch may be the reasons for keratoconus. Interleukin-6(IL-6), tumor necrosis factor-α(TNF-α) and matrix metalloproteinase-9(MMP-9) were over expressed in the tears of patients with keratoconus. Inflammatory cytokines would be increased because of long time eye rubbing, accelerating the development of keratoconus. Lysine oxidase(LOXs) is a copper-dependent amino acid oxidase, as collagen and elastin cross-linking agent, which plays an important role in the body development, structural stability and tissue damage repair. Keratoconus patients expressed lower LOXs tear which hindered the synthesis of extracellular matrix collagen, reducing the mechanical properties of the cornea with aggravating the development of keratoconus. As an important regulator of cell growth, Prostaglandins E2 is also one of the important inflammatory mediators of inflammatory reactions. It was involved in various physiological and pathological processes, playing a key role in inflammation. Prostaglandins E2 is significantly increased in keratocytes of keratoconus, whose production is 10 times greater than in keratocytes from normal corneas, promoting the development of fibroblast. Corneal is a typical bearing tissue, and its mechanical environment may be changed by mechanical stress. Therefore, mechanical stress and inflammatory factors are important factors in the development and progression of keratoconus.In our study, normal cornea and keratoconus fibroblasts were cultured in vivo. Two effects, which were different concentration of PGE2 and mechanical stress, were taken to test the expression of mRNA from LOXs of keratoconus fibroblasts. The experiment was divided into three groups, the first group was the LOXs mRNA change over time in 0h, 6h and 12 h, respectively; the second group was effects of different concentrations(0ng/ml、1ng/ml、10ng/ml and 50ng/ml) of PGE2 on LOXs mRNA expression; the third group was 12h’s tensile stretch with 12% witch magnitude, testing the effects of mechanical stretch and PGE2 on LOXs mRNA expression in keratoconus corneal fibroblasts. In this study, the Flexercell 4000 which was a flexible substrate traction system was used to stretch cells to simulate micro-mechanical environment of damage corneal fibroblasts, using real-time PCR method to detect the LOXs mRNA expression and the enzyme linked immunosorbent assay(ELISA) to detect the content of the LOXs proteins.Some conclusion had been made from our study: LOXs can be expressed by normal cornea and fibroblasts, but the expression of fibroblasts was fewer than normal cornea. LOX、LOXL-2 and LOXL-4 of normal cornea and LOXs of fibroblasts enhanced with low concentration of PGE2(1ng/ml)may increase the expression of mRNA. However, there was no obvious LOXs concentration dependence for the LOXs.Compared with the matched group, the LOXL-2 and LOXL-2 mRNA expression of normal cornea was increased by dependant mechanical stretch, while the expression of LOXL-3 and LOXL-4 mRNA of fibroblasts was decreased. On the other hand, the LOXL-4 mRNA expression of normal cornea was decreased by the union effect between mechanical stretch and PGE2, while only LOXL-1 mRNA of all LOXs was increased under that effect. The overall trend of the protein activity of LOXs is consistent with their genes expression. LOXs mRNA expression versus time has no significant time-dependent.In conclusion, we deduced that the Inflammatory and Inflammatory cytokines and mechanical stretch reduce the expression of LOXs, resulting in corneal collagen cross-linked disorders, corneal structure changes, decreased mechanical stability, under in intraocular pressure is prone to bulging, which may be one of the pathogenic mechanisms and the development of keratoconus.