EGF Cooperating With NS398 Stimulates The Healing Of Corneal Endothelium Freeze Injury In Cat

It is hard for corneal endothelium of humans, primates and cats to heal completely. Injuries to the corneal endothelium of some animals such as rabbits heal completely because of extensive mitosis of rabbit endothelial cells in vivo. By contrast, injuries to the corneal endothelium of adult humans, primates, and cats heal predominantly by migration and enlargement of endothelial cells rather than by mitosis which is inhabited by TGF-P2 in anterior chamber, the ability of the endothelium of these species to compensate adequately after injuries or disease is limited. Thus, In this study, we injected sodium hyaluronate contained EGF and NS398 to anterior chamber, which obtained suitable therapeutic dose and cycle from the assay of corneal endothelial cells cultured, and aimed to evaluate the effect of simultaneous treatment. In the healing of corneal endothelium, we detect variation of contents for phospho-Smad2 (the singnal protein of TGF-β2), a-smooth muscle actin (myofibroblast marker), and proliferating cell nuclear antigen(the marker of new cells)to realize the mechanism of promoting endothelial cells and reducing endothelial clouding.Cat corneal endothelial cells were cultured. First, the inhibiting effect of different concentrations of TGF-P2 on corneal endothelial proliferation was detected by MTT. The optimal concentration of TGF-β2 added to the culture medium to simulate the inhibiting environment of the cat corneal endothelial (FCE) cells in vivo. And we detected the promoting value of different concentrations of EGF and NS398 after the cells inhibited by TGF-P2. Finally, the concentration and reaction time of EGF and NS398 on promoting cell proliferation were detected.Transcorneal freezing was used for destroying portions of the corneal endothelium. After the creation of the wound, the right eye of cat was injected with 0.02 ml of sodium hyaluronate which contained EGF and NS398 three days one time. The left eye was control treatment group. Deparaffinized sections were processed for phospho-Smad2, a-smooth muscle actin (myofibroblast marker), and proliferating cell nuclear antigen (PCNA). The effect of healing on corneal endothelial was evaluated by endothelium stain of alizarin red and trypan blue.TGF-β2 suppressed FCE cell proliferation in a dose-dependent manner with the maximal effect being reached at lOng/ml dose. Weather the endothelium cells inhibited by TGF-P2 or not, EGF promoted FCE cell proliferation in a dose-dependent manner with the maximal effect being reached at lOng/ml dose. after TGF-p2 (10ng/ml) pretreated application for 10 min. the maximal effect of NS398 at 5μmol/ml cooperated with 10ng/ml EGF induce great more growth effect at the 72h time point. In vivo, compared with the control group, cell uncovered zone were greatly reduced in EGF and NS398-treated group, In EGF and NS398-treated group, all kinds of healing index were better than control group, especially in cell transitional zone. Immunohistochemistry showed few Phopsho-Smad2 and a-SMA positive cells were seen in the endothelial layer of injure repaired group compared with control groups, the repaired group had more PCNA positive cells.In vitro, after FCE cells were previous cultured in the presence of lOng/ml of TGF-β2,5μmol/l of NS398 and 10ng/ml of EGF had a maximal effect on FCE cell proliferation at the 72h time point. In vivo, NS398 blocked TGF-βsignaling by targeted deletion of Smad2, restrained the arrest of cell cycle and endothelial mesenchymal transition. Thus, the marker of new cells PCNA was expressed significantly in EGF and NS398 treated group. And the marker of myofibroblast aSMA was not seen in the eyes of the drug group at each time. EGF induced the migration of FCE cells, cooperated with NS398 stimulated FCE healing rapidly.