| Objective Establishing an animal model of PCO by extracapsular cataract extraction in rabbits to detect the contents of DKK1(Dickkopf-1)and Wnt3 a in the aqueous humor.Observe the migration of cells and the occurrence of EMT in DDK1 and control groups after cataract surgery.Effects on Wnt3a-induced EMT and cell migration and exploring possible cellular mechanisms.Seek new targets for effective inhibition of PCO.Strive to provide a better prevention and treatment program for PCO after cataract surgery.Methods Twenty-four New Zealand white rabbits were selected and the right eye was subjected to extracapsular cataract extraction.The concentration of Wnt3 a and Dkk1 in the right eye aqueous humor was measured before operation.The rabbits were randomly divided into two groups(12 in each group),the phosphate buffered saline(PBS)treatment group and the Dkk1 treatment group.Approximately 1 ml Dkkl(30 ng/ml)or 1 ml PBS was injected into the anterior chamber of the right eye,respectively.On the fourteenth day after surgery,the right eye aqueous humor was taken to measure the concentrations of Wnt3 a and Dkk1.The left and right eyes were removed,and formalin fixation,paraffin embedding,enzyme-linked immunosorbent assay,immunohistochemistry(IHC)staining,western blot,immunofluorescence,and cell migration assay were performed for histological analysis.Results On the 14 th day after surgery,most of the right eyes treated with PBS developed dense fibrosis or apparent epithelial turbidity,and the right eye posterior capsule treated with Dkk1 showed mild turbidity.Immunohistochemistry results showed that 14 days after surgery,the right eye treated with PBS showed several features associated with PCO,including significant cell growth and prominent ?-catenin nuclear localization in the center of the posterior capsule.In the Dkk1 treatment group,there was less expression of ?-catenin in the posterior capsule of the right eye than in the PBS treatment group(32.8±6.2% vs.9.2±3.1%,P<0.05).Enzyme-linked immunosorbent assays showed that the average Wnt3 a level in PCO eyes was 173.8 pg/ml.The average Dkk1 concentration in PCO eyes 35.0 pg/ml.Western blot and immunofluorescence analysis showed that down-regulation of E-cadherin and up-regulation of fibronectin and ?-SMA were induced in WLE3 a transfected HLE-B3 cells.However,the addition of Dkk1 to Wnt3a-overexpressing cells detected increased expression of E-cadherin in HLE-B3 cells and decreased fibronectin expression.In addition,immunofluorescence also showed that Wnt/?-catenin signaling pathway is involved in EMT and LEC migration,while Dkk1 inhibits EMT and cell migration by inhibiting nuclear accumulation of Wnt/?-catenin.Transwell migration assay results showed that Wnt3 a induced significant horizontal and vertical migration in HLE-B3 cells.Dkk1 blocked Wnt3a-induced HLE-B3 cell migration.Gelatin zymography results showed that Wnt3 a significantly increased the number of stress fibers,the expression of MMP-1,and the activity of MMP-2 and MMP-9.In contrast,Dkk1 treatment significantly reduced the amount of stress fibers and inhibited Wnt3a-induced MMP-1 expression and increased MMP-2 and MMP-9 activity.Conclusion 1.Dkk1 inhibits PCO formation in rabbit PCO model.2.Dkk1 reverses the expression of EMT-related proteins in HLE-B3 cells up-regulated by Wnt3 a.3.Dkk1 inhibits the migration of Wnt3 a overexpressed HLE-B3 cells.4.Dkk1 inhibits cell migration by inhibiting nuclear accumulation of ?-catenin.5.Dkk1 inhibits cytoskeletal synthesis,MMP1 expression,MMP-2 and MMP-9 activity in Wnt3 a overexpressing HLE-B3 cells. |
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