Lycium Barbarum Polysaccharides as a Potential New therapy to Prevent Corneal Scarring

Abstract

Purpose To the efficacy and optimal concentration of Lycium barbarum polysaccharide (LBP) solution in the suppression of keratocyte myofibroblast-differentiation and proliferation in an in vitro model of cornea epithelial-stromal injury. Methods Cell culture and characterization - Primary human corneal keratocytes were used and cultured using fibroblast medium supplemented with 2% FBS, fibroblast growth supplement and penicillin/streptomycin. Cells were characterized through fibroblast-like morphology, vimentin and fibronectin staining. Passage 4 to 6 cells were used for experiments. Myofibroblast differentiation and fibrotic protein expression- Cells were first pre-treated with LBP for 24 hours then 10ng/ml TGFB1 for 48 hours. Myofibroblast-differentiation was assessed through α-smooth muscle actin immunofluorescence (IF) staining and further quantified by western blot. Presence of type I, II and III collagen are assessed using IF. Scratch wound assay - Keratocytes were pretreated with LBP medium. Upon confluency, the monolayer of keratocytes was wounded with a plastic micropipette tip and the rate of wound closure was determined using time-lapse microscopy. Proliferation Assay - Keratocytes were seeded onto 48-well plates, pre-treated with specific LBP concentrations for 24 hours then TGFB1 induction at 10ng/ml for 48 hours. The number of viable cells was estimated using the MTS assay. Results Characteristic morphology and positive staining for vimentin and fibronectin were identified in our human keratocyte cell line using phase-contrast microscopy and IF microscopy respectively. Under IF, using α-smooth muscle actin staining, we demonstrated that LBP pre-treatment effectively inhibited keratocyte myofibroblast-differentiation in a dose-dependent fashion. With the cell viability assay, we further demonstrated that higher LBP concentrations selectively inhibited myofibroblast proliferation but had no significant effect of keratocyte proliferation. Conclusions Lycium barbarum polysaccharides is a promising therapeutic agent that selectively inhibits myofibroblast differentiation and proliferation after corneal injury. This potentially attenuates corneal scarring by reducing myofibroblast extracellular matrix secretion without inhibiting keratocyte activity, which plays an important role in corneal innate immunity.