The anti-fibrotic and pro-healing effects of topical lycium barbarum polysaccharide (LBP) treatment in an in vivo alkali-induced corneal epithelial-stromal injury model

Abstract

Corneal opacification ranks fourth in the aetiologies of blindness worldwide. In clinical practice, topical pharmacological therapies for corneal opacity - namely mitomycin-C and corticosteroids - could result in unwanted side effects involving increased intraocular pressure and scleral necrosis. Lycium barbarum polysaccharide (LBP) taken from Goji berry demonstrated anti-fibrotic properties in vitro. This suggested its capacity of addressing corneal fibrosis. The present study implemented a mouse epithelial-stromal alkali injury model to evaluate LBP's efficacy in reducing opacity and expediting epithelial healing. C57Bl/6J wild-type mice (n=6) were used in the present study. 1.5 mm-diameter circular filter paper soaked with 0.5M NaOH was placed onto the centre of right cornea under sedation for 45 seconds, followed by irrigation using filtered water. Mice received topical application of 2 or 20 mg/mL LBP while PBS was applied to control group. The topical treatments were applied in 3 modalities: before alkali injury (pre-injury treatment), after alkali injury (post-injury treatment), and both before and after alkali injury (pre-post-injury treatment). Pre-injury treatment lasted for 7 consecutive days prior to injury. Post-injury treatment lasted for 7 consecutive days after injury. Pre-post-injury treatment included 7 days before and 7 days after injury. On Day 0, 1, 2, 3, 7, 14, 21, and 28 post-injury, mice corneas were examined by slitlamp microscopy. Modified Fantes score (0-4) was used to grade opacity, while estimated epithelial defect was calculated from fluorescein staining. Corneal thickness was evaluated by histology and expression of fibrotic markers such as fibronectin was examined by immunohistochemistry and enzyme-linked immunosorbent assay (ELISA). 0.5M NaOH was found to induce consistent corneal opacification with increased central corneal thickness and deposition of fibronectin in stroma. Pre-post-injury treatment with 2 and 20 mg/mL LBP significantly reduced corneal opacification. The modified Fantes score on Day 28 was 2.00 and 1.67 respectively, compared to the score of 3.17 in the PBS control group (p < 0.0245; p < 0.0051). 2 and 20 mg/mL LBP pre-post-injury treatment also accelerated reepithelialisation on Day 1, with estimated epithelial defect area of 0.06 } 0.14 mm2 and 0.05 } 0.11 mm2 respectively, compared to 0.45 } 0.15 mm2 in the PBS control group (p < 0.05). Histology results revealed that 20 mg/mL LBP pre-post-injury treatment reduced total corneal thickness and ELISA results showed reduced corneal fibronectin expression on Day 28. Immunohistochemistry staining was negative for collagen type III, alpha-smooth muscle actin (α-SMA), and vimentin in all corneal samples. Given its anti-fibrotic and pro-healing properties, LBP has the potential of becoming a topical pharmacologic regimen for the treatment and prevention of corneal opacification in relation to alkali injury. Further investigations such as underlying mechanism are required to facilitate LBP’s translation into clinical practice.