A phytochemical, Emodin, attenuates corneal stromal fibroblast differentiation and fibrosis

Abstract

Purpose : Trauma, infection and injury lead to corneal opacity and vision loss.This is a major cause of blindness globally. Limited efficient and safe treatments are available currently. Emodin is a natural phytochemical used in traditional medicine due to potent anti-inflammatory and anti-fibrotic properties. The therapeutic effects of Emodin in the cornea are still unknown. This study tested whether Emodin can inhibit TGFβ1-induced corneal stromal fibroblast (CSF) transformation to corneal myofibroblasts (CMF) known to cause corneal fibrosis using an in vitro model.

Methods : Primary human corneal stromal fibroblasts (hCSF) were generated from donor human corneas for experiments. The hCSF cells were then grown in six-well plates at an initial density of 7.5x10⁴ in MEM medium containing 10% fetal bovine serum (FBS). hCSFs were exposed to 5ng/ml of TGFβ1 for 72h in serum-free conditions to differentiate into human corneal myofibroblasts (hCMF). Emodin toxicity was determined by treating hCSF cultures with Emodin concentrations ranging from 0.5µM to 100µM for 72 hours. Immunofluorescence, western blotting, qPCR, MTT, and 2D/3D confocal microscopy were used to measure differential changes in mRNA and protein levels of profibrotic markers, alpha-smooth muscle actin (αSMA), collagen I, and collagen IV.

Results : A series of dose-dependent (1-50µM) and time-dependent (0-120h) cell viability assays performed by exposing Emodin to hCSFs found no significant decrease in cellular viability by 1-25µM doses of Emodin (0-5%) whereas 30-100µM doses of Emodin showed considerable decrease in cell viability (21-37%). All later experiments were performed at 25µM Emodin. No significant alteration in the proliferation and migration of CSFs was observed at 72h after 25µM Emodin exposure. hCSFs grown in +Emodin and +TGFβ1 under serum-free conditions showed significantly decreased mRNA and protein levels of αSMA, collagen I, and collagen IV compared to the hCSFs grown in -Emodin and +TGFβ1 under serum-free conditions.

Conclusions : Emodin has potential to control fibrosis as a topical ophthalmic solution. Additional studies are warranted.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.