Abstract
Epithelial to Mesenchymal Transition (EMT) of the retinal pigment epithelium is involved in the pathogenesis of proliferative vitreoretinopathy (PVR) that often leads to retinal detachment. In this study, Triphala, an ayurvedic formulation and two of its active ingredients, namely chebulagic acid and chebulinic acid were evaluated for anti-EMT properties based on in vitro experiments in human retinal pigment epithelial cell line (ARPE-19) under TGFβ1 induced conditions. ARPE-19 cells were treated with TGFβ1 alone or co-treated with various concentrations of aqueous extract (AqE) (30 - 300 μg/ml); alcoholic extract (AlE) (50 - 500 μg/ml) of triphala and the active principles chebulagic acid (CA) and chebulinic acid (CI) (CA,CI: 50 - 200 μM). The expression of EMT markers namely MMP-2, αSMA, vimentin and the tight junction protein ZO-1 were evaluated by qPCR, western blot and immunofluorescence. The functional implications of EMT, namely migration and proliferation of cells were assessed by proliferation assay, scratch assay and transwell migration assay. AqE, AlE, CA and CI reduced the expression and activity of MMP-2 at an ED50 value of 100 μg/ml, 50 μg/ml, 100 μM and 100 μM, respectively. At these concentrations, a significant down-regulation of the expression of αSMA, vimentin and up-regulation of the expression of ZO-1 altered by TGFβ1 were observed. These concentrations also inhibited proliferation and migration of ARPE-19 cells induced by TGFβ1. EMT was found to be induced in ARPE-19 cells, through SMAD-3 phosphorylation and it was inhibited by AqE, AlE, CA and CI. Further studies in experimental animals are required to attribute therapeutic potential of these extracts and their active compounds, as an adjuvant therapy in the disease management of PVR.
Highlights
Proliferative Vitreoretinopathy (PVR) is one of the complicated disorders of the eye and a leading cause for the failure of retinal reattachment surgery in rhegmatogenous retinal detachment (RRD) patients
Peaks detected in the aqueous extract of triphala (AqE) (4, 5 and 10) and alcoholic extract (AlE) (2, 3 and 8) show the presence of Chebulagic acid (CA), chebulinic acid (CI) and Gallic acid (GA) in the extracts of triphala (Fig. 1B, C)
AqE, AlE, CA and CI are non-cytotoxic to the ARPE-19 cells
Summary
Proliferative Vitreoretinopathy (PVR) is one of the complicated disorders of the eye and a leading cause for the failure of retinal reattachment surgery in rhegmatogenous retinal detachment (RRD) patients. In PVR, there is characteristic membrane formation on the retina, called as the epi-retinal membrane (ERM). It reflects the wound healing response, elicited by retinal detachment (RD), that ends up in fibrosis which further leads to tractional retinal detachment [1,3,4]. RPE are cuboidal cells that arrange as a monolayer, forming outer blood retinal barrier of the eye. They are mitotically quiescent throughout adult life. RPE undergo Epithelial to Mesenchymal transition (EMT), a process that underlies the pathology of PVR [8]. In vitro studies show that RPE undergo mesenchymal transition upon TGFβ1 induction [13,14]
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