RESUMEN
PURPOSE: Diabetic retinopathy (DR) is characterized by pro-inflammatory, pro-angiogenic and pro-fibrotic environment during the various stages of the disease progression. Basement membrane changes in the retina and formation of fibrovascular membrane are characteristically seen in DR. In the present study the effect of Alcoholic (AlE) extracts of Triphala an ayurvedic herbal formulation and its chief compounds, Chebulagic (CA), Chebulinic (CI) and Gallic acid (GA) were evaluated for TGFß1-induced anti-fibrotic activity in choroid-retinal endothelial cells (RF/6A). METHOD: RF/6A cells were treated with TGFß1 alone or co-treated with AlE, CA, CI or GA. The mRNA and protein expression of fibrotic markers (αSMA, CTGF) were assessed by qPCR and western blot/ELISA. Functional changes were assessed using proliferation assay and migration assay. To deduce the mechanism of action, downstream signaling was assessed by western blot analysis along with in silico docking studies. RESULT: AlE (50⯵g/ml) CA and CI at 10⯵M reduced the expression of pro-fibrotic genes (αSMA and CTGF) induced by TGFß1, by inhibiting ERK phosphorylation. GA did not inhibit TGFß1 mediated changes in RF/6A cells. In silico experiments shows that CA and CI has favourable binding energy to bind with TGFß receptor and inhibit the downstream signaling, while GA did not. CONCLUSION: Hence this study identifies Triphala and its chief compounds CA and CI as potential adjuvants in the management of DR.
Asunto(s)
Benzopiranos/farmacología , Coroides/irrigación sanguínea , Retinopatía Diabética/tratamiento farmacológico , Células Endoteliales/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Glucósidos/farmacología , Taninos Hidrolizables/farmacología , Extractos Vegetales/farmacología , Vasos Retinianos/efectos de los fármacos , Factor de Crecimiento Transformador beta1/toxicidad , Animales , Benzopiranos/metabolismo , Sitios de Unión , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Retinopatía Diabética/enzimología , Retinopatía Diabética/patología , Células Endoteliales/enzimología , Células Endoteliales/patología , Fibrosis , Glucósidos/metabolismo , Taninos Hidrolizables/metabolismo , Macaca mulatta , Simulación del Acoplamiento Molecular , Neovascularización Patológica , Fosforilación , Unión Proteica , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Vasos Retinianos/enzimología , Vasos Retinianos/patología , Transducción de Señal/efectos de los fármacosRESUMEN
Tumor necrosis factor-α (TNFα) a pleiotropic cytokine induces pro-inflammatory and pro-angiogenic changes in conditions such as diabetic retinopathy (DR) and neovascular age related macular degeneration (NV-AMD). Hence, inhibition of TNFα mediated changes can benefit the management of DR and NV-AMD. Triphala, an ayurvedic herbal preparation is known to have immunomodulatry functions. In this study we evaluated the alcoholic extract of triphala (AlE) and its compounds Chebulagic acid (CA), Chebulinic acid (CI) and Gallic acid (GA) for their anti-TNFα activity. TNFα induced pro-inflammatory and pro-angiogenic changes in the retinal-choroid microvascular endothelial cells (RF/6A). Treatment with CA/CI/GA and the whole Triphala extract showed characteristic inhibition of MMP-9, cell proliferation/migration and tube formation as well the expression of IL-6, IL-8 and MCP-1 without affecting cell viability. This was mediated by inhibition of p38, ERK and NFκB phosphorylation. Ex vivo angiogenesis assay using chick chorioallantoic membrane (CAM) model also showed that TNFα-induced angiogenesis and it was inhibited by AlE and its active principles. Further, in silico studies revealed that CA, CI and GA are capable of binding the TNFα-receptor-1 to mediate anti-TNFα activity. This study explains the immunomodulatory function of Triphala, evaluated in the context of retinal and choroid vasculopathies in vitro and ex vivo; which showed that CA, CI and GA can be a potential pharmacological agents in the management of DR and NV-AMD.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Antiinflamatorios/farmacología , Benzopiranos/farmacología , Células Endoteliales/efectos de los fármacos , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Ácido Gálico/farmacología , Glucósidos/farmacología , Taninos Hidrolizables/farmacología , FN-kappa B/metabolismo , Extractos Vegetales/farmacología , Neovascularización Retiniana/prevención & control , Vasos Retinianos/efectos de los fármacos , Factor de Necrosis Tumoral alfa/toxicidad , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Inhibidores de la Angiogénesis/metabolismo , Animales , Antiinflamatorios/metabolismo , Benzopiranos/metabolismo , Línea Celular , Embrión de Pollo , Relación Dosis-Respuesta a Droga , Células Endoteliales/enzimología , Células Endoteliales/patología , Ácido Gálico/metabolismo , Glucósidos/metabolismo , Taninos Hidrolizables/metabolismo , Mediadores de Inflamación/metabolismo , Macaca mulatta , Metaloproteinasa 9 de la Matriz/metabolismo , Simulación del Acoplamiento Molecular , Neovascularización Fisiológica/efectos de los fármacos , Fosforilación , Unión Proteica , Receptores Tipo I de Factores de Necrosis Tumoral/metabolismo , Neovascularización Retiniana/enzimología , Neovascularización Retiniana/patología , Vasos Retinianos/enzimología , Vasos Retinianos/patología , Transducción de Señal/efectos de los fármacos , Factores de TiempoRESUMEN
Background & objectives: Proliferative vitreoretinopathy (PVR) is characterized by the presence of epiretinal membrane (ERM), which exerts traction and detaches the retina. Epithelial to mesenchymal transition (EMT) of the retinal pigment epithelial (RPE) cells underlies ERM formation. Adjuvant therapies aimed at preventing recurrence of PVR after surgery mostly failed in clinical trials. This study was aimed to evaluate the anti-EMT properties of bio-active compounds epigallocatechin gallate (EGCG), curcumin and lycopene as inhibitors of EMT induced by transforming growth factor beta 1 (TGF-ß1) in cultured ARPE-19 cells. Methods: ARPE-19 cells were treated with TGF-ß1 alone or co-treated with EGCG (1-50 µM), lycopene (1-10 µM) and curcumin (1-10 µM). The mRNA and protein expression of EMT markers, alpha-smooth muscle actin, vimentin, zonula occludens-1 and matrix metalloproteinase-2 (MMP-2), were assessed by reverse transcription polymerase chain reaction/quantitative polymerase chain reaction and immunofluorescence/enzyme linked immunosorbent assay. Activity of MMP-2 was assessed by zymography. Functional implications of EMT were assessed by proliferation assay (MTT assay) and migration assay (scratch assay). Western-blot for phosphorylated Smad-3 and total Smad-3 was done to delineate the mechanism. Results: EGCG and curcumin at 10 µM concentration reversed EMT, inhibited proliferation and migration through Smad-3 phosphorylation, when induced by TGF-ß1 in ARPE-19 cells. Lycopene did not prevent EMT in ARPE-19 cells. Interpretation & conclusions: EGCG and curcumin are potent in preventing EMT induced by TGF-ß1 in ARPE-19 cells and therefore, proposed as potential molecules for further pre-clinical evaluation in PVR management.