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Aqueous and alcoholic extracts of Triphala and their active compounds chebulagic acid and chebulinic acid prevented epithelial to mesenchymal transition in retinal pigment epithelial cells, by inhibiting SMAD-3 phosphorylation.

Sivasankar S, Lavanya R, Brindha P, Angayarkanni N - PLoS ONE (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: R.S. Mehta Jain Department of Biochemistry and Cell Biology, Kamalnayan Bajaj Institute for Research in Vision and Ophthalmology, Vision Research Foundation, Chennai, India; School of Chemical and Biotechnology, SASTRA University, Thanjavur, India.

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.

No MeSH data available.


Related in: MedlinePlus

AqE, AlE, CA and CI reduced MMP-2 induced by TGFβ1.Treatment of ARPE-19 cells with TGFβ1 alone or co-treated with various concentrations of AqE, AlE, CA, CI or GA for 36 h. The cell free conditioned media was used for gelatine zymography (A). Untreated ARPE-19 cells showing secretion of MMP-2 (Lane 1: MMP-9 standard, 2: MMP-2 standard, 3,4: ARPE-19 conditioned medium) (a); TGFβ1 (5 ng/ml) inducing MMP-2 activity at 36 h (b); Effect of AqE (c), AlE (d), CA (e), CI (f), GA (g) and vehicle control (h) on MMP-2 activity by zymography. Quantitation of MMP-2 levels by using ELISA (B). Data represents Mean ± SD of at least three independent experiments in triplicates. p value: ###p<0.001 is the comparison between control vs. TGFβ1 and p values: **p<0.01, ***p<0.001are comparison between TGFβ1 and corresponding treatment.
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pone.0120512.g003: AqE, AlE, CA and CI reduced MMP-2 induced by TGFβ1.Treatment of ARPE-19 cells with TGFβ1 alone or co-treated with various concentrations of AqE, AlE, CA, CI or GA for 36 h. The cell free conditioned media was used for gelatine zymography (A). Untreated ARPE-19 cells showing secretion of MMP-2 (Lane 1: MMP-9 standard, 2: MMP-2 standard, 3,4: ARPE-19 conditioned medium) (a); TGFβ1 (5 ng/ml) inducing MMP-2 activity at 36 h (b); Effect of AqE (c), AlE (d), CA (e), CI (f), GA (g) and vehicle control (h) on MMP-2 activity by zymography. Quantitation of MMP-2 levels by using ELISA (B). Data represents Mean ± SD of at least three independent experiments in triplicates. p value: ###p<0.001 is the comparison between control vs. TGFβ1 and p values: **p<0.01, ***p<0.001are comparison between TGFβ1 and corresponding treatment.

Mentions: ARPE-19 cells secreted MMP-2 into the medium (Fig. 3A: a) which was further induced by TGFβ1 (5 ng/ml) at 36h (Fig. 3A: b). Treatment with AqE, AlE, CA and CI, decreased TGFβ1-induced MMP-2 activity (Fig. 3A: c-f). But gallic acid did not reduce MMP-2 activity (Fig. 3A: g). TGFβ1 treatment increased the MMP-2 protein level significantly to 14.9 ± 0.9 ng/ml compared to untreated control 3.8 ± 0.8 ng/ml. Treatment with AqE, AlE, CA and CI down-regulated the MMP-2 protein levels dose-dependently and significantly. GA did not have an effect on MMP-2 levels induced by TGFβ1 (Fig. 3B). The ED 50 value for MMP-2 inhibition was found to be 100 μg/ml for AqE, 50 μg/ml for AlE and 100 μM for both CA and CI.


Aqueous and alcoholic extracts of Triphala and their active compounds chebulagic acid and chebulinic acid prevented epithelial to mesenchymal transition in retinal pigment epithelial cells, by inhibiting SMAD-3 phosphorylation.

Sivasankar S, Lavanya R, Brindha P, Angayarkanni N - PLoS ONE (2015)

AqE, AlE, CA and CI reduced MMP-2 induced by TGFβ1.Treatment of ARPE-19 cells with TGFβ1 alone or co-treated with various concentrations of AqE, AlE, CA, CI or GA for 36 h. The cell free conditioned media was used for gelatine zymography (A). Untreated ARPE-19 cells showing secretion of MMP-2 (Lane 1: MMP-9 standard, 2: MMP-2 standard, 3,4: ARPE-19 conditioned medium) (a); TGFβ1 (5 ng/ml) inducing MMP-2 activity at 36 h (b); Effect of AqE (c), AlE (d), CA (e), CI (f), GA (g) and vehicle control (h) on MMP-2 activity by zymography. Quantitation of MMP-2 levels by using ELISA (B). Data represents Mean ± SD of at least three independent experiments in triplicates. p value: ###p<0.001 is the comparison between control vs. TGFβ1 and p values: **p<0.01, ***p<0.001are comparison between TGFβ1 and corresponding treatment.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4368423&req=5

pone.0120512.g003: AqE, AlE, CA and CI reduced MMP-2 induced by TGFβ1.Treatment of ARPE-19 cells with TGFβ1 alone or co-treated with various concentrations of AqE, AlE, CA, CI or GA for 36 h. The cell free conditioned media was used for gelatine zymography (A). Untreated ARPE-19 cells showing secretion of MMP-2 (Lane 1: MMP-9 standard, 2: MMP-2 standard, 3,4: ARPE-19 conditioned medium) (a); TGFβ1 (5 ng/ml) inducing MMP-2 activity at 36 h (b); Effect of AqE (c), AlE (d), CA (e), CI (f), GA (g) and vehicle control (h) on MMP-2 activity by zymography. Quantitation of MMP-2 levels by using ELISA (B). Data represents Mean ± SD of at least three independent experiments in triplicates. p value: ###p<0.001 is the comparison between control vs. TGFβ1 and p values: **p<0.01, ***p<0.001are comparison between TGFβ1 and corresponding treatment.
Mentions: ARPE-19 cells secreted MMP-2 into the medium (Fig. 3A: a) which was further induced by TGFβ1 (5 ng/ml) at 36h (Fig. 3A: b). Treatment with AqE, AlE, CA and CI, decreased TGFβ1-induced MMP-2 activity (Fig. 3A: c-f). But gallic acid did not reduce MMP-2 activity (Fig. 3A: g). TGFβ1 treatment increased the MMP-2 protein level significantly to 14.9 ± 0.9 ng/ml compared to untreated control 3.8 ± 0.8 ng/ml. Treatment with AqE, AlE, CA and CI down-regulated the MMP-2 protein levels dose-dependently and significantly. GA did not have an effect on MMP-2 levels induced by TGFβ1 (Fig. 3B). The ED 50 value for MMP-2 inhibition was found to be 100 μg/ml for AqE, 50 μg/ml for AlE and 100 μM for both CA and CI.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: R.S. Mehta Jain Department of Biochemistry and Cell Biology, Kamalnayan Bajaj Institute for Research in Vision and Ophthalmology, Vision Research Foundation, Chennai, India; School of Chemical and Biotechnology, SASTRA University, Thanjavur, India.

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.

No MeSH data available.


Related in: MedlinePlus