Table 1.
List of Primers used for PCR.
Fig 1.
HPTLC analysis of aqueous extract of Triphala (AqE) and alcoholic extract of Triphala (AlE).
(A) HPTLC of Standards: Chebulagic acid (S1), Chebulinic acid (S2), Gallic acid (S3), AlE (T1), AqE (T2). (B) and (C) are chromatograms of AlE and AqE respectively.
Fig 2.
Cytotoxicity assay for AqE, AlE, CA and CI in ARPE-19 cells.
MTT assay showing the effect of AqE, AlE, CA, CI and GA on viability of ARPE-19 cells. Data represents Mean ± SD of three independent experiments in triplicates.
Fig 3.
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.
Fig 4.
Effect of AqE, AlE, CA and CI on mRNA expression of EMT markers.
Quantitative real time PCR showing the mRNA expression of MMP-2 (A), vimentin (B), αSMA (C) and ZO-1 (D) in cells treated with TGFβ1 alone and co-treated with AqE, AlE, CA or CI for 36 h. Data represents Mean ± SD of three independent experiments in triplicates. p values:###p<0.001 is the comparison between control vs. TGFβ1 and p values: *p<0.05, **p<0.01, ***p<0.001 are comparisons between TGFβ1 and corresponding treatment.
Fig 5.
Effect of AqE, AlE, CA and CI on EMT markers in ARPE-19 and BRPE cells.
Western blot showing the effect of AqE (30–300μg/ml), AlE (10–500 μg/ml) (A); CA and CI (10–100 μM) (B) on the expression of EMT markers, altered by TGFβ1 in ARPE-19 cells. Western blot (C) and zymography (D) showing the effect of AqE (100 μg/ml), AlE (50 μg/ml), CA (100 μM) and CI (100 μM) on EMT markers, altered by TGFβ1 in BRPE cells. Band intensity was quantified using NIH image-J software and expressed as fold change after normalization with βactin. Data represents Mean ± SD of three independent experiments. p values:#p<0.05, ##p<0.01, ###p<0.001 are comparisons between control and TGFβ1. p values: **p<0.01, ***p<0.001 between TGFβ1 and corresponding treatment.
Fig 6.
Effect of AqE, AlE, CA and CI on protein expression of αSMA, Vimentin and ZO-1 by Immunofluorescence.
ARPE-19 cells were seeded in chamber slides, treated with TGFβ1 or co-treated with TGFβ1 and various concentrations of AqE, AlE, CA and CI as indicated for 48 h. Cells were immunostained for αSMA (Red fluorescence), vimentin and ZO-1 (Green fluorescence). Image magnification 40X, Scale bar 10μm.
Fig 7.
AqE, AlE, CA and CI prevent proliferation of ARPE-19 cells.
Crystal violet dye exclusion showing the effect of TGFβ1 treatment and co-treatment with AqE, AlE, CA or CI on cell proliferation (A). Linear relationship between cell number and absorbance is shown (B). Data represents mean ± SD of three independent experiments done in quadruplicates. p value: ###p<0.001 is a comparison between control vs. TGFβ1 treatment. p value: ***p<0.001 is a comparison between TGFβ1 and corresponding treatment.
Fig 8.
AqE, AlE, CA and CI prevent migration of ARPE-19 cells.
Scratch assay showing migratory capacity of ARPE-19 cells upon TGFβ1 induction and on treatment with AqE, AlE, CA and CI (A). The Transwell migration assay showing the effect of TGFβ1 and co-treatment with AqE, AlE, CA and CI on migration of ARPE-19 cells (B). Enumeration of cell number is given in graph (C). All experiments were done thrice independently in triplicates. p value: ###p<0.001 is a comparison between control vs. TGFβ1 treatment. p value: ***p<0.001 is a comparison between TGFβ1 and corresponding treatment.
Fig 9.
TGFβ1 induces EMT through SMAD-3 phosphorylation, which was prevented by AqE, AlE, CA and CI.
Effect of LY364947 and SIS3 on the expression of EMT markers altered by TGFβ1, seen by Quantitative real time PCR (A), Immunofluorescence (B), ELISA (C), Western blot showing TGFβ1 induction of SMAD-3 phosphorylation at various time points (D) effect of AqE, AlE, CA and CI on TGFβ1 induced SMAD-3 phosphorlyation at 30 min (E). Data represents mean ± SD of three independent experiments. p value: ##p<0.01 is a comparison between control and TGFβ1 treatment. p values: *p<0.05, **p<0.01 are a comparison between TGFβ1 and corresponding treatment.