Molecular properties from Siempreviva plant extracts against pterygium pathogenesis

Abstract

Purpose : Non-surgical approaches to pterygium are limited to descriptive evidence, and fail to associate molecular mechanisms beyond clinical improvements. We demonstrated inhibition of pterygium fibroblast viability with extracts of Sedum spp plant and now explore the extracts modulation of pterygium pathogenesis.

Methods : Human pterygium fibroblasts (HPFs), or NIH3T3 fibroblasts (3T3) were treated with media-only (sham) or Sedum spp extracts: sap, hexane (Hex) or hexane/acetone (Hex/Ac). Cell viability, and LD₅₀, were determined using increasing concentrations (0-2,000 mcg/mL) at 24h post-treatment by cell tittering and Probit analysis or Log regression. ECM remodeling at 2, 6 and 72h post-treatment was assessed in HPFs with release of matrix metalloproteinase 3 (MMP-3), and proliferation signaling was assessed at 6h with VEGF and CTGF by ELISA. Oxidative stress was judged by NO production with Greiss assay in RAW 264.7 cells (RAW) challenged (or not) with LPS (10 mcg/mL).

Results : Reduction in cell viability were found in sap-treated cells in HPFs and 3T3 (LD₅₀=269 and 217mcg/mL), and with Hex/Ac treatment (847mcg/mL and 647mcg/mL respectively). MMP-3 was stable between 2 and 72h with sham, at 6h had a 3-fold increase with sap (p<0.05) against sham, no difference showed for Hex/Ac-treated HPFs. VEGF release increased (3.6-fold, p<0.05) only with sap. CTGF increased (2.3-fold, p<0.05) only with Hex/Ac vs sham. Sap extract promoted significant NO production with (p<0.05) or without (p<0.005) LPS stimulation. No difference was seen between sham- and Hex-treated RAW.

Conclusions : Sap extract is the most anti-proliferative for HPFs and 3T3, but it's associated with increased ECM remodeling, proliferation and oxidation. In addition to mild anti-proliferative effect of Hex/Ac, lack of association with pathogenic events embraces this extract for future therapeutic development.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.

 

(A) LD₅₀ of S. spp. extracts. (B) MMP-3 release. Left: Total protein concentration 2 and 72 h post primary culture. Right: Total protein release fold-change compared to ctl group at 24h post-treatment. (C) VEFG and CTGF in supernatants. Fold-change of total protein release compared to cctl group at 24h post-treatment. (D) Detection of NO- derivates after treatment and challenge (or not) with 10mcg/mL of LPS. (B) and (C) were analyzed with multiple paired T-test. (D) Was analyzed with 2way ANOVA using Tukey's multiple comparison test.