Effects of Resveratrol on IL‐1α and Nitric Oxide Levels in GTM‐3 Cells

Abstract

ObjectivesResveratrol (RSV), an antioxidant polyphenol found in many fruit and plant products, could potentially have a role in reducing the trabecular meshwork (TM) tissue abnormalities observed in primary open‐angle glaucoma (POAG). The mechanisms by which this occurs are not completely understood. This study was designed to evaluate the effects of RSV on the expression and levels of selected nitric oxide synthase (NOS) enzymes, and nitric oxide (NO), respectively, and the inflammatory biomarker interleukin‐1 alpha (IL‐1 α) in human glaucomatous TM (GTM‐3) cells.MethodsGTM‐3 cells were grown to approximately 90% confluence and analyzed by Western blot analysis for the expression of endothelial and inducible nitric oxide synthases (eNOS; iNOS). GAPDH loading control was used to confirm accurate protein loading. The concentration‐related effects of RSV (0.1 – 100 mM) on IL‐1 α and NO levels were assessed. Values were normalized to protein. Cells were pre‐treated with RSV for 24 hours before analysis. Each experiment was performed at least three times.ResultsDensitometry data showed concentration‐related increases in eNOS protein expression and reduction in expression of iNOS at high RSV concentrations in GTM‐3 cells. Significant increases in eNOS expression were observed to be dependent on RSV concentrations. At 10 μM concentration, an average protein value of 1.33 ± 0.23 was obtained, while at 100 μM an average protein value of 1.47 ± 0.09 was obtained as compared to control (0.76 ± 0.09). Our results indicate a significant decrease in iNOS protein expression at RSV concentration of 100 μM. Our results indicated that NO levels increased with increasing concentrations of RSV. NO levels obtained following RSV treatment (0.1, 1, 10 and 100 μM), were 6 ± 0.7, 7 ± 0.8, 7.3 ± 0.7 and 9.5 ± 1 nM/mg protein, respectively. The average value obtained for the control group was 4.8 ± 0.6 nM/mg protein. Significant increases in IL‐1α levels were observed with increasing concentrations of RSV up to a concentration of 10 μM. There was a decrease in IL‐1 levels from 10 μM to100 μM RSV. The values obtained for concentrations of 0.1 – 100 μM were significantly different when compared to the control.ConclusionsOur results confirmed that resveratrol increases NO levels in glaucomatous TM cells. This increase is possibly due to increased eNOS expression in these cells. Decreased NO production in TM cells have been associated as a risk factor for primary open‐angle glaucoma, and increasing NO production as a result of RSV supports the beneficial effects of this antioxidant in these cells. Studies suggest that iNOS activation is a potential contributor to oxidative stress. Therefore, our results showing a reduction in iNOS expression further support RSV's antioxidant capabilities.Support or Funding InformationThis research was supported by NIH RCMI G12 Grant: 8G12MD007602 and Renovation and Research Core Consolidation Grant number 1G20RR031196This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.