ABSTRACT
Gallic acid is claimed to possess antioxidant, antiinflammatory and cytoprotective effects. Since pancreatic islets from Type 2 diabetic patients have functional defects, it was hypothesized that glucolipotoxicity might induce apoptosis in beta-cells and gallic acid could offer protection. To test this, RINm5F beta-cells were exposed to high glucose (25 microM) or palmitate (500 microM) or a combination of both for 24 h in the presence and absence of gallic acid. Cells subjected to glucolipotoxicity in the absence and presence of gallic acid were assessed for DNA damage by comet assay. Apoptosis was inferred by caspase-3 protein expression and caspase-3 activity and changes in Bcl-2 mRNA. RT-PCR was used to analyse PDX-1, insulin and UCP-2 mRNA expression in RINm5F beta-cells and insulin levels were quantified from the cell culture supernatant. NFkappaB signal was studied by EMSA, immunofluorescence and Western blot analysis. While RINm5F beta-cells subjected to glucolipotoxicity exhibited increased DNA damage, apoptotic markers and NFkappaB signals, all these apoptotic perturbations were resisted by gallic acid. Gallic acid dose-dependently increased insulin secretion in RINm5F beta-cells and upregulated mRNA of PDX-1 and insulin. It is suggested that the insulin-secretagogue and transcriptional regulatory action of gallic acid is a newly identified mechanism in our study.
Subject(s)
Apoptosis/drug effects , Gallic Acid/pharmacology , Insulin-Secreting Cells/drug effects , Insulin/metabolism , Caspase 3/metabolism , Cell Line , DNA Damage , Electrophoretic Mobility Shift Assay , Gene Expression Regulation , Glucose/adverse effects , Homeodomain Proteins/metabolism , Humans , In Situ Nick-End Labeling , Insulin Secretion , Ion Channels/metabolism , Mitochondrial Proteins/metabolism , NF-kappa B/metabolism , Palmitic Acid/adverse effects , Proto-Oncogene Proteins c-bcl-2/metabolism , Trans-Activators/metabolism , Uncoupling Protein 2ABSTRACT
Oxidative stress is fast becoming the nutritional and medical buzzword for the twenty-first century. The theoretical importance of oxidative stress in diabetes is highlighted by its potential double impact on metabolic dysfunction on one hand and the vascular system on the other hand. The new concept of oxidative stress, being an important trigger in the onset and progression of diabetes and its complications, emphasizes the need for measurement of markers of oxidation to assess the degree of oxidative stress. While we have been routinely measuring biomarkers in our molecular epidemiology projects, here we discuss the utility of two assays, (a) DNA damage assessment by COMET measurement and (b) telomere length measurement. As DNA damage is efficiently repaired by cellular enzymes, its measurement gives a snapshot view of the level of oxidative stress. The protocol allows for measurement of oxidative DNA damage (FPG-sensitive DNA strand breaks). Telomere length measured by Southern blotting technique allows one to estimate the chronic burden of oxidative stress at the molecular level and is now considered as biomarker of biological aging.
Subject(s)
Biomarkers/metabolism , Comet Assay/methods , DNA Damage , Oxidative Stress , Telomere/metabolism , Blotting, Southern/methods , Cells, Cultured , Humans , Oxidation-Reduction , Reproducibility of ResultsABSTRACT
PURPOSE: The stromal-derived factor (SDF)-1alpha and the CXC receptor (CXCR)-4 jointly regulate the trafficking of various cell types and play a pivotal role in cell migration, proliferation, and survival. The purpose of this study was to assess whether curcumin inhibits human retinal endothelial cell (HREC) migration by interfering with SDF-1alpha/CXCR4 signaling. METHODS: Primary HREC culture was established and maintained in endothelial growth medium. The viability and proliferation of HRECs were assessed by MTT and thymidine uptake assays, respectively. The effect of SDF-1alpha-induced HREC migration (chemotaxis) in the presence and absence of curcumin was determined using the Boyden chamber migration assay. Intracellular Ca(2+) concentration was measured by fluorometric analysis. Immunofluorescence and Western blot analyses were performed to quantify CXCR4, phosphorylated AKT, and PI3-kinase expression levels. RESULTS: HREC migration increased in a dose-dependent manner (1, 10, 50, and 100 ng/mL; P < 0.001) in SDF-1alpha-treated cells. In contrast, AMD3100, an inhibitor of CXCR4 effectively inhibited HREC migration dose dependently. HREC migration was decreased when the cells were exposed to EGTA, a chelator of Ca(2+). Curcumin also blocked Ca(2+) influx, an important signal for HREC migration. In addition, curcumin significantly (P < 0.001) decreased SDF-1alpha-induced HRECs migration and downregulated SDF-1alpha-induced expression of CXCR4, phospho-AKT, phospho-phosphatidylinositol-3-kinase (PI3-K), and eNOS. CONCLUSIONS: This study indicates that curcumin has an inhibitory effect on SDF-1alpha-induced HREC migration. The plausible mechanism of action could be upstream blockage of Ca(2+) influx and the downstream reduction of PI3-K/AKT signals.
