SH-SY5Y cells treated with Quercetin-3-glucoside then H2O2

Flavonoids are polyphenolic compounds with potent anti-oxidant and free radical scavenging activities. Here, we examined the cytoprotective actions of Quercetin-3-glucoside (Q3G) against oxidative stress induced by hydrogen peroxide. Pre-treatment of the neuroblastoma cells, SH-SY5Y with Q3G for 18 h reduced cell death after a brief exposure to hydrogen peroxide. The cytoprotective effects of Q3G were associated with decreased free radical generation suggesting that this mechanism accounts for Q3G-mediated cytoprotection. To address the possibility that Q3G-mediated cytoprotection involved alterations in gene expression, cDNA microarray studies were performed. These studies identified elevated expression of 25 genes and decreased expression of 3 genes only in Q3G pre-treated cells under oxidative stress. The majority of genes up-regulated by Q3G in SH-SY5Y cells under oxidative stress encode enzymes involved in lipid biosynthesis. Quantitative Real time PCR (qRT-PCR) validated changes in gene expression that were identified by cDNA microarray analyses. The induction of multitude of genes involved in cholesterol signaling pathway by Q3G in cells under oxidative stress suggests an important role for elevated cholesterol synthesis in the cytoprotective actions of Q3G. This hypothesis was confirmed by the detection of elevated levels of cholesterol in cells pre-treated with Q3G and subjected to oxidative stress. Moreover, inhibition of cholesterol biosynthesis with mevastatin blocked the cytoprotective effects of Q3G against oxidative stress-induced cell death. Taken together, these studies suggest a novel mechanism for flavonoid-induced cytoprotection involving elevated cholesterol synthesis that may act to reduce lipid peroxidation and promote membrane repair after oxidative injury. Keywords: cytoprotection, oxidative stress The SH-SY5Y cells were treated with either Q3G or vehicle (DMSO) for 6 hours, then both were subject to an H2O2 insult. The labeled cDNA derived from Q3G treated cells plus H2O2 insult represent the "Experimental sample" while the labeled cDNA from DMSO treated plus H2O2 insult samples are the "Reference sample". Two batches of cells were so treated and dye swaps were done for each, giving a total of 4 replicates.