262 - Investigating the anti-oxidant effects of estrogen in RAW cell macrophages

Abstract

The female sex hormone, estrogen (E2) is proposed to have cardioprotective effects due to its anti-oxidant properties. Previous studies have shown that as women enter the post-menopausal period, cardiovascular incidences increase dramatically. This may be due to a decrease in E2 and its protective properties. E2 receptors can be found on the surface of the RAW macrophage cell as well as the nucleus and mitochondria, where E2 can modulate both nuclear and mitochondrial genes in response to inflammatory stimuli. We hypothesize that in the RAW murine macrophage cell line E2 increases cell viability, decreases reactive oxygen species (ROS) and increases mitochondrial function. PCR analysis confirmed ER-α presence in RAW cells. 48 hours after plating RAW cells, they were treated with low (100nM) and high (1mM) levels of E2 in serum and phenol-free DMEM. The cells were pretreated with E2 or vehicle for 15hrs, followed by treatment with the inflammation-inducing endotoxin, Lipopolysaccharide (LPS) for 6hrs. The Calcein AM Assay showed greater cell concentrations when cells were pretreated with E2. ROS production measurement via DCF Fluorescence of post-LPS treatment showed that E2 pretreatment of cells decreased ROS production. Changes in ROS production may be due alterations in mitochondrial membrane potential. Therefore, mitochondrial membrane potential was measured using TMRM Fluorescence and showed that E2 pretreatment increased mitochondrial membrane potential. A greater mitochondrial membrane potential may infer a more efficient mitochondria. Seahorse measurement of mitochondrial function confirmed that low and high levels of E2 pretreatment increases mitochondrial function in RAW cells. We hope to further explore cellular localization of ROS production and the effects of estrogen on mitochondrial function after LPS treatment. This data suggests that estrogen is anti-oxidant and may exert these effects by modulating mitochondrial function. Overall this data suggests that the anti-oxidant properties of estrogen may be beneficial to cardiovascular health.