Education and Human Sciences, College of (CEHS)


Date of this Version

Summer 6-21-2016

Document Type



Buckner, T. (2016). Delta Tocotrienol Attenuates NLRP3 Inflammasome Activation via Inhibition of NF-κB Priming and Reactive Oxygen Species Generation. (Master’s thesis). Available from University of Nebraska-Lincoln Digital Commons.


A THESIS Presented to the Faculty of The Graduate College at the University of Nebraska In Partial Fulfillment of Requirements For the Degree of Master of Science, Major: Nutrition, Under the supervision of Professor Soonkyu Chung. Lincoln, Nebraska: June, 2016

Copyright (c) 2016 Teresa Buckner


Chronic, low-grade inflammation during obesity is associated with the development of metabolic dysfunction. The NLRP3 inflammasome is assembled in response to cellular stressors and leads to cytotoxic cytokines IL-1β and IL-18 production, which implicates the NLRP3 inflammasome in inflammatory conditions, including type 2 diabetes. Tocotrienols are antioxidant and anti-inflammatory forms of vitamin E. Delta-tocotrienol (dT3) displays NF-κB inhibitory and anti-oxidant abilities, and is easily isolated from the Annatto plant. My primary aim was to determine whether dT3 inhibits NLRP3 inflammasome activation and to compare the extent to which dT3 inhibits NLRP3 inflammasome with other tocotrienol forms, i.e. alpha-tocotrienol (aT3) and gamma-tocotrienol (gT3). The second aim was to investigate the role of dT3 in NF-κB priming and reactive oxygen species (ROS) production, two major signaling pathways for NLRP3 inflammasome activation. To determine the inflammasome activation, J774 macrophages (iJ774) that stably overexpress an inflammasome reporter were pretreated with dT3, aT3, and gT3 and stimulated with lipopolysaccharide (LPS)/nigericin. Inflammasome activation was measured through Gaussia luciferase activity (iGLuc), IL-1β production, and caspase-1 and iGLuc immunoblotting. Raw 264.7 macrophages pretreated with dT3, aT3, and gT3 were stimulated with LPS. We measured 1) gene expression of down-stream targets of NF-κB (Nlrp3, Tnf-α, IL-1β, IL-18) by qPCR and 2) degradation of inhibitory protein of NF-κB (IκBα) by Western blot. Raw 264.7 macrophages pretreated with dT3, aT3, and gT3 were stimulated with LPS/nigericin and ROS production was measured using an ROS-sensitive florescent probe. Results show that 1) 0.5-5 μM dT3 pretreatment inhibited NLRP3 inflammasome activation and IL-1β production; 2) 1 μM dT3, aT3, and gT3 pretreatment inhibited NLRP3 inflammasome activation and IL-1β production; 3) 1-2.5 μM dT3 inhibited NF-κB activation; 4) 1 μM dT3, aT3, and gT3 pretreatment diminished NF-κB activation in macrophages; and 5) 1 μM dT3, aT3, and gT3 decreased ROS production in LPS/nigericin treated macrophages. These studies demonstrated that tocotrienols (dT3, aT3, gT3) lowered NLRP3 inflammasome activation in macrophages through inhibition of NF-κB activation and ROS production with an apparent efficacy of gT3>dT3>aT3. It suggests that inclusion of dietary tocotrienols may constitute a novel dietary strategy to attenuate and/or prevent pathogenic progression of obesity.

Advisor: Soonkyu Chung