Title

Anti-inflammatory Effect of Secoisolariciresinol Diglucoside (SDG) in Microglia

Date of Award

Spring 5-15-2017

Degree Type

Thesis

Degree Name

MSOB (Master of Science in Oral Biology)

Primary Advisor

Kelly Jordan-Sciutto

Abstract

Secoisolariciresinol diglucoside (SDG) is the main plant lignan in flaxseed and has been thoroughly researched in the past decades due to its unique health properties. SDG has been shown to have therapeutic benefits for an array of diseases including breast and prostate cancer, hyperlipidemia, atherosclerosis, hypertension, diabetes mellitus, and radiation pneumonopathy. With a number of studies recognizing the various health benefits of SDG, few have focused on its potential anti-inflammatory effect and, specifically, its interaction with the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathway. Thus, the aim of this study is to elucidate the interaction between SDG and NF-κB in microglial cells in vitro.

Mixed rat cortical cells were cultured for 2 weeks and microglia were isolated according to a previously established protocol with slight modifications. Wells were divided between 7 groups based on pre-treatment with vehicle or SDG and treatment with water or TNF-α: Vehicle group (V) pre-treated with 2 µL PBS vehicle and treated with 10 µL H2O; SDG 50 (S50) pre-treated with 50µM and treated with 10 µL H2O; SDG 100 (S100) pre-treated with 100µM SDG and treated with H2O; Vehicle + TNF-α (V+T10) pre-treated with 2 µL PBS vehicle and treated with 10 ng/mL TNF-α; SDG 50 + TNF-α (S50+T10) pre-treated with 50µM SDG and treated with 10 ng/mL TNF-α; SDG 100 + TNF-α (S100+T10) pre-treated with 100µM SDG and treated with 10 ng/mL TNF-α; and SDG 100x2 + TNF-α (S100x2+T10) pre-treated with 100µM SDG and treated with both 100µM SDG and 10 ng/mL TNF-α. Pre-treatment occurred 4 hours prior to treatment. Groups were further divided based on harvest time at 2-, 6-, and 24-hours for different analyses. ELISA, nitrate-nitrite assay, and qPCR analyses probing for IL-1β and IL-6, nitrate and nitrite, and TNF-α and IL-1β, respectively, were performed to measure NF-κB activity.

The qPCR results show a significant increase in mRNA expression of IL-1β and TNF-α after treatment with 10 ng/mL TNF-α (p < .05). For both NF-κB targets, no significant difference was seen between TNF-α treatment groups with or without 50 µM SDG pre-treatment. ELISA results did not show any trends for IL-1β or IL-6. No increase in the concentrations of either NF-κB target was seen after treatment with TNF-α. Nitrate-nitrite assay results did not show significant differences between any groups (p > .05). TNF-α did not elicit an increase in NO metabolite concentration in the positive control group (Veh + T10).

The data obtained suggest that SDG does not have an inhibitory effect on the NF-κB pathway in microglia under these experimental conditions. After TNF-α-induced microglial activation, SDG did not significantly alter the expression of NF-κB targets. The results of this study do not support the hypothesis that SDG has an anti-inflammatory effect in microglia mediated through the NF-κB pathway. Thus, therapeutic effects of SDG in microglia may be attributed to other mechanisms. Changes in protocol may prove helpful in finding an interaction but it is recommended that future investigation of SDG’s effect on microglia focus on other pathways.

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