Search:
Computing and Library Services - delivering an inspiring information environment

Investigation of the Nrf2-Mediated inhibition of neuroinflammation by diosgenin

Abudheir, Anwer (2017) Investigation of the Nrf2-Mediated inhibition of neuroinflammation by diosgenin. Masters thesis, University of Huddersfield.

[img]
Preview
PDF - Accepted Version
Available under License Creative Commons Attribution Non-commercial No Derivatives.

Download (3MB) | Preview

Abstract

Chronic activation of microglia results in neuroinflammation and subsequently
neurodegeneration. Diosgenin is a plant-derived steroidal saponin found in
fenugreek and roots of yam. Some studies have shown that diosgenin possess
anti-inflammatory effect in various experimental models. However, little is known
about the anti-neuroinflammatory effect of diosgenin in the microglia. In this
study, the anti-neuroinflammatory effect of diosgenin (5, 10 and 20 µM) was
investigated in LPS-activated BV2 microglia. Nitrite and PGE2 levels were
measured using a Griess assay and an enzyme immunoassay (EIA),
respectively. In addition, levels of the cytokines TNFα, IL-6, IL-1β and IL-10 were
measured using specific mouse ELISA. Further, LPS-induced ROS generation in
BV2 microglia was tested using a DCFDA assay. Protein levels of COX-2, iNOS,
the main targets in NF-B pathway as well as the levels of p38 MAPK and Akt
were measured with immunoblotting. Additionally, the effect of diosgenin on the
activity of NF-B promoter was examined using a luciferase reporter gene assay
in HEK293 cells. NF-B binding to the DNA was investigated using EMSA. The
role of Nrf2 in the anti-neuroinflammatory effect of diosgenin was investigated in
BV2 microglia by western blot analysis of HO-1, NQO1 and Nrf2. BV2 microglia
were transfected with siRNA for Nrf2, followed by stimulation with LPS in order to
investigate the role of Nrf2 in the anti-inflammatory activity of diosgenin. The
effect of diosgenin on neuroinflammation-induced HT22 neuronal toxicity was
also evaluated using conditioned medium obtained from LPS-activated BV2
microglia.
Results show that diosgenin reduced the production of NO and PGE2 through
inhibition of iNOS and COX-2 expression, respectively in LPS-stimulated BV2
microglia. In addition, diosgenin reduced the secretion of other pro-inflammatory
factors including IL-6, IL-1β, TNFα and ROS. By contrast, the compound
increased IL-10 release in LPS-stimulated BV2 cells. Further results show that
the anti-neuroinflammatory effect of diosgenin is mediated through the inhibition
of NF-B signalling pathway. Furthermore, the compound also attenuated Akt
signalling, but did not inhibit p38 MAPK signalling in LPS-stimulated BV2 cells.
Additionally, diosgenin treatment resulted in an up-regulation of the expression
5
of the antioxidant proteins HO-1 and NQO1, and GSH in BV2 microglia,
suggesting that diosgenin activates Nrf2 signalling. In addition, the compound
increased the nuclear translocation of Nrf2 and its binding to ARE in BV2 microglia. Diosgenin down-regulated the level of the inhibitory protein Keap1,
indicating that diosgenin activates Nrf2/ARE pathway by down-regulation of
Keap1. Transfection of BV2 microglia with Nrf2 siRNA resulted in the loss of antineuroinflammatory
effect of diosgenin. Diosgenin produced a neuroprotective
effect through the inhibition of neuroinflammation-induced neurotoxicity and ROS
generation in HT22 mouse hippocampal neurons. Taken together, these results
demonstrate that diosgenin inhibits LPS-mediated neuroinflammation through
interference with NF-B signalling. The anti-neuroinflammatory effect of
diosgenin is dependent on the activation of Nrf2 signalling.
The results obtained from this study suggest that diosgenin inhibited NF-Bmediated
neuroinflammation through molecular mechanisms that are possibly
closely linked to Nrf2/ARE antioxidant protection system in BV2 microglia cells. It
was demonstrated that diosgenin is neuroprotective in both neuroinflammation
and oxidative stress-mediated neuronal damage, an action that is important in
neurogeneration. The effects of the compound on processes linked to oestrogen
receptors suggests a potential estrogenic activity which needs further
investigation. Activation of microglial AMPK is a property which further
demonstrates that diosgenin may be producing inhibition of neuroinflammation through activation of endogenous systems which block the transcriptional activity
of NF-B.

Item Type: Thesis (Masters)
Subjects: Q Science > Q Science (General)
Schools: School of Applied Sciences
Depositing User: Sharon Beastall
Date Deposited: 05 Sep 2018 14:15
Last Modified: 07 Feb 2019 16:15
URI: http://eprints.hud.ac.uk/id/eprint/34672

Downloads

Downloads per month over past year

Repository Staff Only: item control page

View Item View Item

University of Huddersfield, Queensgate, Huddersfield, HD1 3DH Copyright and Disclaimer All rights reserved ©