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The modulation of neuroinflammation by flavonoids and their in vivo metabolites

Reference	BB/C518222/1
Principal Investigator / Supervisor	Professor Jeremy Spencer
Co-Investigators / Co-Supervisors
Institution	University of Reading
Department	Food and Nutritional Sciences
Funding type	Research
Value (£)	229,722
Status	Completed
Type	Research Grant
Start date	01/08/2005
End date	31/07/2008
Duration	36 months

Abstract

Dietary derived flavonoids have been shown to protect against both age-related cognitive and motoric decline in animals and humans. These beneficial effects have been attributed to their antioxidant properties, although recent findings demonstrate a role of specific flavonoids in interacting selectively within cell signalling cascades that regulate neuronal viability. For example, they may act to limit neuroinflammatory processes, which contribute to the progressive neuronal damage in many brain diseases. This proposal is designed to determine the ability of flavonoids and importantly their in vivo metabolite forms to exert protection against neuronal injury induced by inflammatory processes. To achieve this we will look at their ability to modulate both the production of mediating toxic agents released from glial cells following their activation and also their ability to modulate neuronal signalling and enhance neuronal resistance to these toxic agents. The main objectives will be to: 1. To investigate the extent to which physiologically relevant flavonoid metabolites are able to enter neurons and glial cells in isolation or in co-culture. 2. To determine whether flavonoid glucuronides are cleaved following microglial activation. 3. To examine the modulation of microglial nitric oxide and ROS generation by flavonoids and their metabolites through inhibition of iNOS. 4. To evaluate the mechanism of protection against activated microglial-induced neuronal injury by flavonoids in a physiologically relevant co-culture model. These studies will utilise flavonoid metabolites to make it more relevant to the physiological environment. As these metabolites are not commercially available we will use established techniques for their synthesis and HPLC with diode array detection, LC-MS MS and 1H-NMR for purification and characterisation. The redox potential of new metabolites will be established. The differential uptake of flavonoids into neurons, microglia and astrocytes willbe studied by use of HPLC with diode array detection and LC-MS MS. Then the potential for flavonoid glucuronides to be cleaved in the presence of microglia activated with LPS -1L-gamma will be determined by measurement of beta-glucuronidase release and by monitoring the liberation of flavonoid aglycone forms from glucuronide conjugates by HPLC and LC-MS MS. The ability of the liberated aglycone to protect neurons against damage induced by reactive oxygen- and reactive nitrogen-species will also be investigated. We will examine the potential of flavonoids and their in vivo forms to prevent the production and release of cytotoxic agents from activated glial cells, such as nitric oxide and other reactive intermediates, (ie. superoxide, peroxynitrite). Release of nitric oxide will be monitored by direct measurement of Nitric Oxide in the culture medium and by monitoring nitrite levels using the Griess reagent. The flavonoids possessing the most potent ability to inhibit the activation of inducible nitric oxide synthase (iNOS) will be identified by use of Western immunoblotting of iNOS levels in glial lysates. The cellular and molecular basis of their effects on microglia will be addressed by assessing their ability to modulate glial signalling pathways, in particular the mitogen activated protein kinase (MAPK) signalling cascade. Phosphorylation activation of extracellular signal related kinase (ERK), p38 and transcription factors such as CREB will be measured using standard SDS page Western immunoblotting. Lastly, the ability of flavonoids and their metabolite forms to modulate neuronal signalling through the MAPK and phosphotidylinositol-3-kinase (P13K) Akt signalling cascades will be examined by measurement of the phosphorylation state of specific kinases. The inhibition of pro-apoptotic signalling will be investigated as well as the activation of pro-survival signalling and the up-regulation of protective proteins, such as superoxide dismutase.

Summary

unavailable

Committee	Closed Committee - Agri-food (AF)
Research Topics	Ageing, Diet and Health, Neuroscience and Behaviour
Research Priority	X – Research Priority information not available
Research Initiative	X - not in an Initiative
Funding Scheme	X – not Funded via a specific Funding Scheme

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