Elbakoush, Abdel M. (2018) Modulation of neuroinflammation with the phytoestrogen, formononetin. Doctoral thesis, University of Huddersfield.

Neuroinflammation and pro-inflammatory mediators play key roles in the pathogenesis of neurodegenerative diseases such as Alzheimer disease (AD). Studies have suggested that oestrogen has anti-inflammatory and neuroprotective activities. However, risks of tumour development, heart disease, stroke and blood clots associated with oestrogen replacement therapy limit its application in neurodegenerative disorders. This has switched interest to the use of alternatives such as the selective oestrogen receptor modulators (SERMs) and phytoestrogens which retain the neuroprotective effects of oestrogen with fewer side effects. Formononentin (FMN) is a phytoestrogen which showed significant anti-inflammatory activity through inhibition of nitric oxide (NO) released from lipopolysaccharide-stimulated mouse macrophage RAW cells. However, the ability of formononetin to produce anti-neuroinflammatory activityin LPS-activated microglia is unknown. In addition, the molecular mechanisms involved in the action of the compound in neuroinflammation is unknown. It is also not clear if the anti-inflammatory and neuroprotective effects of formononetin are mediated through estrogen receptors. This study has evaluated the effects of formononetin on neuroinflammation in BV2 microglia. Also the roles of ERβ activation in the anti-inflammatory activity of formononetin in the microglia was investigated.

BV2 microglia were activated with lipopolysaccharide (LPS) with or without formononetin (2.5, 5 and 10μM), followed by measurement of TNFα, IL-6, nitrite and PGE2 levels in culture supernatants. Also, protein levels of iNOS, COX-2, total IkBα, phospho-IkBα, phospho-p65 subunit, phospho-IKKα, phospho-p38,phospho-JNK, phospho-ERK 1/2, phospho-MLK3, phospho-TAK1 and ERβ were detected with immunoblotting. A reporter gene assay was used to evaluate NF-kB and ERβ transcriptional activation in HEK293. Electrophoretic mobility shift assay (EMSA) and DNA binding assays were used to examine the effect of formononetin on DNA binding of NF-kB. HT22 neuronal cells were used to study the neuroprotective activity of formononetin. The role of estrogen receptor in the anti-inflammatory activity of formononetin was investigated with western blotting for ERαand ERβexpression in BV2 microglia The effect of formononetin on transcriptional activity of oestrogen-response element (ERE) was also investigated. Small interfering RNAs (siRNAs) targeted at ERβ were used to silence ERβ geneand levels of TNFα, nitrite and PGE2 were detected in ERβ-silenced BV2 microglia, while immunofluorescence was used to detect NF-κB p65 subunit.

Results show that formononetin significantly suppressed TNFα, IL-6 and IL-1β without affecting viability of BV2 microglia cells. Furthermore, formononetin inhibited the production of nitrite and PGE2, and reduced protein levels of COX-2 and iNOS in LPS-activated microglia. In addition, formononetin prevented HTT22 neuronal toxicity produced by microglia-conditioned medium. Formononetin targeted NF-kB activity by inhibiting the phosphorylation of IKKα, IkBα and nuclear translocation of the p65 subunit. The compound also suppressed NF-kB/DNA binding activity in LPS stimulated BV2 microglia. Further results showed that formononetin inhibited p-38, JNK and ERK1/2 MAPKs activation by targeting upstream proteins TAK-1 and MLK3. The study revealed that BV2 microglia express ERβ, but not ERα. The study also demonstrated that formononetin increased transcriptional activity of ERE in HEK293 cells. Results further showed that this compound increased protein levels of ERβin BV2 microglia. RNA interference experiments revealed that NF-kB-mediated anti-inflammatory activity of formononetin was abolished following transfection of BV2 microglia with ERβ siRNA. Taken together, this study has demonstrated for the first time, that formononetin inhibits neuroinflammationin BV2 microglia, and neuroinflammation-mediated neurotoxicityin HT22 mouse hippocampal neurons by targeting upstream convergence proteins in both the NF-kB and MAPK signalling pathways. This study also established that the ability of the compound to interact with microglial ERβ possibly contributes to the anti-inflammatory action of formononetin.

El-Bakoush THESIS.pdf - Accepted Version
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