Formononetin, A Phytoestrogen In Red Clover Inhibits Neuroinflammation In LPS-activated Microglia

ABDEL ELBAKOUSH, OLUMAYOKUN OLAJIDE. University of Huddersfield, Huddersfield, UK

Inflammation is a common feature in neurodegenerative diseases such Alzheimer\'s disease. Microglial cells, the resident macrophages of the central nervous system, become chronically activated and mediate pathology inducing inflammatory responses. While inflammation may not initiate disease, it has been shown to promote disease progression. Formononetin is an isoflavone present in food supplements like red clover. Formononetin has been shown to improve microcirculation of blood, as well as produce anti-cancer and antioxidant effects . Several studies have suggested that formononetin has an anti-inflammatory activity. However, the molecular mechanism of its action, especially in neuroinflammation remains unknown. This study aimed at evaluating the effect of formononetin on neuroinflammation in LPS-activated BV2 microglia. BV2 cells were stimulated with sterile LPS in the presence or absence of formononetin (2.5, 5 and 10 µM). After 24 h, levels of pro-inflammatory cytokines (TNFα, IL-6) in cell supernatants were determined using ELISA. Nitrite production was determined with the Griess assay, while PGE2 production was measured using an enzyme immunoassay (EIA). Protein expressions of inducible nitric oxide synthase (iNOS) and cyclooxyeganse-2 (COX-2) were determined by western blot analysis. A reporter gene assay was also performed using Human Embryo Kidney

Cells (HEK293) to investigate the effect of formononetin on NF-κB-mediated gene transcription. Results showed that formononetin (10 µM) significantly (p<0.01) suppressed TNFα and IL-6 production in a concentration-dependent manner. The levels of TNFα and IL-6 production were 40±5 % and 62±3%, respectively when compared with LPS control. At this concentration, the compound did not affect the viability of BV2 cells. Also, formononetin (10 µM) significantly (p<0.001) inhibited the production of nitrite and PGE2. Immunoblotting experiments revealed that formononetin reduced the expression of COX-2 (49±5%) and iNOS (20±5%) protein expression, in comparison with LPS control. Further experiments revealed significant (p<0.05) inhibition of NF-κB by 35±5% with 10 μM formononetin. These data suggests that formononetin might be inhibiting neuroinflammation in LPS-activated BV2-microglia cells, through mechanisms involving NF-κB activity.

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