Estrogen Receptor β -mediated Inhibition Of Neuroinflammation In LPS-activated BV2 Microglia By The Phytoestrogen Biochanin A

RAVIKANTH VELAGAPUDI, OLUMAYOKUN OLAJIDE. University of Huddersfield, Huddersfield, UK

In neuroinflammation, hyperactive microglia is known to release variety of pro-inflammatory cytokines like nitrite, prostaglandin E2 (PGE2), TNFα, IL-6 and reactive oxygen species. This activation is closely associated with the pathogenesis of neurodegenerative diseases like Alzheimer’s disease (AD). Biochanin A is a phytoestrogen commonly found in dietary supplements like red clover, sprouts, soy, chickpea and cabbage (1). Earlier studies have shown that Biochanin A inhibited LPS-activated pro-inflammatory mediators in mesencephalic neuron-glia cultures and microglia-enriched cultures (2). The aim of the current study was to investigate whether anti-neuroinflammatory action of Biochanin A is mediated through ERβ signalling in LPS-activated BV2 microglia. Cultured BV2 cells were pre-treated with Biochanin A (6-18 μM) prior to stimulation with LPS (100 ng/ml) for 24 h, supernatants were evaluated for the levels of nitrite usin g Griess assay. The effect of Biochanin A on LPS-activated inducible nitric oxide synthase (iNOS) protein expression was evaluated using immunoblotting. Viability of BV2 cells treated with LPS (100 ng/ml) in the presence or absence of Biochanin A (6-18 μM) was measured by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay Results showed that Biochanin A (18 μM) significantly (p<0.0001) reduced nitrite production by 40±11.0%, and iNOS protein expression by 25±9.2% when compared with LPS (100ng/ml) control without affecting the viability of BV2 microglia. Further experiments showed that the presence of ERβ siRNA significantly reversed the effect of Biochanin A (18 μM) on nitrite production (8.5±7.51%, LPS+Bio-A) vs (18.75±3%, LPS+Bio-A+ ERβ siRNA) and iNOS protein expression (46±7.8%, LPS+ Bio-A) vs (98.2±3.2, LPS+Bio-A+ ERβ siRNA). We have also showed that the effect of 17β-estradiol (10 nM) was significantly reversed in ERβ silenced BV2 cells. Taken together, our results showed that Biochanin A suppressed nitrite and iNOS mediated neuroinflammation in LPS-activated BV2 microglia. We have also proved that these inhibitory actions are ERβ dependent.

1. Middleton Jr et al, The effects of plant flavonoids on mammalian cells: implications for inflammation, heart disease, and cancer. Pharmacol. Rev, 52, 673–751, 2000.

2. Chen H et al., Biochanin A protects dopaminergic neurons against lipopolysaccharide-induced damage through inhibition of microglia activation and proinflammatory factors generation. Neurosci Lett, 417(2):112-7, 2007.