Tiliroside inhibits LPS-induced acetyl-NF-κB-p65 by activating SIRT1 in BV2 microglia.

Nuclear factor kappaB (NF-κB) has to undergo several post-translational modifications to regulate the transcription of the inflammatory genes that are involved in neuroinflammation. Once NF-κBis translocated to the nucleus, it undergoes deactylation by Sirtuin 1 (SIRT1), a class III histone deactyl-transferase (1) and (2). Reversing this process through SIRT1 activation might promote the inhibition of NF-kB signalling in the activated microglia (1). Earlier, we have shown that tiliroside inhibited LPS-activated TRAF-6/NF-κB-mediated neuroinflammation in BV2 microglia (3). In the present study we aimed to assess the effectof tiliroside on SIRT1 activation and LPS-induced acetyl-NF-κB-p65 in BV2 microglia. To measure SIRT1 activation, cultured BV2 cells were incubated with tiliroside. Nuclear extracts were then prepared and subjected to western blot. Levels of acetyl and total NF-κB-p65 were also measured using western blot on nuclear extracts of BV2 microglia pre-treated with tiliroside (2-6 μM) prior to stimulation with LPS (100 ng/ml) and IFNγ (5 ng/ml). Our results show that tiliroside (6 µM) significantly increasedSIRT1 expression by ~33.3-foldafter 24 h. Further, tiliroside (2-6 μM) dose dependentlyand significantly (p<0.001) upregulated SIRT1 protein expression by~31.2-foldafter 24h. Western blot experiments show that LPS (100 ng/ml) and IFNγ (5 ng/ml) increased levels of acetyl-NF-κB-p65 in BV2 microglia. However, treatment with tiliroside (2-6 μM) diminished the levels of acetylation by~2.08-fold respectively, but had no effect on total-NF-κB-p65. Taken together, tiliroside inhibits NF-κBsignalling possibly by promoting deactylation of NF-kB-p65 at lys310 through activation of SIRT1 in BV2 microglia.

1. Chen Lf et al. (2001). Duration of nuclear NF-kappaB action regulated by reversible acetylation. Science, 293(5535): 1653-7.

2. Yeung F et al. (2004). Modulation of NF-kappaB-dependent transcription and cell survival by the SIRT1 deacetylase. EMBO J, 16; 23(12): 2369-80.

3. Velagapudi R et al. (2014). Tiliroside, a dietary glycosidic flavonoid, inhibits TRAF-6/NF-kappaB/p38-mediated neuroinflammation in activated BV2 microglia. Biochim. Biophys. Acta, 1840 pp. 3311–3319.