Nimbolide targets multiple signalling pathways to reduce neuroinflammation in BV2 microglia

Abstract Nimbolide, a limonoid compound found in the neem plant, was investigated for effects on neuroinflammation in BV2 microglia activated with LPS. Cultured BV2 cells were stimulated with LPS (100 ng/ml) followed by treatment with nimbolide (125, 250 and 500 nM). Results showed that nimbolide caused significant reduction in the levels of TNFα, IL-6, IFNγ, NO/iNOS, PGE2/COX-2 in LPS-activated BV2 cells. Further experiments revealed that LPS-induced increased expression of phospho-p65 and phospho-IκBα proteins were reduced in the presence of nimbolide. Also, LPS-induced NF-κB acetylation, increased binding to consensus sites, and transactivation, as well as phosphorylation of p38 and JNK MAPKs were reduced by nimbolide. Reduction of cellular ROS generation by nimbolide was accompanied by reduction in gp91phox protein levels, while antioxidant effects were observed through elevation in protein levels of HO-1 and NQO-1. It was observed that treatment of BV2 microglia with nimbolide resulted in reduced levels of cytoplasmic Nrf2 which was accompanied by increased levels in the nucleus. Furthermore, treatment with this compound resulted in increased binding of Nrf2 to ARE consensus sites accompanied by enhanced ARE luciferase activity. Knockdown experiments revealed a loss of anti-inflammatory activity by nimbolide in cells transfected with Nrf2 siRNA. Treatment with nimbolide resulted in nuclear accumulation of SIRT-1, while siRNA knockdown of SIRT-1 resulted in reversal of anti-inflammatory activity of nimbolide. It is proposed that nimbolide reduces neuroinflammation in BV2 microglia through mechanisms resulting in dual inhibition of NF-κB and MAPK pathways. It is also proposed that activation of Nrf2 antioxidant as well as SIRT-1 deacetylation mechanisms may be contributing to its anti-inflammatory activity.