Neuroinflammation is present in the pathophysiological mechanisms of several diseases that affect the central nervous system (CNS). Microglia have a prominent role in initiating and sustaining the inflammatory process. Epiisopiloturine (EPI) is an imidazole alkaloid obtained as a by-product of pilocarpine extracted from Pilocarpus microphyllus (jaborandi) and has shown promising anti-inflammatory and antinociceptive properties. In the present study, we investigated the effects of EPI on the inflammatory response in microglial cells (BV-2 cells) induced by lipopolysaccharide (LPS) and explored putative underlying molecular mechanisms. Cell viability was not affected by EPI (1-100 mug/mL) as assessed by both LDH activity and the MTT test. Pretreatment with EPI (25, 50, and 100 mug/mL) significantly reduced the proinflammatory response induced by LPS, as observed by a decrease in nitrite oxide production and iNOS protein expression. EPI (25 mug/mL) reduced IL-6 and TNF-alpha production, by 40% and 34%, respectively. However, no changes were observed in the anti-inflammatory IL-10 production. Mechanistically, EPI inhibited the TLR4 expression and phosphorylation of NF-kappaB p65 and MAPKs (JNK and ERK1/2) induced by LPS, but no changes were observed in TREM2 receptor expression in LPS-stimulated cells. In conclusion, our data demonstrated the potent anti-inflammatory properties of EPI in microglial cells. These effects are associated with the reduction of TLR4 expression and inhibition of intracellular signaling cascades, including NF-kappaB and MAPKs (JNK and ERK1/2). CI - Copyright (c) 2023 Joao Antonio Costa de Sousa et al.