Antileishmanial Effect of 18-Glycyrrhetinic Acid Is Mediated by Toll- Like Receptor-Dependent Canonical and Noncanonical p38 Activation

18-Glycyrrhetinic acid (GRA), a natural immunomodulator, greatly reduced the parasite load in experimental visceral leish-maniasis through nitric oxide (NO) upregulation, proinflammatory cytokine expression, and NF-B activation. For the GRA-mediated effect, the primary kinase responsible was found to be p38, and analysis of phosphorylation kinetics as well as studies with dominant-negative (DN) constructs revealed mitogen-activated protein kinase kinase 3 (MKK3) andMKK6 as the immedi-ate upstream regulators of p38. However, detection of remnant p38 kinase activity in the presence of both DNMKK3 andMKK6 suggested alternative pathways of p38 activation. That residual p38 activity was attributed to an autophosphorylation event en-sured by the transforming growth factor -activated kinase 1 (TAK1)-binding protein 1 (TAB1)-p38 interaction and was com-pletely abolished upon pretreatment with SB203580 in DNMKK3/6 double-transfected macrophage cells. Further upstream sig-naling evaluation by way of phosphorylation kinetics and transfection studies with DN constructs identified TAK1, myeloid differentiation factor 88 (MyD88), interleukin 1 receptor (IL-1R)-activated kinase 1 (IRAK1), and tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6) as important contributors to GRA-mediated macrophage activation. Finally, gene knock-down studies revealed Toll-like receptor 2 (TLR2) and TLR4 as the membrane receptors associated with GRA-mediated antileishmanial activity. Together, the results of this study brought mechanistic insight into the antileishmanial activity of GRA, which is dependent on the TLR2/4-MyD88 signaling axis, leading toMKK3/6-mediated canonical and TAB1-mediated nonca-nonical p38 activation