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

18β-Glycyrrhetinic acid (GRA), a natural immunomodulator, greatly reduced the parasite load in experimental visceral leishmaniasis through nitric oxide (NO) upregulation, proinflammatory cytokine expression, and NF-κB 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) and MKK6 as the immediate upstream regulators of p38. However, detection of remnant p38 kinase activity in the presence of both DN MKK3 and MKK6 suggested alternative pathways of p38 activation. That residual p38 activity was attributed to an autophosphorylation event ensured by the transforming growth factor β-activated kinase 1 (TAK1)-binding protein 1 (TAB1)-p38 interaction and was completely abolished upon pretreatment with SB203580 in DN MKK3/6 double-transfected macrophage cells. Further upstream signaling 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 knockdown 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 to MKK3/6-mediated canonical and TAB1-mediated noncanonical p38 activation.