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

Abstract

b 18␤-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-B activation. For the GRAmediated 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 ␤-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. The term leishmaniasis encompasses a spectrum of vectorborne protozoan parasitic diseases affecting 12 million people worldwide with 0.5 million new cases per annum. With no available vaccines for treatment of leishmaniasis, chemotherapy remains the major tool to combat infection (1). The primary treatment for leishmaniasis includes pentavalent antimonials, amphotericin B, pentamidine, miltefosine, and aminosidine, which often pose problems of high toxicity and several adverse effects. In addition, the combination of lengthy treatment schedules and the high cost of the compounds makes the treatment unsuitable in many cases (2). Evidence for a drug that specifically eliminates the infection and is safe enough to be used in the clinical field is still lacking. We have previously shown that 18␤-glycyrrhetinic acid (GRA), a pentacyclic triterpene derivative of the ␤-amyrin type, extracted from the root of the medicinal plant licorice (Glycyrrhizza glabra L.), might completely cure experimental visceral leishmaniasis (3). The pharmacological properties of GRA include antitumorigenic (4), hepatoprotective (5), antiulcerative (6), and immunomodulatory effects (7). Apart from immunomodulation, GRA plays a role in shifting the cellular kinase/phosphatase balance toward kinases during infection (8). This host-favorable act...

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