Anti-leishmanial and immunomodulatory effects of harmaline against Leishmania tropica through molecular docking, gene expression profiling and oxidative stress modulation

Cutaneous leishmaniasis continues to represent a significant health concern in many endemic areas, largely due to the limitations of currently available therapies, including adverse effects, reduced responsiveness, and the growing emergence of drug-resistant parasites. The present investigation explored the antileishmanial potential of harmaline (HAR) against Leishmania tropica through a combination of experimental and computational approaches. Parasite susceptibility was examined in both promastigote and intracellular amastigote stages using MTT-based viability assessment and microscopic enumeration of Giemsa-stained infected macrophages, respectively. The safety profile of HAR was evaluated in THP-1-derived macrophages to calculate IC 50 , CC 50 , and selectivity index values. In addition, transcriptional changes in apoptosis-associated markers (Bax and Bcl-2), cytokines related to Th1/Th2 immune responses (IFN-γ, IL-12, TNF-α, IL-10, and TGF-β), and inducible nitric oxide synthase (iNOS) were quantified by real-time PCR. Reactive oxygen species generation, nitric oxide release, and antioxidant enzyme activities were also determined, while apoptotic alterations were confirmed using Annexin V/PI staining and flow cytometric analysis. Computational docking studies were conducted to evaluate the binding behavior of HAR toward iNOS and selected apoptosis-regulating proteins. Harmaline reduced parasite viability in both developmental stages in a concentration-dependent manner and promoted apoptotic responses, as reflected by an elevated Bax/Bcl-2 ratio and increased Annexin V-positive populations. Exposure to HAR further enhanced ROS and NO production while diminishing SOD and CAT activities, suggesting a shift toward a pro-oxidative cellular environment. Moreover, treatment favored a protective immune profile characterized by increased expression of IFN-γ, IL-12, and TNF-α together with suppression of IL-10 and TGF-β. Docking results supported the possibility of biologically relevant interactions between harmaline and proteins involved in nitric oxide synthesis and apoptosis regulation. Collectively, these findings indicate that harmaline exerts both antiparasitic and immunoregulatory effects and may serve as a promising candidate for further in vivo evaluation as an adjunctive approach for cutaneous leishmaniasis management.