Estudo in vitro da atividade antileishmania e linfoproliferativa do ácido gálico em combinação com o sulfato de paromomicina

Abstract

Cutaneous leishmaniasis is a neglected disease with high morbidity caused by Leishmania, whose progression depends on the balance between innate and adaptive immune responses. Despite the importance of CD4⁺ T lymphocytes in parasite control, pharmacological treatment is still indispensable, but limited due to toxicity, adverse effects, and parasite resistance. In this context, combination therapies, such as the association of paromomycin sulfate with bioactive natural compounds, stand out as promising therapeutic strategies. Therefore, the aim of this study was to evaluate the antileishmania and immunomodulatory activity of the combination of gallic acid (GA) and paromomycin sulfate (PAR) against Leishmania guyanensis. The methodology included in vitro assays to evaluate the inhibitory activity on promastigote and amastigote forms of L. guyanensis, as well as cytotoxicity in RAW 264.7 macrophages. Additionally, PBMCs from healthy volunteers (n = 13) were stimulated with GA, PAR, their combination (GA+PAR), Glucantime®, and phytohemagglutinin (PHA). After 72 hours, lymphoproliferation (BrdU) and cytokine production were analyzed by flow cytometry, with integration of immunological parameters by Spearman's correlation. Regarding the results, GA showed antileishmania activity in promastigotes (IC₅₀ of 142.5–126.5 µM), while PAR and GA+PAR were not active (IC₅₀ >150 µM). GA and PAR were not cytotoxic (CC₅₀ >150 µM), however GA+PAR reduced cell viability at concentrations of 150 and 100 µM (CC₅₀ 131.2 – >150 µM). In macrophages infected with amastigotes, GA and GA+PAR showed high efficacy (IC₅₀ of 11.4 and 20.5 µM at 48 h, respectively), while PAR remained low in activity (IC₅₀ >150 µM). In the immunological evaluation, GLU showed greater lymphoproliferation (61.5%), followed by GA (46.2%) and PAR and AG+PAR (30.8%). AG preferentially modulated the TNF/IL-6 axis, PAR was associated with IL-10, and the AG+PAR combination exhibited an integrated and balanced profile between pro- and anti-inflammatory cytokines, without direct proliferative stimulation, while GLU promoted broad activation. In conclusion, the AG+PAR combination showed promising antileishmania synergy in L. guyanensis and demonstrated a complementary and balanced immunomodulatory pattern, modulating inflammatory pathways without inducing cell proliferation, making it a promising candidate for the development of a possible new drug, and ultimately, to adapt the therapeutic protocol for tegumentary leishmaniasis.