Synergistic Antileishmanial Effect of Oregano Essential Oil and Silver Nanoparticles: Mechanisms of Action on Leishmania amazonensis

Abstract

American tegumentary leishmaniasis, a zoonotic disease caused by the Leishmania genus, poses significant challenges in treatment, including administration difficulty, low efficacy, and parasite resistance. Novel compounds or associations offer alternative therapies, and natural products such as oregano essential oil (OEO), extracted from Origanum vulgare, have been extensively researched due to biological effects, including antibacterial, antifungal, and antiparasitic properties. Silver nanoparticles (AgNp), a nanomaterial with compelling antimicrobial and antiparasitic activity, have been shown to exhibit potent leishmanicidal properties. We evaluated the in vitro effect of OEO and AgNp-Bio association on L. amazonensis and the death mechanisms of the parasite involved. Our results demonstrated a synergistic antileishmanial effect of OEO + AgNp on promastigote forms and L. amazonensis-infected macrophages, which induced morphological and ultrastructural changes in promastigotes. Subsequently, we investigated the mechanisms underlying parasite death and showed an increase in NO, ROS, mitochondrial depolarization, accumulation of lipid-storage bodies, autophagic vacuoles, phosphatidylserine exposure, and damage to the plasma membrane. Moreover, the association resulted in a reduction in the percentage of infected cells and the number of amastigotes per macrophage. In conclusion, our findings establish that OEO + AgNp elicits a late apoptosis-like mechanism to combat promastigote forms and promotes ROS and NO production in infected macrophages to target intracellular amastigote forms.

Keywords: NO; ROS; apoptosis-like; association; cell death; leishmaniasis.

Figure 1

Effect of OEO and AgNp on L. amazonensis promastigotes treated for 24 h. (A) OEO (3.125, 6.25, 12.5, 25, 50, 100 μg/mL) and (B) AgNp (0.1, 0.5, 1, 5, 10 μg/mL) percentage of viable promastigotes. DMSO 0.1% was used as vehicle control. The values represent the mean ± standard error of the mean (SEM) of three independent experiments. Asterisks indicate a significant difference compared to control **** p ≤ 0.0001.

Figure 2

Representative isobologram of in vitro interactions between OEO and AgNp using the method of fixed ratios. Numbers on the axes represent doses calculated from the IC₅₀ and CC₅₀ values of each compound. (A) Activity on promastigotes of L. amazonensis; (B) cytotoxicity on peritoneal macrophages from BALB/c.

Figure 3

Morphological and ultrastructural changes on promastigotes of L. amazonensis treated with 9.6/0.08 μg/mL OEO/AgNp for 24 h. (A–D) SEM images: (A) untreated promastigotes; (B–D) OEO/AgNp treated-promastigotes. (E–H) TEM images: (E) untreated promastigotes; (F–H) OEO/AgNp treated-promastigotes. fp, flagellar pocket; k, kinetoplast; m, mitochondrion; n, nucleus; *, autophagic vacuole; ■, mitochondrial swelling; ▲, lipid-storage bodies; , DNA disorganization in nuclei; →, alteration of the plasma membrane. Scale bars = 5 μm (A–D), 1 μm (E), 0.5 μm (F–H).

Figure 4

Biochemical targets affected by treatment with the association of OEO/AgNp in L. amazonensis promastigotes treated with the dose combination of 9.6/0.08 μg/mL OEO/AgNp. (A) Total ROS were measured by an increase in fluorescence caused by the conversion of nonfluorescent dye, H₂DCFDA, to fluorescent, DCF; (B) Griess method for nitrite levels in supernatant; (C) mitochondrial membrane potential assay using TMRE labeling; (D) lipid-storage bodies using Nile red labeling; (E) autophagy vacuoles using MDC labeling. Data represent the mean ± SEM of three independent experiments performed in duplicate. Asterisks indicate significant difference compared to control * (p ≤ 0.05), ** (p ≤ 0.01), **** (p ≤ 0.0001).

Figure 5

Phosphatidylserine exposure in promastigotes treated with the association at the chosen dose (9.6/0.08 μg/mL OEO/AgNp) using annexin V/FITC and PI. Data acquisition and analysis flow cytometry with a total of 10,000 events. (A) Typical dot plot of three independent experiments. (B) Count of L. amazonensis promastigotes co-staining of annexin V/FITC and PI analyzed by flow cytometry. Asterisks indicate significant difference compared to control * (p ≤ 0.05), ** (p ≤ 0.01). (C) Cell size of L. amazonensis promastigotes treated with OEO + NpAg and (D) quantitative analysis of cell size. FSC-A was considered a function of cell size. The black line corresponds to the control (untreated parasites), and the red area indicates the parasites treated with OEO + NpAg.

Figure 6

Intramacrophagic L. amazonensis death by the association of OEO/AgNp is dependent on NO and ROS. L. amazonensis-infected macrophages submitted to a 24 h of treatment with OEO/AgNp (A) fluorescent probe DCF for reactive oxygen species measurement in culture cells, (B) Griess method for nitrite levels in the culture supernatant. The values represent the mean ± SEM of three independent experiments performed in duplicate. * (p < 0,05), **** (p < 0,0001).

Figure 7

Effect with the association of OEO/AgNp on L. amazonensis-infected macrophages. L. amazonensis-infected macrophages were treated with the association of OEO/AgNp for 24 h, and the percentage of infected macrophages (A) and the number of amastigotes per macrophage (B) were evaluated. AmB 1µM (positive control). The values represent the mean ± SEM of three independent experiments performed in duplicate. Significant difference compared to control ** (p ≤ 0.01) **** (p ≤ 0.0001) photomicrographs of infected macrophages (C) and macrophages infected and treated with the association (D) stained with Leishman dye under 1000x magnification in an optical microscope. Arrows indicate amastigote forms. Scale bar = 10 µm.