Amentoflavone as an Ally in the Treatment of Cutaneous Leishmaniasis: Analysis of Its Antioxidant/Prooxidant Mechanisms

Abstract

Treatment of leishmaniasis is a challenging subject. Although available, chemotherapy is limited, presenting toxicity and adverse effects. New drugs with antileishmanial activity are being investigated, such as antiparasitic compounds derived from plants. In this work, we investigated the antileishmanial activity of the biflavonoid amentoflavone on the protozoan Leishmania amazonensis. Although the antileishmanial activity of amentoflavone has already been reported in vitro, the mechanisms involved in the parasite death, as well as its action in vivo, remain unknown. Amentoflavone demonstrated activity on intracellular amastigotes in macrophages obtained from BALB/c mice (IC₅₀ 2.3 ± 0.93 μM). No cytotoxicity was observed and the selectivity index was estimated as greater than 10. Using BALB/c mice infected with L. amazonensis we verified the effect of an intralesional treatment with amentoflavone (0.05 mg/kg/dose, in a total of 5 doses every 4 days). Parasite quantification demonstrated that amentoflavone reduced the parasite load in treated footpads (46.3% reduction by limiting dilution assay and 56.5% reduction by Real Time Polymerase Chain Reaction). Amentoflavone decreased the nitric oxide production in peritoneal macrophages obtained from treated animals. The treatment also increased the expression of ferritin and decreased iNOS expression at the site of infection. Furthemore, it increased the production of ROS in peritoneal macrophages infected in vitro. The increase of ROS in vitro, associated with the reduction of NO and iNOS expression in vivo, points to the antioxidant/prooxidant potential of amentoflavone, which may play an important role in the balance between inflammatory and anti-inflammatory patterns at the infection site. Taken together these results suggest that amentoflavone has the potential to be used in the treatment of cutaneous leishmaniasis, working as an ally in the control and development of the lesion.

Keywords: Glucantime; amentoflavone; antileishmanial activity; biflavonoid; cutaneous leishmaniasis; intralesional treatment; prooxidant.

Figure 1

In vitro amentoflavone effect on macrophages infected with L. amazonensis. BALB/c mice peritoneal macrophages were infected with L. amazonensis promastigotes and treated with amentoflavone (0–11.14 μM) for 72 h. (A) Total intracellular amastigotes in 200 macrophages and (B) Dose-response curve of amentoflavone. Mock-treated infected cells were used as a control (0 μM). The columns represent the mean ± S.E.M. of quadruplicates. Significant difference (1-way ANOVA, followed by Tukey post-test) in relation to the mock-treated control *p < 0.05, **p < 0.01, ***p < 0.0001.

Figure 2

Effect of intralesional amentoflavone treatment on L. amazonensis-infected BALB/c mice. (A) Lesion size kinetics. The size of the lesions was monitored weekly from the beginning of treatment until 1 week after the last treatment dose by measuring the footpad with a caliper, with 0.1 mm sensitivity. The data shown represents the average of the difference between the infected footpad and the uninfected contralateral footpad ± S.E.M. of 10 animals per group. (B) Parasite load estimated by limiting dilution assay. (C) Parasite load estimated by qPCR. Points and bars represent the mean ± S.E.M. of five animals per group. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001: significant difference in relation to the mock-treated control (1-way ANOVA, followed by Tukey post-test). L.a., Animals infected with L. amazonensis; AMT, Amentoflavone; GLU, Glucantime.

Figure 3

Histopathological analysis of BALB/c mice treated intralesionally. Photomicrographs of the footpad and lymph nodes of BALB/c mice infected with L. amazonensis and treated with vehicle (A, B), amentoflavone 0.5 mg/kg/dose (C, D) or Glucantime 64 mg Sb⁵⁺/kg/dose (E, F). (A) Inflammatory infiltrate with polymorphonuclear cells and intense parasitism (black arrows) in the footpad (hematoxylin and eosin); (B) Infected macrophages (black arrows) in the lymph node (Giemsa). (C) Amastigotes in footpad (black arrow) (hematoxylin and eosin). (D) Hyperplastic germinal center in the lymph node with follicular hyperplasia (Giemsa). (E) Connective tissue thickening among the muscles and inflammatory infiltrate (asterisk) in the footpad (hematoxylin and eosin). (F) Polymorphonuclear cells (mainly eosinophils), parasitism (detail, white arrow) in the lymph node (Giemsa). Representative images (2 animals/group).

Figure 4

Evaluation of NO production and iNOS expression on amentoflavone-treated mice (A) Nitrite quantification in peritoneal macrophages isolated from L. amazonensis-infected BALB/c, treated or not with amentoflavone intralesionally. Macrophages were stimulated with total L. amazonensis antigen (1 µg/µl) and the amount of nitrite in the cell culture supernatant was measured by the Griess reaction. The bars represent the mean ± S.E.M. of five animals per group. *p < 0.05, **p < 0.01 (Test t). (B) Relative quantification of iNOS mRNA in L. amazonensis-infected BALB/c mouse footpads treated or not with amentoflavone or Glucantime, intralesionally. Expression was estimated by 2-ΔΔCT method, using Rplp0 as a reference gene. The bars represent the mean ± S.E.M. of three animals per group. L.a., Animals infected with L. amazonensis; AMT, Amentoflavone; GLU, Glucantime.

Figure 5

Relative quantification of Nrf2-related genes in L. amazonensis-infected BALB/c mice footpads treated or not with amentoflavone or Glucantime, intralesionally. RT-qPCR analyses were performed to quantify the expression of Nrf2 (A), HO-1 (B), and ferritin (C) genes. Expression was estimated by 2-^ΔΔCT method, using Actb as a reference gene. The bars represent the mean ± S.E.M. of three animals per group. Significant differences (1-way ANOVA, followed by Tukey post-test); *p < 0.05; **p < 0.01 in relation to the mock-treated control. Nrf2, nuclear factor, erythroid derived 2, like 2; HO-1, heme oxygenase 1; Feritin, L-ferritin. Actb, actin beta.

Figure 6

Amentoflavone concentration influence on the ROS production in peritoneal macrophages. Murine peritoneal macrophages (2 × 10⁶) were treated with different concentrations of amentoflavone. (A) Uninfected macrophages treated with amentoflavone in concentrations of 1.15 μM (1/2 IC₅₀), 2.3 μM (IC₅₀), 4.6 μM (2× IC₅₀) or 9.2 μM (4× IC₅₀) for 24, 48, and 72 h. Mock-treated macrophages were used as controls. Antimycin B and Glucose/Glucose oxidase (G/GO) were used as a positive control. (B) Macrophages infected with L. amazonensis and treated with different concentrations of amentoflavone for 24, 48, or 72 h. Infected and mock-treated macrophages were used as controls. The generation of ROS was measured using the fluorescent indicator H₂DCFDA. The data were expressed in units of fluorescence intensity (FIU). Significant differences in relation to the control (t test): *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001. AMT, Amentoflavone; L.a., L. amazonensis.