The Therapeutic Potential of Angeli's Salt in Mitigating Acute Trypanosoma cruzi Infection in Mice

Abstract

Chagas disease (CD), caused by Trypanosoma cruzi, is a neglected tropical disease prevalent in Latin America. Infected patients are treated to eliminate the parasite, reduce the cardiomyopathy risk, and interrupt the disease transmission cycle. The World Health Organization recognizes benznidazole (BZ) and nifurtimox as effective drugs for CD treatment. In the chronic phase, both drugs have low cure rates and serious side effects. T. cruzi infection causes intense tissue inflammation that controls parasite proliferation and CD evolution. Compounds that liberate nitric oxide (NO) (NO donors) have been used as anti-T. cruzi therapeutics. Currently, there is no evidence that nitroxyl (HNO) affects T. cruzi infection outcomes. This study investigated the effects of the HNO donor Angeli's salt (AS) on C57BL/6 mice infected with T. cruzi (Y strain, 5 × 10³ trypomastigotes, intraperitoneally). AS reduced the number of parasites in the bloodstream and heart nests and increased the protective antioxidant capacity of erythrocytes in infected animals, reducing disease severity. Furthermore, in vitro experiments showed that AS treatment reduced parasite uptake and trypomastigote release by macrophages. Taken together, these findings from the murine model and in vitro testing suggest that AS could be a promising therapy for CD.

Keywords: Chagas disease; leukopenia; macrophages; nitroxyl; oxidative stress; therapy; thrombocytopenia.

Figure 1

Experimental design. C57BL/6 mice were subjected to daily treatment with varying doses of Angeli’s salt (6, 60, and 600 µg/kg/animal), diluted in phosphate buffer (PBS) with a pH of 7.2 and administered via the intraperitoneal route. This treatment was administered 15 min post-infection for a duration of 12 days. During the acute phase, parasitemia was monitored by counting blood-borne trypomastigotes, while the assessment of survival was observed daily until day 31 post-infection. At 12 days post-infection, plasma nitrite level, oxidative stress, and macrophage infection were measured. All blood analyses and cell counts were conducted using standard methods.

Figure 2

T. cruzi infection course and response to AS therapy. C57BL/6 mice were infected with 5 × 10³ trypomastigotes of T. cruzi. Daily treatment with AS (6–600 µg/kg/mouse) was initiated 15 min after infection and continued for 12 days. Control T. cruzi-infected mice received PBS (n = 8). The mean ± SEM values shown are representative of three independent experiments, and significant differences in parasitemia were observed (* p = 0.0067, Friedman test). (A) treatment with AS at doses of 6 ug/kg (n = 6), (B) 60 ug/kg (n = 8), and (C) 600 ug/kg (n = 7). Overall, parasitemia was also represented as the area under the curve (AUC) analysis. Significance was determined as * p = 0.0025 (D), * p = 0.00190 (E), and * p = 0.0091 (F), applying the unpaired t test.

Figure 3

The effect of AS on anemia in T. cruzi-infected mice. On the 12th day post-infection, (A) hematocrit and (B) hemoglobin were evaluated. C57BL/6 mice were divided into groups of five and infected with 5 × 10³ T. cruzi, treated or not with AS (6–600 µg/kg/mouse). Control T. cruzi-infected mice (Tc group) received PBS. The mean ± SEM values shown are representative of three independent experiments, and significant differences were observed between uninfected and infected groups. * p ≤ 0.05, two-way ANOVA with Tukey post-test, ns = non significance.

Figure 4

AS treatment mitigates leukopenia and thrombocytopenia observed during the acute phase of T. cruzi infection (day 12 p.i). For this investigation, C57BL/6 mice were divided into groups of five and infected with 5 × 10³ T. cruzi, treated or not with AS (6–600 µg/kg/mouse). The control group consisted of T. cruzi-infected mice (Tc group) receiving PBS. The mean ± SEM values shown in the figures are representative of three independent experiments. (A) Leukocytes, * p ≤ 0.05, two-way ANOVA with Tukey post-test (* uninfected vs. experimental groups), (# infected groups treated with AS 60 and 600 µg/kg/mouse group vs. uninfected group and vs. infected group treated with AS 6 µg/kg/mouse group). (B) Platelet levels and (C) reticulocyte counts, * p ≤ 0.05, two-way ANOVA with Tukey post-test (* uninfected vs. experimental groups), ns = non significance.

Figure 5

Effect of AS on heart parasitism. C57BL/6 mice were divided into groups of three to five individuals, and they were infected with 5 × 10³ T. cruzi, treated or not with AS (6–600 µg/kg/mouse). Control T. cruzi-infected mice received PBS. Animals were euthanized 12 days after infection, and sections of the heart from each mouse were collected for histopathological analysis. Tissue fragments were fixed in a 10% buffered formalin solution, dehydrated, cleared, and embedded in paraffin. Chilly tissue samples were sliced into 5 mm thick sections and stained with hematoxylin and eosin (H&E) to assess amastigote nests. (A) Tissue parasitism was scored by counting the total number of amastigote nests in 25 microscope fields (1 × 400 magnification) per histopathological section. The mean ± SEM values shown are representative of two independent experiments, * p ≤ 0.05, two-way ANOVA with Tukey post-test (* control vs. experimental groups). (B) Representative photomicrograph of cardiac tissue from control and experimental groups. * Shows amastigote nest.

Figure 6

AS attenuates erythrocyte oxidative stress on day 12 after T. cruzi infection. (A) Oxygen uptake and (B) induction time. Groups of C57BL/6 mice (n = 5/group) were infected with 5 × 10³ T. cruzi and subsequently treated or not with AS (6–600 µg/kg/mouse). As a control, T. cruzi-infected mice (Tc group) received PBS. The mean ± SEM values shown are representative of two independent experiments, * p ≤ 0.05, two-way ANOVA with Tukey post-test (* uninfected vs. experimental groups), # p ≤ 0.05, two-way ANOVA with Tukey post-test (* control vs. experimental groups). ns = not significant.

Figure 7

Time course curve of t-butyl hydroperoxide-initiated chemiluminescence in erythrocytes. Groups of C57BL/6 mice (n = 5/group) were infected with 5 × 10³ T. cruzi and either treated or not treated with AS (6–600 µg/kg/mouse). Uninfected mice and untreated T. cruzi-infected mice (Tc group) were included as controls. The values presented are the mean ± SEM and represent two independent experiments. Significance was determined as * p ≤ 0.05, using Kruskal–Wallis’s test, indicating a significant difference from the values observed in the controls (uninfected/infected-non-treated group or infected/non-treated group/infected treated group).

Figure 8

Effect of AS therapy on nitrite levels in the plasma (day 12 p.i). C57BL/6 mice were divided into groups and infected with 5 × 10³ T. cruzi, treated or not with AS (6–600 µg/kg/mouse). T. cruzi-infected mice (Tc group, n = 10) and uninfected mice received PBS (n = 9) and were used as controls. The mean ± SEM values shown are representative of two independent experiments, * p ≤ 0.05, two-way ANOVA with Tukey post-test (* uninfected vs. experimental groups), # p ≤ 0.05, two-way ANOVA with Tukey post-test (Tc vs. uninfected group and Tc vs. Tc 60 µg/kg), Ψ p ≤ 0.05, two-way ANOVA with Tukey post-test (Tc + 6 µg/kg vs. Tc 60 µg/kg and Tc + 6 µg/kg vs. Tc 600 µg/kg). Tc + 6 µg/kg (n = 5); Tc 60 µg/kg (n = 7); and Tc 600 µg/kg (n = 9). ns = non-significant.

Figure 9

AS mediates T. cruzi infection in macrophages. (A) Cellular metabolic activity by reducing resazurin in T. cruzi trypomastigotes. (B) Cell viability in macrophages treated with AS (7.5–120 µM) by MTT assay. Controls consisting of H₂O₂ (1 mM) and NaOH (0.01 mM). (C) Internalization index of the interaction process between macrophages, treated with AS (15–60 µM) for 1 h and exposed to T. cruzi (5:1). (D) The effect of AS on trypomastigote release in T. cruzi-infected macrophages. Cells were infected with T. cruzi trypomastigotes and treated daily or not with AS. The release of trypomastigotes into the supernatant was detected and measured from day 4 to day 7 after infection. Values represent the mean ± SEM for triplicate determination and are representative of two independent experiments. * p ≤ 0.05, two-way ANOVA with Tukey post-test (H₂O₂ vs. experimental groups), ★ p ≤ 0.05, two-way ANOVA with Tukey post-test (Tc vs. experimental groups), ♦ p ≤ 0.0001, two-way ANOVA with Tukey post-test (Tc vs. experimental groups), **** p ≤ 0.0001, two-way ANOVA with Tukey post-test (Tc vs. AS 15µM), #### p ≤ 0.0001, two-way ANOVA with Tukey post-test (AS 30 µM vs. AS 60 µM). ns = non-significant.

Figure 10

Effect of AS on nitric oxide (NO) production. Production of NO by macrophages was determined by measuring the level of accumulated nitrite, a metabolite of NO, in the culture supernatant using Griess reagent. Values are the mean ± SEM and are representative of two independent experiments. * p ≤ 0.05, two-way ANOVA with Tukey post-test (* uninfected vs. experimental groups), # p ≤ 0.05, two-way ANOVA with Tukey post-test (control vs. experimental groups), Ψ p ≤ 0.05, two-way ANOVA with Tukey post-test (Tc + 15µM vs. Tc 30 µM and Tc + 15 µM vs. Tc 60 µM). ns = non-significant.