Figure 1.
Chemical structure of RuBzNO2.
Figure 2.
RuBzNO2 is highly effective in killing trypomastigotes and does not show cytotoxicity.
Trypomastigote forms of T. cruzi (A) or spleen cells of mice (B) were cultured with serial dilutions of RuBzNO2 or Bz for 24 h at 37°C. A. Live parasites were counted by microscopy to determine the trypanocidal activity of the compounds. B. Dead cells were stained with propidium iodide and quantified by flow cytometry. The bars indicate means+SEM of duplicates and are representative of three independent experiments.
Figure 3.
RuBzNO2 is able to release NO inside the cell and kills the amastigotes.
A. Vero cells were incubated with DAF-2 DA and the reducing agent phenylephrine in the presence or absence of RuBzNO2. The formation of the fluorescent compound (DAF-2T), which occurs in the presence of NO in the cytoplasm, was assessed by fluorescence microscopy. B. Cells were incubated with trypomastigote forms of T. cruzi for 24 h at 37°C. The remaining parasites were washed from the culture, and the cells were incubated at 37°C for an additional 24 h. Cells were stained with Giemsa dye, and the percentage of infected cells was determined by optical microscopy. The bars indicate means+SEM of triplicates and are representative of two independent experiments. * represents p<0.05.
Figure 4.
Treatment with RuBzNO2 reduces parasitemia and improves the survival of T. cruzi-infected mice.
Mice were infected with 2000 trypomastigotes of T. cruzi and treated orally for 10 days from the first day of patent parasitemia (5 days post-infection) with 4 µmol/kg or 0.4 µmol/kg of Bz or RuBzNO2. A. Parasitemia was evaluated in the blood by counting parasites with an optical microscope. B. Survival was monitored daily. The bars indicate means+SEM of 6–7 mice/experiment and are representative of three independent experiments. * represents p<0.05.
Figure 5.
Treatment with RuBzNO2 ameliorates heart inflammation and lesions induced by T. cruzi infection.
Mice were infected with 2000 trypomastigotes of T. cruzi and treated from day 5 for 10 days with various concentrations of Bz or RuBzNO2. Twenty days after infection, the heart histology (A–B) was evaluated, and the CK-MB level (C) was measured in the serum. A. Representative photomicrograph of heart stained with H&E. B. Quantification of the inflammatory cells shown in A. The bars indicate means+SEM of 4–6 mice/experiment and are representative of two independent experiments. * represents p<0.05.
Figure 6.
Heart parasitism is reduced after treatment with RuBzNO2.
Mice were infected with 2000 trypomastigotes of T. cruzi and treated with various concentrations of Bz or RuBzNO2. After 20 dpi, the DNA from the heart was isolated, and T. cruzi was quantified with real-time PCR. Each dot indicates an individual mouse, and the graph is representative of two independent experiments. * represents p<0.05.