Treatment of Trypanosoma cruzi-infected mice with propolis promotes changes in the immune response

Abstract

Ethanol extract of Bulgarian propolis (Et-Blg) was administered by oral route in doses ranging from 25 to 100 mg/kg body weight in experimental Trypanosoma cruzi-infected Swiss mice. Treatment with 50 mg Et-Blg/kg body weight/day led to a decrease in parasitemia and showed no hepatic or renal toxic effect. Treatment with Et-Blg led to a decrease in the spleen mass and modulated the initial inflammatory reaction as demonstrated by analysis of the leukocyte profile in peripheral blood, quantification of T cells subsets, and phenotypic markers in the spleen. Preferential expansion of CD8⁺ and partial inhibition in the increase of CD4⁺CD69⁺ and CD8⁺CD69⁺ in CD4⁺CD44⁺ and CD8⁺CD44⁺ and in the decrease of CD8⁺CD62L in Trypanosoma cruzi-infected mice were also observed. Taken together, our data indicate that treatment of Trypanosoma cruzi-infected mice with Et-Blg interferes with the basic properties of immune cells.

Introduction

The flagellate protozoan Trypanosoma cruzi is the etiological agent of Chagas disease, an endemic Latin America parasitosis which has infected 16–18 million infected people (WHO, 2002). This disease is characterized by an acute phase with detectable parasitemia and a long-lasting asymptomatic phase (Laguens et al., 1994). The immune system is also involved in the parasite persistence (Zhang and Tarleton, 1999), the lymphoid system being a possible target and an active component of the Trypanosoma cruzi infection. The development of alternative drugs to replace nifurtimox and benznidazole, currently used for the treatment of chagasic patients, is urgent (Coura and De Castro, 2002). In this context, our laboratory is involved in the investigation of new natural and synthetic agents for the treatment of experimental Trypanosoma cruzi-infected animals (De Castro and Higashi, 1995, Pereira et al., 1998, Olivieri et al., 2002, Garzoni et al., 2004).

Propolis is a bee product, made from plant exudates, used for the construction and repair of the hive, as well as protection against micro-organisms. It is a complex mixture, with more than 200 compounds already identified in different samples (Bankova et al., 2000). Propolis presents a variety of biological activities, and its effect as anti-inflammatory, analgesic, and tissue regenerative agent has been demonstrated by different groups in in vivo models (De Castro, 2001). Such effects have been associated with the presence of phenolic compounds, such as flavonoids, and aromatic acids, and to their anti-oxidative properties (Heim et al., 2002, Ichikawa et al., 2002). Propolis extracts present low toxicity to experimental animals, with LD₅₀ higher than 7 g/kg for mice (Arvouet-Grand et al., 1993). In relation to pathogenic protozoa, ethanol extracts showed activity against Toxoplasma gondii, Trichomonas spp., Giardia lamblia, and Trypanosoma cruzi (Starzyk et al., 1977, Torres et al., 1990, Higashi and De Castro, 1994, De Castro and Higashi, 1995, Marcucci et al., 2001, Cunha et al., 2004).

We have already reported the in vitro activity of the ethanol extract of Bulgarian propolis (Et-Blg), with a known chemical composition, against Trypanosoma cruzi and different species of fungi and bacteria of medical importance (Prytzyk et al., 2003, Salomão et al., 2004). In different mouse models, this extract also exhibited analgesic and anti-inflammatory effects which were associated with its high content of flavonoids (Paulino et al., 2003). These results encouraged us to assay the effect of Et-Blg on mice experimentally infected with Trypanosoma cruzi, and to verify the potential toxicity and immunomodulatory properties of this standardized propolis extract.