Biotherapic of Trypanosoma cruzi 17d increases apoptosis in experimentally infected mice
DOI:
https://doi.org/10.51910/ijhdr.v10i36.467Keywords:
Biotherapic, Apoptosis., Chagas' diseaseAbstract
Introduction: the mechanism of action of ultradiluted medicines has not yet been established[1,3]. Many basic research studies have focused on isopathic models using in vitro or in vivo designs [4,5]. Recent studies indicate that an ultradiluted (isopathic) antigen can transfer signals to the immune system and modulate its response when an organism is challenged against this same antigen [6]. Some studies on experimental infection of mice by T. cruzi identified apoptotic cells and showed that the increase of their number is associated with an increase also in the number of parasites in the blood of the infected animals, while blockage of apoptosis can be the target of therapeutic intervention [7,8]. Aim: to evaluate the development of apoptosis in mice treated with biotherapic of Trypanosoma cruzi in dilution 17d through in situ detection of fragmented DNA. Method: in a blind randomized controlled trial, 36 male Swiss mice age 4 or 8 weeks were distributed in groups control - treated with 7% hydroalcoholic solution(CI-4=9 animals or CI-8=9 animals); and treated with biotherapic 17d (BIOT-4=9 animals or BIOT-8=9 animals). Infection was performed with 1,400 trypomastigotes T. cruzi-strain Y via intraperitoneal. Biotherapic 17d was prepared through the addition of 0.9ml of concentrated T. cruzi (10E+7 trypomastigotes/ml) to 9.1 ml of distilled water. The following dilutions were prepared in 86% hydroalcoholic solution until dilution 16d. Dilution 17d was prepared with 7% hydroalcoholic solution. It was performed microbiological control and biological risk in vivo. Treatment: 0.2 ml in 3 consecutive days, oral route, from the moment infection was verified. Animals were sacrificed on the 3rd day of treatment in a chamber saturated with ether. The liver and spleen were removed and fixated in 4% paraformaldehyde for 24 hours and then included in paraffin. Apoptosis was evaluated through DNA fragmentation - TUNEL technique (TdT dUTP-biotin Nick End Labeling (ApopTag® Peroxidade-Chemicon). For statistical analysis software Statistica 8.0 was used. This study was approved by the Ethics Committee for Animal Experimentation of UEM. Results and Discussion: in the samples of liver of animals age 4 and 8 weeks either treated or not with biotherapic 17d it was found cells parasitized by amastigotes of T. cruzi with apoptotic bodies, or phagocytic cells with phagocytic vacuole with apoptotic marked material inside them. The number of cells in apoptosis in animals age 4 weeks was not significantly (p=0.03) larger in treated group BIOT-C4 than in control group CI-4 (Figure 1). In animals age 8 weeks, the number of cells in apoptosis was significantly (p<0.001) larger in the treated group BIOT-8 than in control group CI-8 (Figure 1). Figure 1. Average number of cells in apoptosis observed in slices of liver of male Swiss mice age 4 and 8 weeks infected by 1,400 blood trypomastigotes Y strain treated (biotherapic) or not (control) with biotherapic of Trypanosoma cruzi 17d. Technique employed: Terminal deoxynucleotidyl transferase-mediated UTP nick end labelling - APOPTAG (Millipore). * p <0,05 regarding the corresponding control. These results point to a mechanism of action for the rise in the peak of parasitemia caused by ultradiluted substances in low dilutions in Swiss mice experimentally infected by T. cruzi, as it was observed in several studies [9-12]. It is known that homeopathic preparations are able to increase communication between cells [13-14]. Affected organisms treated with ultradilutions would resort to apoptosis as mechanism of cure, removing altered cells[15]. Literature confirms that the use of homeopathic medicines increases apoptosis[16]. The idea to be assessed is that, when attempting to eliminate infection by T. cruzi, parasitized cells enter into apoptosis and consequently release parasites to the circulation rising the peak of parasitemia up. This hypothesis was demonstrated here, since it was shown that the number of cells in apoptosis and the number of cells containing phagocytized apoptotic material inside them (Figure 2) was larger in the group treated with biotherapic of T. cruzi 17d than in the control group. Figure 2. Average number of cells with phagocytic vacuole with apoptotic material observed in slices of liver of male Swiss mice age 4 and 8 weeks infected by 1,400 blood trypomastigotes Y strain treated (biotherapic) or not (control) with biotherapic of Trypanosoma cruzi 17 d. Technique employed: Terminal deoxynucleotidyl transferase-mediated UTP nick end labelling - APOPTAG (Millipore). * p <0,05 regarding the corresponding control. According to the law of similitude, a homeopathic medicine must cause symptoms similar to the ones observed in disease [17] in order to awaken in the organism a reaction fit to combat the affection and promote the re-equilibrium of health in the treated individual. In this study in particular, the administration of biotherapic of T. cruzi 17d increased apoptosis and acted according to the principle of similarity, since Dos Reis et al (2007) state that apoptotic cells were detected in both experimental infection of mice by T. cruzi and the heart of patients with chronic Chagas' disease[8]. In another article, members of the same group state that an increase of apoptotic cells during infection by T. cruzi might contribute to the increase of parasitemia in infected animals [7]. According to Francisco (2007)[18], treatment of mice infected by T. cruzi with benznidazole - the only medicine available in Brazil for the etiological treatment of Chagas' disease - elicits an accumulation of CD8 T lymphocytes due to inhibition of apoptosis. Conclusion: these results show that apoptosis is increased in animals treated with biotherapic of T. cruzi 17d.
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Copyright (c) 2021 Patrícia Flora Sandri, Gislaine Janaina Sanchez Falkowski, Luzmarina Hernandes, Márcia Machado de Oliveira Dalálio, Denise Lessa Aleixo, Mônica Lúcia Gomes, Anélio Dias Nascimento Júnior, Ricardo Alberto Moliterno, Silvana Marques de Araújo
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