Avaliação do tratamento da paracoccidioidomicose experimental com uso do anti-fúngico itraconazol associado com aplicação de laser de baixa potência

Abstract

Paracoccidioidomycosis (PCM) is a systemic mycosis caused by the fungus Paracoccidioides spp. (Pb), which requires prolonged treatment, justifying studies that expand therapeutic options. Itraconazole (Itra) is effective for PCM and requires shorter therapy than other drugs. Low Level LASER Therapy (LLLT) is a new application for the treatment of PCM, which can improve inflammatory aspects of the disease. Here we propose to to study the effect of simultaneous administration of the antifungal drug Itra and complementary therapy with LLLT in an experimental murine model. We used different experimental models, the first tests were done in an “in vitro” model to evaluate the use of different Itra concentrations, thus 3 concentrations (3, 10 and 50mg/Kg) evaluating their antifungal activity of concentrations of the drug, toxicity and its action on cells and fungi together. Then, we used the subcutaneous “air pouch” model, inoculating the virulent Pb18 isolate of P. brasiliensis in mice, and after 8 days, “air pouch” cells constituted mostly of PMNs were collected , to evaluate the effects of antifungal and LASER therapy. In this model, cell viability, differential cell count, content of oxidative oxygen metabolism products (ROS), and nitrogen (NO) metabolism products, proteins, and cytokines GM-CSF, TNF-α, INF-y, IL-10, IL-12, IL-4, IL-17 and KC (IL-8). Finally, we used intraperitoneal infection with P. brasiliensis (Pb18) intraperitoneally to evaluate the infection/cure profile after oral treatment with the highest concentration of Itraconazole (50mg/Kg) for 120 days, in which we evaluated the profile of humoral immunity (production of specific IgG antibodies) Delayed type hypersensibility (DTH), as well as the effects of treatment on organs such as lungs, liver, spleen and epipplon, analyzing the NO, protein and cytokines GM-CSF, Il-17, IL-4, IL-12 and KC (IL-8) content. The results of this “in vitro” model showed that Itra has fungicidal activity against a virulent strain of Pb (Pb18), with no toxic effect on splenocytes. This "in vitro" model allowed us to conclude that the responsible for the lysis of the fungus was the drug, with the concentration of 50mg/Kg being more efficient. In this way, the results of the model of subcutaneous infection by “air pouch” showed that LLLT has effects on cells (PMNs) increasing their production of oxygen (ROS) and nitrogen (NO) metabolites, their metabolic activity, besides increasing the production of KC, INF-y, TNF-α, IL-10, GM-CSF, and IL-4 cytokines. The association with Itra attenuates the effect of LLLT, with less cell activation being observed as we increase the concentrations of the antifungal drug increases. The combination of the two treatments (Itra and LLLT) results in less cell activation, as verified by lower production of ROS and decreased synthesis of inflammatory cytokines, in parallel with increased fungicidal activity. These results indicate that in this experimental situation it is less necessary to activate immune cells to fight the fungus. Histopathological analysis of the "air pouch" showed a decrease in the number of inflammatory cells, in addition to an improvement in the wound healing profile, with an increase in the number of fibrocytes, in parallel with a decrease in the number of fungi. With the increase in the concentrations of the antifungal drug, the number of fungi at the site of infection was lower, a fact that was enhanced by the use of LLLT. The results obtained from the intraperitoneal infection with the treatment of Itra orally (gavage) showed that this treatment model with the highest concentration of the antifungal (Itra) decreases the inflammation in the epipplon, which is the shock organ in the intraperitoneal infection. by Pb18, possibly by decreasing the amount of viable fungi at the infection local.