Title : Anti-proliferative and metabolic activity of the compounds caffeic acid and oregano essential oil in human trophoblast cells infected with the RH strain of toxoplasma gondii
Abstract:
Introduction: Toxoplasmosis, an infection caused by the protozoan Toxoplasma gondii, becomes serious in immunocompromised individuals, ocular toxoplasmosis, and, especially, in cases of congenital infection. Treatment of toxoplasmosis is hampered by the toxicity of conventional medications, sulfadiazine, and pyrimethamine. Therefore, other compounds have been evaluated as alternative treatments for this infection, such as caffeic acid (CA), a phenolic compound found abundantly in many plants and foods, with proven antimicrobial, antioxidant, anti-leishmanial, and anti-trypanosomal effects. Oregano (Origanum vulgare) essential oil (OEO) presents a promising alternative due to its antibacterial, antifungal, and antiparasitic potential. The objective of this study was to analyze the antiproliferative capacity and the direct mechanisms of action of OEO and CA on T. gondii tachyzoites (RH strain).
Materials and methods: To evaluate the cytotoxicity of the compound, HTR8/SVneo and BeWo cells were cultured in 96-well plates for 24 hours in an incubator at 37°C and 5% CO2. Subsequently, the cells were treated, respectively, with AC at concentrations of 10-10000 μg/mL and OEO at concentrations of 0.7 to 150 μg/mL for 24 hours. To investigate the direct effect of the compound on the free forms of T. gondii tachyzoites, a viability test was performed using the trypan blue exclusion method. Tachyzoites (5x105) were treated with AC (1-200 μg/mL) and OEO (0.7-150 μg/mL) for 1 hour, stained and counted under an optical microscope. Finally, to verify the effect of the compound on T. gondii infection, HTR8/SVneo cells were maintained in 24-well plates containing 13-mm round coverslips for 24 hours and then infected with 5x105 tachyzoites of the RH strain of T. gondii. After 3 hours of infection, the cells were washed and treated with AC at concentrations of 5, 10, 25, and 50 μg/mL and OEO at concentrations of 6.2, 12.5, 25, and 50 μg/mL for 24 hours. Subsequently, the coverslips were stained and mounted for analysis under a light microscope (E100, Nikon-LED, 100X objective). Subsequently, to determine the mechanisms leading to parasite death, mitochondrial membrane potential and lipid droplets were evaluated, as well as autophagy by monodansylcadaverine. Finally, scanning and transmission electron microscopy analyses were performed.
Results: After treatment, the results showed that both compounds exhibited low cytotoxicity in cells and reduced tachyzoite viability. There was a reduction in infection and intracellular proliferation of 55% and 84% at concentrations of 25 and 50 μg/mL of OEO, respectively, and 76.5% and 80.5% at concentrations of 25 μg/mL and 50 μg/mL of AC. In free tachyzoites, both treatments caused mitochondrial membrane depolarization and lipid droplet formation, as well as mitochondrial swelling, observed by TEM. These changes are linked to the induction of autophagy and plasma membrane permeabilization, with leakage of cytoplasmic contents and nuclear alterations, confirmed by TEM and SEM, culminating in parasite death.
Conclusion: Treatments with OEO and AC demonstrated anti-T. gondii activity, directing the death of the parasite through metabolic alterations without causing toxicity to cells, and can be considered promising compounds in the treatment of congenital toxoplasmosis in the future.
