[The Effect of Oleuropein on The Mitochondrial Membrane Potential and Generation of Reactive Oxygen Species on Leishmania tropica Promastigotes]. / Oleuropein'in Leishmania tropica Promastigotlari Üzerinde Mitokondri Membran Potansiyeli ve Reaktif Oksijen Türlerinin Olusumuna Etkisi.

Leishmania parasites, which are reported to be endemic in 98 countries around the world, infect humans as well as wild and domestic carnivores and small mammals, and are transmitted by sand flies (Phlebotomus, dwarf sandflies). It is reported that 350 million people are at risk and two million new cases are seen in the world every year. It has been reported that different drugs (topical paromomycin, oral miltefosine, ketoconazole, rifampin, and zinc) have been tried in studies especially in endemic regions in the treatment of cutaneous leishmaniasis, and response to treatment has been obtained at different rates. Today, the search for alternative treatments continues and many studies have been carried out for this purpose. For centuries, olive leaf extracts have been used to maintain health. Oleuropein has numerous health benefits, including antioxidant, antimicrobial, anti-inflammatory, antiatherogenic, anticarcinogenic, antiviral activities, cardio- and neuroprotective, hepatoprotective effects. The aim of this study was to determine and understand the mode of action of oleuropein, the cell death mechanisms caused by oleuropein in L.tropica promastigotes. In this study, the phenolic and flavonoid content of oleuropein was determined by HPLC method. The antioxidant capacity and the amount of oleuropein were determined. Afterwards, morphological and physiological (mitochondrial membrane potential, formation of reactive oxygen species, Annexin V binding) changes triggered by oleuropein in L.tropica promastigotes were investigated using flow cytometry. Our studies revealed that apoptotic properties such as mitochondrial dysfunction, production of reactive oxygen species, flip-flop action of phosphatidylserine could induce cell death in L.tropica promastigotes. It has been observed that oleuropein induced typical apoptotic morphological features in L.tropica promastigotes. Total phenolic content and total flavonoid content values of oleuropein extract were determined as 33 mg/g and 229 mg/g. The radical removal method was used to investigate the antioxidant capacity of methanol extracts against free radicals. Total antioxidant content of oleuropein extract was determined as 87%. In addition, the amount of oleuropein in the oleuropein extract was determined as 21. 1% by HPLC. The oleuropein dose that killed 50% of L.tropica promastigotes, that is the IC50 value, was detected as 46.6 µg/mL after 24 hours. It was observed that the parasites in the control group preserved their typical morphological features with a single nucleus, flagella, kinetoplast and narrow cell body at both 24 and 48 hours. It was observed that as oleuropein concentrations increased, the and kinetoplasts of L.tropica promastigotes could not be distinguished from each other, they moved away from the narrow cell body structure, they lost their flagella and turned into a round form, and they moved away from the typical form of the parasite. The percentage of Annexin V+ apoptotic cells was found to be 2.9 ± 0.4% in the untreated control group, and 38.1 ± 6.9% in the oleuropein-treated group. Polarization in the mitochondrial membrane of healthy promastigotes caused an approximately 1.7-fold change in the direction of depolarization in oleuropein-treated promastigotes. According to these findings, oleuropein triggered mitochondria-related death in L.tropica promastigotes. Moreover, 1.4 ± 0.2 fold increase in reactive oxygen species production was detected in oleuropein-treated promastigotes compared to the untreated control group. Comparisons between groups were made using the independent sample t test method. In conclusion, phenolic compounds of olive leaf extract oleuropein induced apoptotic cell death in L.tropica promastigotes. Our results support that olive products such as oleuropein may have anti-parasitic effects.

[An In Vitro Study on Sonodynamic Therapy of Leishmania tropica Using Curcumin]. / Kurkumin Kullanilarak Leishmania tropica'nin Sonodinamik Tedavisi Üzerine Bir In Vitro Çalisma.

Leishmaniasis is an infectious disease that is transmitted by Phlebotomus, 400 thousand new cases appearing every year, and approximately 350 million people are at risk, and accepted by the World Health Organization as one of the six important tropical diseases. Cutaneous leishmaniasis is a disease that occurs on exposed areas of the body and is characterized by long-term non-healing skin lesions. Although the treatment methods applied today vary according to the clinical picture of the patient, the immune system of the person and the causative agent Leishmania species, there is still no standard treatment scheme that has few side effects and can be used in the treatment of leishmaniasis. Therefore, alternative treatment methods with less side effects are being tried. Sonodynamic therapy (SDT) has also emerged as an active antimicrobial research area in recent years. SDT, a new modality for antibacterial therapy, aims to increase antibacterial effects with the simultaneous combination of low-intensity ultrasound and sonosensitizer. There is no information in the literature about the effect of SDT on parasites. In this study, it was aimed to demontrate the anti-leishmanial effect and possible mechanisms of curcumin mediated SDT on L.tropica promastigotes in vitro. Parasites were incubated with 0.25, 1.0, 4.0 and 15.6 micromolar (µM) of curcumin for one hour and subjected to 1 MHz frequency, 50% duty cycle and 3 W/cm2 intensity ultrasound irradiation. XTT assay was used to evaluate the viability of the cells and morphological changes were analyzed by Giemsa staining. Flow cytometry was used to quantify the fluorescence emitted by intracellular reactive oxygen species (ROS) signal, JC-1, cell cycle, Annexin V/PI staining reagents. With the combination of curcumin (15.6 µM) and ultrasound (3 W/cm2 intensity, seven minutes), L.tropica promastigote viability was found to be significantly decreased compared to the control group. Giemsa staining results showed that 15.6 µM curcumin mediated SDT induced several morphological alterations in L.tropica promastigotes typical for apoptosis. Late apoptosis was observed in 15.6 µM curcumin combined SDT treated parasites according to Annexin/PI staining. Besides, curcumin mediated SDT caused mitochondrial membrane potential (∆á´ªm) loss. Cell cycle analysis data indicated that curcumin based SDT caused an subG1 arrest in the cell cycle of L.tropica promastigotes. The generation of intracellular ROS detected by flow cytometry was increased in L.tropica promastigotes treated with curcumin mediated SDT. This study provided new data elucidating the molecular mechanism underlying the anti-leishmanial effect of curcumin mediated SDT. Curcumin mediated SDT has the potential to inactivate L.tropica promastigotes. However, further testing with amastigote or animal models is needed.

In vitro Evaluation of the Effect of Sonodynamic Therapy Using Rose Bengal on Leishmania tropica Promastigotes'

Objective: There is a need for new treatment options for treating Leishmaniasis, since there is no standard treatment scheme with few side effects. Sonodynamic therapy (SDT) is also a candidate to be one of these options. SDT is a treatment method based on the simultaneous combination of low-intensity ultrasound and a sonosensitizer, and the generation of reactive oxygen species in cells in the presence of molecular oxygen. Sonosensitizer, ultrasound, and molecular oxygen individually, these components are not toxic, but when combined form cytotoxic reactive oxygen species In this study, we evaluated the effect of rose bengal (RB)-mediated SDT on Leishmania tropica (L. tropica) promastigotes. Methods: SDT was performed using different concentrations of RB (20, 40, and 80 µM) and ultrasound at a frequency of 1 MHz with an intensity of 1, 1.5, and 2 W/cm2 for 10, 20, and 30 min. Results: Incubation with different RB concentrations applied alone had no effect on L. tropica promastigotes. Ultrasound application time for L. tropica promastigotes alone was determined as 10 min. Ultrasound application intensity showed more significant results at 2 W/cm2. It was determined that the number of promastigotes was lower than that of the control group after 10 min of exposure to ultrasound at 2 W/cm2 at 1 MHz frequency for 10 min with RB (80 µM). Morphologically, round, swollen, atypical forms of the parasite with indistinguishable nuclei are observed, but typical narrow cell body forms have also been detected. Conclusion: These results showed that RB-mediated SDT on L. tropica could be a candidate treatment approach. This approach can be used for both superficial and deeply located lesions. This study emphasized the biophysical mechanisms, ultrasound exposure strategies, reliability and difficulties in the clinical practice of RB-mediated SDT on L. tropica promastigotes.